Molecular Characterization of Prostate Hyperplasia …...Molecular Characterization of Prostate...

Molecular Characterization of Prostate Hyperplasia in Prolactin Transgenic Mice

Karin Dillner

Department of Physiology and Pharmacology Sahlgrenska Academy, Göteborgs University

Sweden 2003

All previously published papers were reproduced with permission from the publishers. Printed by Svenska Tryckpoolen AB © Karin Dillner, 2003 ISBN 91-628-5652-9


- 1 -

ABSTRACT Benign prostatic hyperplasia (BPH) and prostate cancer are age-related diseases, affecting a majority of elderly men in the western world, and are known to be influenced by several different hormones, including sex hormones. Although the hormone prolactin (PRL) is well known to exert trophic effects on prostate cells, its involvement in the pathophysiology is still poorly characterized. In order to evaluate the potential role of PRL in promoting prostate growth, we used PRL-transgenic mouse models that develop prostate phenotypes.

The Mt-PRL transgenic mouse model, ubiquitously overexpressing the rat PRL transgene, develops a dramatic prostate hyperplasia with concurrent chronic hyperprolactinemia and elevated serum androgen levels. In a castration and androgen-resubstitution study, we demonstrated that supraphysiological serum androgen levels are not required for the progress of prostate hyperplasia in adult Mt-PRL transgenic mice. Furthermore, androgen treatment does not induce prostate hyperplasia in wildtype mice. To address the role of local PRL action in the prostate, a new transgenic mouse model (Pb-PRL) was generated using the prostate-specific probasin minimal promoter to drive expression of the rat PRL gene. The androgen-dependency of the probasin promoter resulted in onset of the PRL transgene expression at puberty. The Pb-PRL transgenic mice also develop a significant prostate hyperplasia, evident from 10 weeks of age and the hyperplasia increases with age. In contrast to the Mt-PRL transgenic mice, the Pb-PRL transgenic mice display normophysiological serum androgens levels throughout animal life span. The prostates of both the Mt- and Pb-PRL transgenic mice display a prominent stromal hyperplasia with mild epithelial dysplastic features, leading to an increased stromal/epithelial ratio. Accumulation of secretory material is also a major characteristic. Immunohistochemistry analysis of both the PRL transgenic models’ prostates showed an increased androgen receptor distribution in both the epithelial and stromal cells. Microdissections demonstrated an increased ductal morphogenesis in the Mt-PRL prostate compared to Pb-PRL and controls, indicating that PRL stimulates, directly or indirectly via increased androgen action, prostate ductal morphogenesis in the developing prostate gland. The use of differential gene expression technologies enabled characterization of the molecular mechanisms involved in the prostate hyperplasia. Of particular interest is the potential significance of reduced apoptosis for the development/progression of the prostate phenotype. This finding was further confirmed by immunohistochemical analysis using two different apoptosis markers. Moreover, in line with the prominent expansion of the stromal compartment, were the identified changes in gene expression seen in the PRL transgenic prostate, suggesting that activation of the stroma is important for the development of the prostate hyperplasia.

Altogether, there are histological and molecular similarities between the prostate hyperplasia of PRL-transgenic mice and human prostate pathology, including both BPH and prostate cancer. Key words: Prolactin-transgenic, mouse, prolactin, prostate hyperplasia, gene expression analysis

Karin Dillner

- 2 -

TABLE OF CONTENTS ABSTRACT....................................................................................................... 1 TABLE OF CONTENTS................................................................................. 2 ORIGINAL PAPERS ....................................................................................... 4 LIST OF ABBREVIATIONS.......................................................................... 5 INTRODUCTION ............................................................................................ 6

The Prostate Gland...........................................................................................6 Prostate development ....................................................................................6 Prostate anatomy and structure in human and rodents.................................7

Prostate disorders .............................................................................................8 Benign Prostatic Hyperplasia........................................................................8

Possible theories of BPH etiology .................................................................9 Premalignant lesions of the prostate...........................................................11 Prostate carcinoma ......................................................................................11

Possible theories of prostate cancer etiology .............................................12 Prolactin...........................................................................................................13

Gene, structure, and variants.......................................................................13 Control of prolactin synthesis, secretion and regulation............................15 The prolactin receptor .................................................................................15 Prolactin signal transduction.......................................................................16

Action of prolactin in the prostate gland .....................................................16 Proliferation.................................................................................................17 Apoptosis.....................................................................................................17 Citrate production........................................................................................18

Prolactin in prostate pathophysiology .........................................................18 Prolactin in human prostate cancer and BPH.............................................18 Experimental animal data ...........................................................................19

Rodent models of prostate disease................................................................20 Transgenic prostate hyperplasia models.....................................................21 Rodent models of prostate cancer...............................................................21 Other genetically engineered mouse models with prostate phenotype .....22

Mouse models genetically engineered in the prolactin signaling pathway22 Mouse models genetically engineered in other hormones..........................23

Hormone/growth factor regulation of the prostate ....................................24 Action of androgens in the prostate............................................................24 Interactions between prolactin and androgens in the prostate gland.........25 Action of estrogens in the prostate .............................................................26 Interactions between prolactin and estrogens in the prostate gland ..........27 Action of other peptide hormones and growth factors in the prostate ......27

Functional genomics in the study of the prostate gland ............................28 AIMS OF THE THESIS ................................................................................ 29 METHODOLOGICAL CONSIDERATIONS ........................................... 30

Transgenic animals.........................................................................................30


- 3 -

Approaches to gene expression analysis ......................................................32 cDNA representational difference analysis (RDA) ...................................32

Sequence analysis ........................................................................................ 34 cDNA microarray analysis .........................................................................35

Array design and Printing ........................................................................... 36 Target preparation....................................................................................... 37 Hybridization ............................................................................................... 37 Image analysis and Normalization.............................................................. 38 Data Analysis and Statistical Evaluation.................................................... 39 Experimental design .................................................................................... 40 Microarray databases.................................................................................. 41

Comparisons between cDNA RDA and cDNA microarray analyis .........42 Verification strategies .................................................................................43

cDNA microarray analysis .......................................................................... 43 Real-time RT-PCR ....................................................................................... 43

Assessment of apoptotic activity ...................................................................46 RESULTS AND TECHNICAL COMMENTS........................................... 47

Paper I..............................................................................................................47 Paper II ............................................................................................................49 Paper III...........................................................................................................53 Paper IV...........................................................................................................55

DISCUSSION .................................................................................................. 58 CONCLUSIONS ............................................................................................. 67 ACKNOWLEDGEMENTS........................................................................... 68 REFERENCES................................................................................................ 70

Karin Dillner

- 4 -

ORIGINAL PAPERS This thesis is based on the following papers, which are referred to in the text by their Roman numbers (I-IV);

I. Kindblom J, Dillner K, Ling C, Törnell J and Wennbo H Progressive Prostate Hyperplasia in Adult Prolactin Transgenic Mice is Not Dependent on Elevated Androgen Serum Levels. Prostate 2002 Sep 15;53(1):24-33.

II. Dillner K, Kindblom J, Flores-Morales A, Pang ST, Törnell J, Wennbo H and Norstedt G Molecular Characterization of Prostate Hyperplasia in Prolactin-Transgenic Mice Using cDNA Representational Difference Analysis. Prostate 2002 Jul 1;52(2):139-49.

III. Kindblom J, Dillner K, Sahlin L, Robertson F, Ormandy CJ, Törnell J and Wennbo H Prostate Hyperplasia in a Transgenic Mouse with Prostate-Specific Expression of Prolactin Endocrinology, 2003, in press.

IV. Dillner K, Kindblom J, Flores-Morales A, Shao R, Törnell T, Norstedt G and Wennbo H Gene Expression Analysis of Prostate Hyperplasia In Mice Overexpressing the Prolactin Gene Specifically in the Prostate. Submitted for publication.


- 5 -

LIST OF ABBREVIATIONS -/- aa AP AR BPH cDNA Cy DLP DP DP1, 2, 3 ECM ER EST FDR GH hPRL LP MMP mRNA Mt-1 Mt-PRL Pb Pb-PRL PIF PIN PL PRL PRLR PSA RDA rPRL RT-PCR SAM ssDNA Stat SMA TUNEL TURP UGM UGS UTR VP

homozygous gene-deficiency amino acids anterior prostate androgen receptors benign prostatic hyperplasia complementary deoxyribonucleic acid cyanine dorsolateral prostate dorsal prostate difference products 1, 2, 3 extracellular matrix estrogen receptor expressed sequence tags false discovery rate growth hormone human PRL lateral prostate matrix metalloproteinase messenger ribonucleic acid metallothionein-1 gene The metallothionein-1 promoter - rat prolactin gene probasin gene The minimal probasin promoter - rat prolactin gene prolactin inhibiting factors Prostatic intra-epithelial neoplasia placental lactogen prolactin prolactin receptor prostate specific antigen representational difference analysis rat prolactin reverse transcription polymerase chain reaction Significance Analysis of Microarrays single stranded DNA signal transducers and activators of transcription Statistics of Microarrays Analysis terminal deoxynucleotidyl transferase dUTP nick end labeling transurethral resection of the prostate urogenital mesenchyme urogenital sinus untranslated region ventral prostate

Karin Dillner

- 6 -

INTRODUCTION THE PROSTATE GLAND The prostate gland is an exocrine gland that is found only in mammals. The main function of the gland is to produce a major fraction of the seminal fluid, including enzymes, amines, lipids and metal ions. One unique function of the prostate gland is the capacity to produce, accumulate and secrete high levels of citrate [1]. The prostate varies in its anatomy, biochemistry and pathology between different species. The mature mammalian prostate is a glandular organ consisting of epithelial and stromal cell types that are hormonally regulated. The epithelium consists of a single layer of polarized columnar epithelial cells together with basal and neuroendocrine cells. The epithelial cells supply secretions that empty through ducts into the urethra to form the major component of the seminal plasma of the ejaculate. The surrounding stromal compartment comprises of fibroblasts, smooth muscle cells and loose collagenous extracellular matrix (ECM), in addition to neuronal, lymphatic and vascular components. Interest in understanding the biology of the prostate has largely been driven by the high incidence of prostate diseases, including benign prostatic hyperplasia (BPH) and prostate cancer. PROSTATE DEVELOPMENT The development of the male reproductive tract is dependent upon androgens and mesenchymal-epithelial interactions [2]. The initial event in prostatic morphogenesis is the outgrowth of solid cords of epithelial cells, so-called prostatic buds, from the urogenital sinus epithelium into the surrounding urogenital sinus mesenchyme. In rodents, this occurs in a precise spatial pattern that establishes the lobar subdivisions of the prostate [2, 3]. In rodents, the critical time period for ductal budding and the consequent process of ductal growth and branching initiate around day 15 of gestation and conclude approximately 4-5 weeks postpartum [4-6]. The branching morphogenesis is almost entirely complete by 2 weeks after birth in the mouse [4]. At this time, serum testosterone levels are still low and the increase in prostatic wet weight is modest. As shown by neonatal castration studies, the neonatal prostatic ductal morphogenesis is sensitive to, but does not require, chronic androgen stimulation [7]. The prepubetal growth of the prostatic ductal network is considered non-uniform, where the growth is


- 7 -

highest in the distal region, at the ductal tips, and much lower in the proximal region closest to the urethra [8, 9]. At puberty, the testosterone levels raise significantly, and the rodent prostatic wet weight and DNA content increase more rapidly [7]. In contrast, the human prostate morphogenesis occurs entirely during the fetal period, with ductal development primarily occurring in the first half of gestation [10]. PROSTATE ANATOMY AND STRUCTURE IN HUMAN AND RODENTS There are fundamental differences between the prostate anatomy in human, dog and other primates and non-primates, e.g. rodents. The human prostate is associated with the urethra contiguously below the urinary bladder and prostatic ducts emanate from the urethra radiating towards the periphery completely surrounding the urethra. The adult human gland can be divided into four zones based on morphology; the anterior fibromuscular stroma, the central zone, the peripheral zone and the transition zone. The two latter are of more clinical interest because prostatic carcinoma arises nearly exclusively from the peripheral zone and BPH from the transition zone [11]. In contrast, the process of branching morphogenesis in rodents ultimately gives rise to three distinct bilaterally symmetrical prostatic lobes: the anterior prostate (AP; also known as the coagulating gland), the dorsolateral prostate (DLP), and the ventral prostate (VP). The DLP is sometimes further divided into the dorsal prostate (DP) and the lateral prostate (LP). Individual lobes are located in specific positions around the urethra, but not completely circumscribing it [2]. This explains why rodents, in contrast to most humans, do not suffer from urinary tract symptoms following prostate enlargement. The ducts of each of the rodent prostatic lobes have a characteristic branching pattern [4]. The VP and LP lobes are attached to the urethra by two or three main ducts that show extensive so-called “oak tree” branching, whereas the DP lobe demonstrates multiple main urethral ducts with less extensive so-called “palm tree” branching morphology [4]. Furthermore, the ductal system also shows regional variation in morphology and functional activity [12] and therefore ductal system of each lobe can be further subdivided into regional segments, defined as proximal, intermediate and distal with respect to the urethra [13]. The VP has no clear homologous counterpart in the prostate of higher animals, whereas the DLP are considered the most homologous to the human prostate [14, 15]. The prostate tissue can be divided into epithelial and stromal parts and the proportion between epithelial and stromal compartments differs between species. In the adult rat the stromal:epithelial ratio is 1:5, whereas in humans,

Karin Dillner

- 8 -

the stromal and epithelial cells are present in approximately the same number in the normal prostate [16, 17]. Another important species difference between rodent and human prostate is the presence of the androgen-regulated serine protease, prostate specific antigen (PSA) in human. PSA is produced by both prostate epithelial cells and prostate cancer cells and is the most commonly used serum marker for prostate cancer as well as to monitor responses to therapy. Genes related to human PSA have been detected in several non-human primate species, but not in other mammalian species, including rodents [18]. PROSTATE DISORDERS Prostate gland disorders are age-related diseases affecting a majority of elderly men in the western world. Among mammals with a prostate gland, humans and dogs are the only species known to suffer from BPH and prostate carcinoma [19]. BENIGN PROSTATIC HYPERPLASIA BPH is characterized as a slow, progressive enlargement of the prostate gland, which eventually causes obstruction and subsequent problems with urination. However, BPH is believed to be neither a premalignant lesion nor a precursor of prostate cancer. The incidence of BPH is increasing dramatically with age from about 50% at 50 year of age to 90 % by the ninth decade of life [20]. The BPH progression is characterized by hyperplasia of both the stromal and epithelial compartments. When calculating the stromal:epithelial ratio, clinical reports have firmly established a dominance of the stromal compartment in BPH tissues, which is in contrast to the balanced epithelial and stromal distribution in normal prostate tissue [21-24]. Furthermore, in symptomatic BPH patients the stromal:epithelial ratio has been reported to be significantly higher than in asymptomatic patients [22]. Testosterone is the principal circulating androgen. In men, it is secreted primarily by the testis, with the adrenal glands providing a minor contribution. To be maximally active in the prostate, testosterone must first be converted to dihydroxy testosterone (DHT) by the enzyme 5-alpha reductase. DHT is about five times more potent as an androgen within cells than testosterone, and it binds readily to the androgen receptors (AR) in the nucleus. Androgens are clearly required for development of BPH and


- 9 -

reduction of androgenic effects through 5-alpha reductase inhibitors is utilized in the pharmacotherapy of BPH. Treatment with 5-alpha reductase inhibitors rapidly reduces DHT serum levels and over time results in an average decrease in prostate volume [25]. In addition, alpha-1 adrenoreceptor antagonists are increasingly used, either given in combination with a 5-alpha reductase inhibitor or separately [26]. The mechanism of action of the alpha-1 adrenoreceptor antagonists is primarily to reduce the contractility of the smooth muscle cells in the bladder neck and prostatic urethra which result in an improved urinary flow. The traditional surgical techniques such as transurethral resection of the prostate are still appropriate for some patients, although with improved medical treatments now available, the number of men undergoing surgery is most likely declining [27]. Possible theories of BPH etiology

Despite of BPH’s obvious importance as a major health problem, little is known in terms of the biological processes that contribute to the pathogenesis of BPH. However, a number of theories have been proposed over recent years to explain the etiology of the pathological phase of BPH and the most typical will be described briefly below. Although they may show some degree of contradictions, they most likely contribute together to the pathogenesis of BPH. One of the theories, the dihydroxy testosterone theory, was originally based on the failure of BPH to develop in men castrated prior to puberty. Although controversy still exists, a decreased testosterone/DHT ratio, due to both decrease in plasma testosterone levels and possibly an increase in DHT levels, in elderly men with BPH, may be involved in the etiology of BPH [28, 29]. DHT levels in BPH may be higher than in normal prostate tissue. The local levels of DHT may be increased by age, testicular endocrine function declines steadily with age and at 75 years of age, mean plasma testosterone levels are reported to be around 65% of levels in young males [30] and the decrease in bio-active (non sex hormone-binding globulin (SHBG)-bound) testosterone levels is even more pronounced [31]. This is likely due in part to the recognized increase in SHBG binding capacity associated with ageing. [32-34]. The DHT theory proposes that there is a shift in prostatic androgen metabolism that occurs with aging, which leads to an abnormal accumulation of the more potent DHT in the prostate, thus producing the enlarged prostate. Although the level of DHT in BPH tissue might not be elevated compared to normal tissue, it is very likely that the 5-alpha-reductase activity and AR levels are greater in BPH tissue than in controls. It is the binding of DHT to the AR which is important in

Karin Dillner

- 10 -

stimulating cell proliferation, and prostatic cells may therefore gradually become more and more sensitive to androgens with ageing [35]. Moreover, the reduction in prostate size upon suppression of androgen-mediated action, either by blocking secretion of circulating testosterone and adrenal androgen, inhibiting 5 alpha-reductase to prevent DHT formation, or blocking DHT binding to AR, have further proved the DHT theory [25, 36]. Another theory, the embryonic reawakening theory, was originally based on BPH histopathological features from which McNeal concluded that the prostate stroma undergoes an “embryonic reawakening”, resulting in inductive effects of the local stroma, which in turn induces hyperplastic changes in the epithelium through stromal-epithelial interactions. Somehow a shift of stromal-epithelial interactions occurs with aging, which leads to the inductive effect on prostatic growth. A further theory is the stem cell theory [37]. The stem cell is a proliferative cell but the number of these cells within the prostate is unknown but believed to be very low. The normal behavior of stem cells include: (i) relatively undifferentiated; (ii) their numbers are preserved; (iii) unlimited proliferating potential; (iv) easily adapt to the environment; and (v) finally, but maybe the most important, they are pluripotent, which means that they can give rise to a number of different cell types. According to this theory, BPH could occur as a result from changed properties of the stem cells giving rise to a clonal expansion of cell populations [38]. One more theory, the estrogen-androgen imbalance theory, suggests that an age-associated imbalance between circulating estrogens and testosterone plays a role in the pathogenesis of BPH [39]. In humans, the serum testosterone and free-testosterone levels decrease with age, but the serum estradiol level is constant throughout life. Therefore, with age, creating an estrogen-dominant status compared to that at younger ages. These endocrine changes at mid-life have been extensively investigated through the past 30 years, and are commonly referred to as the “andropause” [40]. This results in a gradual, but significant, increase in the ratio of estradiol/testosterone in the serum [41]. Estrogen plays an important role in prostate pathophysiology (for more information, see section “ACTION OF ESTROGENS IN THE PROSTATE”). An additional theory, the reduced apoptosis theory, suggests that a reduced rate of apoptosis is involved in the etiology of BPH [42], based on the observations of reduced apoptotic activity in BPH tissue compared to control [43, 44]. A homeostasis appears to exist after the prostate has reached its adult size, whereby the rates of prostatic cell growth and prostatic apoptosis are in equilibrium. This ensures that neither involution nor overgrowth takes


- 11 -

place, so that prostate size is constant. The reduced apoptosis theory suggests that the increased prostate volume in BPH is a function of a decrease in the rate of cell death perhaps in parallel with an increase in cell proliferation. PREMALIGNANT LESIONS OF THE PROSTATE Prostatic intra-epithelial neoplasia (PIN) is associated with various alterations in prostatic cellular architecture such as dysplastic foci present in the prostatic ducts and acini [45]. Other histological or biological changes that have been reported include: decreased secretory differentiation, nuclear and nucleolar abnormalities, neovascularity, increased proliferative potential and genetic instability with variation of DNA content. Based on the morphological features, PIN can be divided into low and high grades. PIN is most commonly found in the peripheral zone of the human prostate. Genetic events in PIN have been linked to the development of prostatic carcinoma. However, detailed analysis of the genetic alterations in PIN and matched cancer samples has been limited by the small size of foci of PIN, as well as by the marked morphologic heterogeneity and multi-focality of both lesions [46, 47]. Although, it seems like high-grade PIN is a precursor lesion to prostate carcinoma, the lack of adjacent high-grade PIN in many early cancers indicates the contradictory. PROSTATE CARCINOMA Prostate carcinoma remains one of the most common malignant diseases and is a leading cause of cancer-related deaths among men in the industrialized world. However, the vast majority of men harboring pathologic evidence of prostate cancer are not clinically diagnosed with this disease and it is far more common to die with prostate cancer than as a direct result of the disease. The development of new capillary blood vessels (angiogenesis) might well be one of the first steps in cancer progression. This may be induced by the abnormal tumor expression of growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF) [48]. Further tumor progression and eventual metastasis may result from the fact that malignant cells are less adhesive to one another than normal cells. Cadherins are cell surface glycoproteins that are required for cell adhesion. Changes in the gene which controls cadherins could well be involved in progression and metastasis [49]. Extension of the tumor into the ECM is probably a complex alteration involving mediators between the malignant cells and the adhesive proteins of the ECM, e.g. integrins and fibronectin [50].

Karin Dillner

- 12 -

Possible theories of prostate cancer etiology

The exact cause of prostate cancer is unknown, and part of the problem is the variability and heterogeneity of the tumor within the prostate gland. However, the most established risk factors for development of prostate cancer include ageing, race, diet and a family history of prostate cancer. In addition, a number of theories for its pathogenesis have been suggested over recent years and together these theories most likely contribute to development of the disease. One theory suggests an imbalance in growth regulation in the prostate. As in BPH, stromal-epithelial interactions and growth factors may also play a role in the pathogenesis of malignant disease of the prostate. These important local regulatory factors are involved in a balance which controls, not only cell growth, but also apoptosis. Inappropriate regulation of growth factors, which are produced not only by the target cells themselves, but also by neighboring cells, could develop a significant imbalance which, if prolonged, would be an important step in the genesis of the cascade of events which ultimately leads to prostate cancer. Another possible theory proposes that the stroma undergoes an activation process, resulting in a formation of a so-called reactive stroma. There are considerable evidence that neoplastic stroma is different from the stroma of normal tissue. In an effort to maintain tissue homeostasis, the stromal compartment reacts to tumorigenic epithelium in a process similar to the generation of granulation tissue in wound repair stroma [51]. This activation of the stroma, resulting in a so-called “reactive stroma” and includes phenotypical changes of the stroma cells to a more myofibroblast-like phenotype (transient form between fibroblasts and smooth-muscle cells). The formation of reactive stroma is known to occur in many human cancers, including prostate, and is likely to promote tumorigenesis [52]. Furthermore, it is characterized by ECM remodeling, elevated protease activity, increased angiogenesis and an influx of inflammatory cells. An additional theory involves the possibility of genetic instability in the growing tumor. This genetic instability refers to accumulation of several genetic defects that can occur either at the nucleotide level (e.g., insertion, deletion, or base substitution) or at the chromosomal level (such as, loss or gain of an entire chromosome or small portions) [53]. The genetic instability may result in the stimulation of proto-oncogene and/or inactivation of tumor suppressor genes. Carcinogenesis may develop when the genetic restraint and control in the growth of the cell is lost. Abnormal intracellular behavior can be


- 13 -

induced by oncogene activation or by a change in activity or character of the tumor suppressor genes. Proto-oncogenes are normal cellular genes involved in the regulation of growth and cellular differentiation, for example c-ras and c-myc. The simultaneous activation of these oncogenes could override the inhibitory restraints of neighboring cells and allow tumor proliferation. In parallel with oncogenes, normal cell also contain genes which protect against cancer, so-called tumor suppressor genes, for example the p53 and retinoblastoma (Rb) genes. The normal role of these genes include control of cell division, cell cycle check points and DNA repair, all to reduce and control the proliferation activity of the cells. It is known that loss of these genes may result in cancer, and it seems probable that the prostate tumors that occur in younger men, which appear to have a familial basis, may also be the result of specific gene deletions [53]. Furthermore, it is suggested that increased genetic instability is associated with decreased androgen-responsiveness and progressive behavior of human prostate tumors. Changes may take place which allow the development of androgen-insensitive cells and the death of androgen-sensitive cells. This would provide a further movement away from the modulating influence of androgens on the growth factors associated with normal cell regulation. However, it remains unclear whether this genomic instability is causing the progression of cancer or is the consequence of cancer [53]. PROLACTIN Prolactin (PRL) has classically been considered as a pituitary-derived peptide hormone but over the last decade expression of the PRL gene has also been demonstrated in several extrapituitary tissues [54]. More than 70 years ago, PRL was found to be a pituitary factor that stimulates mammary gland development and lactation in rabbits, but since then PRL has been demonstrated to regulate more than 300 different biological functions, including reproduction, lactation growth, development, metabolism, immunomodulation, osmoregulation and behaviour [55]. GENE, STRUCTURE, AND VARIANTS PRL is a member of the PRL/PL/GH hormone family, to which among others growth hormone (GH) and placental lactogen (PL) also belong to. They all share genomical, structural, biological and immunological features [56, 57]. More recently, this family has been linked to a still more extended family of proteins, referred to as hematopoietic cytokines [58].

