Cancer as a Disease of DNA Organization and Dynamic Cell ... · .^-dimensional organization and...

9
[CANCER RESEARCH 49, 2525-2532, May 15, 1989] Perspectives in Cancer Research Cancer as a Disease of DNA Organization and Dynamic Cell Structure1 Kenneth J. Pienta, Alan W. Partin, and Donald S. Coffey The Johns Hopkins Oncology Center and the Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Cancer cells develop resistance to all known natural and synthetic drugs; normal cells do not. This resistance is a reflec tion in part of the wide diversity of functions expressed by cancer cells within a tumor. This variation in function (pleio- tropism) is accompanied by variation in structure (pleomor- phism), and together they form the basis for tumor cell heter ogeneity. This tumor cell heterogeneity provides malignant tumors with a tremendous biological diversity, which enables them to succeed in a competitive environment that includes therapeutic manipulations. The driving force in the develop ment of tumor cell heterogeneity is thought to be genetic instability (1-4). However, it is unknown whether cell structure determines this instability or whether the instability of the DNA itself produces the instability in structure (5, 6). It is well recognized that chromatin structure can regulate DNA function within the cell (7-11). The purpose of this "Perspectives" article is to provide an overview of the importance of nuclear and cell structure in DNA organization and suggest how these may be altered in the cancer cell. The Nuclear Matrix How the vast array of DNA is arranged within the nucleus in an organized fashion is difficult to comprehend. For example, if the nucleus were magnified in size to a sphere 3 feet in diameter, the DNA molecule would extend as a filament for 100 miles. Following replication and before mitosis, this fila ment length would double to 200 miles. This vast amount of DNA must be spatially organized in order to avoid any entan glement during replication and subsequent mitosis. This could not be accomplished by free-floating or soluble DNA but must require a precise 3-dimensional organization and topological considerations. The organization of interphase DNA is believed to be accomplished by the interaction of the DNA at specific sites to a nuclear matrix system. The nuclear matrix is defined as the dynamic structural subcomponent of the nucleus that directs the 3-dimensional organization of DNA into loop domains and provides sites for the specific control of nucleic acid intranuclear and particulate transport (12). Conceptually, the nuclear matrix can be viewed as the nuclear equivalent to the cytomatrix. These matrix struc tures had also been termed "skeletal" or "scaffolding" compo nents, until it became apparent that they exhibited dynamic properties and were not simply rigid framework structures. The nuclear matrix is not a single structural entity but a complex that contains specific subcomponents such as the pore-complex- lamina, residual nucleoli, and internal ribonucleoprotein parti cles attached to a dynamic fibrous network of proteins, RNA, and polysaccharides (13-20). The nuclear matrix structure is essentially devoid of histones and lipids and represents less than 15% of the mass of the intact nucleus (Fig. 1). The nuclear matrix is an important structural component in a variety of nuclear functions reviewed in Table 1. Primarily, the nuclear matrix serves an important role in DNA organiza tion and nuclear structure. DNA loop domains are attached at their bases to the nuclear matrix (21, 22), and this organization is maintained throughout both interphase and metaphase (23- 27). These loops are 50-150 kbp2 long and are equivalent in size to the replicón, i.e., the amount of DNA replicated as a unit during DNA synthesis that resides between adjacent rep licating forks (21). There are approximately 50,000 to 100,000 of these DNA loops per nucleus. Constrained at their bases to the matrix, the DNA loops are often supercoiled (22), which may control in part the chromatin structure through changed DNA topology. Topoisomerase II, one of the enzymes that modulates DNA topology, is associated with the interphase nuclear matrix (28, 29) and with the mitotic chromosome scaffold (30). The nuclear matrix also plays an important role in DNA replication (31). The matrix contains fixed sites for DNA synthesis (21, 32-34) located at the base of the loops. During DNA synthesis the loop domains are reeled down through the attached replicating complexes. When the total DNA attached to the nuclear matrix is treated with EcoRl restriction enzyme, a minute fraction of the DNA still remains on the matrix that is enriched in replicative forks (35). Vogelstein et al. (22) have been able to visualize and follow the rate of movement of labeled DNA into these loop domains as they are replicated. Further more, Tubo et al. (36) have reported that matrix-bound DNA synthesis /;/ vitro continued from replication sites being used for DNA synthesis in intact nuclei in vivo at the time of isolation. The DNA-replicating complex located at the base of the loop has been isolated and termed the reputase (37). The reputase is a 24-30-nm-diameter particle with a molecular weight of approximately 5 million and contains at least eight enzymes which include ribonucleoside diphosphate reductase, thymidylate synthetase, dihydrofolate reductase, DNA meth- ylase, topoisomerase, and DNA polymerase (37). This large multienzyme complex appears to be under allosteric control (38). The DNA replication fork, DNA polymerase a, and newly replicated DNA have all been closely associated with the nuclear matrix during DNA synthesis (32-36, 39). Earnshaw and Heck (30) have shown that the scaffold or matrix of the metaphase chromosome contains topoisomerase II, which is also a com ponent of the interphase nuclear matrix during the time of DNA synthesis (28, 29). Nelson et al. (40) have reported that newly synthesized DNA can be covalently attached to topoisom erase II of the interphase nucleus. With time the newly labeled DNA moves away from the topoisomerase, which indicates that the topoisomerase II is located at the base of the DNA loops in close proximity to, but in the wake of, the replicating fork. This is consistent with the observations of Noguchi et al. (41) that topoisomerase is part of the replicase particle that forms the replisome complex for a fixed site for DNA synthesis. Recently, Dijkwel and Hamlin (42) have identified specific matrix DNA attachment regions that are positioned near rep lication initiation sites and interamplicon junctions in the am- Received 1/15/89; accepted 2/21/89. 1This work was supported by NIH Grants CA 15416 and AM 22000. 2The abbreviations used are: kbp, kilobase pairs; ECM, extracellular matrix. 2525 on June 30, 2020. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Transcript of Cancer as a Disease of DNA Organization and Dynamic Cell ... · .^-dimensional organization and...

