Mutations of the p16 Gene Product Are Rare inProstate Cancer

Jaya P. Gaddipati,1,2 David G. McLeod,1,2 Isabell A. Sesterhenn,3Christopher J. Hussussian,4 Yue Ao Tong,1,2 Prem Seth,5

Nicholas C. Dracopoli,4 Judd W. Moul,1,2 and Shiv Srivastava1,2*1Center for Prostate Disease Research, Department of Surgery, Uniformed Services

University of the Health Sciences, Bethesda, Maryland2Urology Service, Walter Reed Army Medical Center, Washington, DC

3Armed Forces Institute of Pathology, Washington, DC4National Center for Human Genome Research, National Institutes of Health,

Bethesda, Maryland5Medical Breast Section, Medicine Branch, National Cancer Institute, National Institutes

of Health, Bethesda, Maryland

BACKGROUND. The p16 gene product is a negative regulator of cell cycle and has beenshown to be deleted or mutated in a number of tumor cell lines and primary tumors. Therole of p16 in prostate cancer is not defined. Prostate cancer tissues and cell lines wereevaluated for p16 gene alterations.METHODS. Five metastatic prostate cancer cell lines were analyzed for p16 gene structureand its expression by Southern and Northern blot analyses. Forty-one DNA specimens from18 microdissected primary tumor specimens, adjacent normal tissues, and cell lines wereamplified by polymerase chain reaction for p16 protein coding and splice junction se-quences. Mutations were analyzed by single strand conformation polymorphism and DNAsequencing.RESULTS. DU 145 cell line exhibited a missense mutation in codon 84 (GAC to TAC). Withthe exception of previously reported polymorphism, no mutation was detected in p16 cod-ing or splice junction sequences in primary prostate cancer specimens.CONCLUSIONS. Inactivation of p16 gene by mutations in the protein coding sequencedoes not play a major role in the genesis of primary prostate cancer. Prostate 30:188–194,1997. © 1997 Wiley-Liss, Inc.

KEY WORDS: prostate cancer; p16; microsatellite; polymerase chain reaction; singlestrand conformation polymorphism

INTRODUCTION

Prostate cancer is the most frequently diagnosedsolid tumor and the second leading cause of cancerdeath in men in the United States [1]. Alterations ofoncogenes and tumor suppressor genes representone of the key molecular defects analyzed in humancancer [2]. Despite recent progress in the character-ization of molecular alterations in prostate cancer,much remains to be learned about the role of a spe-cific oncogene or tumor suppressor gene in the gen-esis and progression of human prostate cancer [3–5].

The tumor suppressor gene p16 (MTS1) has beenshown to be altered in a variety of tumor cell linesandprimary tumors [6–10]. The p16 gene is located

The opinions and assertions contained herein are the private viewsof the authors and should not be construed as reflecting the viewsof the Department of Defense or the United States Army.*Correspondence to: Shiv Srivastava, Ph.D., Center for ProstateDisease Research, Department of Surgery, Uniformed ServicesUniversity of the Health Sciences, Bethesda, MD 20814-4799.Received 6 September 1995; Accepted 8 January 1996

The Prostate 30:188–194 (1997)

© 1997 Wiley-Liss, Inc.

on chromosome 9p21–22 and encodes a cell cycle reg-ulatory protein which binds to cyclin dependent ki-nase 4 (CDK4) and inhibits cyclin D/CDK4 mediatedphosphorylation of the protein encoded by the retin-oblastoma (RB) tumor suppressor gene [11]. In someglioma cell lines which harbor wild type p16 gene,CDK4 gene amplification and higher levels of CDK4expression were observed, suggesting CDK4 amplifi-cation and/or overexpression as an alternative mech-anism to p16 gene inactivation [12,13]. The role of p16or CDK4 genes remains to be understood in the gen-esis of prostate cancer. To assess the role of the p16gene in prostate cancer tumorigenesis, we have ana-lyzed five established metastatic prostate cancer celllines and 18 microdissected primary tumor specimensand corresponding adjacent normal tissue for dele-tions and mutations. We have further examined p16and CDK4 expression in metastatic prostate cancercell lines. The data presented in this report suggestthat mutations of p16 gene in the protein coding se-quence are rare in prostate cancer.