Karin Dillner

- 14 -

The gene encoding PRL is unique and is found in all vertebrates [55]. The rat PRL (rPRL) gene is located on chromosome 17, is approximately 10 kb long and composed of five exons and four introns. The human PRL (hPRL) gene is also approximately 10 kb, but located on chromosome 6 and contains an additional exon at the 5'-end [59]. This extra exon is only transcribed in extrapituitary sites, generating a 134 bp longer transcript differing in the 5´-untranslated region, compared to the pituitary transcripts [54]. The mature form of the protein contains 199 residues (23 kDa) and is folded into an all-α-helix protein. Although the tertiary structure has not been determined, PRL is predicted to adopt the four-helix bundle folding described for the GHs [55, 60]. Extrapituitary PRL protein is identical to pituitary PRL, but different promoters drive the expression of PRL in pituitary and extrapituitary sites in humans [61]. Pituitary PRL is controlled by a proximal promoter, which requires the Pit-1 transcription factor for trans-activation. In human, the promoter is divided into a proximal region and a distal enhancer, both of which are necessary for optimal pituitary-specific expression. The pituitary-type promoter and its regulation by dopamine, estrogens, neuropeptides and some growth factors have been well characterized [58]. In contrast, the synthesis of extrapituitary PRL is driven by a superdistal promoter, located 5.8 kb upstream of the pituitary start site. This promoter is silenced in the pituitary, does not bind Pit-1 and is not affected by dopamine or estrogens [60]. The superdistal promoter contains binding sites for several transcription factors but its regulation is poorly understood [62]. The PRL isoform 16K, was discovered more than 20 years ago as the N-terminal 16-kDa fragment resulting from the proteolysis of rat PRL by acidified mammary extracts [63]. The protease responsible for the cleavage of rat PRL into 16K PRL was identified as cathepsin D, whose implication in tumor progression is relevant [64]. 16K PRL was shown to have lost PRLR binding ability but otherwise to have acquired the ability to specifically bind another membrane receptor [65] through which it exerts anti-angiogenic activity [66]. Although this receptor is still not identified, some of its downstream signaling targets have been elucidated [67-70]. Moreover, a PRL-related hormone called proliferin (also known as mitogen-regulated protein (MRP)) [71] has been identified as a growth factor-inducible gene in immortalized mouse fibroblasts [72, 73], but in vivo it is produced primarily by the trophoblast giant cells [74]. Interestingly, reactivation of the proliferin gene expression has been associated with increased angiogenesis, as shown in a cell culture model of fibrosarcoma tumor progression [75].


- 15 -

CONTROL OF PROLACTIN SYNTHESIS, SECRETION AND REGULATION In contrast to what is seen with all the other pituitary hormones, the hypothalamus tonically suppresses PRL secretion from the pituitary. If the pituitary stalk is cut, PRL secretion increases, while secretion of all the other pituitary hormones falls dramatically due to loss of hypothalamic releasing hormones. Dopamine serves as the major inhibiting factor on PRL secretion. Dopamine is secreted into portal blood by hypothalamic neurons, binds to receptors on lactotrophs, and inhibits both the synthesis and secretion of PRL. Agents and drugs that interfere with dopamine secretion or receptor binding lead to changes in secretion of PRL. In addition to tonic inhibition by dopamine, PRL secretion is positively regulated by several hormones, including thyroid-releasing hormone (TRH), oxytocin, gonadotropin-releasing hormone (GrRH) and vasoactive intestinal polypeptide (VIP) [76, 77]. Moreover, estrogens provide a well-studied positive regulation of PRL synthesis and secretion [78, 79]. THE PROLACTIN RECEPTOR The PRL receptor (PRLR) belongs to the class 1 cytokine receptor superfamily and they all share a homology in their extracellular regions, characterized by the conserved cysteine residues and the tryptophan-serine-x-tryptophan-serine motif [55]. The cytoplasmic domain of the PRLR lacks any intrinsic enzymatic activity; however, it includes a proline-rich motif (‘box 1’) that couples to protein kinase signaling molecules which in turn activate downstream effectors. A single PRLR gene exists from which several PRLR isoforms derive. The PRLR isoforms differ in the length and composition and are referred to as long, intermediate or short PRLR with respect to their size. In human, one long, one intermediate and two short isoforms have been identified (reviewed in [55]). In rat, all three isoforms are present, whereas, in mice, one long and three short isoforms have been identified [80, 81]. Regardless of post-transcriptional splicing events, the extracellular ligand-binding domain is identical in all isoforms. The PRLR binds to at least three types of ligands: PRL, PL, and primate GHs [57]. Activation of the cell surface receptor involves dimerization of two PRLR molecules [57], which is mediated by a single molecule of ligand [82]. The ligand binds in a two-step process in which site 1 on the PRL ligand molecule binds to one receptor molecule, after which a second receptor molecule binds to site 2 on the hormone, forming a homodimer

Karin Dillner

- 16 -

consisting of one molecule of PRL and two receptor molecules. [57]. Once bound to one of its ligand, PRLR triggers intracellular signaling cascades. Like all cytokine receptors, PRLR lacks intrinsic enzymatic activity and therefore transduces its signal inside the cell via a wide number of associated kinases. PRLR is virtually expressed in all tissues [55]. However, because of the extremely broad distribution of PRLR, it is currently difficult to propose a general overview of its regulation of expression [55]. PROLACTIN SIGNAL TRANSDUCTION The main and best-known cascades involve the Jak/Stat pathway, the Ras-Raf-MAPK pathway, and the Src tyrosine kinases (e.g. Fyn), but other transducing proteins are also involved [55, 83]. Site-directed mutational studies have identified specific tyrosine residues within the PRLR cytoplasmic domain that can be phosphorylated and participate in recruiting Stats, insulin receptor substrates (IRS), and adaptor proteins to the receptor complex [55]. Depending on the presence or absence of these features, the various PRLR isoforms are expected to exhibit different signaling properties. For example, the short PRLR is not tyrosine-phosphorylated, which prevents this isoform from interacting directly with SH2-containing proteins, such as Stat factors. However, these interactions may also be mediated by certain adaptor proteins [84]. The PRLR signaling pathways can be negatively regulated by protein tyrosine phosphatases, although their mechanism of action is still poorly understood [84, 85]. Recently, the SOCS (suppressor of cytokine signaling) gene family was identified and they function by negatively regulating the Jak/Stat pathway at the level of activation [86]. Finally, another emerging field in PRLR signaling is the occurrence of cross talk with members of other receptor families, such as tyrosine kinases [87, 88] or nuclear receptors [89]. ACTION OF PROLACTIN IN THE PROSTATE GLAND PRL-mediated effects in the prostate are well described and supported by both in vivo and in vitro studies in rodent and human tissues. The presence of PRLR in both human and rodent prostate are well known [90-93]. Moreover, the PRL ligand has been demonstrated to be locally expressed both in human and rat prostate epithelium [93, 94]. The expression of PRL ligand in the rat DP and LP was found to be androgen dependent in vivo as well as in organ cultures [94]. These results could indicate a role for PRL as an


- 17 -

autocrine/paracrine growth factor, regulated by androgen, as well as mediating androgenic downstream effects in the rat prostate. Most of the described PRL prostatic effects have been studied in intact animals. However, several reports indicate that PRL exert many androgen-independent effects [95, 96]. PROLIFERATION In human BPH organ cultures, human primary prostate epithelium and in the androgen refractory human prostate cancer cell lines PC-3 and DU145, PRL has been shown to stimulate growth and significantly increase the cell proliferation rate [93, 97-99]. In one of these studies, DHT, estrogen and progesterone were assessed in parallel with PRL, but they all were found to exert weaker proliferating effects than PRL [99]. Moreover, PRL has been found to up-regulate ornithine decarboxylase (ODC) in the LP of rat. ODC is a rate-limiting enzyme in polyamine biosynthesis, and polyamines have been classified as growth mediators due to their effects on DNA- and RNA-synthesis in somatic cells [100, 101]. Several in vivo studies in rodents, have demonstrated the growth-promoting effects of PRL on the prostate [102-104]. To add to these studies are our own group’s generated PRL-transgenic mice, which develop a dramatic prostate enlargement [105, 106] (see the section RODENT MODELS OF PROSTATE DISEASE). APOPTOSIS The concept of PRL regulation of target tissue size by controlling not only proliferative activity, but also apoptosis, is relatively new. PRL has been shown to significantly inhibit apoptosis in vitro in androgen deprived DP and LP prostate cultures, as assessed by nuclear morphology and in situ DNA fragmentation analysis [107]. This indicates a possible physiological role for PRL as a survival factor for prostate epithelium. In earlier in vivo work, a significant delay of castration-induced regression of the rat LP was noted in pituitary graft bearing animals [95, 108, 109]. In addition, these studies also indicated that AR did not mediate PRL actions on the prostate gland, as evidenced by the failure of flutamide, an AR antagonist, to inhibit the delay in prostatic regression. These results also reveal a lobe-specific response to PRL in the androgen-deprived prostate. Taken together, these observations suggest that in addition to known trophic actions in target tissues, PRL may regulate cell number by prolonging survival through anti-apoptotic mechanisms.

Karin Dillner

- 18 -

CITRATE PRODUCTION The major function of the prostate gland is to accumulate and secrete extraordinarily high levels of citrate. In addition to citrate, the normal and BPH prostate also accumulate the highest levels of zinc in the body. In prostate cancer the capability for citrate production has been found to be lost and the ability for high zinc accumulation diminished [110, 111]. In several different species and model systems, PRL has been shown to androgen-independently stimulate citrate production, by direct regulation on enzymes involved in the citrate production, including mitochondrial aspartate aminotransferase, m-AAT, [112, 113], pyruvate dehydrogenase, PDH E1α [114, 115], m-aconitase [116] and aspartate transporter [14]. In addition, studies have revealed that the accumulation of zinc in the prostate also is regulated by PRL, independently of androgens. PRL increases both cellular and mitochondrial zinc levels of citrate-producing prostate cells [117]. Moreover, the regulation of the ZIP-type plasma membrane zinc uptake transporter has been reported to be regulated by PRL [118]. It is suggested that this ZIP-type zinc transporter is responsible for the ability to accumulate and transport high amounts of zinc in prostate cells. PROLACTIN IN PROSTATE PATHOPHYSIOLOGY Although PRL is well known to exert trophic effects on prostate cells, its role in the development and regulation of the age-dependent disorders, BPH and prostate cancer, is still poorly characterized. In order study the participation of PRL in the regulation of proliferative prostatic disorders several different experimental animal models have been used. PROLACTIN IN HUMAN PROSTATE CANCER AND BPH The role of PRL in human prostate biology and pathophysiology is not well known. The altered endocrine status of aging men is likely to be of importance for development of prostate pathophysiology. Testosterone and GH levels decrease while estrogen levels increase with age. Conflicting data exists whether the circulating PRL levels increase or not with increasing age in the human male [32, 119-122]. Moreover, in a subset of aged men, an increase of TRH-stimulated PRL secretion together with an increase in circulating PRL level have been demonstrated [123, 124].


- 19 -

There is no clear correlation between serum PRL levels and risk of BPH or prostate cancer. More than 20 years ago, a beneficial effect of hypophysectomy in combination with castration compared to castration alone was observed in patients with disseminated prostate cancer [125, 126]. This indicated a role for one or more pituitary hormones, such as PRL, in advanced prostate cancer. Furthermore, significantly higher PRL serum levels have been reported in patients with prostate cancer [127, 128] and patients with BPH [129]. However, other studies report no differences in serum PRL levels between prostatic carcinoma patients and age-matched controls [121, 130]. Moreover, there is evidence that elevated PRL serum levels correlate with poorer prognosis in patients with advanced prostate [131, 132]. Although there are conflicting data, some clinical trials of advanced prostate cancer treatment have indicated a significant improvement in clinical response when combining conventional treatment with PRL suppression [128, 133-136]. There are clinical studies that have indicated increased prostatic tissue levels of PRL in patients with BPH [137] and prostate cancer [138]. Interestingly, PRL serum levels have been reported to transiently decrease following prostatectomy or transurethral resection of the prostate, TURP, [129, 139, 140], indicating loss of local PRL production or a prostatic influence on pituitary PRL secretion. Similar results have been presented in rodents [141]. Using immunohistochemistry, Nevalainen et al. reported local production of PRL in human prostate tissue [93]. Moreover, this study showed the presence of PRLR in the human prostate. The staining of the receptor was localized mainly to the secretory epithelium, but faint staining was also noted in the prostatic stroma. Collectively, these data provide significant support for the existence of an autocrine/paracrine loop of PRL in the human prostate. Furthermore, using in situ hybridization and immunohistochemistry Leav et al [91] demonstrated an increased PRLR expression levels in dysplastic lesions, whereas in lower grade carcinomas the receptor expression levels approximated those found in normal prostatic epithelium. Results from this study suggest that PRL plays a role in the development and maintenance of the human prostate and may participate in early neoplastic transformation of the gland. EXPERIMENTAL ANIMAL DATA Enhanced growth of rodent prostate lobes after pituitary grafting under the renal capsule [102], or local grafting to a specific lobe [103, 104] has been reported. In rat, anterior pituitary grafting to the LP results in significant growth specifically in the LP compared to controls [103]. These results indicated a local direct effect of PRL on the LP. In mice, implantation of a

Karin Dillner

- 20 -

single anterior pituitary into the VP of intact mice results in a significant increase in VP weight and the area occupied by the glands of VP associated with the elevation of circulating PRL. Furthermore, hyperplastic lesions were noted in the grafted prostate lobes of these animals [104]. In another work, hyperprolactinemia has been reported to induce prostatic dysplasia in vivo. Noble rats, treated with testosterone and estradiol-17β2 for a prolonged time period, develop DLP dysplasia, a pre-neoplastic lesion. In these rats, the dysplasia was mediated via estradiol-induced hyperprolactinemia, as evidenced by effective inhibition of dysplastic evolution through the co-treatment of bromocriptin (a dopamine antagonist) [142]. Furthermore, animals which are exposed to a transient increase in PRL secretion prior to puberty have been shown to develop LP inflammation (prostatitis) as adults [143]. A recent study reports that early lactational exposure to atrazine, a toxic agent that suppresses suckling-induced PRL release, leads to altered PRL regulation and subsequent prostatitis in the male offspring. The mechanistic explanation is that without early lactational exposure to PRL (postnatal day 1-9), tuberoinfundibular neuronal growth is impaired and as a consequence prepubertal PRL levels become elevated. This results in higher incidence and severity of LP inflammation in the offspring, evident at 120 days of age [144]. In addition to the abovementioned short PRL-treatment studies, also prolonged treatment of PRL has been shown to induce dramatic enlargement of the prostate as shown in our PRL transgenic mice which ubiquitously express the rat PRL transgene (Mt-PRL) [105] (see the following section). RODENT MODELS OF PROSTATE DISEASE Because the rodent prostate does not spontaneously develop prostate carcinoma and benign hypertrophy or hyperplasia, the usefulness of studying the mouse prostate as a model of human disease is frequently addressed. However, the known heterogeneity of pathological prostate changes in the human prostate gland and the multifaceted nature of prostate disease have prompted the development of less complex, complementary model systems to study the etiology of prostate disease. Both prostate cancer and benign hypertrophy or hyperplasia can be induced in the rodent prostate through genetic modulation or chemical induction and several such models have been established. The advent of transgenic techniques in mice have put increasing


- 21 -

focus on the mouse as a model organism for in vivo studies aiming at understanding gene function and by this gain insights into human pathophysiological conditions. Moreover, the mouse genome project will soon be completed which will enable a direct comparison between the mouse and human genes. TRANSGENIC PROSTATE HYPERPLASIA MODELS Male mice overexpressing the rat PRL gene, Mt-PRL transgenic mice, develop a dramatic enlargement of the prostate gland, which shows similarities prostatic hyperplasia in humans. These animals were generated using a construct consisting of the rat PRL gene under the control of the ubiquitous metallothionein (Mt) promoter, which gives the transgene a general transcription in virtually all cell types. Expression of transgene was detected in all parts of the prostate (DP, VP, LP, AP). The prostate enlargement is mainly characterized by an expansion of the stromal compartment and areas of glandular hyperplasia with accumulation of secretory material [105]. Although dysplastic epithelial features were detected in individual prostates from older PRL-transgenic animals, no development of prostate carcinoma has been observed. The PRL-transgenic animals display, in addition to high serum levels of PRL, approximately a three-fold increase in serum androgen levels compared to wildtype littermates. The degree of prostate enlargement showed no correlation to circulating levels of PRL or testosterone. RODENT MODELS OF PROSTATE CANCER There are several rodent models for human prostate cancer. One of the most well known is the Dunning-3327 rat prostatic adenocarcinoma model [145]. There are several recently established transgenic mouse models for use in prostate cancer studies [146]. The purpose of utilizing these animal models is to identify specific molecular changes in early malignant disease. As the mouse does not spontaneously develop prostate malignancy, different transgenic strategies for in vivo tumor induction have been developed including the use of the the SV40 early genes, such as the tumorigenic T antigen (Tag). Transgenes are usually under the control of a prostate-specific promoter region such as probasin or C3, directing expression to prostate epithelial cells. The transgenic models of prostate cancer can be divided into two main types. The first consists of models resulting from enforced expression of SV40

Karin Dillner

- 22 -

early genes. Two frequently used models are the TRAMP (transgenic mouse model for prostate carcinoma) model and the C3(1)-Tag transgenic model, which utilizes the minimal rat probasin promoter to drive the expression of the Tag gene. In addition, a number of transgenic lines use the long probasin promoter to express large SV40 early genes. These models are well characterized and widely distributed, displaying progressive disease ranging from epithelial hyperplasia or PIN to adenocarcinoma and development of metastases [147]. The second type of transgenic mice utilizes the promoters mentioned above to express various “natural” molecules that have previously been suggested to play a role in development of prostate cancer. The list is extensive but includes c-myc, Bcl-2 and dominant negative transforming growth factor beta (TGFß). Interestingly, the majority of these models only display a relatively mild phenotype, primarily epithelial hyperplasia or PIN. Moreover, these phenotypes usually not arise until the mice are of advanced age. OTHER GENETICALLY ENGINEERED MOUSE MODELS WITH PROSTATE PHENOTYPE Mouse models genetically engineered in the prolactin signaling pathway

Null mutated mice have been generated both for the PRL ligand, PRL-/-[148], and the PRLR-/- [149]. PRL-/- males are reported fertile [148], whereas studies of male PRLR-/- mice have demonstrated both a subset of completely infertile males and a general latency to first successful mating [150]. Moreover, the studies of the prostate gland in PRLR-/- males did reveal only subtle histological alterations and the PRL-/- prostate has not been very well characterized. Taken together, the data from these two knockout mouse models indicate that PRL action is not of essential importance for male fertility and normal anatomical development of the prostate gland. However, studies of more functional aspects of the gland need to be carried out in these animals. PRL can activate several of the Stat proteins, including Stat 1, 3, 5a, and 5b, but the two latter acts as the major mediator [55]. Stat5a-/- and Stat5b-/- knockout mice have confirmed these molecules as the major transducers of PRL signaling in both prostate and mammary gland [151], and also shown similar phenotype to those of the PRL-/- and PRLR-/- knockout mouse models, mainly emphasizing the irreplaceable role of PRL in reproduction and mammary gland development. PRL signaling in rat prostate tissue is primarily transduced via Stat5a and Stat5b, likely supporting the viability of


- 23 -

prostate epithelial cells during long-term androgen deprivation [152]. In the prostate, studies in Stat5a-/- knockout mice have provided evidence for a direct role of Stat5a in the maintenance of normal tissue architecture and function of the mouse prostate [153]. Lack of Stat5a function results in a distinct prostatic phenotype characterized by an increased occurrence of cyst formation with disorganization and detachment of prostate epithelial cells. In addition to PRL, other polypeptide factors, such as GH, insulin-like growth factor I (IGF-I), epidermal growth factor (EGF) and interleukin-6 (IL-6) are known to activate Stat5. Mouse models genetically engineered in other hormones

The AR transgenic mice overexpress the AR specifically in prostate secretory epithelium [154]. The earliest alteration observed in the AR transgenic mouse prostates was an extensive 5-fold increase in the proliferation of secretory epithelial cells, as evidenced by immunostaining of the proliferating marker Ki-67, in the absence of histological abnormalities. Proliferation in these glands was associated with increased apoptosis, possibly accounting for the absence of hyperplasia. Older AR transgenic mice developed focal areas of intraepithelial neoplasia, resembling human high-grade PIN, but no further malignancy has been observed. A certain resistance to malignant transformation in the mouse prostate compared to humans has been suggested. No reports of any tumorigenic effects of exogenously added androgens in these models are available. The recent generation and characterization of the various estrogen modulated mouse models (αERKO, βERKO, αβERKO and ArKO) have provided new insights regarding the role of estrogens in prostate growth and development [155]. A specific direct response to estrogens is the induction of changes in the prostatic epithelium, termed squamous metaplasia [156-159]. Tissue recombinant studies using epithelium and stroma from wildtype and transgenic mice lacking a functional ERα (αERKO) or ERβ (βERKO) have demonstrated that the development of squamous metaplasia is mediated through stromal ERα [160, 161]. Furthermore, a distinct phenotype of focal epithelial hyperplasia in the VP has been reported in aging mice lacking functional ERβ (βERKO) [162, 163], while no apparent prostate pathology or enlargement has yet been reported in αERKO or the double knockout αβERKO [155]. Altogether, these findings indicate an anti-proliferative role for epithelial ERβ and also suggest that an unbalanced stromal ERα in action could contribute to the phenotype observed. The ArKO (aromatase knockout) mouse lacks endogenous estrogen

Karin Dillner

- 24 -

production due to a non-functional aromatase enzyme. In the ArKO mouse, the combined effects of estrogen absence and elevated androgen and PRL levels result in a moderate prostate enlargement with hyperplasia evident in all lobes and tissue compartments [161]. Moreover, an associated up-regulation of epithelial AR was demonstrated in the ArKO mouse and has been suggested to contribute to the observed phenotype. In the absence of endogenous estrogen (ArKO) or ERs (αERKO and βERKO), prostate development occurs normally, suggesting that intact estrogen signaling is not essential for the initiation of neonatal prostate growth. The histological appearance of the prostate hyperplasia in ArKO male mice is strikingly similar to that of the Mt-PRL-transgenic mice. In contrast, the AROM+ mice, which overexpress the aromatase gene, resulting in elevated estrogens levels, combined with significantly reduced testosterone and FSH levels, and elevated levels of PRL and corticosterone [164]. AROM+ males present a multitude of severe structural and functional alterations in the reproductive organs. Furthermore, squamous metaplasia has been seen in the prostatic collecting ducts, consistent with high levels of endogenous estrogens. Some of the abnormalities, such as non-descended testes and undeveloped prostate, resemble those observed in animals exposed perinatally to high levels of exogenous estrogen, indicating that the elevated aromatase activity results in excessive estrogen exposure during early phases of development. HORMONE/GROWTH FACTOR REGULATION OF THE PROSTATE

All lobes are responsive to both estrogens and to androgens, but to varying degrees; the VP is more sensitive to androgens and the AP more sensitive to estrogens [159, 165]. In rat prostate, both testosterone and estrogen have been shown to regulate the level of the long PRL receptor mRNAs in a tissue-specific manner [92]. In addition to steroid hormones, several different growth factors and other pituitary hormones have been shown to regulate cellular growth, differentiation and apoptosis. ACTION OF ANDROGENS IN THE PROSTATE Androgen is a critical factor for the survival of prostatic epithelial cells. Underdeveloped prostate gland is seen in eunuchs who lack androgen stimulation since childhood [166]. Castration-induced androgen-withdrawal


- 25 -

regress the the number of epithelial cells in the prostate gland via an active process of apoptosis [167, 168]. Apoptosis can be observed within one day after castration and nearly 2/3 of epithelial cells are lost in the VP by seven days of castration [169]. In contrast, testosterone replacement to castrated rats stimulates the re-growth of the gland to its normal size via proliferation of new epithelial cells from basal cells [170]. INTERACTIONS BETWEEN PROLACTIN AND ANDROGENS IN THE PROSTATE GLAND PRL has been shown to potentiate the action of androgens in the support and stimulation of prostatic growth and metabolism [171-173]. This has been hypothezised to be accomplished through increasing prostate receptivity to androgens, mainly by affecting AR levels and 5-alpha reductase activity. Results suggest that PRL is involved in regulating AR synthesis, at least partially by direct action on the prostate gland. In immature, hypophysectomized male rats, PRL treatment can significantly increase AR mRNA levels [174]. Findings in adult, castrated and pituitary grafted rats suggest that PRL promotes LP growth via an increase in nuclear AR levels, and thus optimizes tissue response to circulating testosterone [175]. Furthermore, pituitary grafting in immature rats can produce a significant increase in the weight of the seminal vesicles and the VP and AP [176]. In the VP, nuclear AR content increased, whereas the cytosolic AR content decreased, suggesting increased translocation of the AR to the nucleus. In a study on human BPH patients, cytosolic and nuclear levels of AR were shown to be proportional to plasma PRL levels [177]. These findings indicate plasma PRL involvement in the regulation of AR content also in the benign human prostate. Recently the existence of crosstalk between the signal transduction systems of steroid hormones and peptide hormones/growth factors were recognized [178-180] which provides a mechanism for locally produced growth factor influence on AR activation. In the progression of prostate cancer to an androgen-independent state, local growth factors, such as PRL, may prove instrumental in regulation of cell growth. In rat, hyperprolactinemia by pituitary grafting can lead to increased 5-alpha reductase activity in the testis [181] but indications of a PRL-induced increase in 5-alpha reductase activity in the prostate is limited [182]. PRL mediation of steroid uptake through alterations of the plasma membrane permeability in human BPH tissue has also been reported [183].