Page 1: Cancer as a Disease of DNA Organization and Dynamic Cell ... · .^-dimensional organization and shape. DNA organization: DNA loop domains are attached to 21-30 nuclear matrix at their

[CANCER RESEARCH 49, 2525-2532, May 15, 1989]

Perspectives in Cancer Research

Cancer as a Disease of DNA Organization and Dynamic Cell Structure1

Kenneth J. Pienta, Alan W. Partin, and Donald S. CoffeyThe Johns Hopkins Oncology Center and the Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Cancer cells develop resistance to all known natural andsynthetic drugs; normal cells do not. This resistance is a reflection in part of the wide diversity of functions expressed bycancer cells within a tumor. This variation in function (pleio-tropism) is accompanied by variation in structure (pleomor-phism), and together they form the basis for tumor cell heterogeneity. This tumor cell heterogeneity provides malignanttumors with a tremendous biological diversity, which enablesthem to succeed in a competitive environment that includestherapeutic manipulations. The driving force in the development of tumor cell heterogeneity is thought to be geneticinstability (1-4). However, it is unknown whether cell structuredetermines this instability or whether the instability of the DNAitself produces the instability in structure (5, 6). It is wellrecognized that chromatin structure can regulate DNA functionwithin the cell (7-11). The purpose of this "Perspectives" article

is to provide an overview of the importance of nuclear and cellstructure in DNA organization and suggest how these may bealtered in the cancer cell.

The Nuclear Matrix

How the vast array of DNA is arranged within the nucleusin an organized fashion is difficult to comprehend. For example,if the nucleus were magnified in size to a sphere 3 feet indiameter, the DNA molecule would extend as a filament for100 miles. Following replication and before mitosis, this filament length would double to 200 miles. This vast amount ofDNA must be spatially organized in order to avoid any entanglement during replication and subsequent mitosis. This couldnot be accomplished by free-floating or soluble DNA but mustrequire a precise 3-dimensional organization and topologicalconsiderations. The organization of interphase DNA is believedto be accomplished by the interaction of the DNA at specificsites to a nuclear matrix system.

The nuclear matrix is defined as the dynamic structuralsubcomponent of the nucleus that directs the 3-dimensionalorganization of DNA into loop domains and provides sites forthe specific control of nucleic acid intranuclear and particulatetransport (12). Conceptually, the nuclear matrix can be viewedas the nuclear equivalent to the cytomatrix. These matrix structures had also been termed "skeletal" or "scaffolding" compo

nents, until it became apparent that they exhibited dynamicproperties and were not simply rigid framework structures. Thenuclear matrix is not a single structural entity but a complexthat contains specific subcomponents such as the pore-complex-lamina, residual nucleoli, and internal ribonucleoprotein particles attached to a dynamic fibrous network of proteins, RNA,and polysaccharides (13-20). The nuclear matrix structure isessentially devoid of histones and lipids and represents less than15% of the mass of the intact nucleus (Fig. 1).