MATERIALS AND METHODS

Cell Lines and Tumor Specimens

LNCaP, DU 145, and PC-3 cells derived from met-astatic lesions of human prostate cancer, and MCF-7derived from a human breast adenocarcinoma wereobtained from American Type Culture Collection(ATCC), Rockville, MD. DuPro-1 cells, derived fromathymic nude mice metastasis of prostate adenocar-cinoma [14] and 1LN PC3-1A (1LN) cells, nudemouse metastatic variant of PC-3 cells [15] were gen-erous gifts of Dr. David Paulson, Duke UniversityMedical Center, Durham, NC.

Tissue specimens obtained from radical prostatec-tomy, at Walter Reed Army Medical Center, Wash-ington, DC, were embedded in O.C.T. compound(Miles Inc., Elkhart, IN) and then frozen immediatelyat –80°C. Tumor regions representing over 80% neo-plastic cells and corresponding normal region fromthe same tissue were identified by hematoxylin andeosin staining of a 10 micron serial section of frozenspecimens by one of us (I.A.S.). Eighteen specimensselected for this study were microdissected for tumorand normal regions from two 10 micron sections.

DNA/RNA Extractions

Genomic DNA was extracted from microdissectedtissue sections and from cell lines by proteinase Kdigestion followed by phenol extraction and ethanolprecipitation [16]. Poly A+ RNA was extracted fromcell lines by using FasTrack mRNA isolation kit (In-vitrogen, San Diego, CA).

Southern Blot Analysis

High molecular weight DNA from cell lines wasdigested with restriction enzyme EcoRI, fractionatedby electrophoresis on 0.6% agarose gel and trans-ferred onto nylon membranes. The nylon membraneswere hybridized with random primer labeled p16cDNA probe [11].

Northern Blot Analysis

Poly A+ RNA from cell lines was electrophoresedon formaldehyde agarose gels and transferred to ny-lon membrane by electro-blotting. Blots were sequen-tially hybridized with p16 cDNA [11], CDK4 [17], andglyceraldehyde-3-phosphate dehydrogenase (GAP-DH) probes [18]. CDK4 probe was generated by PCRamplification of genomic DNA using primers fromCDK4 coding sequence [17] [sense: 58- CATGTAGAC-CAGGACCTAAGG-38 (510–530 nucleotides); an-tisense: 58-GGAGCTCGGTACCAGAGTG-38 (756–775 nucleotides)] which yielded an expected size bandon agarose gel.

SSCP and DNA Sequence Analysis

PCR reactions (20 ml) were carried out using 10 ngof genomic DNA. Conditions for PCR, SSCP, andsequencing strategy were essentially same asdescribed previously by Hussussian et al. [8]. PCRprimers for different p16 exons were: exon 1 (sense:58-GGGAGCAGCATGGAGCCG-38, antisense: 58-A-GTCGCCCGCCATCCCCT-38); overlapping primersets for exon 2 ([a] sense: 58-AGCTTCCTTTCCGTCA-TGC-38, antisense: 58-GCAGCACCACCAGCGTG-38,[b] sense: 58-AGCCCAACTGCGCCGAC-38, anti-sense; 58-CCAGGTCCACGGGCAGA-38, [c] sense: 58-TGGACGTGCGCGATGC-38, antisense: 58-GGAAG-CTCTCAGGGTACAAATTC-38); and exon 3 (sense:58CCGGTAGGGACGGCAAGAGA-38, antisense:58-CTGTAGGACCCTCGGTGACTGATGA-38). Onetwentieth of PCR product was analyzed by SSCP, us-ing MDE gels (AT Biochem, Malven, PA) and the gelswith and without glycerol were run at 6–8 W for 10–12hr at room temperature. DNA extracted from SSCPvariant bands were ligated with pCRIITA-cloning vec-tor (Invitrogen) and transformed into DH5a compe-tent cells (Life Technologies, Gaithersburg, MD).DNA was isolated using RPM kit (Bio 101, Inc., LaJolla, CA) and both strands were sequenced with SP6and T7 primers using Sequenase version 2.0 (UnitedStates Biochemicals, Cleveland, OH).