Karin Dillner

- 26 -

To interpret findings in rodent versus human studies, one needs to be aware of the important differences in influence of PRL on circulating androgen levels. In man, PRL is known to decrease circulating androgen levels through depression of gonadotrophine release from the pituitary gland [184], whereas in rodents, PRL can elevate circulating androgen levels by increasing the response to luteinizing hormone in the testis [185]. ACTION OF ESTROGENS IN THE PROSTATE A hierarchy of estrogen responsiveness in the three prostatic lobes has been revealed in male mice, with the AP being the most responsive, the dorsolateral lobe less responsive, and the ventral lobe the least responsive. [159]. The expression of both known estrogen receptor subtypes in adult human and rodent prostate is now well established, with expression of ERα described primarily in a subset of stromal cells and ERβ restricted to the ductal epithelium [186-188]. Although the newly discovered ERβ shares many of the functional characteristics of ERα, the molecular mechanisms regulating the transcriptional activity of ERβ may be distinct from those of ERα. For example, the growth effects of estrogens during fetal development are mediated primarily by ERβ in the human prostate, which can be immunodetected in the nuclei of nearly 100% of epithelial and in the majority of stromal cells throughout gestation. However, ERα has been shown to contributes to postnatal glandular development [156]. Estrogen plays an important role both in prostate physiology and pathophysiology. The developing prostate is particularly sensitive to estrogenic exposure. During prostate morphogenesis, elevated levels of endogenous (maternal or excess local production) or exogenous (diethylstilbestrol or environmental chemicals) estrogens induce permanent changes in prostate growth in rodents. Fetal and neonatal exposure to estrogens results in pathological and functional changes of the prostate [189]. High-dose of testosterone together with estradiol stimulates prostatic carcinogenesis in adult male rats [190]. In mice, these effects are dose-related as low-dose estrogen exposure may increase the adult prostate size whereas high-dose exposure reduces prostate size [189]. An increase in AR levels has been associated with low-dose estrogen-induced increases in prostate size [190]. Neonatal exposure of rodents to high doses of estrogen is known to permanently imprint the growth and function of the prostate and predispose the gland to hyperplasia and severe dysplasia analogous to PIN with aging [160]. Following neonatal exposure of rats to high doses of


- 27 -

estrogen on days 1-5 of life, a permanent reduction in prostate growth and responsiveness to androgen occurs relative to a reduction in AR expression in adult animals [165]. Moreover, exogenous estrogen administration in adult rodents leads to squamous metaplasia of the AP [157, 159]. As mentioned earlier, development of squamous metaplasia has been shown to be mediated through stromal ERα [160, 161]. INTERACTIONS BETWEEN PROLACTIN AND ESTROGENS IN THE PROSTATE GLAND Estrogen are known to act directly on pituitary lactotrophs and indirectly on the hypothalamic dopaminergic system and several studies suggest that neonatal estrogen treatment can induce long-term alterations in pituitary synthesis and release of PRL [191-193]. Moreover, estrogens are well-known to promote PRL release resulting in elevated PRL levels systemically [78, 79]. It is thus quite possible that the prostate effects of estrogen imprinting are in fact partially PRL-mediated. Furthermore, PRL is able to stimulate expression of both ERα and ERβ in corpus luteum and decidua during pregnancy [194-196] as well as stimulate estradiol binding activity or mRNA levels in the mammary gland [197] and liver [198]. In the prostate, effects of estrogen treatment appear to be in part mediated by increased PRL levels [199], something that is further demonstrated in the aforementioned dysplastic prostate model of estrogen-treated Noble rats [142]. ACTION OF OTHER PEPTIDE HORMONES AND GROWTH FACTORS IN THE PROSTATE Growth factors regulate cellular growth, differentiation and apoptosis. In addition to steroid hormones, an array of positive and negative growth factors controls the balance between cell proliferation and apoptosis in the prostate. Several oncogene products that contribute to neoplastic proliferation have been found to be homologues to growth factors, growth factor receptors, or molecules in the signal-transducing pathways of these receptors. There are numerous growth factor families that have been implicated in normal, neoplastic and malignant prostate growth and it is far beyond this thesis to review the action of all reported hormones and growth factors. The in the literature mentioned growth factors include, the IGF family, EGF, TGF, FGF family, platelet-derived growth factor (PDGF) and VEGF, which all are the main stimulatory regulators of proliferation in the prostate [200]. Furthermore, the pituitary hormones, GH and luteinizing

Karin Dillner

- 28 -

hormone (LH), play physiologically significant roles in the normal prostate, either alone or synergistically with androgens [201]. Nevertheless, the involvement of these hormones in the development of BPH and prostatic carcinoma is an issue that needs to be addressed. The TGF-family is the main inhibitory regulator of proliferation acting on the epithelial cells. However, recent studies have demonstrated proliferative and anti-apoptotic effects of TGFβ in stromal cells [202]. Altogether, the growth factors exert autocrine and paracrine effects upon stromal and epithelial cells and interact with other factors and binding proteins to control prostate growth [203]. FUNCTIONAL GENOMICS IN THE STUDY OF THE PROSTATE GLAND

The network of action of different hormones and growth factors on the prostate gland and their involvement in prostate pathophysiology are unquestionable complex. The recent completion of the human [204], and the draft of the mouse [205], genome sequence together with the improvement of high-throughput technologies, such as gene expression profiling, will hopefully provide a basis for rational determination of which pathways and molecular targets that are appropriate to further study. The unveiling of a detailed genetic map of the main species and models of prostate research promise to dramatically increase our understanding in the genetic basis of prostate disorders together with the basic mechanism of the action and involvement of hormones and growth factors for the induction of prostate disease.


- 29 -

AIMS OF THE THESIS The overall aim of this thesis was to study the consequences of chronic exposure to extraordinary high levels of PRL in the development of prostate hyperplasia as well as to characterize the molecular mechanisms present in the hyperplastic prostates. The specific aims were:

• To investigate the role of circulating androgen in the promotion of prostate hyperplasia in PRL transgenic mice with transgene onset during early prostate development (Paper I)

• To characterize a new PRL-transgenic model of prostate hyperplasia in

which the PRL transgene was overexpressed specifically in the prostate with onset at puberty (Paper III)

• To compare the ductal development in two models of prostate

hyperplasia; one with fetal onset and the other with pubertal onset of the PRL transgene expression (Paper III)

• To evaluate the use of differential gene expression analysis in

characterization of the molecular mechanisms of the prostate hyperplasia in PRL transgenic mice (Paper II and IV)

Karin Dillner

- 30 -

METHODOLOGICAL CONSIDERATIONS TRANSGENIC ANIMALS The establishment of the transgenic technology has introduced new and invaluable techniques to study and understand the function of a specific gene in biological processes. There are basically two types of transgenic animals based on the technique used to generate them. The first method to be established in 1980 was the microinjection technique allowing overexpressing of a gene product by injection of foreign DNA into a one cell mouse zygote [206]. The incorporation of the foreign DNA is completely random using this approach and it is only possible to overexpress a gene product and not to mutate a certain gene. In contrast, the other embryonic stem cell (ES)-cell technique (also known as gene knockout) was established in 1987 and this method made it possible to interact with the mouse genome at a specific position and to mutate a specific gene [207]. A wide range of transgenic and knockout mouse models have now been established and further technical improvements have made both temporal and spatial overexpression/gene deletion possible. These accomplishments have given unique insights into the specific biological properties and functions of specific genes and furthermore provided valuable models for investigating the functional in vivo role of target genes. In this thesis we utilized two different transgenic mice models. The rat PRL transgene where used in both constructs, in parallel with two different promoters to direct spatial (where) and temporal (when) expression of the transgene, resulting in two different PRL transgenic mouse models. In paper I and II, the metallothionein (Mt) promoter was used to drive the PRL transgene. The Mt gene is expressed in virtually all cell types. Activation of the Mt-1 promoter during the early embryonic stage is well described, with abundant expression already by day 12 of gestation reported [208, 209]. Thus, the PRL expression was considered general in the Mt-PRL transgenic mice. In contrast to the general expression of a transgene, a cell-specific promoter can be used that direct the expression of the transgene to a certain cell type. In paper III and IV, the probasin (Pb)-PRL transgenic mice were utilized. The construct of these mice include the minimal Pb promoter to direct the expression of the rPRL transgene to the epithelial cells of the DP, LP, and VP [210]. Pb is an androgen-dependent basic secretory protein, abundantly localized in the lumen and acinal regions of the rat prostate epithelium [211]. Studies have demonstrated that the Pb minimal promoter (458 bp) can target heterologous gene expression specifically to the prostate


- 31 -

in a developmentally and hormonally regulated fashion [210]. In contrast to the Mt-1 promoter, activation of the Pb promoter is androgen dependent, and it is thus activated by the increasing androgen levels seen in late prepubertal stage in the animals [210]. The Pb promoter is consequently not active until after the most essential period of ductal morphogenesis in the neonate prostate gland has occurred [4]. The integration of the transgene in the genome is considered a random event and the number of copies inserted can not be regulated. In the majority of zygotes injected, the integration will occur at a single position on one of the chromosomes. As a consequence, the resulting animal will be heterozygous for the integrated transgene. To rule out the possibility that the transgenic phenotype is being a consequence of a heterozygous mutation introduced by the integration of the transgene, it is preferable to generate more than one line of transgenic animals, allowing comparisons. Identification of transgenic animals takes place in several steps. The founder animals are first identified at the DNA level. Lines of transgenic animals are then generated from founders and expression of the transgene is characterized at RNA or protein level. Detection of transgenic expression in the desired tissues denotes the successful establishment of a new transgenic animal. The founder animals are analyzed at the DNA level by obtaining a tail biopsy at two weeks of age followed by DNA preparation. Either southern blot hybridization or PCR, using one primer located in the promoter and the other in the structure gene of the construct, is used to identify the transgene. Southern blot verification is more reliable and therefore preferred at least in the identification of founder animals. Thereafter, PCR screening is accepted for identification of the transgene in the subsequent transgenic offspring generated from the founder animals. In case of cDNA constructs, it is important to allow discrimination between the mRNA expression generated by the transgene and the contaminating cDNA construct. This can be done by introducing intron sequences in the DNA construct. Moreover, it is important to be able to distinguish between mRNA expression and protein production of the transgene and the corresponding endogenous gene’s products.

Karin Dillner

- 32 -

APPROACHES TO GENE EXPRESSION ANALYSIS During the last ten years, the development of more and more powerful techniques for differential gene expression studies have provided entirely new insights into molecular mechanisms underlying biological processes. In this thesis, we applied two different methods, cDNA RDA and cDNA microarray analysis, to molecular characterize the prostate hyperplasia of PRL transgenic mice and those two methods will be discussed in more detail in the following chapter. CDNA REPRESENTATIONAL DIFFERENCE ANALYSIS (RDA) In paper II, we used the method of cDNA-RDA to identify differentially expressed transcripts in the hyperplastic prostate of the Mt-PRL transgenic mice compared to wildtype control littermates. RDA has been successfully adapted to identify genes that are differentially expressed between two populations of cells [212]. Representative cDNA fragments from each population are first generated by restriction endonuclease digestion of cDNAs followed by PCR amplification. The resulting mixtures, termed ‘representations’, are then subject to successive rounds of subtractive cross-hybridization followed by differential PCR amplification. This leads to progressive enrichment of cDNA fragments that are more abundant in one population than the other. Figure 1 shows a schematic description of the RDA procedure. The PCR products after each RDA round are termed differential products (DP). Theoretically, consecutive DP should contain more stringently selected gene fragments and less noise from non-differentially expressed genes. To allow isolation of both up- and down-regulated genes, both samples are used as tester and driver, respectively, in two parallel procedures. In paper II, we aimed to identify both up- and down-regulated transcripts in the hyperplastic prostates of Mt-PRL transgenic mice compared to controls, and we therefore used both samples as tester and driver, in two parallel procedures. cDNA-RDA is a powerful technique for isolation of differentially expressed genes, but it also has limitations in that not all of the differentially expressed genes are necessarily enriched during the procedure. The lack of four-base pair restriction sites in the messenger RNA may result in the generation of <100% coverage of expressed genes in the representations. In contrast, the restriction fragments may be too big for efficient amplification by PCR. The PCR amplification step to generate the starting representations in the RDA procedure is a very critical step for a successful RDA. In order to generate representations that truly represent the original cDNA pool with respect to


- 33 -

mRNA

cDNA

PRL-transgenicprostate

Controlprostate

Linkerligation

PCR

Representations

Restrictiondigestion

Samples

Tester Driver

Digest and ligatenew linker Digest

Mix, melt and hybridize

Tester : Tester Tester : Driver Driver : Driver

Exponential amplification

Linear amplification

Noamplification

PCR

Repeatprocedure

Figure 1. A schematic description of the cDNA-RDA procedure. The first step is to synthesize cDNA using purified mRNA as template. The double-stranded cDNA is cut with a 4-basepair restriction enzyme. A linker, complementary to the generated overhangs, is ligated onto the cDNA fragments. This generates a pool, which is amplified by PCR, using primers complementary to the linker. This procedure generates a representation and one such representation is made from each of the two mRNA pools to be compared. The linker is then removed, using the same restriction enzyme as before. A new adapter is ligated onto the tester fragments only. The tester is then mixed with driver in excess, the mix is heat denatured and allowed to hybridize. A PCR amplification using primers complementary to the new adapters is performed. During this step, only tester: tester hybrids are amplified exponentially. Tester: driver hybrids are linearly amplified, and can be removed by nuclease treatment. Driver: driver hybrids are not amplified at all. The procedure is then repeated with increasing ratios of fresh driver. After a few rounds, distinct bands can be visualized on an agarose gel. These bands are isolated, and the products are cloned into vectors and characterized. Reproduced from Hubank et al [212].

Karin Dillner

- 34 -

fragment distribution while avoiding a size bias, the PCR needs to be carefully titrated for each sample. The sensitivity of RDA remains to be defined. To allow detection of a transcript, the relative differences in expression levels between tester and driver populations are thought to be the major determinant. The flexibility of the RDA methodology can be employed to overcome this issue, as variation in the stringency of hybridization will influence the detection of small differences in gene expression between tester and driver populations. By lowering the stringency of subtraction (increase the amount of tester cDNA relative to the driver cDNA), cDNA-RDA can enrich for genes with subtle differences in gene expression [212]. However, too much driver cDNA can cause insufficient enrichment of the targets, rendering differences invisible whereas too little driver cDNA may cause insufficient exhaustion of common (but differentially expressed) sequences in the tester cDNA, generating background. The risk of cloning non-differentially expressed genes is obviously also higher in the less stringent enrichment case. Furthermore, the relative expression level of the corresponding gene may affect the degree of enrichment on the cDNA-RDA. Not all differentially expressed genes are equally enriched in the process, which favors fragments with high levels of differential expression, especially if RDA is performed for 3-4 rounds (DP3 and DP4). There is an inverse relation between the degree of enrichment for differentially expressed genes and the complexity of the output of RDA. If RDA is performed for 1-2 rounds, a broader spectrum of differentially expressed genes (including those with lower levels of differential expression) are obtained together with many non-differentially expressed genes, necessitating large-scale screening of the output, for example by using microarray, to remove those transcripts. Sequence analysis

To follow up the RDA output, sequence analysis of RDA clones were performed by routine sequence analysis using cycle sequencing with dye-labeled nucleotides followed by running of purified products on an automated sequencing machine. Subsequently, the Staden sequence analysis package [213] was used for vector clipping, redundancy, and assembly analysis. Sequences were annotated and given an accession number by analyzing for homologies with published sequences in the non-redundant and expressed sequence tags (EST) divisions of the public databases of NCBI (National center for Biotechnology Information) by using the BLAST (N/X) software [214]. More than 85% homology over at least 50 base pairs region was required to annotating sequences based on homology to known genes or


- 35 -

ESTs. Clones that failed to match any existing database entry in BLAST (N/X) search were denoted unknowns. Functional prediction was performed in silico by using the information at UniGene, The Institute for Genomic Research (TIGR)-EGAD, Online Mendelian Inheritance in Man (OMIM), and Medline databases.

CDNA MICROARRAY ANALYSIS The DNA microarray, also called chip, has become an important tool in gene expression studies, monitoring RNA expression levels, but can also be utilized to study mutations and polymorphisms at the DNA level. In this thesis, cDNA micorarray technology was used to characterize the molecular mechanisms of importance for the prostate hyperplasia in PRL-transgenic mice compared to controls. However, we made use of this method in different ways in the two studies. In paper II, we applied the cDNA microarray technology to verify the cloned RDA products, isolated as differentially expressed between the Mt-PRL transgenic prostates compared to controls. In contrast, in paper IV, we used the cDNA microarray technology to screen for differentially expressed transcript in the Pb-PRL transgenic model of prostate hyperplasia. Basically, there are two main types of microarrays. The first type is the one composed of oligonucleotides which are synthesized in situ by photolithography [215]. These chips are also available commercially and form the basis of GeneChip™ technology sold by Affymetrix. The other type of microarray, cDNA microarray, was originally developed by Brown and colleagues at Stanford University [216]. This form of microarrays usually comprises PCR-amplified inserts from cDNA clones representing known genes and ESTs [217]. cDNA microarrays are generally used for comparative analysis where the two samples to be compared are hybridized onto a single chip. In contrast when using Affymetrix arrays, each sample is hybridized on separate arrays. Although, this results in the use of increased numbers of chips, it also provides the advantage that post hoc comparisons not planned in the original experiment can be more easily made. Another advantage of using short oligonucleotide probes on an array is the built-in ability to distinguish close members of a gene family. However, the current Affymetrix oligonucleotide expression platform is still significantly more expensive than cDNA arrays and lack flexibility when it comes to producing custom-designed arrays. Newer platforms using 50-70-mer spotted oligonucleotides allow for rapid array design and implementation. Experience with this technology is still limited, but it may offer the best alternative.

Karin Dillner

- 36 -

The microarray technology is advancing at an impressive rate and this thesis will only describe the most important methodological characteristics. All steps mentioned need to be carefully optimized for the successful application of cDNA microarray analysis. Figure 2 shows a schematic description of the DNA microarray procedure. Array design and Printing

Customized cDNA microarrays are fabricated by first selecting the genes to be printed/immobilized onto the array from public databases/repositories or institutional sources. Control clones can help to validate the microarray-derived data. Selected cDNA clones may be spotted twice at different locations on the chip to serve as “within slide” reproducibility controls. A set of negative controls including repetitive DNA, polyA sequences, genomic DNA and non-cross-reactive gene sequences from different organisms may be utilized to ensure specific hybridization. In addition so-called spiking controls (positive controls) may be used by adding RNA that will hybridize specifically to spots included on the array. High throughput DNA preparation is performed in either 96- or 384-well format by PCR amplification of the selected clones/gene sequences. Subsequently, the DNA is purified by ethanol precipitation and resuspended in an appropriate “spotting” solution. Moreover, the purity of each gene is checked on an

Figure 2. Schematic of microarray experiments. From Duggan DJ et al. [218].


- 37 -

agarose gel. Spotting is carried out by a robot, which deposits a nanoliter of PCR product onto an aminosilane-coated glass slide in serial order to produce circular spots of about 90-200 µm in diameter. Spotted DNA is cross linked to the matrix by ultraviolet irradiation and denatured by exposure to heat. Target preparation

The total RNA or mRNA samples, that are to be compared, are extracted from two tissues or cell groups and labeled with different fluorescent dyes in a reverse transcription reaction generating fluorescent dye-incorporated cDNA. Most often, the cyanine-3, Cy3 (green), and Cy5 (red) dyes are used as they have well separated emission spectra which enable efficient channel separation in the signal detection. The labeling cDNA synthesis reaction is rapid, but the bulky Cy-dye molecules may reduce the incorporation efficiency of labeled nucleotides. In order to eliminate dye specific effects caused by a labeling bias, resulting in an uneven labeling of the two dyes for a specific gene sequences, a dye-swap design is recommended. Each hybridization is then performed twice but with switched colors during labeling. Finally, purification of samples is performed to remove unincorporated dye. This is often performed by spin column purification. The amount of total RNA required for one microarray experiment is currently approximately 15 µg for each sample and this is considered one of the bottlenecks in microarray analysis. Although a number of amplification strategies have been developed, which aim to reduce the amount of starting material, [219-222], the limitations of all these strategies are reproducibility and unbiased amplification which is necessary to preserve the relative expression levels from the two starting RNA samples that are to be compared. Hybridization

Hybridization of the labeled target is ideally linear (i.e. proportional to the amount of labeled targets), sensitive so that low abundance genes are detected, and specific so that probes hybridize only to the desired gene in the complex target mixture. The large size of the cDNA probes is also helpful in enabling stringent hybridization conditions and lowering cross-hybridization of unrelated genes, although closely related gene families will still be able to anneal to some extent. Procedures to reduce background (a step commonly called pre-hybridization) include inactivation of free reactive groups on the glass slide surface before hybridization. This can be performed either by

Karin Dillner

- 38 -

chemical inactivation [223] or by treatment with biomolecules such as bovine serum albumin (BSA) [224] to block the reactive groups. The hybridization temperature and buffer will determine the stringency of the hybridization. Salmon sperm DNA, polyA, tRNA, sodium dodecyl sulfate (SDS), and Cot1 DNA are added to the hybridization to eliminate nonspecific hybridization due to repetitive sequences [225]. After hybridization, the chip is washed in multiple steps, to wash away disturbing particles and loosely bound target DNA. Image analysis and Normalization

The fluorescent signal of the hybridized probes is measured with a laser scanner capable of detecting emission from the Cy3 and Cy5 channels (showing green and red signals, respectively) to monitor the spots where target DNA has bound. Laser intensity and detector gain should be adjusted to yield images with non-saturated spots and approximately similar overall signal intensities for the red and green channels. An overlay of the red and green images will therefore allow a relative comparison, where the intensity of the signals from the two different samples is directly correlated with the original concentration of mRNA in the cell or tissue. Calculation of the expression ratio for each clone (red/green channel), enables the assignment of up-regulated, down-regulated, non-differentially or absent expression. The image processing and subsequent data analysis from the microarray experiments are crucial for extraction of useful information. Image analysis in paper II and IV was performed by using GenePix Pro software. First, a grid describing the array design is aligned on the image to localize and link a clone identity to each spot. The software extracts intensity and background measurement for each probe. Automatic flagging localizes absence of a spot or very weak spots (≤1.4 (paper II) or ≤2 (paper IV) times above background) and manual flagging is used to eliminate artifacts. The value of the signal from each spot is calculated as the average intensity minus the background. To allow for inter-array comparisons, each array needs to be normalized to remove systematic sources of variation. Normalization between the two fluorescent images was performed using ‘LOWESS’ normalization method in the SMA (Statistics of Microarrays Analysis) package [226, 227]. SMA is an add-on library written in the public domain statistical language R [228] and can be used to analyze simple replicated experiments. The LOWESS (Locally Weighted Scatter Plot Smoother) algorithm performs a local fit to the data in an intensity-dependent manner. The intensity value for each spot


- 39 -

is normalized based on data distribution in the immediate neighborhood of the spot’s intensity. Bias in spatially defined sub-sets of the data can also be compensated for by normalization strategies (‘Pin-wise LOWESS’) e.g. when clear biases caused by pin-to-pin variations during array printing or uneven hybridizations are observed. Data Analysis and Statistical Evaluation

cDNA microarrays is now becoming used in a more or less standardized fashion and it has become increasingly clear that simply generating the data is not enough; one must be able to extract meaningful information about the system being studied. Despite the combined efforts of biologists, computer scientists, statisticians and software engineers, there is no one-size-fits-all solution for the analysis and interpretation of genome-wide expression data. There are now numbers of tools available for interpreting the data and choosing among them is challenging. The most basic question one can ask in a transcriptional profiling experiment is which genes’ expression levels changed significantly. Highly abundant genes with great differences in expression will normally not cause any problems as they will display expression ratios above experimental noise and measurement variations. However, for the detection of subtle expression differences and low abundance genes, a statistically justified experimental design and data evaluation is crucial. The many sources of variation in a microarray experiment can be divided into three different parts. First, the biological variation, which is intrinsic to all organisms; it may be influenced by genetic or environmental factors, as well as by whether the samples are pooled or individual. Second, the technical variation, which might have been introduced to the samples during the extraction, labeling or hybridization procedures. Third, measurement error, which is associated with reading the fluorescent signals, which may be affected by factors such as dust on the array. Technical replicates generally involve a smaller degree of variation in measurements than the biological replicates. Replication is essential in experimental design because it allows accounting for different sources of variability. It is more difficult to say how many replicates should be done, although Lee et al indicates that three replicates are sufficient to account for technical variability [229]. The ability to assess such variability allows identification of biologically reproducible changes in gene expression levels. Standard analyses of t-like tests assume that the data are

Karin Dillner

- 40 -

sampled from normal populations with equal variances. Although log transformation of the expression ratios can improve normality and help equalize variances [230], ultimately the best estimates of the data’s distribution come from the data themselves. Permutation tests, generally carried out by repeatedly scrambling the samples’ class labels and computing t statistics for all genes in the scrambled data, best capture the unknown structure of the data [226, 231]. These types of tests do not assume normal distribution of the data set. One advantage of permutation methods is that they allow more reliable correction for multiple testing. The issue of multiple tests is crucial, as microarrays typically monitor the expression levels of thousands of genes.