The nuclear matrix is an important structural component ina variety of nuclear functions reviewed in Table 1. Primarily,

the nuclear matrix serves an important role in DNA organization and nuclear structure. DNA loop domains are attached attheir bases to the nuclear matrix (21, 22), and this organizationis maintained throughout both interphase and metaphase (23-27). These loops are 50-150 kbp2 long and are equivalent in

size to the replicón, i.e., the amount of DNA replicated as aunit during DNA synthesis that resides between adjacent replicating forks (21). There are approximately 50,000 to 100,000of these DNA loops per nucleus. Constrained at their bases tothe matrix, the DNA loops are often supercoiled (22), whichmay control in part the chromatin structure through changedDNA topology. Topoisomerase II, one of the enzymes thatmodulates DNA topology, is associated with the interphasenuclear matrix (28, 29) and with the mitotic chromosomescaffold (30).

The nuclear matrix also plays an important role in DNAreplication (31). The matrix contains fixed sites for DNAsynthesis (21, 32-34) located at the base of the loops. DuringDNA synthesis the loop domains are reeled down through theattached replicating complexes. When the total DNA attachedto the nuclear matrix is treated with EcoRl restriction enzyme,a minute fraction of the DNA still remains on the matrix thatis enriched in replicative forks (35). Vogelstein et al. (22) havebeen able to visualize and follow the rate of movement of labeledDNA into these loop domains as they are replicated. Furthermore, Tubo et al. (36) have reported that matrix-bound DNAsynthesis /;/ vitro continued from replication sites being usedfor DNA synthesis in intact nuclei in vivo at the time ofisolation. The DNA-replicating complex located at the base ofthe loop has been isolated and termed the reputase (37). Thereputase is a 24-30-nm-diameter particle with a molecularweight of approximately 5 million and contains at least eightenzymes which include ribonucleoside diphosphate reductase,thymidylate synthetase, dihydrofolate reductase, DNA meth-ylase, topoisomerase, and DNA polymerase (37). This largemultienzyme complex appears to be under allosteric control(38). The DNA replication fork, DNA polymerase a, and newlyreplicated DNA have all been closely associated with the nuclearmatrix during DNA synthesis (32-36, 39). Earnshaw and Heck(30) have shown that the scaffold or matrix of the metaphasechromosome contains topoisomerase II, which is also a component of the interphase nuclear matrix during the time ofDNA synthesis (28, 29). Nelson et al. (40) have reported thatnewly synthesized DNA can be covalently attached to topoisomerase II of the interphase nucleus. With time the newly labeledDNA moves away from the topoisomerase, which indicates thatthe topoisomerase II is located at the base of the DNA loopsin close proximity to, but in the wake of, the replicating fork.This is consistent with the observations of Noguchi et al. (41)that topoisomerase is part of the replicase particle that formsthe replisome complex for a fixed site for DNA synthesis.Recently, Dijkwel and Hamlin (42) have identified specificmatrix DNA attachment regions that are positioned near replication initiation sites and interamplicon junctions in the am-

Received 1/15/89; accepted 2/21/89.1This work was supported by NIH Grants CA 15416 and AM 22000. 2The abbreviations used are: kbp, kilobase pairs; ECM, extracellular matrix.

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Fig. 1. Isolation of the nuclear matrix. Thenuclear matrix of a normal rat liver nuclei isisolated by sequential extractions using non-ionic detergent, brief DNase I digestion, and ahypertonic salt buffer. These extractions remove over 98% of the DNA, 70% of the RNA,and 90% of the nuclear proteins resulting in aresidual structure that is essentially devoid ofhistones and lipids (12-20). NUCLEUS

SEQUENTIALEXTRACTION1.NONIONICDETERGENT3.

HYPERtONIC SALT

REMOVED90%

PROTEIN98% PHOSPO.LIPID95%DNA70% RNA

NUCLEARMATRIX

Table 1 Nuclear matrix is the dynamic structural subcomponent of the nucleusthat directs the functional organization of DNA into loop domains and provides

organizational sites for many of the functions involving DNA

Reported functions of the nuclear matrix Refs.

Nuclear morphology: The nuclear matrix contains struc- 12-20turai elements of the pore complexes, lamina, internalnetwork, and nucleoli which give the nucleus its overall.^-dimensional organization and shape.

DNA organization: DNA loop domains are attached to 21-30nuclear matrix at their bases and this organization ismaintained during both interphase and metaphase. Nuclear matrix shares some proteins with the chromosome scaffold including topoisomerase II, an enzymewhich modulates DNA topology.

DNA replication: The nuclear matrix has fixed sites for 21, 22, 31-42DNA replication, containing the replisome complexfor DNA replication that includes polymerase andnewly replicated DNA.