PCR Amplification and Deletion Analysis

Genomic DNA (30 ng) from normal and tumor tis-sue specimens was co-amplified in a single PCR re-

p16-Prostate Cancer 189

action mixture using b-actin primers (Research Ge-netics Inc., Huntsville, AL) and p16 exon 2 primersspanning codons 69 to 118. After amplification, thePCR products were separated by electrophoresis on2.5% agarose gel and the p16 and b-actin primer de-rived products were visualized by ethidium bromidestaining.

Analysis of Chromosome 9p 21–23 Loci byMicrosatellite Polymorphism

Ten nanograms of genomic DNA was PCR ampli-fied either by using a [g-32P] end labeled primer or byincluding [a-32P]dCTP in PCR reaction, for threepolymorphic microsatellite loci on 9p: D9S157 (9p22–23) [19], IFNa (9p21), and D9S171 (9p21) [20]. ThePCR products were analyzed on 7% acrylamide/urea/formamide gels as described [21].

RESULTS

Analysis of p16 Gene Structure and Expression inProstate Cell Lines

To analyze for any major defects of p16 gene in-cluding deletions and/or rearrangements in prostatecancer cell lines, Southern blot analysis of the ge-nomic DNA was performed on five established met-astatic prostate cancer cell lines (DU 145, DuPro-1,PC-3, LNCaP, and 1LN). No detectable change wasobserved by Southern blot analysis of cell line DNAsstudied (Fig. 1; data not shown for DuPro-1 and1LN). Under similar experimental conditions, weconfirmed the previously reported homozygous de-letion of p16 gene in the breast cancer cell line MCF-7[22]. We next analyzed the expression of the p16 genein these five metastatic prostate cancer cell lines [DU145, DuPro-1, PC-3, 1LN, and LNCaP] (Fig. 2). p16mRNA levels expressed in these cell lines varied con-siderably with high p16 mRNA levels in cell lines: DU145, DuPro-1, and 1LN, moderate level in PC-3,barely detectable levels in LNCaP, and none inMCF-7.

Five prostate cancer cell line DNAs describedabove were further analyzed for subtle alterations bySSCP analysis of PCR fragments representing com-plete p16 protein coding sequence. A single primerset was used for screening exons 1 and 3 and theirflanking intron sequences, whereas three sets ofoverlapping primers were used to cover exon 2 andits flanking intron sequences. SSCP analysis of twooverlapping fragments of exon 2 revealed SSCP vari-ant in DNA specimens from DU 145 cell line (Fig.3B,C) that was not present in the control human pla-centa DNA. DNA sequencing analysis of the 10 plas-mid DNA clones of the SSCP bands from DU 145

exhibited a missense mutation in codon 84 (GAC toTAC, asp to tyr; Fig. 3F, Table I). This result stronglysuggests that only mutant p16 allele is present in theDU 145 cell line DNA. A second SSCP variant wasalso detected in exon 3 of DU 145 DNA (Fig. 3E), andfurther sequence analysis (Table I) revealed a previ-ously reported polymorphism (C/G) in base 494 of the38 untranslated region of p16 gene [23].

Analysis of p16 Gene in Primary Prostate CancerSpecimens Derived From Radical Prostatectomy

Normal and tumor DNAs from 18 radical pros-tatectomy specimens were examined for the status ofp16 gene alterations. Co-amplification of these DNAsusing primers for exon 2 of the p16 gene and for theb-actin gene at two different PCR cycles (25 and 30cycles) resulted in two amplicons of the expectedsizes (Fig. 4). None of the tumor DNAs showed ap-parent p16 gene deletion and comparison of p16 andb-actin bands exhibited a uniform intensity ratio, re-flecting uniform makeup of template DNA.

Eleven paired normal and primary tumor DNAswere also analyzed for the loss of heterozygosity forthree 9p loci with microsatellite markers: D9S157(9p22–23), IFNa (9p21), and D9S171 (9p21), and theinformative specimens (five for D9S157, seven for

Fig. 1. Southern blot analysis of p16 gene in human metastaticprostate cancer cell lines DU 145, LNCaP, and PC-3, human pla-centa (HP) and testicular tumor (T1, T2) derived DNAs. DNAswere digested with EcoRI, and Southern blots were hybridizedwith p16 cDNA probe.

190 Gaddipati et al.

IFNa, and nine for D9S171) did not exhibit deletion orloss of heterozygosity (data not shown).