In paper II and IV, we used the permutation-based statistical method, Significance Analysis of Microarrays (SAM) software, adapted specifically for microarrays. Today, SAM is a well accepted statistical method for estimating the variability of the repeated experiment [231]. Briefly, SAM assigns a score to each transcript on the basis of change in gene expression relative to the standard deviation of multiple independent measurements. Thereby, SAM allows selection of differentially regulated genes based on estimation of the percentage of genes identified as differentially regulated by chance, the so-called false discovery rate (FDR). To each of the genes in the array a q-value is assigned. This value is similar to the familiar p-value and measures the lowest FDR at which the gene is called significant. Experimental design

The expression ratio obtained from a microarray experiment is relative, i.e. no absolute values of the number of mRNA molecules per cell can be obtained. The key issue in designing a cDNA microarray experiment is to decide whether to use direct or indirect comparisons; that is, whether to make the comparison within or between slides [232]. Figure 3a show a direct design where the comparison of two different samples is made within one slide using the same orientation of dye labeling. In this design, dye bias may affect the final result [233, 234]. To avoid this, a technical replication may be performed by comparing each sample using two arrays in a dye-swap design (Figure 3b). If more than two samples are to be compared, a series of hybridizations that can be correlated among them have to be performed and a so-called indirect design has to be set up. A common strategy is to use a reference sample (e.g. a pool of all samples, a common control or a zero time point) that is hybridized to each array with one of the other sample (Figure 3c). Finally, the loop-design may be applied, with sometimes very complex setups, which increase the specificity in measurements and provides a more economical use of resources. In this strategy every sample is compared to two other samples in a


- 41 -

fashion that finally relates all samples in a close loop where the number of measurements per sample is automatically doubled (Figure 3c). Although, several advantages of the loop design, the interpretation may be problematic to a non-statistician. A concern is that this design is sensitive to failed experiments. If one of the links in the loop is missing due to for example a failed hybridization or too little starting RNA material, the entire set of hybridizations will yield less valuable data. The experimental design of the microarray experiments in paper II and IV were according to the direct dye-swap experimental design of Figure 3b. This design was used as we aimed to compare two different samples, the hyperplastic prostates of PRL-transgenic mice versus the prostates of control mice.

Figure 3. Experimental designs of cDNA microarray analysis. Letters represent RNA samples, and arrows represent microarray hybridizations. A. Microarray design where the two test samples (A and B) is directly compared. B. A variation of A using a dye swap for each comparison. C. The standard reference design uses a single array to compare each test sample to the reference RNA. D). Loop design. Microarray databases

The importance of public access to microarray data and the possibility of comparing different experiments using a common platform face a true challenge. In an attempt to standardize the microarray procedures and data handling, the international working group MIAME (the Minimum Information About a Microarray Experiment) has been established to set up

Reference

A B C

a. b.

c.A

B

C

D

d.

A B A B

Karin Dillner

- 42 -

certain guidelines. These guidelines include details of: (1) how the experiment was designed, (2) the design of the arrays or the name and location of spots on arrays, (3) sample name, extraction and labeling, (4) hybridization protocols, (5) methods for image measurements, and (6) the controls used. Meanwhile, several local as well as public available data bases have been created for gene expression data. COMPARISONS BETWEEN CDNA RDA AND CDNA MICROARRAY ANALYIS In paper II, cDNA-RDA was used to isolate differentially expressed transcripts in prostate of the Mt-PRL transgenic mice compared to controls. In contrast, in paper IV, the cDNA microarray technology was used to identify differentially expressed transcripts in prostatic hyperplasia of the transgenic mice compared to controls. The similarity of these two methods relies in the fact that both methods are applied to identify differentially expressed transcripts between the two groups that are to be compared. Depending of the aim of the study both of the methods can be successfully used. The main difference between cDNA microarray and cDNA-RDA analysis is that cDNA-RDA is a differential cloning method to isolate differentially expressed transcripts, while cDNA microarray technology only detect those sequences that have been previously identified and fixed to the support matrices. One might argue that when the human and mouse genome projects soon will be completed, cloning methods such as RDA, will not be of particular use. That is true for using the method as a pure cloning method, but not when it comes to its use for identifying differentially expressed genes. One advantage of RDA is that this method it is more sensitive than that of cDNA microarray. The PCR amplification steps in the RDA procedure makes this method superior in terms of identifying rare transcripts with large differential expression. Even though the PCR amplification steps should make differences greater, the RDA procedure may fail to detect abundant transcripts that have small differential expression. Although the method of cDNA microarray becoming more and more standardized, this method is still very costly and consequently its use are restricted to a small number of specialized laboratories. Moreover, most laboratories are able to handle the bioinformatics associated with the sequenced RDA output which is far from the sophisticated bioinformatics following a cDNA microarray experiment.


- 43 -

VERIFICATION STRATEGIES cDNA microarray analysis

Coupling of RDA subtraction with microarray analysis creates an efficient method for detection of unique, differentially expressed genes. The RDA may be halted at an early round of subtraction, which lessens the loss of differentially expressed cDNAs, perhaps due to PCR amplification preferences, and maintains diversity. However, this probably also contribute to a higher proportion of non-differentially expressed species in RDA output compared to RDAs performed using a greater number of rounds. This more complex output of RDA may be efficiently verified using cDNA microarrays. This verification may be performed in two variants. First, microarrays built with RDA-derived clones are hybridized to Cy3- and Cy5-labeled RNA samples that served as starting material. This use of cDNA microarray analysis might be inefficient for verification of low expressed transcripts or to detect small expression differences. For those transcripts, an alternative methodology that includes PCR amplification steps, such as Real-Time RT-PCR, might need to be used. Alternatively, the arrayed RDA output could be hybridized to Cy3- and Cy5-labeled RDA representations, as well as with the differential products from the different RDA rounds (DP1, DP2 etc). This will allow identification of those inserts that are differentially represented in the starting populations and the subsequent DP products. Clones that fail to hybridize at all to the representation targets but hybridize differentially to the DP pools should likewise be selected for further confirmation using an alternative method such as real-time RT-PCR as these likely include rare transcripts that are truly differentially expressed. Those clones that hybridize with the same intensity to the representation targets are highly likely to be false positives, even if they show differential hybridization with labeled DP targets. Therefore, the latter strategy might be the most efficient in allowing the advantage of the sensitivity of the RDA method in its ability to identify very rare and low abundant transcripts. In paper II, we haltered the cDNA-RDA after two rounds of subtraction and amplification rounds (DP2). The RDA output, DP2-hyperplastic and DP2-control, was verified using cDNA microarray analysis. Cy3- and Cy5-labeled RNA samples was used to verifiy the cDNA-RDA. Real-time RT-PCR

The RT-PCR approach can be successfully applied to validate the results of

Karin Dillner

- 44 -

any primary differential gene expression screening method once the sequence of the candidate gene is known. The main three advantages of RT-PCR is its sensitivity for the detection of low-abundance mRNA, the use of a very small amount of starting RNA samples and the possibility of discrimination between different splice forms of the transcript of interest. The conventional RT-PCR technique is not considered to be quantitative, as the final amount of PCR product is related not just to the initial template concentration but also to primer-dimer accumulation, PCR product re-annealing, and DNA polymerase binding to primers [235, 236] The development of kinetic RT-PCR (often referred to as real-time RT-PCR) has revolutionized the possibilities for quantitating mRNA [237]. In real-time RT-PCR, the accumulation of PCR products is monitored at the end of each cycle by fluorescence. During early cycles, the fluorescence is indistinguishable from background, but after a subsequent number of cycles the fluorescence increases exponentially. The PCR cycle number at which the fluorescence crosses a threshold, which is within the exponential phases, can be related to the amount of starting material; samples with more starting template will achieve the threshold fluorescence level more rapidly than those with less starting template. There are two general methods for the quantitative detection of the amplicon: (a) fluorescent probes and (b) DNA-binding agents. In the first “fluorescent probes” method, a specific probe, the so-called “TaqMan” probe [238], for the PCR product of interest is designed and labeled with a reporter dye at the 5’-end and a quencher dye at the 3’- end. During the extension phase of the PCR, the TaqMan probe will be cleaved by the endonucleolytic activity of the Taq polymerase, which allows the quencher and the reporter dye to be separated, and fluorescence emitted from the reporter dye (Figure 4a). In the second “DNA-binding agents” method, a non-sequence specific fluorescent DNA-binding dye, such as SYBRGreen I, is used which possess the ability to incorporate into double stranded DNA [239]. The unbound dye exhibits little fluorescence in solution, but during elongation increasing amounts of dye bind to the newly synthesized double-stranded DNA (Figure 4b). Its greatest advantage is that it can be applied with any pair of primers for any target, making its use less expensive than that of probe. While the sensitivity is usually quite good, such dyes bind to any double stranded DNA and thus do not distinguish between the PCR product of interest and alternate products, primer-dimers, etc. The product of interest should therefore be validated in assay development by stopping the kinetic PCR reaction after various numbers of cycles and performing electrophoresis on the products in an agarose gel. In addition, this problem can be overcome by generating a melting curve of the amplicon [240].


- 45 -

FluorophoreQuencher

RV Primer

Quencher

PolymeraseFluorophorePolymerase

i

ii

SYBRGreen I

Target

Fluorescent dye

A. Fluorescent probes B. DNA binding agents

i

ii

FW Primer

Figure 4. A. Fluorescent probes (e.g. TaqMan system). (i) After denaturation, primers and probe anneal to the target. Fluorescence does not occur because of the proximity between fluorophore and quencher. (ii) During the extension phase, the probe is cleaved by the 5’ to 3’ enzymatic activity of Taq polymerase. Thereby quencher and fluorophore are separated, allowing fluorescence emission from the reporter dye. FW, forward; RV, reverse. B. DNA-binding agents (e.g. SYBR Green I). (i) The dyes free in the solution do not emit fluorescence light. (ii) As soon as the SYBR Green binds to the dsDNA, target fluorescence occurs. Target RNA can be quantified using either absolute or relative quantification. Absolute quantification determines the absolute amount of target (expressed as copy number or concentration), whereas relative quantification determines the ratio between the amount of target and a reference transcript, usually a suitable housekeeping gene. This normalized value can then be used to compare, for example, differential gene expression in different samples. An important consideration is to ensure that the housekeeping gene is expressed at constant levels in the two different samples to be compared. In paper IV, we verified a set of differentially expressed transcripts subsequent to the identification using cDNA micorarray analysis by using the SYBR Green real-time RT-PCR approach. To quantitate the target RNA the relative quantification method was used with the acidic ribosomal phosphoprotein PO (Arbp) as the internal standard. In this approach, the ratio between the amount of target molecule and the internal standard molecule within the same sample is calculated. This normalized value was subsequently used to compare the relative expression ratio obtained with the real-time RT-PCR method with that obtained in the cDNA microarray analysis.

Karin Dillner

- 46 -

ASSESSMENT OF APOPTOTIC ACTIVITY In paper IV, the results from the microarray experiments indicated a reduction in apoptotic activity in the Pb-PRL transgenic mice compared to controls. Therefore we used two different apoptotic markers to assess apoptotic activity between the prostates of transgenic and control mice. Apoptosis is distinct from necrosis in both the biochemical and the morphological changes that occur [241-245]. In contrast to necrotic cells, apoptotic cells are characterized morphologically by compaction of the nuclear chromatin, shrinkage of the cytoplasm and production of membrane-bound apoptotic bodies. Biochemically, apoptosis is distinguished by fragmentation of the genome and cleavage or degradation of several cellular proteins. As with cell viability, no single parameter fully defines apoptosis; therefore, it is often advantageous to use several different approaches when studying apoptosis. Several methods have been developed to distinguish live cells from early and late apoptotic cells and from necrotic cells. The method of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) is widely used for detecting DNA nicks in apoptotic cells. An alternative method is the detection of single stranded DNA (ssDNA) which signifies the downstream event of DNA fragmentation. DNA fragmentation may be detected in both histologically defined apoptotic cells and morphologically intact apoptotic cells. The immunohistochemical method involving an antibody specific for ssDNA protein in cells allows accurate assessment of apoptosis [246, 247]. Furthermore, detection of ssDNA is considered more apoptosis-specific than the widely used TUNEL method for detection of DNA fragmentation and also detects apoptotic cells at an earlier stage than TUNEL [248, 249]. Apoptosis is mediated by a proteolytic cascade. The caspases, a family of cysteine proteases, play an essential role in the initiation, regulation, and execution of the downstream proteolytic events occurring during apoptosis [250-252]. Upon activation through proteolytic processing, caspases trigger substrate proteolysis and other changes that result in chromatin condensation, DNA fragmentation, and ultimately the apoptotic phenotype [251, 253, 254]. Caspase-3 is a key effector in the apoptosis pathway, amplifying the signal from initiator caspases (such as caspase-8) and for apoptosis-associated chromatin margination, DNA fragmentation, and nuclear collapse during apoptosis [253]. The detection of activated caspase-3 could therefore be a valuable and specific tool for identifying apoptotic cells in tissue sections, even before all the morphological features of apoptosis occur.


- 47 -

RESULTS AND TECHNICAL COMMENTS PAPER I Progressive prostate hyperplasia in adult Mt-PRL transgenic mice is not dependent on elevated serum androgen levels Transgenic mice overexpressing the rat PRL gene under control of the ubiquitous Mt-1 promoter develop a dramatic prostatic enlargement with parallel chronic hyperprolactinemia and elevated serum androgen levels. Histologically the prostate enlargement is mainly characterized by an expansion of the stromal compartment and areas of glandular hyperplasia with an accumulation of secretory material [105]. In paper I, we aim to clarify the role of circulating androgen levels in the promotion of abnormal prostate growth in the adult Mt-PRL transgenic mouse prostate. Separate groups of 12 weeks old animals (age-matched wild-type and Mt-PRL transgenic males) were surgically castrated followed by subcutaneous implantation of slow-release testosterone pellets containing 7.5 mg testosterone or placebo substance. The testosterone dose of 7.5 mg, was chosen to give as normophysiological levels of testosterone as possible, and was found to not significantly differ from the circulating testosterone serum levels of wildtype controls. After 8 weeks of hormone/placebo pellet treatment, animals were killed followed by serum sampling and prostate dissection. As an additional control, prostates from age-matched groups of non-treated wildtype and Mt-PRL transgenic mice were collected at both start and endpoint of the experiment. Results revealed that progression of prostate hyperplasia in adult Mt-PRL transgenic males was not affected by normalization of circulating testosterone levels. Immunohistochemical studies revealed a significantly increased proportion of AR positive epithelial cells in all prostate lobes of the Mt-PRL transgenic compared to wild-type. The increased distribution of epithelial AR remained high in the group of animals that were castration and substitution to normophysiological androgen levels. In addition, the Mt-PRL transgenic males possess more prominent stromal AR positivity than wildtype controls. The present study demonstrates that progressive prostate hyperplasia in adult Mt-PRL transgenic mice is not dependent on the elevated serum androgen levels present in the animals. In addition, our results suggest that prolonged hyperprolactinemia results in changes in prostate epithelial and stromal cell AR distribution. The increased AR distribution in both epithelial and stromal

Karin Dillner

- 48 -

cells in the Mt-PRL transgenic prostate lobes may increase the androgen sensitivity and thereby also influence the development of the observed prostate phenotype. Prolonged androgen treatment has no significant effect on prostate growth in wildtype adult mice The data about the importance of prolonged exposure to extraordinary high levels of androgens on prostate growth in rodents is conflicting, showing both unaffected prostate size [255] and induction of hyperplasia [256]. To determine the long-term effects of elevated circulating androgen levels on the prostate gland of wild-type male mice, a separate group of 12-week-old wildtype mice were sham-operated and subcutaneously implanted with 30 mg of testosterone slow-releasing pellet. The high dose of 30 mg testosterone was selected to give the treated group of wildtype animals a comparable levels of circulating testosterone as the Mt-PRL transgenic male mice have. After 8 weeks of treatment, prostates were dissected and serum samples obtained. On average, these animals displayed a 4-fold increase in serum testosterone levels compared to untreated wildtypes. These testosterone levels did not significantly differ from levels found in Mt-PRL transgenic males. Prostate wet weight in testosterone-treated wildtype did not significantly differ from that in untreated wildtype males neither as separate lobe weight nor as total organ weight. Histological appearance of the prostate lobes was not either different from that observed in wildtypes. These findings establish that prolonged androgen stimulation of adult male mice (C57BL/6JxCBA-strain) has no significant effects on prostate growth or histological appearance. This also supports the conclusions drawn from the results in castrated and androgen substituted Mt-PRL transgenic males, indicating that the hyperplastic process in transgenic prostate is not dependent on an elevated state of circulating androgens. Like the Mt-PRL transgenic mice, wildtype males treated with 30 mg of testosterone exhibited significantly higher numbers of AR-positive epithelial cells compared to untreated wildtypes. However in contrast to the Mt-PRL transgenic mice, stromal AR content was unaffected in testosterone-treated wildtypes. Taken together, these results show that prolonged androgen stimulation of young adult male mice has no significant effects on prostate growth or histological appearance. These data further support the findings in castrated and androgen substituted Mt-PRL transgenic males, that progression of prostate hyperplasia is not dependent on elevated levels of circulating androgens. Moreover, the results from the immunohistochemical analysis


- 49 -

suggest that the prostate hyperplasia of Mt-PRL transgenic mice is not primarily mediated via increased epithelial AR contents. Comparison of post-castrational regression patterns in the prostate lobes of Mt-PRL transgenic mice compared to wildtype mice Involution of the prostate after androgen-deprival by testicular castration is a well characterized process. Regression of the epithelial cell population, through an active process of apoptosis, occurs rapidly after castration. To establish the androgen dependency of this PRL transgenic prostate model, separate groups of 12-week-old male Mt-PRL transgenic and wildtype mice were castrated and subcutaneously implanted with placebo pellets. After 8 weeks, prostates were dissected and serum samples obtained. DLP and VP were significantly reduced after castration, in both Mt-PRL transgenic and wildtype prostates. In addition, similar histological appearance, with marked loss of both glandular epithelium and interductal stroma, were observed in both groups. Post-castrational VP weights in Mt-PRL transgenic males did not significantly differ from those of wildtype, whereas post-castrational DLP weight was significantly higher in Mt-PRL transgenic than in wildtype. However, considering the small but significant weight difference already at 12 weeks of age, the relative rate of reduction in DLP weight after castration was similar in Mt-PRL transgenic and wildtype, -66% and -78%, respectively. Altogether, these data show that androgens are clearly required for maintaining the transgenic phenotype as demonstrated by the similar patterns of prostatic regression seen in Mt-PRL transgenic and wildtype mice after androgen withdrawal. In the DLP, some weight differences were maintained after androgen-deprival; this difference may be attributable to the existing difference in glandular size at the time of castration. This finding could also partly be due to lobular differences in PRL responsiveness reported earlier [171, 175]. PAPER II Isolation of differentially expressed transcripts in the enlarged prostates of Mt-PRL transgenic mice compared to controls The objective of this study was to characterize the molecular mechanisms in the prostate of importance for the prostate hyperplasia seen in Mt-PRL

Karin Dillner

- 50 -

transgenic mice. Therefore, the method of cDNA representational difference analysis (cDNA RDA) was used which allow identification of novel genes that were differentially expressed in the enlarged prostates of the Mt-PRL transgenic mice compared to controls. The cDNA RDA was performed on prostatic tissue (DLP and VP) from mice of an age of four to six months. To generate samples as representative as possible, reflecting the prostate phenotype, RNA samples were pooled from four transgenic mice and five control littermates, respectively. Representations, generated from control and transgenic cDNA, respectively, were used as driver (control) and tester (hyperplastic), or vice versa, to generate both up- and down-regulated transcripts in the hyperplastic prostates compared to controls (see METHODOLOGICAL CONSIDERATIONS). Two successive rounds of subtraction and amplification were performed, generating libraries containing two sets of difference products, DP2-hyperplastic and DP2-control. Upon gel electrophoresis of the Sau3AI-cut RDA products of DP2-hyperplastic and DP2-control, six distinct bands were visualized. To subclone as many different products as possible from each library, each band were excised from the agarose gel and subcloned individually. 384 bacterial colonies, 192 from each RDA library, were picked, plasmid DNA was prepared followed by routine sequencing. After sequence alignments, the sequences were analyzed for homologies with published sequences in the non-redundant and EST divisions of the public databases of NCBI. This reduced the complexity of the RDA output so that the 384 clones sequenced, was reduced to 152 different unique sequences having a length longer than 50 base pairs. 69 of these, 37 DP2-hyperplastic and 32 DP2-control, were identified as previously annotated transcripts, whereas 83 were novel sequences not found in the public databases (referred to as unknowns) at the time when the study was performed. Verification of the RDA output by using cDNA Microarray Analysis To confirm that the obtained RDA difference products represented truly differentially expressed transcripts, the 152 non-redundant RDA products were selected for further verification using cDNA microarray analysis. 28 of the different RDA products were printed in duplicates, at different locations on the chip, to serve as “within slide” reproducibility controls (see METHODOLOGICAL CONSIDERATIONS). The RDA-derived microarrays were co-hybridized with labeled control and transgenic total RNA from a new set of animals. In order to eliminate dye specific effects caused by a labeling bias, dye-swap design of targeting labeling was used. The hybridizations were performed four independent times, twice Cy3-labeling the control and Cy5-labeling the hyperplastic total RNA, and twice with opposite colors (dye-


- 51 -

swapped). Probes rendering weaker signals than 1.4 times the background were eliminated and not considered for further analysis. Using this criterion, 48 of the 152 uniquely printed RDA clones could be detected in the Mt-PRL transgenic and control prostatic total RNA. To identify the significant differentially expressed transcripts, the data from the four repeated microarray experiments were statistically analyzed using the SAM algorithm [231]. Genes with average fold changes of more than 50% (correspond to a fold change of 1.5) were counted as differentially expressed. With an estimated FDR of less than 2%, 15 out of the 48 detected RDA products were identified as differentially regulated (of which 5 were unknowns). In terms of fold regulation, previous results from our laboratory have shown that this level can be reproduced, as shown by independent validation using RNase protection assay [257, 258]. Overall, the complexity of the RDA output could be largely reduced by: i) the annotation step (from 384 to 152), ii) the detection limitation of cDNA microarray technique (from 152 to 48), and iii) verification step (from 48 to 15) – resulting in 15 significantly differentially regulated transcripts, by an average fold-change of least 1.5, between the Mt-PRL transgenic and control prostates. One might reflect on the low number of significantly differentially regulated transcripts that were identified in this study. It has to be clarified that the final outcome of RDA differential cloning method depends largely on how extensive one makes the cloning. In our study, we cloned a number of 384 transcripts and certainly the more bacterial colonies that are cloned the higher probability there is to isolate and cover the complete set of differentially expressed transcripts that there are between the two groups that are compared. This may contribute to missing out important transcripts. Another reflection might be the obvious detection limitations of cDNA microarray analysis. There are several possible explanations for the relatively small number of detected transcripts. First, the ideal length of the cDNAs to be printed on cDNA microarrays is approximately 1000 base pairs. The lengths of our RDA products were between 200-500 base pairs, which probably contribute to reduce the sensitivity for detection by decreasing the hybridization. Second, a classical hybridization-based method like that of cDNA microarray depends on the specific activity of probes. Third, the PCR amplification steps in the RDA makes small expression differences greater as well as enables detection of low expressed transcripts. To enable verification of low expressed transcripts or small expression differences, an alternative method including PCR amplification steps, such as real-time RT-PCR, might need to be used. A further reflection is the relatively few significantly differentially regulated transcripts that were found at last. Most likely this is a consequence of the detection limitations of the method of cDNA microarray, but there are also other possible

Karin Dillner

- 52 -

explanations. A relative large proportion of the RDA output was expected to be so-called “false positive” clones as the RDA procedure was haltered after two rounds to. The reason for stopping the RDA subtraction and amplification steps at an early round is to diminish the loss of differentially expressed cDNAs and thereby maintain the RDA output diversity [259]. The verified differentially expressed RDA clones in hyperplastic versus control prostates In the present study, 152 non-redundant transcripts were differentially cloned in the Mt-PRL transgenic prostate compared to control. Although, not all of these transcripts could be detected and/or verified using cDNA microarray analysis, we still think they together may contribute to an interesting result as many of them are new transcripts to be cloned in the prostate. Therefore, several of the 152 differentially expressed transcripts most likely hold information of differentially expressed transcripts between the Mt-PRL transgenic and control prostates which will be found to be significantly differentially regulated if another verification method than cDNA microarray is used. Regarding the 10 annotated and verified differentially expressed transcripts, a number of them gave interesting information of possible molecular mechanisms involved in the development/ progression of the prostate hyperplasia of Mt-PRL transgenic mice. Of particular interest were the up-regulation of vimentin and the down-regulation of cytokeratin 8 which may indicate the importance of the “embryonic reawakening theory” in the development of the prostate phenotype of the Mt-PRL transgenic mice. Furthermore, the down-regulation of aldose reductase may be a sign of involvement of reduced apoptosis for development of the hyperplasia of the Mt-PRL transgenic mice. In addition the down-regulation of the candidate tumor-suppressor, the transcript coding for the RIL protein may further contribute to the prostate phenotype of Mt-PRL transgenic mice. In summary, the identified differentially expressed transcripts supports molecular similarities between the prostate hyperplasia of the Mt-PRL-transgenic mice and human BPH. Furthermore, the finding of new prostate hyperplasia related transcripts, both previously annotated and unknown transcripts, might be of large use both as potential biomarkers and to understand the underlying cause of benign growth of the prostate gland.