RNA synthesis: Actively transcribed genes are associated 47-65with the nuclear matrix. The nuclear matrix containstranscriptional complexes, newly synthesized heterogeneous nuclear RNA, and small nuclear RNA. RNA-processing intermediates are bound to the nuclear matrix.

Nuclear regulation: The nuclear matrix has specific sites 43-46, 66-72, 74,for steroid hormone receptor binding. DNA viruses are 75, 81synthesized in association with the matrix. The nuclearmatrix is a cellular target for transformation proteins,some retrovirus products like the large T antigen, andEIA protein. Many of the nuclear matrix proteins arephosphorylated at specific times in the cell cycle.

plifíeddihydrofolate domain of Chinese hamster ovary cells.Therefore, the nuclear matrix is well positioned to play animportant structural role in the organization and biologicalcontrol of DNA eukaryotic replication. Furthermore, whenDNA viruses replicate in a mammalian cell they are also synthesized in association with the nuclear matrix of the host cell(43-46). It is easy to visualize how alteration in the nuclearmatrix structures could impinge on the regulation of DNAsynthesis.

Many investigations have reported that the nuclear matrix isassociated with nuclear RNA and RNA synthesis (47-57).Transcriptional complexes have been identified on the nuclearmatrix (47). Newly labeled heterogeneous nuclear RNA andsmall nuclear RNA are enriched on the nuclear matrix (48-55).Ciejek et al. (56) observed that 95% of unprocessed mRNAprecursor for several genes, which include ovalbumin, are associated with the nuclear matrix of the chick oviduct. When theintron portions of the primary transcript were processed out,the mature mRNA was formed and released from the nuclearmatrix. The snRNAs are also associated with the matrix andbind to the intron regions of the RNA transcript and may beresponsible for the attachment. Mariman and Van Venrooij(57) reported that all RNA cleavage products and RNA proc

essing intermediates were firmly bound to nuclear matrix. Manystudies with a wide variety of genes have demonstrated thatactive genes are associated with the nuclear matrix while tran-scriptionally inactive genes are not, providing further evidencethat the matrix may play an important organizing role intranscriptional functions (58-65).

Several other observations indicate that the nuclear matrix isan important modulator of nuclear regulation. The nuclearmatrix is a major site of steroid hormone receptor binding (66-72). Barrack et al. (66-68) have shown that in the presence ofspecific steroids, 40-60% of all nuclear steroid receptors (estrogens or androgens) are associated with this matrix. In addition,the nuclear acceptor for the steroid-bound receptor appears tobe part of the nuclear matrix in steroid target tissues (69, 70).New properties of the nuclear matrix have recently been identified. Fey and Penman (73) have reported that nuclear matrixproteins, localized to the interior of the nucleus, vary in a celltype-specific manner, suggesting that the nuclear matrix mayplay an important role in development and tissue organization.

In cancer cells transformation proteins appear to be associated with the nucleus, and many of these appear to be involvedwith the matrix. For example, the nuclear matrix is reported tobe one of the targets for retrovirus myc oncogene protein (74,75), adenovirus ElA-transforming protein (76), and polyomalarge T antigen (77-79). Numatrin, a nuclear matrix protein,has been associated with the induction of mitogenesis (80).Nuclear matrices from various cells contain binding sites formyb proteins (81), and the nuclear matrix has been shown tobe altered during transformation (82-85). Since steroid receptors and transforming proteins such as the nuclear oncogeneproducts affect DNA function in target cells, more insight isrequired into how they alter the functions of the nuclear matrix.

DNA Organization

The packaging and 3-dimensional organization of DNAwithin chromatin, metaphase chromosomes, and the interphasenucleus remain largely unsolved. Dynamic reorganization ofchromatin is visually apparent in the cell cycle from interphaseto metaphase. How the DNA loop domains are maintainedthroughout these transitions of the cell cycle is still unclear.The actual structure of these loop domains has only recentlybegun to be understood. Three higher order levels of DNAorganization have been identified: nuclesomes, 30 nm chroma-tin fibers, and DNA loop domains. Although controversy stillexists about the exact nature of each of these structures, theyare well accepted as basic units of DNA organization. The DNAloop domain was first proposed by Cook et al. (86) in 1976when they suggested that loop structures are involved in thesuperhelical organization of eukaryotic DNA. In 1980, Vogel-stein et al. (22) reported that DNA loop domains were attached

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at their base to the nuclear matrix and that these loops weretopologically constrained by that attachment. Furthermore, thenuclear matrix contained fixed sites for replication of DNAloops that Pardoll et al. (21) showed were the structural equivalents of replicons, the basic lengths of DNA synthesized ascontinuous units (87). The DNA loop domain defines a basicunit of higher order DNA structure which is present throughoutthe cell cycle in eukaryotic cells (88,89). These loops have beenestimated to be between 10 and 180 kbp pairs with an averageof 63 ±14 kbp (90). An average loop would be large enough tocontain 300 nucleosomes wound with 6 nucleosomes per solenoid turn into a 30-nm fiber utilizing the model proposed byFinch and Klug (91) and supported by the observations of others(92-94). Williams et al. (95) have demonstrated that the 30-nm fiber could also be constructed with crossed-linker nucleo-soma! organization; however, the DNA packing ratio of both30-nm fibers is similar. The 30-nm fiber forms the filament ofa loop, a basic structure of both interphase and metaphaseDNA. If a human diploid nucleus contains 6x10'' base pairs,

there would be approximately 100,000 of these DNA loopdomains within a single nucleus. The full organization of theseloop domains within the interphase nucleus and metaphasechromatid has still not been elucidated. In interphase, severallines of evidence support a model that has the DNA loopsattached to the inner portions of the nuclear matrix. Manyinvestigators have demonstrated that newly synthesized DNAoccurs throughout the interior of the nucleus and not just atthe periphery or lamina areas as was once believed (21, 96-99).

In metaphase, DNA loop domains are preserved in chromosomes (100), structures devoid of nuclear envelope and laminaproteins. The higher order structure of metaphase chromosomes remains controversial. Several different models havebeen described which include radial loop (100-102), foldedfiber (103), unit-fiber (104), spiral coil (105), and coiled-coil(106) models. We compared the amounts of DNA in a chromatid of the No. 4 human chromosome with the measuredchromatid dimensions at maximum condensation. DNA loopscan be packed into actual chromatid dimensions only when aradial loop model and current concepts of higher order DNAstructure are used (See Fig. 2 and Table 2) (90). This modelfeatures loops wrapped radically around the central axis of thechromatid as they stack to achieve overall chromosome length.Our analysis revealed that there would be 18 DNA loops perradial turn of the chromatid. This 18-loop unit forms an as yettheoretical higher order structure of DNA organization that wehave termed the "miniband" because it represents the smallest

achievable band of a chromosome (90). The miniband is equivalent to one full radial turn of 18 loops, each of 60 kbp, aroundthe central axis of the chromatid to form minibands of approximately 1 million base pairs of DNA (18 x 60 kbp).

The nuclear matrix provides a dynamic framework for DNAorganization. Insight into the dynamic process of the matrixhas been provided by fluorescent antibody studies of changes inthe distribution of nuclear matrix antigens at different timesduring the mitotic cell cycle (107-109) (see Fig. 3). The DNAloop domains present in the metaphase chromosome maintaintheir association with the nuclear matrix in the interphasenucleus. The telomere regions on the end of the chromosomeare attached to the peripheral lamina. As the nucleus approaches metaphase, the nuclear lamina proteins are phospho-rylated and have been shown to diffuse as small vesicles intothe cytoplasmic area as the nuclear envelope disintegrates (110,111). The chromosomes, now free of the lamina, collapse intocondensed mitotic structures. At the end of telophase, the ends

The Formation Of The Radial Loop Chromosome

Doubl« Stranded DNA

I10 nm Nucleosomes

30 nm Solenoid

Loops

Miniband

Chromosome

XXDOOOOOOOOC }2nm

~ i, .<=*. .

hOnm

V 6 Nucleosomes/Turn

Matrix(Topoisomerase)

so Mm•¿�

(Side View)

Bow PairsperlObp80

bp1160iii-.hjrntl1.20Gb

p(t*lurnleo.ooobpl(xr

loop)I.I

xio'bp<p«ln,,n,t.or.l18

loops/MinibandPochinq

Roho16-74068012

«10*I.2»IO'

Fig. 2. Schematic of the levels of organization within a chromatid of achromosome. Approximately 160 base pairs (b.p.) of 2-nm DNA helix is woundtwice around the histone octamers to form the 10-nm nucleosomes. Thesenucleosomes form a "beads on a string" Tiber which winds in a solenoid fashionwith 6 nucleosomes per turn to form the 30-nm chromatin filament. The 30-nmfilament forms the 60-kbp DNA loops that are attached at their bases to thenuclear matrix structure. The loops are then wound into the 18 radial loops thatform a miniband unit. The minibands are continuously wound and stacked alonga central axis to form each chromatid. Variations in interchromosomal length areachieved by altering the number of minibands in each chromatid. Variations inintrachromosomal length are achieved by the winding, unwinding, and compacting of the minibands (90).