- 53 -

PAPER III Generation of transgenic mice overexpressing the PRL transgene specifically in the prostate under normophysiological androgen levels To address the role of local PRL action in the prostate, a new transgenic mouse model (Pb-PRL) was generated using the prostate-specific rat probasin (Pb) minimal promoter to drive expression of the rat PRL gene. Pb-PRL transgenic males developed a significant enlargement of both the DLP and VP lobes evident from 10 weeks of age and increasing throughout animal life span. In addition, the DNA content was measured in the prostate gland at 20 weeks of age, showing a significant three-fold increase in the DLP and VP, respectively, indicating a true hyperplasia with increased number of cells. Expression of the transgene was restricted to the prostate (DLP, VP, and AP) and present from 4 weeks of age. Also, a weak expression of the transgene could be observed in seminal vesicles at this age. Moreover, transgenic rPRL was detectable at low levels in the circulation of transgenic animals from 10 weeks of age, most likely associated with the continuing increase in prostate size. In contrast to the ubiquitous Mt-PRL transgenic mice, serum androgen levels did not significant differ from that of wild-type mice at any time point. The Pb-PRL prostate is histologically characterized by a significant stromal hyperplasia and secretion-filled distended ducts and focal areas of epithelial dysplasia. The glandular dysplastic foci had several morphological characteristics in common with low-grade prostatic intraepithelial neoplasia (PIN) lesions previously reported in other genetically engineered mouse models [260]. No high-grade PIN or prostate tumor formation were detected in Pb-PRL transgenic prostate. In addition, focal areas of mild to moderate chronic inflammation, exhibiting stromal mononuclear (primarily lymphocytes and macrophage) infiltrate, were frequently observed in both VP and LP lobes in Pb-PRL transgenic mice. Furthermore, immunohistochemical analysis revealed a significant increase in stromal cell distribution of androgen receptors (AR) and estrogen receptors alpha (ERα). In contrast, distribution of estrogen receptor beta (ERβ) was nearly uniform in both Pb-PRL transgenic and wildtype prostate.

Karin Dillner

- 54 -

Comparative analysis of prostate ductal branching morphogenesis and quantitative analysis of prostate cellular composition in Mt-PRL and Pb-PRL transgenic mice compared to controls To reveal possible phenotypic differences in ductal architecture due to different onset of transgenic rPRL expression, microdissection technique was used to examine branching morphogenesis of individual lobes in Mt-PRL and Pb-PRL transgenic prostate. Quantification was made by counting primary urethral ducts as well as duct branchpoints and terminal ductal tips at 12 weeks of age. In 12-weeks-old Pb-PRL prostate, no statistically significant differences were detected in the number of branch points per duct and the number of ductal tips present in each lobe compared to wild-type controls. However, marked ductal dilation and elongation was seen in the Pb-PRL from an early age, and complete microdissection was not achievable in animals over 20 week of age due to the formation of a densely fibrous interductal stroma that abrogated its normally high susceptibility to collagenase. In contrast, counting of ducts and tips in Mt-PRL VP and LP lobes at the same age demonstrated a significant increase, with approximately a doubling in the number of branching points and terminal tips compared to wildtype, whereas the number of main urethral ducts remained unchanged. Like the Pb-PRL transgenic prostate, the ducts were elongated and more dilated compared to controls and microdissection was also prevented by formation of a densely fibrous stroma in prostate lobes of older Mt-PRL animals. Quantitative analysis of prostatic tissue cellularity demonstrated a marked increase in the stromal to epithelial ratio in all lobes of both Mt-PRL and Pb-PRL transgenic prostates compared to controls. In wild-type controls, the lobe-specific stromal:epithelial ratio varied between 1:2.5 and 1:10, whereas in all lobes of Mt-PRL and Pb-PRL, transgenic prostate stromal and epithelial cells were present in approximately equal numbers. Overall, the Pb-PRL transgenic represents a new model for the study of PRL effects in the prostate. Most significantly, the development of Pb-PRL hyperplasia occurs mainly post-pubertally and in a setting of normal androgen levels, thereby resembling the situation in the adult human prostate. This study indicates the ability of PRL to promote, directly or indirectly, ductal morphogenesis in the developing prostate and further to induce abnormal growth primarily of the stroma in the adult gland in a setting of normal androgen levels.


- 55 -

PAPER IV Global analysis of gene expression in the enlarged prostate lobes of Pb-PRL transgenic mice The objective of this study was to characterize the molecular mechanisms involved in the prostate hyperplasia seen in the Pb-PRL transgenic mice overexpressing the PRL gene specifically in the prostate. Global changes of gene expression were analyzed by using a cDNA microarray chip containing about 6250 cDNA probes of rat and mouse origin. The gene expression analysis was performed in DLP and the VP, separately. We have chosen to denote genes as differentially regulated if their level of expression was changed by 70% (corresponding to a fold change of 1.7) or more in a statistically significant fashion. This level of change has previously been shown to be valid when compared to other direct methods, such as Northern Blot, ribonuclease protection assay, and RT-PCR [257, 258, 261, 262], as well as in the present study. Of the 6250 cDNA clones analyzed, 2344 showed hybridization in all independent determinations (DLP=2003 and VP=1962). Of those, 266 non-redundant transcripts were found to be differentially expressed (175 up-regulated and 91 down-regulated) in the enlarged prostate of PRL transgenic mice compared to controls in at least one of the lobes. 159 were differentially expressed in the DL lobe (111 up and 48 down) and 224 differentially expressed in the VP (159 up and 65 down). Of those, 117 transcripts were commonly differentially expressed in both DLP and VP (95 up and 22 down). 84 of the 266 non-redundant differentially expressed transcripts were transcripts with unknown function, identified as ESTs. The differences between the gene expression of VP and DLP can reflect biological differences and/or being a consequence of the detection sensitivity associated with the technique of cDNA microarray. Consequently, we have not paid specific attention to these differences. Functional classification of the differentially expressed transcripts, based on their known or suggested functions, revealed that virtually all cellular processes were affected in the prostate hyperplasia compared to control prostate. The two largest functionally categorized groups of both the DLP and VP were those of signal transduction and cell tissue structure. Moreover, a number of immune system-associated transcripts were found to be up-regulated which is in line with previous observations of areas of mild to moderate chronic inflammation, exhibiting stromal mononuclear (primarily lymphocytes and macrophage) infiltrate, in both the Pb-PRL VP and LP (Paper III). Subsequential real-time RT-PCR, using mouse specific primers for the orthologous mouse genes, did verify 10 of the differentially regulated transcripts, indicating the validity of using cDNA microarray technology in

Karin Dillner

- 56 -

combination with statistical methods for identifying differentially regulated genes. Moreover, the verification using mouse specific primers for the orthologous mouse genes to the cDNA clone of rat origin on the cDNA microarray further supports the use of rat probes to measure orthologous mouse genes. Assessment of apoptosis activity in Pb-PRL transgenic and control prostates In order to assess possible differences in apoptotic activity in the Pb-PRL transgenic and control prostates, two well accepted apoptosis markers where used (activated caspese-3 and single stranded (ss) DNA). In the Pb-PRL transgenic prostate, no activation of caspase-3 was detected using immunofluorescence. In control prostate, distinct clusters of apoptotic epithelial cells were occasionally detected in distal regions of the ductal system. Furthermore, no detectable levels of ssDNA were present in any lobes of Pb-PRL transgenic prostate using immunohistochemistry. In contrast, control littermate prostate focally displayed distinct nuclear ssDNA immunoreactivity in numerous epithelial cells, located almost exclusively in the distal ductal regions of all the prostate lobe types. Taken together, the ss-DNA and caspase-3 immunohistochemistry results, clearly indicate an overall diminished apoptotic activity in all prostate lobes of the Pb-PRL transgenic mice compared to controls. Differentially expressed transcripts in the enlarged prostates of Pb-PRL transgenic mice compared to control prostates Interestingly, a number of the identified differentially expressed transcripts in Pb-PRL transgenic compared to control prostate gave information of possible molecular mechanisms involved in the development/progression of the prostate hyperplasia. Among others a group of transcripts with pro-apoptotic activity were found to be down-regulated (Bok (Bcl-2-related ovarian killer protein), CIPAR-1 (castration induced prostatic apoptosis related protein-1) and Nuclear protein 1) in parallel with some transcripts with anti-apoptotic activity that were up-regulated (clusterin (also known as testosterone repressed prostate message-2 or sulfated glycoprotein-2) and SARP-1 (Secreted apoptosis-related protein 1), in the Pb-PRL transgenic prostate compared to controls. Together with the results of diminished apoptotic activity in all prostate lobes of the Pb-PRL transgenic assessed by immunohistohemistry, this likely indicates the importance of reduced apoptosis activity in the pathogenesis of prostate hyperplasia in Pb-PRL transgenic mice.


- 57 -

Moreover, numerous differentially expressed transcripts, between the enlarged prostates of Pb-PRL transgenic and control mice, were transcripts associated with tissue remodeling. The increased stromal:epithelial ratio of the Pb-PRL transgenic prostate, together with differential regulation of a significant fraction of genes involved in tissue remodeling activity, including synthesis and degradation of the ECM and changes in protease activity, suggests that activation of the stroma is involved in the development of the prostate phenotype. The obvious importance of the stromal compartment in the development of the prostate phenotype supports the “embryonic reawakening theory” of BPH etiology. Overall, the differentially expressed transcripts identified in this study, show many molecular similarities between the prostate hyperplasia of PRL-transgenic mice and human prostate pathology including both BPH and prostate cancer.

Karin Dillner

- 58 -

DISCUSSION PRL is a factor which, alone or synergistically with androgens, exerts trophic effects in the mature gland in rodents [201] and in human prostatic cells in vitro [14], besides it’s all other demonstrated functions. Moreover, growth-promoting effects of PRL on the prostate are well known in rodents made hyperprolactinemic by pituitary grafting [102-104, 109, 263]. To add to these findings, is the ubiquitous transgenic expression of the rPRL gene in Mt-PRL male mice, demonstrating that long-term exposure to PRL leads to prostate hyperplasia. In addition to chronic hyperprolactinemia, the Mt-PRL transgenic mice display elevated serum androgen levels [105]. Extrapituitary production of PRL has raised interest in the past few years, although its secretory control and functional relevance remain largely unknown. Moreover, the recent detection of locally produced PRL in prostate epithelium, together with the presence of the PRLR, have indicated a possible auto/paracrine action of PRL in prostate tissue [93, 94]. In an attempt to further explore the in vivo effects of enhanced PRL action in the prostate gland, but without the possible systemic alterations resulting from a prolonged hyperprolactinemic state, a prostate-specific PRL transgenic was generated which allow us to study the role of PRL action locally in the prostate. As shown in clinical reports, PRL may be elevated locally in BPH tissue, without any significant increase in serum PRL levels [137]. Although the contribution of local PRL production to circulating PRL levels is presumably low, it may be sufficient to exert significant activity on its local environment [264]. Local overexpression of the PRL transgene in the prostate of Pb-PRL transgenic male mice results in a significant prostate hyperplasia with a predominantly stromal phenotype. From this study we concluded that local, rather than circulating, elevated PRL levels are sufficient to induce prostate hyperplasia. Ductal branching morpholology in the rodent prostate gland has been extensively studied, showing that ductal formation is initiated around embryonic day 15 and considered to be essentially completed at 4-5 weeks (day 35) postpartum [4-6]. Due to the strong androgen-dependency of the Pb promoter, expression of the PRL transgene was first detected at 4 weeks of age. Thus, the PRL transgene is not expressed during the essential time period of ductal development. Consequently, the PRL-induced prostate hyperplasia of Pb-PRL transgenic mice arises from a normally developed ductal structure, under normophysiological circulating androgen levels, thereby resembling the situation in the adult human prostate.


- 59 -

The use of prostate lobe microdissections can effectively demonstrate changes in ductal structure resulting from neonatal exposure to developmentally active factors. In paper III, the use of the microdisection method revealed a significant increase of ductal morphogenesis in Mt-PRL transgenic prostate compared to littermate controls, with approximately a doubling in branching points and ductal tips evident in the DP, LP, and VP lobes. Activation of the Mt-1 promoter during the early embryonic stage is well described, with abundant expression already by day 12 of gestation reported [208, 209]. In contrast to the Mt-PRL transgenic mice, the Pb-PRL transgene expression initiates subsequent to the period when the ductal formation and branching are essentially terminated. Consequently, the Pb-PRL prostate exhibited no significant changes in mature ductal architecture when compared to controls. However, marked ductal dilation and elongation was evident in both transgenic models, and the formation of a dense fibrous and cellular interductal stroma appeared equally pronounced in the Pb-PRL as in the Mt-PRL prostates. The differences in ductal architecture of the PRL transgenic models can be explained by the temporal differences in expressin of the transgene. Alternatively, the altered androgen status in the Mt-PRL transgenic males may have an impact on the early ductal development in the prostate. The involvement of elevated androgen levels in increased branching morphogenesis has in fact been demonstrated previously in the VP of hypogonadal mice, where a single neonatal dose of androgens caused an increase in VP branching and lobe weight at adulthood [265]. However, in another study neonatal castration experiments demonstrated that significant branching morphogenesis occurs in the absence of androgens [7]. Furthermore, androgen replacement following neonatal castration results in precocious ductal formation, but final numbers of ductal tips and branchpoints do not exceed those seen in adult control males [7]. From this study Donjacour and Cunha concluded that neonatal prostatic ductal morphogenesis is sensitive to, but does not require, chronic androgen stimulation. Taken together, these findings demonstrate that PRL can, directly or indirectly through androgen stimulation, induce a significant increase in neonatal prostate morphogenesis. Comparative analysis of relative tissue areas and cellular area density confirmed the histological similarities of the Mt-PRL and Pb-PRL transgenic models, with marked expansion of the stromal compartment, resulting in a marked increase in the stromal:epithelial ratio. The stromal phenotype is of special interest because of its resemblance to human BPH which most commonly also present a significant increase in primarily the stromal compartment rather than epithelial [266, 267]. In addition, morphometric quantitation of human BPH tissue firmly established a dominance of the

Karin Dillner

- 60 -

stromal component, resulting in an increased stromal:epithelial ratio [21-24]. The implications of our findings in the PRL-transgenic mice models with respect to a clinical setting have yet to be determined. Furthermore, in symptomatic BPH patients, the stromal:epithelial ratio has been reported significantly higher than in asymptomatic patients [22]. However, it is not presently clear how the histological composition contributes to the pathophysiology of clinical symptoms associated with BPH. The histological resemblance of BPH and our PRL transgenic mice models, regarding the importance of the stromal compartments for the development of the prostate hyperplasia, is in line with the “embryonic reawakening theory” of BPH etiology proposed by McNeal. This theory emphasizes that BPH represents a reawakening of the embryonic and inductive potential of prostatic stroma, which in turn induces hyperplastic changes in the epithelium through stromal-epithelial interactions. Several studies have proved the importance of the epithelial-stromal interactions both in normal prostate development as well as the influence of abnormal reciprocal interaction between epithelial cells and the embryonic mesenchyme or adult stroma in the progression of neoplastic growth in the human prostate gland [268]. Although the exact mechanisms of such tissue interactions are not fully understood, there is growing evidence that they may operate through cell-ECM interactions, remodeling of ECM and auto-/paracrine growth factors [269]. Interestingly, the molecular patterns obtained from the gene expression analysis using the methods of cDNA-RDA and cDNA microarray further indicated a potential importance and possible activation of the stromal compartment for the development and/or progression of the prostate phenotype of the PRL transgenic mice. Although a few transcripts were commonly found to be differentially expressed in both the Mt-PRL and Pb-PRL transgenic mice models, the more extensive nature of the cDNA microarray, compared to that of the cDNA-RDA, enabled a greater insight into the molecular mechanism behind the prostate phenotype of the Pb-PRL than that of the Mt-PRL transgenic mice model. By this mean we do not exclude the possible molecular similarities of these two models of inducing the prostate phenotype, but a more extensive gene expression profiling of the Mt-PRL transgenic mice await in order to be able to assign any more specific molecular conclusions of this model’s phenotype. In addition, the differences that these two models hold in terms of circulating androgen levels and ductal morphogenesis further makes a direct comparison of these two models’ prostate phenotype and the identified differentially expressed transcripts rather challenging.


- 61 -

The broad molecular characterization made in the Pb-PRL transgenic mice gave us interesting clues of possible molecular mechanisms of importance for the prostate phenotype, including both the prostate hyperplasia and the dysplastic lesions resembling PIN I and PIN II displayed in this mouse model. Interestingly, numerous of the identified differentially expressed transcripts are directly associated with stromal cells and the ECM proteins that are secreted from stromal cells, including fibronectin, vimentin, laminin, osteonectin and collagens. This may reflect altered activity of the prostatic stromal cells in the prostates of the transgenic mice. In addition to their structural role, ECM proteins have a pronounced influence on tissue remodeling regulating cell growth, differentiation, communication, and migration [269]. Moreover, degradation of the ECM, mediated by a variety of proteolytic enzymes, such as matrix metalloproteinases (MMPs) and other proteases, have significant roles in normal and pathological tissue remodeling, including wound repair and tumorigenesis [51]. A set of transcripts, including members of the families of cathepsins, MMPs and TIMPs, were differentially regulated in the enlarged prostate of Pb-PRL transgenic mice compared to controls. These transcripts may serve as potential actors that modify the tissue homeostasis, possibly by changing the reciprocal stromal-epithelial interactions, which eventually contributes to promote the pathological tissue growth observed in the model. The differential regulation of several of these candidate transcripts has previously been associated with prostate disorders in both humans and animal models indicating a relevance of these transcripts in the prostate pathogenesis of PRL transgenic mice. The phenomenon of tumorigenesis promotion by an activated stroma (generation of a so-called “reactive stroma”) has previously been associated with prostate pathology and other human cancers [52]. The reactive stroma is characterized by ECM remodeling, elevated protease activity, increased angiogenesis and an influx of inflammatory cells [52]. The list of differentially regulated transcripts in the Pb-PRL transgenic prostate have much in common with the processes involved in what is in the literature described as characteristics of the reactive stroma (reviewed in [52]). The up-regulation of vimentin together with a down-regulation of desmin, suggest a myofibroblastic-like nature of the stroma cells, which is in line with the described phenotype of reactive stroma. In addition, reactive stroma cells typically express high levels of ECM components, such as collagen type I and III, fibronectin and proteoglycans, as well as proteases that degrade the ECM, observations that are in accordance with our present results. One possible hypothesis might be that PRL influences the initial induction of prostatic hyperplasia by modulating the stromal-epithelial interaction that, in one way or the other, results in an activation of the stroma

Karin Dillner

- 62 -

with the phenotypical features of reactive stroma, which fits well with the stromal expansion as dominant feature of human BPH [21, 23, 24]. In contrast to the Pb-PRL transgenic mice, the Mt-PRL transgenic mice exhibit an increased (in average about 3-fold) androgen serum level compared to controls. Certainly, this could influence the phenotype of these mice, but in what sense and to what degree is questionable. Nevertheless, significant individual variations of circulating testosterone levels were seen in Mt-PRL transgenic males (3.7-34 nmol/L of testosterone) without correlation to the degree of prostate enlargement in the individual animals [105]. With the intention to clarify the role of circulating androgen levels in the promotion of abnormal prostate growth in the adult Mt-PRL transgenic mouse prostate, we designed a castration and testosterone re-substitution study using age-matched transgenic and wildtype mice (Paper I). The aim was to normalize the circulating testosterone levels in young adult Mt-PRL transgenic males for a prolonged time period of 8 weeks. From this study we concluded that elevated serum androgen levels are not required for the progress of prostate hyperplasia in adult Mt-PRL transgenic males. Furthermore, these findings are supported by earlier reports in rodents using pituitary grafts, indicating a proliferative effect of PRL on the prostate regardless of androgen status [102, 270]. The role of androgens as the causative factor for human BPH is debated. However, they are certainly required to allow BPH development as indicated by the facts that there are no reports of BPH occurring in castrated males. This was in line with the observations in our castration study (8 week), showing comparable post-castrational regressive changes in Mt-PRL transgenic and control prostate (Paper I), clearly showing the androgen-sensitivity of the models. However, one should be aware of the differences in long-term effects of androgen-withdrawal, causing prostate regression, and the short-term effects of androgen-withdrawal, where PRL has been shown to slow down the regression and act as a survival factor [107]. In rodents the effects of prolonged androgen treatment on prostate growth is conflicting. Previous studies have showed both unaffected prostate size [255] and induction of hyperplasia [256]. However, in paper I in this thesis, we could not find any significant effect of prolonged androgen treatment on prostate growth in wildtype adult mice. Moreover, the castration and re-substitution studies in the Mt-PRL transgenic mice demonstrated that progressive prostate hyperplasia in adult Mt-PRL transgenic mice is not dependent on elevated serum androgen levels. Furthermore, the Pb-PRL transgenic mice display normophysiological serum androgens levels throughout animal life span, which further support the hypothesis of ours that the elevated circulating androgen levels are not responsible for the


- 63 -

hyperplastic prostate phenotype seen in the Mt-PRL transgenic mice. Prolonged hyperprolactinemia results in an increase in the prostate AR distribution in our PRL transgenic mice models. Several modes of PRL-influence on prostatic androgen sensitivity have been described earlier, including up-regulation of ARs [175], increased activity of the enzyme 5-alpha reductase [181] and increase in the uptake of testosterone into prostate cells [183]. Conversion of testosterone to the more active androgen, DHT, is primarily located to the stroma, due to the presence of type 2 5-alpha reductase isoform in the stroma [271]. It is likely that 5-alpha-reductase activity and AR distribution could play a role in the development of BPH. The importance of stromal AR in the prostate is well known, and mediation of some androgenic effects, such as ductal morphogenesis and epithelial growth, has been proposed not to require intraepithelial AR [8, 272]. In our study we demonstrated that testosterone treatment of adult wild-type mice did not result in any significant prostate hyperplasia but resulted in an up-regulation of epithelial AR, in contrast to unchanged stromal AR distribution (Paper I). In both the Mt- and Pb-PRL transgenic models, we demonstrated an increased precense of stromal AR, as detected by immunohistrochemical analysis. Taken together, an increased stromal AR distribution may contribute to the phenotype observed in our model. Interestingly, this suggestion is further supported by the findings in the AR transgenic mice. The AR transgenic mice, overexpressing the AR specifically in the prostate secretory epithelium of DLP and VP, develop focal areas of intraepithelial neoplasia, but no further progression into malignant lesions [154]. These mice do not develop any signs of prostate hyperplasia, which the authors suggested to be a result of the parallel increase of both proliferation and apoptosis. Furthermore, regarding the similarities of the AR transgenic and PRL transgenic mice in developing PIN formations, one might speculate that the altered levels of epithelial AR are partially responsible. It would be very interesting to make a parallel comparison of the AR distribution and expression levels to further understand the similarities and differences between these models. In parallel with increased stromal AR, an increased stromal ERα content was demonstrated (Paper III), although the relevance of this increase remains to be determined. Recent work has established that both initiation and progression of squamous metaplasia in the prostate after estrogen administration are mediated through stromal ERα [158, 160]. Furthermore, a distinct phenotype of focal epithelial hyperplasia in the VP has been reported in aging mice lacking functional ERβ [162, 163], whereas no apparent prostate pathology or enlargement has yet been reported in αERKO or

Karin Dillner

- 64 -

αβERKO [155]. These findings are indicative of an anti-proliferative role for epithelial ERβ and also suggest that an unbalanced stromal ERα action could contribute to the phenotype observed. In the normal adult prostate, a homeostasis appears to exist, whereby the rates of prostatic cell growth and prostatic apoptosis are in equilibrium. A changed balance between proliferative and apoptotic activity in the aging prostate has been proposed as a mechanism for BPH as well as prostate neoplasm formation and progression. Indeed, one of the proposed theories behind the etiology of BPH, suggests an involvement of reduced rate of apoptosis [42], based on the observations of reduced apoptotic activity in BPH tissue compared to control [43, 44, 273]. Other studies have suggested a potential role for the anti-apoptotic gene bcl-2 in BPH. In benign prostatic tissues, bcl-2 expression is predominantly seen in basal epithelial cells and has been associated with resistance to androgen ablation in BPH epithelium [274]. The identified differentially expressed transcripts, both from the cDNA-RDA and cDNA microarray analysis, gave us interesting insight into possible molecular mechanisms that might contribute to the development/progression of the prostate phenotype of the PRL-transgenic mice. In addition to the large fraction of transcripts involved in the aforementioned activation of the stromal cells, were transcripts associated with apoptosis. To further validate those results, we used immunohistochemical analysis for detection of two established apoptosis markers, activated caspase-3 and presence of ssDNA. In the Pb-PRL transgenic prostate, there were no immuno detectable levels of either activated caspase-3 or ssDNA. In contrast, distinct clusters of apoptotic epithelial cells were occasionally detected in distal regions of the ductal system in all prostate lobes of control samples. These results clearly indicated an overall diminished apoptotic activity in all prostate lobes of the Pb-PRL transgenic mice compared to controls, results which correlate well with the accepted participation of apoptosis in the BPH pathogenesis. In addition, the results from the gene expression analysis in the Pb-PRL transgenic mice demonstrate down-regulation of transcripts with pro-apoptotic activity and up-regulation of transcripts with anti-apoptotic activity. Overall, this supports the importance of reduced apoptotic activity in the pathogenesis of prostate hyperplasia in Pb-PRL transgenic mice. Again, findings in the AR transgenic mice give interesting input to the hypothesis of reduced apoptosis in the etiology of prostate hyperplasia. By the presence of increased proliferation together with an elevated frequency of apoptosis the authors explain the absence of hyperplasia phenotype in the prostate of the AR-transgenic mice [154].