of the chromosome serve as an organizing center for the condensation of lamina proteins to reform the lamina of the nucleus(111-113). In this model some nuclear matrix structures at thebase of the DNA loops will be maintained as the core scaffoldingor matrix within the chromosome. In support of this theory,the nuclear matrix has been shown to share many commonproteins, including topoisomerase II, with the chromosomescaffold (23-30, 114). In the future much work will be neededto determine how DNA loops interact with the nuclear matrix,how the replicating complex is formed during S phase, and howthe matrix-organizing centers control and reestablish nuclearstructure. Additionally, it remains to be seen how the nuclearmatrix-DNA complex is organized and altered with the development of cancer that is associated with distorted nuclearstructures.

DNA and Chromosomal Rearrangements in Cancer

Chromosome translocations have been proposed to be acommon factor in many types of human neoplasias (115, 116).When large translocations occur, there is also a transfer ofchromosomal banding patterns. In the model in Fig. 2, structural elements of the core of the chromosome and the nuclearmatrix that anchor the DNA loops must be transferred in thetranslocation process. Since sister chromatid exchanges occurat the site of DNA replication that is on the matrix, the nuclearmatrix components may be involved in these rearrangements(117). These types of DNA reorganization in cancer that include

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Table 2 Comparison of experimentally observed values of human chromosome 4 with those predicted from the proposed modelHuman Chromosome 4 (1.15 x 10* base pairs) of DNA/chromatid

Observed experimentallyModel predictionsBase

pairs/loop(kbp)63

±14"

60Loop

dimensionsLength

of DNAloop(//in)21.4*

20.4Chromosome

4dimensionsDNA

loops/miniband16.9±

1.9e

18DNApacking

ratio12400*

12260Condensed

length(cm)3.15'

3.19Chromatid

diameter(¡um)0.85'

0.84" Average value ±SEM from Refs. 163-166.* 63,000 base pairs x 3.4 angstroms/base pair =21.4 /im.' Average number of loops per turn on chromâtids counted from micrographs published in Refs. 100, 103, 167, and 168.'' Length of DNA double strand divided by chromatid length (3.91 x IO4firn divided by 3.15 /im).' Measured by Dr. G. F. Bahr, Armed Forces Institute of Pathology, Washington, DC.

INTERPHASE(DIPLOID)

Expansion ofChrom at in

S-PHASE(TETRAPLOID)

Replication ofDNAin Loops

PROPHASE

Slater Chromatid

METAPHASE

ChromosomeCondensation

04-LunluLo V

DNA Loops'

Fig. 3. Schematic diagram of the concept of the role of the nuclear matrix inorganizing a single chromatid in the interphase nucleus. During S phase the DNAloops replicate. During prophase the matrix separates and disengages the telomerefrom the lamina. The lamins are phosphorylated and disperse into the cytoplasmin small vesicles. The matrix attached to the DNA loops condenses duringmetaphase to organize the chromosome (107-113).

gene amplification (118), sister chromatid exchange, and aseries of different subtypes of rearrangements and deletions(119) have all been demonstrated in both animal and humantumors. Each of these processes involves a change in the orderof arrangement of the DNA sequence and as such has beenproposed to be involved in the initiation of carcinogenesis and/or increase progression. Any of these rearrangements whichbestows a growth advantage on the cells could cause a hyper-plasia or tumor formation. If a DNA rearrangement places anerror within the genomic apparatus such that further geneticinstability ensues, a wide variety of cells would result. This typeof genetic instability may be the basis of progression and theformation of tumor cell heterogeneity (1-5, 120). Elucidationof the molecular events which produce this genetic instabilityis critical to the understanding of cancer. It will be necessary todefine the relationship between genetic instability and the alterations of cell structure, which is the morphological hallmark ofcancer diagnosis, in order to further our understanding of thecancer process. The concept of a dynamic tissue matrix systemmay form a basis for this understanding.

The Tissue Matrix System

The nuclear matrix forms an interlocking network with thecytomatrix that extends throughout the cell and makes externalcontact with the ECM (Fig. 4). The cytomatrix is composed inpart of networks of actin microfilaments, intermediate filaments, and microtubules. The ECM includes the basementmembrane and ground substance of the stroma and is composedin part of collagens, laminins, fibronectin, and proteoglycans.Several investigators have proposed that these structural ele

/GLYCOCALYXXINTEGRAL

V PROTEINS /

CYTOMATRIX

/MICROTUBULESA/MICROFILAMENTSAI INTERMEDIATEV FILAMENTS /

EXTRACELLULARMATRIX

COLLAGENS, LAMININS, \( FIBRONECTINS, PROTEOGLYCANS^

INORMALCELL! [CANCERCELL|Fig. 4. The tissue matrix system. The shape of the cell is dependent on

dynamic interactions of its structural components. The nuclear matrix is connected to the cytomatrix. The cytomatrix in turn is attached to the extracellularmatrix via the membrane matrix. Virtually every subcomponent of this matrixsystem has been shown to be altered in the cancer cell (121-137).

ments form a tissue matrix that works in concert to provide adynamic matrix system connected throughout the tissue (121-123). Fey et al. (123) proposed that the nuclear matrix is directlylinked to an intermediate filament complex that functions as astructural unit within the cell. They provided convincing 3-dimensional electron micrographs that show that the nuclearmatrix is contiguous with intermediate filaments that extendedvia the desmosomes over the entire epithelial cell colony. Mostimportant, they have shown that the nuclear matrix-interme

diate filament system retains proteins that are specific to itscell type and that this is unique to this substructure of the cellsystem (124). Fey and Penman (125) demonstrated that tumorpromoters induce a specific morphological signature of a nuclear matrix-intermediate filament scaffold of kidney cells. Intheir studies, the nuclear matrix-intermediate filament complexwas profoundly reorganized in a specific manner after exposureto tumor-promoting agents. These changes fit with Penman's

earlier observations that modulation of cell metabolism is modulated by cell shape and external surface contact (126). Manystudies have shown that much of the macromolecular metabolism of the cell, including DNA, RNA, and protein synthesis,responds to changes in cell shape (127-129). Progressive lossof shape-responsive controls may be an important factor intumor progression. Ben Ze'ev (130) has provided a comprehen

sive review of the changes in the cytoskeleton associated withcancer cells and has proposed that growth-regulated cellularfunctions are regulated by signals which are transmittedthrough an organized cytoskeleton that has been disrupted bythe carcinogenic process. Microfilaments, intermediate filaments, and microtubules have all been documented to be alteredin many transformed cells. Furthermore, it is possible thatsome of the oncogene proteins are tyrosine kinases, which may

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induce some of the cytoskeleton changes that occur with transformation. Observations on the importance of external surfacecontact and anchorage-independent growth of tumor cells havefocused much attention on the importance of the cytoskeletonand its relationship to the ECM in tumor progression. In classicstudies, Folkman and Moscona (131) controlled the shape ofnormal cells in vitro by varying the substratum adhesiveness ofthe culture plates to which the cells were attached. Gospoda-

rowicz et al. (132) observed that cell shape determines themitogenic response of a given cell. Additionally, the ECM hasbeen implicated in the control of genetic expression (133), andReid (134) has demonstrated that the ECM components gly-cosaminoglycans and proteoglycans can induce morphologicalchanges, induce gap junction synthesis, and regulate tissue-specific gene expression. It is well recognized that the ECMclearly plays an important inductive function in embryonicdevelopment and may also modulate adult cells. For example,Cìinhaet al. (135) demonstrated that the ECM can be responsible for functional differentiation in development when theyshowed that urogenital sinus mesenchyme induces urinary bladder epithelial cells to form prostatic epithelial cells and acini.Reddi and Anderson (136) observed that mature fibroblastsunderwent redifferentiation to form new chondroblasts andchondrocytes when they were exposed to demineralized bonecollagen matrix. All of these and many other experimentsdemonstrate that what a cell touches is important in determining what it becomes and how it functions (121). We still knowvery little about the integration and control of these cytoskeleton and extracellular matrix processes and how the informationis transmitted to the nucleus. In summary, the cell may transmitsignals by direct mechanical linkages via the tissue matrixsystem that can regulate DNA function (119-121,137). We doknow that in the cancer cell the cell matrix can be highlydynamic. This is exemplified by cell motility.

Cancer Cell Motility

In 1940, George Gey (138) was the first to use time lapsecinemicroscopy to study the activity of cancer cells derived fromspontaneous transformation of normal cells in tissue culture.In 1966, Sumner Wood wondered whether cell motility wasimportant in the pathogenesis of cancer or simply a cell cultureartifact. He used a transparent rabbit ear chamber to study thein vivo motility of V2 carcinoma cells (139-141). V2 carcinomacells migrated at velocities comparable to those of leukocytesand 200 times faster than macrophages. Coman (142) demonstrated heterogeneity in the motility of tumor cells and suggested that the degree of motility correlated with histológica!differentiation and invasive potential.