- 65 -

The importance of PRL in regulation of apoptotic activity in the prostate is yet to be determined. It is possible that PRL acts preferentially as a survival factor rather than a growth factor. Earlier work has demonstrated PRL-induced delay in castration-induced prostatic regression [108]. In line with this, PRL was recently shown to significantly inhibit the androgen withdrawal-induced apoptosis in DP and LP rat prostate cultures [107]. Moreover, hyperprolactinemia has been shown to induce the synthesis of anti-apoptotic Bcl-2 in rat prostate [275], which is in line with induced Bcl-2 expression in human BPH tissue compared to control [274]. Furthermore, PRL has been shown to inhibit TRAIL-induced apoptosis in the PC-3 prostatic cell line [276]. TRAIL is a member of the TNF family that is known to induce apoptosis in prostate cells [277]. This inhibition was suggested to be mediated by increased phosphorylation of Akt/PKB, a critical regulator of cell survival. The Akt pathway provides the survival signal that involves several pro-apoptotic proteins such as Bad [278, 279] and possible also other members of the Bcl-2 family. PRL has been shown to possess an anti-apoptotic effect in the rat decidua, and this was shown to involve inhibition of caspase-3 activity mediated by the Akt-pathway [280]. In that study, PRL was able to down-regulate both caspase-3 mRNA levels as well as its activity. Taken together, our results suggest an importance of reduced apoptotic activity in the development of prostate hyperplasia in the PRL transgenic mice. The role of PRL in this regulation needs further investigation, as well as the involvement of diminished apoptotic activity for the development of prostate disease. Overall, we conclude that the progression of the prostate hyperplasia in adult Mt-PRL and Pb-PRL transgenic male mice does not require elevated circulating androgen levels. Furthermore, the prostate phenotype of the two PRL transgenic mouse models shares interesting histological characteristics with human BPH. The use of differential gene expression technologies in our studies has enabled us to find molecular similarities between the prostate hyperplasia of the PRL transgenic mouse models and human prostate disorders. Of particular interest is the potential significance of reduced apoptosis for the development/progression of the prostate phenotype. Another interesting observation is the importance and possible activation of the stromal compartment for the development and/or progression of the prostate phenotype of the PRL transgenic mice. There are striking resemblance of the molecular pattern obtained in the PRL transgenic prostate to that previously described in the literature as the “embryonic reawakening theory” of BPH etiology and the theory of “reactive stroma” in prostate cancer etiology. The Pb-PRL transgenic model does resemble the situation in BPH better than the Mt-PRL transgenic model since the prostate hyperplasia develops in a

Karin Dillner

- 66 -

mature gland under normophysiological androgen levels. This in combination with the molecular and histological similarities between the Pb-PRL transgenic model and human prostate pathology illustrates the potential use of this model as valuable tool in the study of BPH.


- 67 -

CONCLUSIONS • Prolonged hyperprolactinemia (Mt-PRL) or prostate-specific PRL (Pb-

PRL) overexpression leads to prostate hyperplasia • The PRL transgenic prostate is histologically characterized by a

prominent stromal hyperplasia with mild epithelial dysplastic features, leading to an increased stromal/epithelial ratio. Accumulation of secretory material is also a major characteristic.

• Pb-PRL transgenic mice with normal circulating testosterone levels

develop prostate hyperplasia • Supraphysiological serum androgen levels are not required for the

progress of prostate hyperplasia in adult Mt-PRL transgenic mice and do not induce prostate hyperplasia in androgen-treated wildtype mice.

• PRL stimulates, directly or indirectly via increased androgen action,

prostate ductal morphogenesis in the developing prostate gland • Reduction of apoptotic activity might be involved in the development of

prostate hyperplasia in PRL transgenic mice • The changes in gene expression pattern seen in Pb-PRL transgenic

prostate suggest that activation of the stroma is important for the development of prostatic hyperplasia

• Histological and molecular similarities exist between the prostate

hyperplasia of PRL-transgenic mice and human prostate pathology, including both BPH and prostate cancer

• The use of differential gene expression analysis shows a great promise in

elucidation of molecular mechanisms behind the diseases of the prostate gland

Karin Dillner

- 68 -

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude to everyone who has helped and contributed to this thesis in one way or another. I would especially like to thank: My outstanding supervisor and friend, Håkan Wennbo, for taking me on as a PhD student. Your contribution to this thesis has been incredibly valuable due to your continuous support and enthusiasm for this project, regardless of the physical distance between Stockholm and Göteborg. Moreover, for all the superb dinners you have served during these years. You are the absolutely best supervisor one can ever dream of and I hope we will always stay in contact! My supervisor, Gunnar Norstedt, for inviting me to work in your laboratory at the Center for Molecular Medicine (CMM), Karolinska institutet. It has been an inspiring place to work in and has enabled me to learn exciting differential gene expression methods and bioinformatics. Furthermore, for interesting scientific discussions and sharing your unlimited scientific visions. My supervisor, Jan Törnell for giving me the opportunity to work at the Dept of Physiology, Göteborg University. Olle G. Isaksson, for your generosity. My co-author and colleague Jon Kindblom, for your important scientific input and contribution in all possible ways to this thesis, and for guiding me as a PhD student throughout these years. Furthermore, for all your work with the transgenic mice and for your skilful handling with the knife, dissecting out the mouse prostates. Also for being a splendid travel companion in Australia and in USA. I hope we will maintain our collaboration. Amilcar Flores-Morales, for distributing some of your infinite energy and teaching me your enormous scientific knowledge in differential gene expression technologies and accompanying bioinformatics. It has really been a pleasure to work with you. See-Tong (Jacob) Pang, for being one of the few at CMM understanding the excitement in hormonal regulation of the prostate. For answering all my clinical questions and for being a great traveling companion in France and England. I hope we will keep in touch in the future! All former, present and associated members of GN’s group, CMM, for your scientifical and non-scientifical contribution: Amilcar Flores-Morales, Christina von Gertten, Kåre Hultén, Ingmarie Höidén-Guthenberg, Eva Johansson, Kristina Linder, Roxana Merino, See-Tong (Jacob) Pang, Elizabeth Rico-Bautista, Nina Ståhlberg, Petra Tollet-Egnell, Åsa Tellgren, Parisa Zarnegar – It has always been a pleasure to go to work when you are all around creating a cheerful atmosphere! All former and present colleagues at the Endocrine Division, Göteborg University, especially: Håkan Billig, Jan Oscarsson and Staffan Edén for your contribution. Moreover, Mohammad Bohlooly, Ola Brushed, Emil Egecioglu, Anders Friberg, Fredrik Frick (baby elephant ☺), Maria Gebre-Medhin, Jenny Kindblom, Jon Kindblom, Joakim Larsson, Daniel Lindén, Charlotte Ling, Emilia Markström, Bob Ohlsson, Ruijin Shao, Klara Sjögren, and Louise Svensson for making me feel very


- 69 -

welcome on my short visits at my home department, and some of you, for all the fun we had traveling. All the former and present members of ATCG and the Dept of Molecular Biology, AstraZeneca R&D Mölndal, for all your help. A special thank to Harriet Thelander, for your patience and assistance during my first year as a PhD student, and Maria Umaerus for all your help in teaching me the technique of real-time RT-PCR. My additional co-authors and co-workers: Chris Ormandy and Fiona Robertsson, Garvan Institute in Sydney, Australia; Ruijin Shao and Charlotte Ling, Endocrine Division, Göteborg University; Sophie Bernichtein and Vincent Goffin, INSERM, Paris, France; Åke Pousette, Andrology Center, and Lena Sahlin, Dept of Woman and Child Health, Karolinska institutet; Jan-Erik Damber, Dept of Urology, Sahlgrenska Hospital; Thank you all for your fantastic help, interesting scientific discussions, great collaboration, and providing me with tissues. All former and present colleagues and friends at CMM, especially the members of Mats Perssons’s group, Catharina Larsson’s group, Lars Terenius’s group, Georgy Bakalkin’s group and Tomas Ekström’s group, for creating a great work environment. For skillful and brilliant technical support: Kåre Hultén, Eva Johansson, and Britt Masironi. For excellent secretarial service: Lena Olofsson at Göteborg University and Christina Bremer, Delphi Post and Britt-Marie Witasp at Karolinska institutet. Brita & Lasse, Eva, Nina Thérese and Åsa, for all your terrific dinners and all pleasant time we have spent together, especially outside the CMM building. Erika, Hanna, Linda, Sanna and Sara, for your support and all great laughs and memories we have shared since our first day as undergraduates. Although we do not live in the same cities anymore, I hope from all my heart that we will always stay updated and keep in touch. Special thanks to Erika and Magnus for you endless hospitality during my visits in Göteborg – it is always a pleasure to stay with such nice friends! All my other friends outside the laboratory. In particular, my “bästis” Camilla, for your long and valuable friendship. The Family Larsson for your warm hospitality and your understanding when I left early in the mornings to the lab on our short weekend visits. My fantastic grand-mother, Anna – in every way you are my source of inspiration! My parents Sven and Agneta, and my brother Fredrik, for always believing in me and supporting me with all your love – it means so much to me! Pär, my love, my life companion and my very best friend, for your endless support and understanding. For driving me to work all mornings and being so patient waiting in the car for hours when picking me up... You are the best and no one makes me as happy as you do! This work was supported by grants from the Medical Faculty at Sahlgrenska Academy, Swedish Society for Medical Research, Assar Gabrielsson’s Fund, Lars Hiertas Memorial Fund, Foundation Clas Groschinsky's Memorial Fund, Wilhelm and Martina Lundgrens Vetenskapsfond, King Gustav V Jubilee Clinic Cancer Research Foundation (Sahlgrenska University Hospital), Konrad and Helfrid Johansson’s Foundation, Emil and Maria Palms Foundation, Rådman och Fru Ernst Colliander’s Foundation, and AstraZeneca R&D.

Karin Dillner

- 70 -

REFERENCES 1. Costello, L.C. and R.B. Franklin. Prostate epithelial cells utilize glucose and

aspartate as the carbon sources for net citrate production. Prostate, 1989. 15(4): p. 335-42.

2. Cunha, G.R., A.A. Donjacour, P.S. Cooke, S. Mee, R.M. Bigsby, S.J. Higgins, and Y. Sugimura. The endocrinology and developmental biology of the prostate. Endocr Rev, 1987. 8(3): p. 338-62.

3. Timms, B.G., T.J. Mohs, and L.J. Didio. Ductal budding and branching patterns in the developing prostate. J Urol, 1994. 151(5): p. 1427-32.

4. Sugimura, Y., G.R. Cunha, and A.A. Donjacour. Morphogenesis of ductal networks in the mouse prostate. Biol Reprod, 1986. 34(5): p. 961-71.

5. Hayashi, N., Y. Sugimura, J. Kawamura, A.A. Donjacour, and G.R. Cunha. Morphological and functional heterogeneity in the rat prostatic gland. Biol Reprod, 1991. 45(2): p. 308-21.

6. Singh, J., Q. Zhu, and D.J. Handelsman. Stereological evaluation of mouse prostate development. J Androl, 1999. 20(2): p. 251-8.

7. Donjacour, A.A. and G.R. Cunha. The effect of androgen deprivation on branching morphogenesis in the mouse prostate. Dev Biol, 1988. 128(1): p. 1-14.

8. Cunha, G.R., A.A. Donjacour, and Y. Sugimura. Stromal-epithelial interactions and heterogeneity of proliferative activity within the prostate. Biochem Cell Biol, 1986. 64(6): p. 608-14.

9. Lee, C. Role of androgen in prostate growth and regression: stromal-epithelial interaction. Prostate Suppl, 1996. 6: p. 52-6.

10. Xue, Y., G. Sonke, C. Schoots, J. Schalken, A. Verhofstad, J. de la Rosette, and F. Smedts. Proliferative activity and branching morphogenesis in the human prostate: a closer look at pre- and postnatal prostate growth. Prostate, 2001. 49(2): p. 132-9.

11. McNeal, J.E. Normal histology of the prostate. Am J Surg Pathol, 1988. 12(8): p. 619-33.

12. Nemeth, J.A. and C. Lee. Prostatic ductal system in rats: regional variation in stromal organization. Prostate, 1996. 28(2): p. 124-8.

13. Sherwood, E.R., L.A. Berg, N.J. Mitchell, J.E. McNeal, J.M. Kozlowski, and C. Lee. Differential cytokeratin expression in normal, hyperplastic and malignant epithelial cells from human prostate. J Urol, 1990. 143(1): p. 167-71.

14. Costello, L.C. and R.B. Franklin. Effect of prolactin on the prostate. Prostate, 1994. 24(3): p. 162-6.

15. Price, D., Comparative aspects of development and structure in the prostate. Biology of the Prostate and Related Tissues, ed. AH. 1963, Bethesda, MD, U.S.: Department of Health, Education, and Welfare, Public Health Serves, National Cancer Institute. p. 1-27.

16. DeKlerk, D.P. and D.S. Coffey. Quantitative determination of prostatic epithelial and stromal hyperplasia by a new technique. Biomorphometrics. Invest Urol, 1978. 16(3): p. 240-5.

17. Bartsch, G. and H.P. Rohr. Comparative light and electron microscopic study of the human, dog and rat prostate. An approach to an experimental model for human benign prostatic hyperplasia (light and electron microscopic analysis)--a review. Urol Int, 1980. 35(2): p. 91-104.

18. Karr, J.F., J.A. Kantor, P.H. Hand, D.L. Eggensperger, and J. Schlom. The presence of prostate-specific antigen-related genes in primates and the expression of recombinant human prostate-specific antigen in a transfected murine cell line. Cancer Res, 1995. 55(11): p. 2455-62.

19. Maini, A., C. Archer, C.Y. Wang, and G.P. Haas. Comparative pathology of benign prostatic hyperplasia and prostate cancer. In Vivo, 1997. 11(4): p. 293-9.


- 71 -

20. Berry, S.J., D.S. Coffey, P.C. Walsh, and L.L. Ewing. The development of human benign prostatic hyperplasia with age. J Urol, 1984. 132(3): p. 474-9.

21. Doehring, C.B., M.G. Sanda, A.W. Partin, J. Sauvageot, H. Juo, T.H. Beaty, J.I. Epstein, G. Hill, and P.C. Walsh. Histopathologic characterization of hereditary benign prostatic hyperplasia. Urology, 1996. 48(4): p. 650-3.

22. Shapiro, E., M.J. Becich, V. Hartanto, and H. Lepor. The relative proportion of stromal and epithelial hyperplasia is related to the development of symptomatic benign prostate hyperplasia. J Urol, 1992. 147(5): p. 1293-7.

23. Robert, M., P. Costa, F. Bressolle, N. Mottet, and H. Navratil. Percentage area density of epithelial and mesenchymal components in benign prostatic hyperplasia: comparison of results between single biopsy, multiple biopsies and multiple tissue specimens. Br J Urol, 1995. 75(3): p. 317-24.

24. Deering, R.E., S.A. Bigler, J. King, M. Choongkittaworn, E. Aramburu, and M.K. Brawer. Morphometric quantitation of stroma in human benign prostatic hyperplasia. Urology, 1994. 44(1): p. 64-70.

25. Edwards, J.E. and R.A. Moore. Finasteride in the treatment of clinical benign prostatic hyperplasia: A systematic review of randomised trials. BMC Urol, 2002. 2(1): p. 14.

26. Price, D. Potential mechanisms of action of superselective alpha(1)-adrenoceptor antagonists. Eur Urol, 2001. 40(Suppl 4): p. 5-11.

27. Brown, C.T. and G. Das. Assessment, diagnosis and management of lower urinary tract symptoms in men. Int J Clin Pract, 2002. 56(8): p. 591-603.

28. Ishimaru, T., L. Pages, and R. Horton. Altered metabolism of androgens in elderly men with benign prostatic hyperplasia. J Clin Endocrinol Metab, 1977. 45(4): p. 695-701.

29. Horton, R., P. Hsieh, J. Barberia, L. Pages, and M. Cosgrove. Altered blood androgens in elderly men with prostate hyperplasia. J Clin Endocrinol Metab, 1975. 41(4): p. 793-6.

30. Vermeulen, A. Andropause. Maturitas, 2000(Jan 15;34(1)): p. 5-15. 31. Harman, S.M., E.J. Metter, J.D. Tobin, J. Pearson, and M.R. Blackman.

Longitudinal effects of aging on serum total and free testosterone levels in healthy men. Baltimore Longitudinal Study of Aging. J Clin Endocrinol Metab, 2001. 86(2): p. 724-31.

32. Davidson, J.M., J.J. Chen, L. Crapo, G.D. Gray, W.J. Greenleaf, and J.A. Catania. Hormonal changes and sexual function in aging men. J Clin Endocrinol Metab, 1983. 57(1): p. 71-7.

33. Lecomte, P., N. Lecureuil, M. Lecureuil, C. Osorio Salazar, and C. Valat. Age modulates effects of thyroid dysfunction on sex hormone binding globulin (SHBG) levels. Exp Clin Endocrinol Diabetes, 1995. 103(5): p. 339-42.

34. Gray, A., H.A. Feldman, J.B. McKinlay, and C. Longcope. Age, disease, and changing sex hormone levels in middle-aged men: results of the Massachusetts Male Aging Study. J Clin Endocrinol Metab, 1991. 73(5): p. 1016-25.

35. Coffey, D.S. and P.C. Walsh. Clinical and experimental studies of benign prostatic hyperplasia. Urol Clin North Am, 1990. 17(3): p. 461-75.

36. Geller, J. Nonsurgical treatment of prostatic hyperplasia. Cancer, 1992. 70(1 Suppl): p. 339-45.

37. Bosch, R.J. Pathogenesis of benign prostatic hyperplasia. Eur Urol, 1991. 20(Suppl 1): p. 27-30.

38. Xue, Y., F. Smedts, A. Verhofstad, F. Debruyne, J. de la Rosette, and J. Schalken. Cell kinetics of prostate exocrine and neuroendocrine epithelium and their differential interrelationship: new perspectives. Prostate Suppl, 1998. 8: p. 62-73.

39. Trachtenberg, J., L.L. Hicks, and P.C. Walsh. Androgen- and estrogen-receptor content in spontaneous and experimentally induced canine prostatic hyperplasia. J Clin Invest, 1980. 65(5): p. 1051-9.

40. Vermeulen, A., R. Rubens, and L. Verdonck. Testosterone secretion and metabolism in male senescence. J Clin Endocrinol Metab, 1972. 34(4): p. 730-5.

Karin Dillner

- 72 -

41. Suzuki, K., S. Inaba, H. Takeuchi, Y. Takezawa, Y. Fukabori, T. Suzuki, K. Imai, H. Yamanaka, and S. Honma. [Endocrine environment of benign prostatic hyperplasia--relationships of sex steroid hormone levels with age and the size of the prostate]. Nippon Hinyokika Gakkai Zasshi, 1992. 83(5): p. 664-71.

42. Thompson, T.C. and G. Yang. Regulation of apoptosis in prostatic disease. Prostate Suppl, 2000. 9: p. 25-8.

43. Kyprianou, N., H. Tu, and S.C. Jacobs. Apoptotic versus proliferative activities in human benign prostatic hyperplasia. Hum Pathol, 1996. 27(7): p. 668-75.

44. Xia, S.J., C.X. Xu, X.D. Tang, W.Z. Wang, and D.L. Du. Apoptosis and hormonal milieu in ductal system of normal prostate and benign prostatic hyperplasia. Asian J Androl, 2001. 3(2): p. 131-4.

45. Bostwick, D.G. and M.K. Brawer. Prostatic intra-epithelial neoplasia and early invasion in prostate cancer. Cancer, 1987. 59(4): p. 788-94.

46. McNeal, J.E. and D.G. Bostwick. Intraductal dysplasia: a premalignant lesion of the prostate. Hum Pathol, 1986. 17(1): p. 64-71.

47. Bostwick, D.G., A. Pacelli, and A. Lopez-Beltran. Molecular biology of prostatic intraepithelial neoplasia. Prostate, 1996. 29(2): p. 117-34.

48. Sugamoto, T., N. Tanji, K. Sato, H. Fujita, S. Nishio, M. Sakanaka, and M. Yokoyama. The expression of basic fibroblast growth factor and vascular endothelial growth factor in prostatic adenocarcinoma: correlation with neovascularization. Anticancer Res, 2001. 21(1A): p. 77-88.

49. Giroldi, L.A. and J.A. Schalken. Decreased expression of the intercellular adhesion molecule E-cadherin in prostate cancer: biological significance and clinical implications. Cancer Metastasis Rev, 1993. 12(1): p. 29-37.

50. Danen, E.H. and K.M. Yamada. Fibronectin, integrins, and growth control. J Cell Physiol, 2001. 189(1): p. 1-13.

51. Mauch, C., T. Krieg, and E.A. Bauer. Role of the extracellular matrix in the degradation of connective tissue. Arch Dermatol Res, 1994. 287(1): p. 107-14.

52. Tuxhorn, J.A., G.E. Ayala, and D.R. Rowley. Reactive stroma in prostate cancer progression. J Urol, 2001. 166(6): p. 2472-83.

53. Karan, D., M.F. Lin, S.L. Johansson, and S.K. Batra. Current status of the molecular genetics of human prostatic adenocarcinomas. Int J Cancer, 2003. 103(3): p. 285-93.

54. Ben-Jonathan, N., J.L. Mershon, D.L. Allen, and R.W. Steinmetz. Extrapituitary prolactin: distribution, regulation, functions, and clinical aspects. Endocr Rev, 1996. 17(6): p. 639-69.

55. Bole-Feysot, C., V. Goffin, M. Edery, N. Binart, and P.A. Kelly. Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice. Endocr Rev, 1998. 19(3): p. 225-68.

56. Miller, W.L. and N.L. Eberhardt. Structure and evolution of the growth hormone gene family. Endocr Rev, 1983. 4(2): p. 97-130.

57. Goffin, V., K.T. Shiverick, P.A. Kelly, and J.A. Martial. Sequence-function relationships within the expanding family of prolactin, growth hormone, placental lactogen, and related proteins in mammals. Endocr Rev, 1996. 17(4): p. 385-410.

58. Horseman, N.D. and L.Y. Yu-Lee. Transcriptional regulation by the helix bundle peptide hormones: growth hormone, prolactin, and hematopoietic cytokines. Endocr Rev, 1994. 15(5): p. 627-49.

59. Truong, A.T., C. Duez, A. Belayew, A. Renard, R. Pictet, G.I. Bell, and J.A. Martial. Isolation and characterization of the human prolactin gene. Embo J, 1984. 3(2): p. 429-37.

60. Goffin, V., J.A. Martial, and N.L. Summers. Use of a model to understand prolactin and growth hormone specificities. Protein Eng, 1995. 8(12): p. 1215-31.

61. Sinha, Y.N. Structural variants of prolactin: occurrence and physiological significance. Endocr Rev, 1995. 16(3): p. 354-69.

62. Posner, B.I., P.A. Kelly, R.P. Shiu, and H.G. Friesen. Studies of insulin, growth hormone and prolactin binding: tissue distribution, species variation and characterization. Endocrinology, 1974. 95(2): p. 521-31.


- 73 -

63. Mittra, I. A novel "cleaved prolactin" in the rat pituitary: Part II. In vivo mammary mitogenic activity of its N-terminal 16K moiety. Biochem Biophys Res Commun, 1980. 95(4): p. 1760-7.

64. Rochefort, H. and E. Liaudet-Coopman. Cathepsin D in cancer metastasis: a protease and a ligand. Apmis, 1999. 107(1): p. 86-95.

65. Clapp, C. and R.I. Weiner. A specific, high affinity, saturable binding site for the 16-kilodalton fragment of prolactin on capillary endothelial cells. Endocrinology, 1992. 130(3): p. 1380-6.

66. Clapp, C., J.A. Martial, R.C. Guzman, F. Rentier-Delure, and R.I. Weiner. The 16-kilodalton N-terminal fragment of human prolactin is a potent inhibitor of angiogenesis. Endocrinology, 1993. 133(3): p. 1292-9.

67. Martini, J.F., C. Piot, L.M. Humeau, I. Struman, J.A. Martial, and R.I. Weiner. The antiangiogenic factor 16K PRL induces programmed cell death in endothelial cells by caspase activation. Mol Endocrinol, 2000. 14(10): p. 1536-49.