This relationship between cell motility and metastatic potential is starting to be explored. Platelet-derived growth factor(143), transforming growth factors (144), insulin (145), andepidermal growth factor (146) have all induced motility innormally quiescent cells. Normal cells demonstrated transientenhanced motility after treatment with phorbol ester (147) andruffling, which was observed with time lapse cinematography(148,149,150). Injection of the p21ras protein product directlyinto the cell induced a transient motility (151). Recently Liottaand Schiffman (152) have identified an autocrine motility factorsecreted by tumor cells which increases tumor cell motility.Hosaka (153, 154) used time lapse videomicroscopy to showdifferences in cell motility between various rat hepatoma celllines that exhibited different propensities for metastasis. Haem-merli and Strauli (155) extended this technique to the study of

human neoplastic cells and suggested that their in vitro motilityreflected their invasive behavior in vivo.

Recently, time lapse videomicroscopy and a visual gradingsystem have been utilized to evaluate cell membrane ruffling,undulation, pseudopodal extension, vectorial translation, andirregularity of the pathway of translation in normal and malignant cells (156, 157). The Dunning R3327 rat prostatic ade-nocarcinoma model provides many histologically indistinguishable sublines of varying metastatic potential which originatedfrom a single animal (158). No biological, biochemical, ormorphological discriminator has previously been capable ofidentifying the individual sublines or predicting their metastaticpotential (158). Mohler et al. (156, 157) demonstrated that fivesublines and normal rat prostate cells could be identified by avisual grading system of cell motility. More recently, Partin etal. (159) have developed a new system for quantitating allaspects of cell motility. This new quantitative method was ableto correlate cell motility changes with an increase in metastaticability in the Dunning tumors (159). Furthermore, Partin et al.(160) demonstrated that motility and metastatic potential canbe induced in the Dunning tumors by transfection and expression of the V-Harvey-ros oncogene. Thus motility of individualcancer cells in vitro has been shown to be sufficiently characteristic to allow accurate assessment of their metastatic potentialin vivo. Current pathological grading systems depend upon theappreciation of cytological and architectural features of dead,fixed histological sections of malignant tissues. A grading system of motility of live cancer cells may better predict thebehavior of a live tumor system.

Tensegrity

The types of mechanical systems that can transfer information within a cell are just beginning to be resolved. Ingber andJameson (161) have suggested a tensegrity model to explainhow cells composed of structural elements may be capable ofsuch information transfer. Tensegrity was defined by Buck-minster Fuller in 1948 as a structural system composed ofdiscontinuous compression elements connected by continuoustension cables, which continually interacted in a dynamic fashion. This structure allows great motility as each part is incoupled equilibrium so that mechanical forces can be transferred throughout the entire system. Recently, Dennerll et al.(162) have observed and measured a tension and compressionsystem in neuntes. They suggest a complimentary force interaction between an actin network under tension and the micro-tubule network under compression. A tension-derived tensegrity structure may be a more appropriate way to view cellstructure than a rigid scaffold framework system. These changesprogress to a cell with increased motility and an ability tometastasize.

Conclusion

The pioneering biophysicist Aaron Kachalsky stated in 1962that "life may be defined as a chemomechanical engine." The

highly motile yet structured cancer cell may also be viewed asa chemomechanical engine in which structure and function areintimately interrelated. Disruptions or changes in the matrixsystem may help explain genetic instability and tumor cellheterogeneity. Our present view of this process is depicted inFig. 5. It is now obvious that cell transformation is a multistepprocess involving genomic changes that can involve oncogenesacting at different sites within the cell. These changes progress

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NORMAL TRANSFORMATION - PROGRESSION

CELL SIGNALINGONCOGKNBS

ACTING ATCYTOSDLITON.PLASMAMEMBRANE

(e.g. r»s)

STRUCTURAL INSTABILITY / /DNA INSTABILITY j /

TUMOR CELL HETEROGENEITY MOTILITYIMMORTALIZATION („eta.taaes)

Fig. 5. The transformation process. Transformation from a normal cell to amalignant one appears to involve multiple steps. These steps are usually considered in terms of "initiation" and "progression." This process may be viewed at

the mechanism/site of action of the different oncogenes involved in tumorprogression. One class of the oncogenes, those acting on the nucleus, e.g., myc,alter the structural stability of cells. This leads to immortalization and concomitant DNA and structural instability. A second class of oncogenes, those acting onthe periphery of the cell, e.g., ras, induce motility into the cell and may impartthe ability to metastasize.

to a cell with increased motility and an ability to metastasize.Understanding this carcinogenic process will require a morecomplete knowledge of the interlocking matrix systems thatextend from the extracellular matrix to the DNA and thatgovern cell shape and function.

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