68. D'Angelo, G., J.F. Martini, T. Iiri, W.J. Fantl, J. Martial, and R.I. Weiner. 16K human prolactin inhibits vascular endothelial growth factor-induced activation of Ras in capillary endothelial cells. Mol Endocrinol, 1999. 13(5): p. 692-704.

69. Struman, I., F. Bentzien, H. Lee, V. Mainfroid, G. D'Angelo, V. Goffin, R.I. Weiner, and J.A. Martial. Opposing actions of intact and N-terminal fragments of the human prolactin/growth hormone family members on angiogenesis: an efficient mechanism for the regulation of angiogenesis. Proc Natl Acad Sci U S A, 1999. 96(4): p. 1246-51.

70. Corbacho, A.M., G. Nava, J.P. Eiserich, G. Noris, Y. Macotela, I. Struman, G. Martinez De La Escalera, B.A. Freeman, and C. Clapp. Proteolytic cleavage confers nitric oxide synthase inducing activity upon prolactin. J Biol Chem, 2000. 275(18): p. 13183-6.

71. Nilsen-Hamilton, M., R.T. Hamilton, and E. Alvarez-Azaustre. Relationship between mitogen-regulated protein (MRP) and proliferin (PLF), a member of the prolactin/growth hormone family. Gene, 1987. 51(2-3): p. 163-70.

72. Nilsen-Hamilton, M., J.M. Shapiro, S.L. Massoglia, and R.T. Hamilton. Selective stimulation by mitogens of incorporation of 35S-methionine into a family of proteins released into the medium by 3T3 cells. Cell, 1980. 20(1): p. 19-28.

73. Linzer, D.I. and D. Nathans. Nucleotide sequence of a growth-related mRNA encoding a member of the prolactin-growth hormone family. Proc Natl Acad Sci U S A, 1984. 81(14): p. 4255-9.

74. Lee, S.J., F. Talamantes, E. Wilder, D.I. Linzer, and D. Nathans. Trophoblastic giant cells of the mouse placenta as the site of proliferin synthesis. Endocrinology, 1988. 122(5): p. 1761-8.

75. Toft, D.J., S.B. Rosenberg, G. Bergers, O. Volpert, and D.I. Linzer. Reactivation of proliferin gene expression is associated with increased angiogenesis in a cell culture model of fibrosarcoma tumor progression. Proc Natl Acad Sci U S A, 2001. 98(23): p. 13055-9.

76. Slater, E.P., G. Redeuihl, K. Theis, G. Suske, and M. Beato. The uteroglobin promoter contains a noncanonical estrogen responsive element. Mol Endocrinol, 1990. 4(4): p. 604-10.

77. Darwish, H., J. Krisinger, J.D. Furlow, C. Smith, F.E. Murdoch, and H.F. DeLuca. An estrogen-responsive element mediates the transcriptional regulation of calbindin D-9K gene in rat uterus. J Biol Chem, 1991. 266(1): p. 551-8.

78. Song, J.Y., L. Jin, and R.V. Lloyd. Effects of estradiol on prolactin and growth hormone messenger RNAs in cultured normal and neoplastic (MtT/W15 and GH3) rat pituitary cells. Cancer Res, 1989. 49(5): p. 1247-53.

79. Asscheman, H., L.J. Gooren, J. Assies, J.P. Smits, and R. de Slegte. Prolactin levels and pituitary enlargement in hormone-treated male-to-female transsexuals. Clin Endocrinol (Oxf), 1988. 28(6): p. 583-8.

80. Davis, J.A. and D.I. Linzer. Expression of multiple forms of the prolactin receptor in mouse liver. Mol Endocrinol, 1989. 3(4): p. 674-80.

Karin Dillner

- 74 -

81. Clarke, D.L. and D.I. Linzer. Changes in prolactin receptor expression during pregnancy in the mouse ovary. Endocrinology, 1993. 133(1): p. 224-32.

82. Elkins, P.A., H.W. Christinger, Y. Sandowski, E. Sakal, A. Gertler, A.M. de Vos, and A.A. Kossiakoff. Ternary complex between placental lactogen and the extracellular domain of the prolactin receptor. Nat Struct Biol, 2000. 7(9): p. 808-15.

83. Yu-Lee, L.Y., G. Luo, M.L. Book, and S.M. Morris. Lactogenic hormone signal transduction. Biol Reprod, 1998. 58(2): p. 295-301.

84. Ali, S. Recruitment of the protein-tyrosine phosphatase SHP-2 to the C-terminal tyrosine of the prolactin receptor and to the adaptor protein Gab2. J Biol Chem, 2000. 275(50): p. 39073-80.

85. Ali, S., Z. Chen, J.J. Lebrun, W. Vogel, A. Kharitonenkov, P.A. Kelly, and A. Ullrich. PTP1D is a positive regulator of the prolactin signal leading to beta-casein promoter activation. Embo J, 1996. 15(1): p. 135-42.

86. Starr, R. and D.J. Hilton. Negative regulation of the JAK/STAT pathway. Bioessays, 1999. 21(1): p. 47-52.

87. Yamauchi, T., K. Ueki, K. Tobe, H. Tamemoto, N. Sekine, M. Wada, M. Honjo, M. Takahashi, T. Takahashi, H. Hirai, et al. Tyrosine phosphorylation of the EGF receptor by the kinase Jak2 is induced by growth hormone. Nature, 1997. 390(6655): p. 91-6.

88. Fenton, S.E. and L.G. Sheffield. Prolactin inhibits epidermal growth factor (EGF)-stimulated signaling events in mouse mammary epithelial cells by altering EGF receptor function. Mol Biol Cell, 1993. 4(8): p. 773-80.

89. Stoecklin, E., M. Wissler, D. Schaetzle, E. Pfitzner, and B. Groner. Interactions in the transcriptional regulation exerted by Stat5 and by members of the steroid hormone receptor family. J Steroid Biochem Mol Biol, 1999. 69(1-6): p. 195-204.

90. Aragona, C. and H.G. Friesen. Specific prolactin binding sites in the prostate and testis of rats. Endocrinology, 1975. 97(3): p. 677-84.

91. Leav, I., F.B. Merk, K.F. Lee, M. Loda, M. Mandoki, J.E. McNeal, and S.M. Ho. Prolactin receptor expression in the developing human prostate and in hyperplastic, dysplastic, and neoplastic lesions. Am J Pathol, 1999. 154(3): p. 863-70.

92. Nevalainen, M.T., E.M. Valve, P.M. Ingleton, and P.L. Harkonen. Expression and hormone regulation of prolactin receptors in rat dorsal and lateral prostate. Endocrinology, 1996. 137(7): p. 3078-88.

93. Nevalainen, M.T., E.M. Valve, P.M. Ingleton, M. Nurmi, P.M. Martikainen, and P.L. Harkonen. Prolactin and prolactin receptors are expressed and functioning in human prostate. J Clin Invest, 1997. 99(4): p. 618-27.

94. Nevalainen, M.T., E.M. Valve, T. Ahonen, A. Yagi, J. Paranko, and P.L. Harkonen. Androgen-dependent expression of prolactin in rat prostate epithelium in vivo and in organ culture. Faseb J, 1997. 11(14): p. 1297-307.

95. Smith, C., D. Assimos, C. Lee, and J.T. Grayhack. Metabolic action of prolactin in regressing prostate: independent of androgen action. Prostate, 1985. 6(1): p. 49-59.

96. Reiter, E., S. Lardinois, M. Klug, B. Sente, B. Hennuy, M. Bruyninx, J. Closset, and G. Hennen. Androgen-independent effects of prolactin on the different lobes of the immature rat prostate. Mol Cell Endocrinol, 1995. 112(1): p. 113-22.

97. de Launoit, Y., R. Kiss, V. Jossa, M. Coibion, R.J. Paridaens, E. De Backer, A.J. Danguy, and J.L. Pasteels. Influences of dihydrotestosterone, testosterone, estradiol, progesterone, or prolactin on the cell kinetics of human hyperplastic prostatic tissue in organ culture. Prostate, 1988. 13(2): p. 143-53.

98. Janssen, T., R. Kiss, and C. Schulman. [Organ culture of human tissue as study model of hormonal and pharmacological regulation of benign prostatic hyperplasia and of prostatic cancer]. Acta Urol Belg, 1995. 63(1): p. 7-14.

99. Janssen, T., F. Darro, M. Petein, G. Raviv, J.L. Pasteels, R. Kiss, and C.C. Schulman. In vitro characterization of prolactin-induced effects on proliferation in the neoplastic LNCaP, DU145, and PC3 models of the human prostate. Cancer, 1996. 77(1): p. 144-9.


- 75 -

100. Rui, H. and K. Purvis. Independent control of citrate production and ornithine decarboxylase by prolactin in the lateral lobe of the rat prostate. Mol Cell Endocrinol, 1987. 52(1-2): p. 91-5.

101. Russell, D.H. Ornithine decarboxylase as a biological and pharmacological tool. Pharmacology, 1980. 20(3): p. 117-29.

102. Negro-Vilar, A., W.A. Saad, and S.M. McCann. Evidence for a role of prolactin in prostate and seminal vesicle growth in immature male rats. Endocrinology, 1977. 100(3): p. 729-37.

103. Schacht, M.J., C.S. Niederberger, J.E. Garnett, J.A. Sensibar, C. Lee, and J.T. Grayhack. A local direct effect of pituitary graft on growth of the lateral prostate in rats. Prostate, 1992. 20(1): p. 51-8.

104. Mori, T. and H. Nagasawa. Effects of pituitary grafting and CB-154 treatment on the growth of prostates in mice. Acta Anat (Basel), 1984. 120(4): p. 180-4.

105. Wennbo, H., J. Kindblom, O.G. Isaksson, and J. Tornell. Transgenic mice overexpressing the prolactin gene develop dramatic enlargement of the prostate gland. Endocrinology, 1997. 138(10): p. 4410-5.

106. Kindblom, J., K. Dillner, C. Ling, J. Tornell, and H. Wennbo. Progressive prostate hyperplasia in adult prolactin transgenic mice is not dependent on elevated serum androgen levels. Prostate, 2002. 53(1): p. 24-33.

107. Ahonen, T.J., P.L. Harkonen, J. Laine, H. Rui, P.M. Martikainen, and M.T. Nevalainen. Prolactin is a survival factor for androgen-deprived rat dorsal and lateral prostate epithelium in organ culture. Endocrinology, 1999. 140(11): p. 5412-21.

108. Kolbusz, W.E., C. Lee, and J.T. Grayhack. Delay of castration induced regression in rat prostate by pituitary homografts. J Urol, 1982. 127(3): p. 581-4.

109. Assimos, D., C. Smith, C. Lee, and J.T. Grayhack. Action of prolactin in regressing prostate: independent of action mediated by androgen receptors. Prostate, 1984. 5(6): p. 589-95.

110. Costello, L.C. and R.B. Franklin. Novel role of zinc in the regulation of prostate citrate metabolism and its implications in prostate cancer. Prostate, 1998. 35(4): p. 285-96.

111. Costello, L.C. and R.B. Franklin. Bioenergetic theory of prostate malignancy. Prostate, 1994. 25(3): p. 162-6.

112. Franklin, R.B. and L.C. Costello. Prolactin directly stimulates citrate production and mitochondrial aspartate aminotransferase of prostate epithelial cells. Prostate, 1990. 17(1): p. 13-8.

113. Franklin, R.B., J. Zou, E. Gorski, Y.H. Yang, and L.C. Costello. Prolactin regulation of mitochondrial aspartate aminotransferase and protein kinase C in human prostate cancer cells. Mol Cell Endocrinol, 1997. 127(1): p. 19-25.

114. Costello, L.C., Y. Liu, and R.B. Franklin. Prolactin specifically increases pyruvate dehydrogenase E1 alpha in rat lateral prostate epithelial cells. Prostate, 1995. 26(4): p. 189-93.

115. Costello, L.C., Y. Liu, J. Zou, and R.B. Franklin. The pyruvate dehydrogenase E1 alpha gene is testosterone and prolactin regulated in prostate epithelial cells. Endocr Res, 2000. 26(1): p. 23-39.

116. Costello, L.C., Y. Liu, J. Zou, and R.B. Franklin. Mitochondrial aconitase gene expression is regulated by testosterone and prolactin in prostate epithelial cells. Prostate, 2000. 42(3): p. 196-202.

117. Liu, Y., R.B. Franklin, and L.C. Costello. Prolactin and testosterone regulation of mitochondrial zinc in prostate epithelial cells. Prostate, 1997. 30(1): p. 26-32.

118. Costello, L.C., Y. Liu, J. Zou, and R.B. Franklin. Evidence for a zinc uptake transporter in human prostate cancer cells which is regulated by prolactin and testosterone. J Biol Chem, 1999. 274(25): p. 17499-504.

119. Vekemans, M. and C. Robyn. Influence of age on serum prolactin levels in women and men. Br Med J, 1975. 4(5999): p. 738-9.

120. Nankin, H.R. and J.H. Calkins. Decreased bioavailable testosterone in aging normal and impotent men. J Clin Endocrinol Metab, 1986. 63(6): p. 1418-20.

Karin Dillner

- 76 -

121. Hammond, G.L., M. Kontturi, P. Maattala, M. Puukka, and R. Vihko. Serum FSH, LH and prolactin in normal males and patients with prostatic diseases. Clin Endocrinol (Oxf), 1977. 7(2): p. 129-35.

122. Yamaji, T., K. Shimamoto, M. Ishibashi, K. Kosaka, and H. Orimo. Effect of age and sex on circulating and pituitary prolactin levels in human. Acta Endocrinol (Copenh), 1976. 83(4): p. 711-9.

123. Blackman, M.R., M.A. Kowatch, R.E. Wehmann, and S.M. Harman. Basal serum prolactin levels and prolactin responses to constant infusions of thyrotropin releasing hormone in healthy aging men. J Gerontol, 1986. 41(6): p. 699-705.

124. Mongioi, A., E. Vicari, and R. D'Agata. The prolactin-secreting system in relation to aging. J Endocrinol Invest, 1985. 8 Suppl 2: p. 33-9.

125. Brendler, H. Adrenalectomy and hypophysectomy for prostatic cancer. Urology, 1973. 2(2): p. 99-102.

126. Tindall, G.T., N.S. Payne, and D.W. Nixon. Transsphenoidal hypophysectomy for disseminated carcinoma of the prostate gland. Results in 53 patients. J Neurosurg, 1979. 50(3): p. 275-82.

127. Farnsworth, W.E. and M.J. Gonder. Prolaction and prostate cancer. Urology, 1977. 10(1): p. 33-4.

128. Tasar, C., A. Akdas, and Z. Kirkali. The effect of ergobromocriptine on serum testosterone and prolactin levels in patients with carcinoma of the prostate. Int Urol Nephrol, 1986. 18(1): p. 71-4.

129. Turkolmez, K., M. Bozlu, K. Sarica, H. Gemalmaz, E. Ozdiler, and O. Gogus. Effects of transurethral prostate resection and transurethral laser prostatectomy on plasma hormone levels. Urol Int, 1998. 61(3): p. 162-7.

130. Jacobi, G.H., G.H. Rathgen, and J.E. Altwein. Serum prolactin and tumors of the prostate: unchanged basal levels and lack of correlation to serum testosterone. J Endocrinol Invest, 1980. 3(1): p. 15-8.

131. Mee, A.D., O. Khan, and K. Mashiter. High serum prolactin associated with poor prognosis in carcinoma of the prostate. Br J Urol, 1984. 56(6): p. 698-701.

132. Morales, A. and J.C. Nickel. Clinical relevance of plasma testosterone and prolactin changes in advanced cancer of prostate treated with diethylstilbestrol or estramustine phosphate. Urology, 1985. 26(5): p. 477-81.

133. Jeromin, L. The serum levels of testosterone and prolactin in patients with prostatic carcinoma treated with various doses of Fostrolin and bromocriptin. Int Urol Nephrol, 1982. 14(1): p. 51-6.

134. Rana, A., F.K. Habib, P. Halliday, M. Ross, R. Wild, R.A. Elton, and G.D. Chisholm. A case for synchronous reduction of testicular androgen, adrenal androgen and prolactin for the treatment of advanced carcinoma of the prostate. Eur J Cancer, 1995. 31A(6): p. 871-5.

135. Jeromin, L., J. Wisniewski, M. Rozniecki, Z. Janiak, and J. Borkiewicz. Five-year clinical follow-up of prostatic cancer patients treated with fosfestrol and bromocriptine. Int Urol Nephrol, 1987. 19(1): p. 81-5.

136. Jacobi, G.H., J.E. Altwein, and R. Hohenfellner. Adjunct bromocriptine treatment as palliation for prostate cancer: experimental and clinical evaluation. Scand J Urol Nephrol Suppl, 1980. 55: p. 107-12.

137. Ron, M., A. Fich, A. Shapiro, M. Caine, and M. Ben-David. Prolactin concentration in prostates with benign hypertrophy. Urology, 1981. 17(3): p. 235-7.

138. Yatani, R., I. Kusano, T. Shiraishi, S. Miura, H. Takanari, and P.I. Liu. Elevated prolactin level in prostates with latent carcinoma. Ann Clin Lab Sci, 1987. 17(3): p. 178-82.

139. Sasagawa, I., T. Nakada, H. Suzuki, Y. Adachi, and M. Adachi. Effect of transurethral resection of prostate on plasma hormone levels in benign prostatic hyperplasia. Br J Urol, 1993. 72(5 Pt 1): p. 611-4.

140. Phadke, M.A., G.R. Vanage, and A.R. Sheth. Circulating levels of inhibin, prolactin, TSH, LH, and FSH in benign prostatic hypertrophy before and after tumor resection. Prostate, 1987. 10(2): p. 115-22.


- 77 -

141. Hurkadli, K.S., R. Joseph, M.S. Kadam, and A.R. Sheth. Involvement of prostate in the regulation of serum levels of FSH, LH, and prolactin in male rats. Prostate, 1986. 9(4): p. 411-6.

142. Lane, K.E., I. Leav, J. Ziar, R.S. Bridges, W.M. Rand, and S.M. Ho. Suppression of testosterone and estradiol-17beta-induced dysplasia in the dorsolateral prostate of Noble rats by bromocriptine. Carcinogenesis, 1997. 18(8): p. 1505-10.

143. Stoker, T.E., C.L. Robinette, B.H. Britt, S.C. Laws, and R.L. Cooper. Prepubertal exposure to compounds that increase prolactin secretion in the male rat: effects on the adult prostate. Biol Reprod, 1999. 61(6): p. 1636-43.

144. Stoker, T.E., C.L. Robinette, and R.L. Cooper. Maternal exposure to atrazine during lactation suppresses suckling- induced prolactin release and results in prostatitis in the adult offspring. Toxicol Sci, 1999. 52(1): p. 68-79.

145. Dunning, W. Natl Cancer Inst Monogr, 1963(12): p. 351-69. 146. Greenberg, N.M. Androgens and growth factors in prostate cancer: a transgenic

perspective. Prostate Cancer Prostatic Dis, 2000. 3(4): p. 224-228. 147. Kasper, S., P.C. Sheppard, Y. Yan, N. Pettigrew, A.D. Borowsky, G.S. Prins,

J.G. Dodd, M.L. Duckworth, and R.J. Matusik. Development, progression, and androgen-dependence of prostate tumors in probasin-large T antigen transgenic mice: a model for prostate cancer. Lab Invest, 1998. 78(3): p. 319-33.

148. Horseman, N.D., W. Zhao, E. Montecino-Rodriguez, M. Tanaka, K. Nakashima, S.J. Engle, F. Smith, E. Markoff, and K. Dorshkind. Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene. Embo J, 1997. 16(23): p. 6926-35.

149. Ormandy, C.J., A. Camus, J. Barra, D. Damotte, B. Lucas, H. Buteau, M. Edery, N. Brousse, C. Babinet, N. Binart, et al. Null mutation of the prolactin receptor gene produces multiple reproductive defects in the mouse. Genes Dev, 1997. 11(2): p. 167-78.

150. Robertson, F., J. Harris, M. Naylor, S. Oakes, J. Kindblom, K. Dillner, H. Wennbo, J. Törnell, P.A. Kelly, J. Green, et al. Prostate Development and Carcinogenesis in Prolactin Receptor Knockout Mice. Endocrinology, 2003(Accepted for publication).

151. Akira, S. Functional roles of STAT family proteins: lessons from knockout mice. Stem Cells, 1999. 17(3): p. 138-46.

152. Ahonen, T.J., P.L. Harkonen, H. Rui, and M.T. Nevalainen. PRL signal transduction in the epithelial compartment of rat prostate maintained as long-term organ cultures in vitro. Endocrinology, 2002. 143(1): p. 228-38.

153. Nevalainen, M.T., T.J. Ahonen, H. Yamashita, V. Chandrashekar, A. Bartke, P.M. Grimley, G.W. Robinson, L. Hennighausen, and H. Rui. Epithelial defect in prostates of Stat5a-null mice. Lab Invest, 2000. 80(7): p. 993-1006.

154. Stanbrough, M., I. Leav, P.W. Kwan, G.J. Bubley, and S.P. Balk. Prostatic intraepithelial neoplasia in mice expressing an androgen receptor transgene in prostate epithelium. Proc Natl Acad Sci U S A, 2001. 98(19): p. 10823-8.

155. Jarred, R.A., S.J. McPherson, J.J. Bianco, J.F. Couse, K.S. Korach, and G.P. Risbridger. Prostate phenotypes in estrogen-modulated transgenic mice. Trends Endocrinol Metab, 2002. 13(4): p. 163-8.

156. Adams, J.Y., I. Leav, K.M. Lau, S.M. Ho, and S.M. Pflueger. Expression of estrogen receptor beta in the fetal, neonatal, and prepubertal human prostate. Prostate, 2002. 52(1):: p. 69-81.

157. Andersson, H. and L.E. Tisell. Morphology of rat prostatic lobes and seminal vesicles after long-term estrogen treatment. Acta Pathol Microbiol Immunol Scand [A], 1982. 90(6): p. 441-8.

158. Risbridger, G., H. Wang, P. Young, T. Kurita, Y.Z. Wong, D. Lubahn, J.A. Gustafsson, and G. Cunha. Evidence that epithelial and mesenchymal estrogen receptor-alpha mediates effects of estrogen on prostatic epithelium. Dev Biol, 2001. 229(2): p. 432-42.

Karin Dillner

- 78 -

159. Risbridger, G.P., H. Wang, M. Frydenberg, and G. Cunha. The metaplastic effects of estrogen on mouse prostate epithelium: proliferation of cells with basal cell phenotype. Endocrinology, 2001. 142(6): p. 2443-50.

160. Prins, G.S., L. Birch, J.F. Couse, I. Choi, B. Katzenellenbogen, and K.S. Korach. Estrogen imprinting of the developing prostate gland is mediated through stromal estrogen receptor alpha: studies with alphaERKO and betaERKO mice. Cancer Res, 2001. 61(16): p. 6089-97.

161. McPherson, S.J., H. Wang, M.E. Jones, J. Pedersen, T.P. Iismaa, N. Wreford, E.R. Simpson, and G.P. Risbridger. Elevated androgens and prolactin in aromatase-deficient mice cause enlargement, but not malignancy, of the prostate gland. Endocrinology, 2001. 142(6): p. 2458-67.

162. Krege, J.H., J.B. Hodgin, J.F. Couse, E. Enmark, M. Warner, J.F. Mahler, M. Sar, K.S. Korach, J.A. Gustafsson, and O. Smithies. Generation and reproductive phenotypes of mice lacking estrogen receptor beta. Proc Natl Acad Sci U S A, 1998. 95(26): p. 15677-82.

163. Weihua, Z., S. Makela, L.C. Andersson, S. Salmi, S. Saji, J.I. Webster, E.V. Jensen, S. Nilsson, M. Warner, and J.A. Gustafsson. A role for estrogen receptor beta in the regulation of growth of the ventral prostate. Proc Natl Acad Sci U S A, 2001. 98(11): p. 6330-5.

164. Li, X., E. Nokkala, W. Yan, T. Streng, N. Saarinen, A. Warri, I. Huhtaniemi, R. Santti, S. Makela, and M. Poutanen. Altered structure and function of reproductive organs in transgenic male mice overexpressing human aromatase. Endocrinology, 2001. 142(6): p. 2435-42.

165. Prins, G.S. and L. Birch. The developmental pattern of androgen receptor expression in rat prostate lobes is altered after neonatal exposure to estrogen. Endocrinology, 1995. 136(3): p. 1303-14.

166. Wu, C.P. and F.L. Gu. The prostate in eunuchs. Prog Clin Biol Res, 1991. 370: p. 249-55.

167. Kerr, J.F. and J. Searle. Deletion of cells by apoptosis during castration-induced involution of the rat prostate. Virchows Arch B Cell Pathol, 1973. 13(2): p. 87-102.

168. Sandford, N.L., J.W. Searle, and J.F. Kerr. Successive waves of apoptosis in the rat prostate after repeated withdrawal of testosterone stimulation. Pathology, 1984. 16(4): p. 406-10.

169. Kyprianou, N. and J.T. Isaacs. Activation of programmed cell death in the rat ventral prostate after castration. Endocrinology, 1988. 122(2): p. 552-62.

170. Evans, G.S. and J.A. Chandler. Cell proliferation studies in the rat prostate: II. The effects of castration and androgen-induced regeneration upon basal and secretory cell proliferation. Prostate, 1987. 11(4): p. 339-51.

171. Thomas, J.A., M.S. Manandhar, E.J. Keenan, W.D. Edwards, and P.A. Klase. Effects of prolactin and dihydrotestosterone upon the rat prostate gland. Urol Int, 1976. 31(4): p. 265-71.

172. Keenan, E.J., E.D. Kemp, E.E. Ramsey, L.B. Garrison, H.D. Pearse, and C.V. Hodges. Specific binding of prolactin by the prostate gland of the rat and man. J Urol, 1979. 122(1): p. 43-6.

173. Jones, R., P.R. Riding, and M.G. Parker. Effects of prolactin on testosterone-induced growth and protein synthesis in rat accessory sex glands. J Endocrinol, 1983. 96(3): p. 407-16.

174. Reiter, E., P. Bonnet, B. Sente, D. Dombrowicz, J. de Leval, J. Closset, and G. Hennen. Growth hormone and prolactin stimulate androgen receptor, insulin-like growth factor-I (IGF-I) and IGF-I receptor levels in the prostate of immature rats. Mol Cell Endocrinol, 1992. 88(1-3): p. 77-87.

175. Prins, G.S. Prolactin influence on cytosol and nuclear androgen receptors in the ventral, dorsal, and lateral lobes of the rat prostate. Endocrinology, 1987. 120(4): p. 1457-64.

176. Coert, A., H. Nievelstein, H.J. Kloosterboer, P. Loonen, and J. van der Vies. Effects of hyperprolactinemia on the accessory sexual organs of the male rat. Prostate, 1985. 6(3): p. 269-76.


- 79 -

177. Odoma, S., G.D. Chisholm, K. Nicol, and F.K. Habib. Evidence for the association between blood prolactin and androgen receptors in BPH. J Urol, 1985. 133(4): p. 717-20.

178. Griffiths, K., P. Davies, C.L. Eaton, M.E. Harper, W.B. Peeling, A.O. Turkes, A. Turkes, D.W. Wilson, and C.G. Pierrepoint. Cancer of the prostate: endocrine factors. Oxf Rev Reprod Biol, 1987. 9: p. 192-259.

179. Reinikainen, P., J.J. Palvimo, and O.A. Janne. Effects of mitogens on androgen receptor-mediated transactivation. Endocrinology, 1996. 137(10): p. 4351-7.

180. Stocklin, E., M. Wissler, F. Gouilleux, and B. Groner. Functional interactions between Stat5 and the glucocorticoid receptor. Nature, 1996. 383(6602): p. 726-8.

181. Takeyama, M., T. Nagareda, D. Takatsuka, M. Namiki, K. Koizumi, T. Aono, and K. Matsumoto. Stimulatory effect of prolactin on luteinizing hormone-induced testicular 5 alpha-reductase activity in hypophysectomized adult rats. Endocrinology, 1986. 118(6): p. 2268-75.

182. Yamanaka, H., R.Y. Kirdani, J. Saroff, G.P. Murphy, and A.A. Sandberg. Effects of testosterone and prolactin on rat prostatic weight, 5alpha-reductase, and arginase. Am J Physiol, 1975. 229(4): p. 1102-9.

183. Farnsworth, W.E. Prolactin effect on the permeability of human benign hyperplastic prostate to testosterone. Prostate, 1988. 12(3): p. 221-9.

184. Segal, S., H. Yaffe, N. Laufer, and M. Ben-David. Male hyperprolactinemia:effects on fertility. Fertil Steril, 1979. 32(5): p. 556-61.

185. Dombrowicz, D., B. Sente, J. Closset, and G. Hennen. Dose-dependent effects of human prolactin on the immature hypophysectomized rat testis. Endocrinology, 1992. 130(2): p. 695-700.

186. Cooke, P.S., P. Young, R.A. Hess, and G.R. Cunha. Estrogen receptor expression in developing epididymis, efferent ductules, and other male reproductive organs. Endocrinology, 1991. 128(6): p. 2874-9.

187. Couse, J.F., J. Lindzey, K. Grandien, J.A. Gustafsson, and K.S. Korach. Tissue distribution and quantitative analysis of estrogen receptor- alpha (ERalpha) and estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and ERalpha-knockout mouse. Endocrinology, 1997. 138(11): p. 4613-21.

188. Makela, S., L. Strauss, G. Kuiper, E. Valve, S. Salmi, R. Santti, and J.A. Gustafsson. Differential expression of estrogen receptors alpha and beta in adult rat accessory sex glands and lower urinary tract. Mol Cell Endocrinol, 2000. 164(1-2): p. 109-16.

189. vom Saal, F.S., B.G. Timms, M.M. Montano, P. Palanza, K.A. Thayer, S.C. Nagel, M.D. Dhar, V.K. Ganjam, S. Parmigiani, and W.V. Welshons. Prostate enlargement in mice due to fetal exposure to low doses of estradiol or diethylstilbestrol and opposite effects at high doses. Proc Natl Acad Sci U S A, 1997. 94(5): p. 2056-61.

190. Prins, G.S. Neonatal estrogen exposure induces lobe-specific alterations in adult rat prostate androgen receptor expression. Endocrinology, 1992. 130(6): p. 3703-14.

191. Kalland, T., J.G. Forsberg, and Y.N. Sinha. Long-term effects of neonatal DES treatment on plasma prolactin in female mice. Endocr Res Commun, 1980. 7(3): p. 157-66.

192. Lopez, J., L. Ogren, and F. Talamantes. Effects of neonatal treatment with diethylstilbestrol and 17 alpha-hydroxyprogesterone caproate on in vitro pituitary prolactin secretion. Life Sci, 1984. 34(23): p. 2303-11.

193. Khurana, S., S. Ranmal, and N. Ben-Jonathan. Exposure of newborn male and female rats to environmental estrogens: delayed and sustained hyperprolactinemia and alterations in estrogen receptor expression. Endocrinology, 2000. 141(12): p. 4512-7.

194. Gibori, G., J.S. Richards, and P.L. Keyes. Synergistic effects of prolactin and estradiol in the luteotropic process in the pregnant rat: regulation of estradiol receptor by prolactin. Biol Reprod, 1979. 21(2): p. 419-23.

195. Tessier, C., S. Deb, A. Prigent-Tessier, S. Ferguson-Gottschall, G.B. Gibori, R.P. Shiu, and G. Gibori. Estrogen receptors alpha and beta in rat decidua cells:

Karin Dillner

- 80 -

cell-specific expression and differential regulation by steroid hormones and prolactin. Endocrinology, 2000. 141(10): p. 3842-51.

196. Frasor, J., K. Park, M. Byers, C. Telleria, T. Kitamura, L.Y. Yu-Lee, J. Djiane, O.K. Park-Sarge, and G. Gibori. Differential roles for signal transducers and activators of transcription 5a and 5b in PRL stimulation of ERalpha and ERbeta transcription. Mol Endocrinol, 2001. 15(12): p. 2172-81.

197. Edery, M., W. Imagawa, L. Larson, and S. Nandi. Regulation of estrogen and progesterone receptor levels in mouse mammary epithelial cells grown in serum-free collagen gel cultures. Endocrinology, 1985. 116(1): p. 105-12.

198. Norstedt, G., O. Wrange, and J.A. Gustafsson. Multihormonal regulation of the estrogen receptor in rat liver. Endocrinology, 1981. 108(4): p. 1190-6.

199. Belis, J.A., L.B. Adlestein, and W.F. Tarry. Influence of estradiol on accessory reproductive organs in the castrated male rat. Effects of bromocriptine and flutamide. J Androl, 1983. 4(2): p. 144-9.

200. Hellawell, G.O. and S.F. Brewster. Growth factors and their receptors in prostate cancer. BJU Int, 2002. 89(3): p. 230-40.

201. Reiter, E., B. Hennuy, M. Bruyninx, A. Cornet, M. Klug, M. McNamara, J. Closset, and G. Hennen. Effects of pituitary hormones on the prostate. Prostate, 1999. 38(2): p. 159-65.

202. Bretland, A.J., S.V. Reid, C.R. Chapple, and C.L. Eaton. Role of endogenous transforming growth factor beta (TGFbeta)1 in prostatic stromal cells. Prostate, 2001. 48(4): p. 297-304.

203. Byrne, R.L., H. Leung, and D.E. Neal. Peptide growth factors in the prostate as mediators of stromal epithelial interaction. Br J Urol, 1996. 77(5): p. 627-33.

204. Venter, J.C., M.D. Adams, E.W. Myers, P.W. Li, R.J. Mural, G.G. Sutton, H.O. Smith, M. Yandell, C.A. Evans, R.A. Holt, et al. The sequence of the human genome. Science, 2001. 291(5507): p. 1304-51.

205. Waterston, R.H., K. Lindblad-Toh, E. Birney, J. Rogers, J.F. Abril, P. Agarwal, R. Agarwala, R. Ainscough, M. Alexandersson, P. An, et al. Initial sequencing and comparative analysis of the mouse genome. Nature, 2002. 420(6915): p. 520-62.

206. Brinster, R.L., H.Y. Chen, M. Trumbauer, A.W. Senear, R. Warren, and R.D. Palmiter. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell, 1981. 27(1 Pt 2): p. 223-31.

207. Thomas, K.R. and M.R. Capecchi. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell, 1987. 51(3): p. 503-12.

208. Andrews, G.K., E.D. Adamson, and L. Gedamu. The ontogeny of expression of murine metallothionein: comparison with the alpha-fetoprotein gene. Dev Biol, 1984. 103(2): p. 294-303.

209. Koropatnick, J. and J.D. Duerksen. Nuclease sensitivity of alpha-fetoprotein, metallothionein-1, and immunoglobulin gene sequences in mouse during development. Dev Biol, 1987. 122(1): p. 1-10.

210. Greenberg, N.M., F.J. DeMayo, P.C. Sheppard, R. Barrios, R. Lebovitz, M. Finegold, R. Angelopoulou, J.G. Dodd, M.L. Duckworth, J.M. Rosen, et al. The rat probasin gene promoter directs hormonally and developmentally regulated expression of a heterologous gene specifically to the prostate in transgenic mice. Mol Endocrinol, 1994. 8(2): p. 230-9.

211. Matuo, Y., N. Nishi, Y. Muguruma, Y. Yoshitake, N. Kurata, and F. Wada. Localization of prostatic basic protein ("probasin") in the rat prostates by use of monoclonal antibody. Biochem Biophys Res Commun, 1985. 130(1): p. 293-300.

212. Hubank, M. and D.G. Schatz. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res, 1994. 22(25): p. 5640-8.

213. Staden, R., K.F. Beal, and J.K. Bonfield. The Staden package, 1998. Methods Mol Biol, 2000. 132: p. 115-30.

214. Altschul, S.F., W. Gish, W. Miller, E.W. Myers, and D.J. Lipman. Basic local alignment search tool. J Mol Biol, 1990. 215(3): p. 403-10.


- 81 -

215. Lockhart, D.J., H. Dong, M.C. Byrne, M.T. Follettie, M.V. Gallo, M.S. Chee, M. Mittmann, C. Wang, M. Kobayashi, H. Horton, et al. Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat Biotechnol, 1996. 14(13): p. 1675-80.

216. Schena, M., D. Shalon, R.W. Davis, and P.O. Brown. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 1995. 270(5235): p. 467-70.

217. Cirelli, C. and G. Tononi. Differences in gene expression during sleep and wakefulness. Ann Med, 1999. 31(2): p. 117-24.

218. Duggan, D.J., M. Bittner, Y. Chen, P. Meltzer, and J.M. Trent. Expression profiling using cDNA microarrays. Nat Genet, 1999. 21(1 Suppl): p. 10-4.

219. Wang, E., L.D. Miller, G.A. Ohnmacht, E.T. Liu, and F.M. Marincola. High-fidelity mRNA amplification for gene profiling. Nat Biotechnol, 2000. 18(4): p. 457-9.

220. Dorris, D.R., R. Ramakrishnan, D. Trakas, F. Dudzik, R. Belval, C. Zhao, A. Nguyen, M. Domanus, and A. Mazumder. A highly reproducible, linear, and automated sample preparation method for DNA microarrays. Genome Res, 2002. 12(6): p. 976-84.

221. Hertzberg, M., M. Sievertzon, H. Aspeborg, P. Nilsson, G. Sandberg, and J. Lundeberg. cDNA microarray analysis of small plant tissue samples using a cDNA tag target amplification protocol. Plant J, 2001. 25(5): p. 585-91.

222. Hertzberg, M., H. Aspeborg, J. Schrader, A. Andersson, R. Erlandsson, K. Blomqvist, R. Bhalerao, M. Uhlen, T.T. Teeri, J. Lundeberg, et al. A transcriptional roadmap to wood formation. Proc Natl Acad Sci U S A, 2001. 98(25): p. 14732-7.

223. Diehl, F., S. Grahlmann, M. Beier, and J.D. Hoheisel. Manufacturing DNA microarrays of high spot homogeneity and reduced background signal. Nucleic Acids Res, 2001. 29(7): p. E38.

224. Hegde, P., R. Qi, K. Abernathy, C. Gay, S. Dharap, R. Gaspard, J.E. Hughes, E. Snesrud, N. Lee, and J. Quackenbush. A concise guide to cDNA microarray analysis. Biotechniques, 2000. 29(3): p. 548-50, 552-4, 556 passim.

225. Nguyen, C., D. Rocha, S. Granjeaud, M. Baldit, K. Bernard, P. Naquet, and B.R. Jordan. Differential gene expression in the murine thymus assayed by quantitative hybridization of arrayed cDNA clones. Genomics, 1995. 29(1): p. 207-16.

226. Dudoit, S., Y.H. Yang, M.J. Callow, and T.P. Speed. Statistical methods for identifying differentially expressed genes in replicated cDNA microarray experiments. 2000, Department of Statistics: UC Berkeley.

227. Yang, Y.H., S. Dudoit, P. Luu, and T.P. Speed. Normalization for cDNA microarray data, in Microarrays: Optical Technologies and Informatics, M.L. Bittner, Y. Chen, A.N. A. N. Dorsel, and E.R. Dougherty, Editors. 2001, Proceedings of SPIE BiOS: San Jose CA. p. 141–152.

228. Ihaka, R. and R. Gentleman. R: A language for data analysis and graphics. Journal of Computational and Graphical Statistics, 1996. 5: p. 299-314.

229. Lee, M.L., F.C. Kuo, G.A. Whitmore, and J. Sklar. Importance of replication in microarray gene expression studies: statistical methods and evidence from repetitive cDNA hybridizations. Proc Natl Acad Sci U S A, 2000. 97(18): p. 9834-9.

230. Black, M.A. and R.W. Doerge. Calculation of the minimum number of replicate spots required for detection of significant gene expression fold change in microarray experiments. Bioinformatics, 2002. 18(12): p. 1609-16.

231. Tusher, V.G., R. Tibshirani, and G. Chu. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A, 2001. 98(9): p. 5116-21.

232. Churchill, G.A. Fundamentals of experimental design for cDNA microarrays. Nat Genet, 2002. 32 Suppl: p. 490-5.

233. Kerr, M.K. and G.A. Churchill. Statistical design and the analysis of gene expression microarray data. Genet Res, 2001. 77(2): p. 123-8.

Karin Dillner

- 82 -

234. Yang, Y.H., M.J. Buckley, and T.P. Speed. Analysis of cDNA microarray images. Brief Bioinform, 2001. 2(4): p. 341-9.

235. Brownie, J., S. Shawcross, J. Theaker, D. Whitcombe, R. Ferrie, C. Newton, and S. Little. The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res, 1997. 25(16): p. 3235-41.

236. Wittwer, C.T., M.G. Herrmann, A.A. Moss, and R.P. Rasmussen. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques, 1997. 22(1): p. 130-1, 134-8.

237. Orlando, C., P. Pinzani, and M. Pazzagli. Developments in quantitative PCR. Clin Chem Lab Med, 1998. 36(5): p. 255-69.

238. Heid, C.A., J. Stevens, K.J. Livak, and P.M. Williams. Real time quantitative PCR. Genome Res, 1996. 6(10): p. 986-94.

239. Morrison, T.B., J.J. Weis, and C.T. Wittwer. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques, 1998. 24(6): p. 954-8, 960, 962.

240. Ririe, K.M., R.P. Rasmussen, and C.T. Wittwer. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem, 1997. 245(2): p. 154-60.

241. Lincz, L.F. Deciphering the apoptotic pathway: all roads lead to death. Immunol Cell Biol, 1998. 76(1): p. 1-19.

242. Darzynkiewicz, Z., G. Juan, X. Li, W. Gorczyca, T. Murakami, and F. Traganos. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry, 1997. 27(1): p. 1-20.

243. Majno, G. and I. Joris. Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol, 1995. 146(1): p. 3-15.

244. Kroemer, G., P. Petit, N. Zamzami, J.L. Vayssiere, and B. Mignotte. The biochemistry of programmed cell death. Faseb J, 1995. 9(13): p. 1277-87.

245. Williams, G.T. and C.A. Smith. Molecular regulation of apoptosis: genetic controls on cell death. Cell, 1993. 74(5): p. 777-9.

246. Kobayashi, S., H. Iwase, Y. Kawarada, N. Miura, T. Sugiyama, H. Iwata, Y. Hara, Y. Omoto, and T. Nakamura. Detection of DNA Fragmentation in Human Breast Cancer Tissue by an Antibody Specific to Single-stranded DNA. Breast Cancer, 1998. 5(1): p. 47-52.

247. Maeda, M., T. Sugiyama, F. Akai, I. Jikihara, Y. Hayashi, and H. Takagi. Single stranded DNA as an immunocytochemical marker for apoptotic change of ischemia in the gerbil hippocampus. Neurosci Lett, 1998. 240(2): p. 69-72.

248. Frankfurt, O.S., J.A. Robb, E.V. Sugarbaker, and L. Villa. Monoclonal antibody to single-stranded DNA is a specific and sensitive cellular marker of apoptosis. Exp Cell Res, 1996. 226(2): p. 387-97.

249. Watanabe, I., M. Toyoda, J. Okuda, T. Tenjo, K. Tanaka, T. Yamamoto, H. Kawasaki, T. Sugiyama, Y. Kawarada, and N. Tanigawa. Detection of apoptotic cells in human colorectal cancer by two different in situ methods: antibody against single-stranded DNA and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) methods. Jpn J Cancer Res, 1999. 90(2): p. 188-93.

250. Cryns, V. and J. Yuan. Proteases to die for. Genes Dev, 1998. 12(11): p. 1551-70. 251. Thornberry, N.A. and Y. Lazebnik. Caspases: enemies within. Science, 1998.

281(5381): p. 1312-6. 252. Thornberry, N.A. Caspases: a decade of death research. Cell Death Differ, 1999.

6(11): p. 1023-7. 253. Slee, E.A., C. Adrain, and S.J. Martin. Executioner caspase-3, -6, and -7 perform

distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem, 2001. 276(10): p. 7320-6.

254. Goyal, L. Cell death inhibition: keeping caspases in check. Cell, 2001. 104(6): p. 805-8.

255. Berry, S.J. and J.T. Isaacs. Comparative aspects of prostatic growth and androgen metabolism with aging in the dog versus the rat. Endocrinology, 1984. 114(2): p. 511-20.


- 83 -

256. Banerjee, P.P., S. Banerjee, R. Dorsey, B.R. Zirkin, and T.R. Brown. Age- and lobe-specific responses of the brown Norway rat prostate to androgen. Biol Reprod, 1994. 51(4): p. 675-84.

257. Tollet-Egnell, P., A. Flores-Morales, N. Stahlberg, R.L. Malek, N. Lee, and G. Norstedt. Gene Expression Profile of the Aging Process in Rat Liver: Normalizing Effects of Growth Hormone Replacement. Mol Endocrinol, 2001. 15(2): p. 308-318.

258. Flores-Morales, A., N. Stahlberg, P. Tollet-Egnell, J. Lundeberg, R.L. Malek, J. Quackenbush, N.H. Lee, and G. Norstedt. Microarray analysis of the in vivo effects of hypophysectomy and growth hormone treatment on gene expression in the rat. Endocrinology, 2001. 142(7): p. 3163-76.

259. Welford, S.M., J. Gregg, E. Chen, D. Garrison, P.H. Sorensen, C.T. Denny, and S.F. Nelson. Detection of differentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray hybridization. Nucleic Acids Res, 1998. 26(12): p. 3059-65.

260. Park, J.H., J.E. Walls, J.J. Galvez, M. Kim, C. Abate-Shen, M.M. Shen, and R.D. Cardiff. Prostatic intraepithelial neoplasia in genetically engineered mice. Am J Pathol, 2002. 161(2): p. 727-35.

261. Pang, S.T., K. Dillner, X. Wu, A. Pousette, G. Norstedt, and A. Flores-Morales. Gene expression profiling of androgen deficiency predicts a pathway of prostate apoptosis that involves genes related to oxidative stress. Endocrinology, 2002. 143(12): p. 4897-906.

262. Iyer, V.R., M.B. Eisen, D.T. Ross, G. Schuler, T. Moore, J.C. Lee, J.M. Trent, L.M. Staudt, J. Hudson, M.S. Boguski, et al. The transcriptional program in the response of human fibroblasts to serum. Science, 1999. 283(5398): p. 83-7.

263. Tripathi, Y. and A.K. Mukhopadhyay. Acid phosphatase activity and fresh tissue weights of accessory sex organs in adult male rats following pituitary transplantation. Indian J Physiol Pharmacol, 1988. 32(4): p. 271-7.

264. Goffin, V., N. Binart, P. Touraine, and P.A. Kelly. Prolactin: the new biology of an old hormone. Annu Rev Physiol, 2002. 64: p. 47-67.

265. Singh, J. and D.J. Handelsman. Imprinting by neonatal sex steroids on the structure and function of the mature mouse prostate. Biol Reprod, 1999. 61(1): p. 200-8.

266. Bartsch, G., H.R. Muller, M. Oberholzer, and H.P. Rohr. Light microscopic stereological analysis of the normal human prostate and of benign prostatic hyperplasia. J Urol, 1979. 122(4): p. 487-91.

267. Bartsch, G., J. Frick, I. Ruegg, M. Bucher, O. Holliger, M. Oberholzer, and H.P. Rohr. Electron microscopic stereological analysis of the normal human prostate and of benign prostatic hyperplasia. J Urol, 1979. 122(4): p. 481-6.

268. Hayward, S.W., M.A. Rosen, and G.R. Cunha. Stromal-epithelial interactions in the normal and neoplastic prostate. Br J Urol, 1997. 79 Suppl 2: p. 18-26.

269. Liotta, L.A. and E.C. Kohn. The microenvironment of the tumour-host interface. Nature, 2001. 411(6835): p. 375-9.

270. Nonomura, M., K. Hoshino, T. Harigaya, H. Hashimoto, and O. Yoshida. Effects of hyperprolactinaemia on reproduction in male mice. J Endocrinol, 1985. 107(1): p. 71-6.

271. Steers, W.D. 5alpha-reductase activity in the prostate. Urology, 2001. 58(6 Suppl 1): p. 17-24; discussion 24.

272. Cunha, G.R. Androgenic effects upon prostatic epithelium are mediated via trophic influences from stroma. Prog Clin Biol Res, 1984. 145: p. 81-102.

273. Claus, S., R. Berges, T. Senge, and H. Schulze. Cell kinetic in epithelium and stroma of benign prostatic hyperplasia. J Urol, 1997. 158(1): p. 217-21.

274. Cardillo, M., G. Berchem, M.A. Tarkington, S. Krajewski, M. Krajewski, J.C. Reed, T. Tehan, L. Ortega, J. Lage, and E.P. Gelmann. Resistance to apoptosis and up regulation of Bcl-2 in benign prostatic hyperplasia after androgen deprivation. J Urol, 1997. 158(1): p. 212-6.

Karin Dillner

- 84 -

275. Van Coppenolle, F., C. Slomianny, F. Carpentier, X. Le Bourhis, A. Ahidouch, D. Croix, G. Legrand, E. Dewailly, S. Fournier, H. Cousse, et al. Effects of hyperprolactinemia on rat prostate growth: evidence of androgeno-dependence. Am J Physiol Endocrinol Metab, 2001. 280(1): p. E120-9.

276. Ruffion, A., K.A. Al-Sakkaf, B.L. Brown, C.L. Eaton, F.C. Hamdy, and P.R. Dobson. The Survival Effect of Prolactin on PC3 Prostate Cancer Cells. Eur Urol, 2003. 43(3): p. 301-8.

277. Munshi, A., G. Pappas, T. Honda, T.J. McDonnell, A. Younes, Y. Li, and R.E. Meyn. TRAIL (APO-2L) induces apoptosis in human prostate cancer cells that is inhibitable by Bcl-2. Oncogene, 2001. 20(29): p. 3757-65.

278. Datta, S.R., H. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh, and M.E. Greenberg. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell, 1997. 91(2): p. 231-41.

279. del Peso, L., M. Gonzalez-Garcia, C. Page, R. Herrera, and G. Nunez. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science, 1997. 278(5338): p. 687-9.

280. Tessier, C., A. Prigent-Tessier, S. Ferguson-Gottschall, Y. Gu, and G. Gibori. PRL antiapoptotic effect in the rat decidua involves the PI3K/protein kinase B-mediated inhibition of caspase-3 activity. Endocrinology, 2001. 142(9): p. 4086-94.

Molecular Characterization of Prostate Hyperplasia …...Molecular Characterization of Prostate...

Documents

Transcript of Molecular Characterization of Prostate Hyperplasia …...Molecular Characterization of Prostate...