Mutational analysis of the p16 gene coding se-quences of the radical prostatectomy specimen DNAsby SSCP, detected no alteration in the prostate tumorspecimen DNAs. However, both normal and tumorDNAs of one radical prostatectomy specimen showeda C/G polymorphism at base 494 which is identical tothat found in the DU 145 cell line DNA (Table I).

CDK4 Expression in Prostate Cancer Cell Lines

Higher levels of CDK4 expression in associationwith gene amplification have been observed in someglioma cell lines lacking alterations in the p16 gene.As frequent p16 gene mutations were not observed inprostate cell lines, Northern blot analysis was per-formed to determine CDK4 mRNA overexpression, ifany, in these cell lines. While DuPro-1 cell line re-vealed a relatively higher level of CDK4, other pros-tate cancer cell lines exhibited minor variations intheir CDK4 expression (Fig. 5). The same blot showedequal levels of GAPDH mRNA expression in thesecell lines (data not shown).

DISCUSSION

This report analyzes the status of the p16 gene inhuman prostate cancer and the data reveal the fol-lowing findings: a) Of five metastatic prostate cancercell lines examined, only one revealed a mutation inthe p16 coding sequence; b) no mutations were iden-tified in the coding region of p16 gene in 18 micro-dissected primary prostate tumor specimens; c) a sin-gle-base polymorphism was observed in one cell lineand in one primary tumor specimen; and d) varyinglevels of p16 and CDK4 expression were observed inthe prostate cell lines examined.

Unlike melanomas, gliomas, and leukemias, ho-mozygous deletion of the p16 protein coding se-quence was not seen in both primary tumors andhuman cell lines derived from metastatic prostate car-cinoma. Furthermore, no evidence of loss of het-erozygosity was observed at three 9p21–23 loci(D9S157, IFNa, and D9S171) in 11 primary prostatetumor specimens. While this manuscript was beingreviewed for publication, we have noted two veryrecent reports which have also evaluated the status ofp16 gene in prostate cancer. The conclusions of ourstudy are in agreement with the report of Komiya etal. describing infrequent mutations of p16 gene inprostate cancer [24]. However, Cairns et al. reporthomozygous deletion (40%) and loss of heterozygos-ity (33%) of 9p chromosome loci neighboring/flankingp16 gene in 15 prostate cancer specimens as charac-terized by newly developed microsatellite markers[25]. The apparent discrepancy between our studyand the report by Cairns et al. are most likely due tothe use of new microsatellite markers used in the lat-ter study. However, quantitative evaluation of p16deletions in tumor specimens can also be com-pounded by the fact that tumor specimens alwayscontain some normal cells despite microdissectionand DNA extraction. For example, amplification ofp16 gene by sensitive PCR methods might encounterthe situation where small fraction of normal cell de-rived DNA in microdissected tumor DNAs give afalse picture of intact p16 gene in the tumor DNAlacking p16 gene. For this reason, in situ-based futureanalysis of p16 gene may be most informative con-cerning the status of p16 gene in prostate cancer spec-imens. The G to T missense mutation in codon 84 ofp16 gene detected in the DU 145 cell line caused anamino acid change in the third ankyrin repeat [26].Furthermore, only the mutant allele was detected inthe cell line. This observation is in agreement withthose of Komiya et al. [24] and Liu et al. [27]. Inter-estingly, the same mutation has also been describedin one of 33 squamous cell carcinomas [10].

The prostate cancer cell lines examined for p16

Fig. 2. p16 mRNA analysis in metastatic prostate cancer cells DU145, PC-3, LNCaP, DuPro-1, and 1LN PC3-1A (1 LN). Poly A+

RNA on Northern blot was analyzed with p16 cDNA probe.LNCaP exhibited severely reduced p16 mRNA in several experi-ments on longer exposure of gel. GAPDH expression was de-tected at equal levels in all cell lines (data not shown).

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mRNA expression utilizing full length p16 cDNAprobe revealed varying levels of p16 mRNA withbarely detectable expression of p16 in LNCaP cells.However, recent studies have revealed that p16 geneexpression involves complex regulatory mechanismsinvolving methylation of CpG sites upstream of exon1 [28] and generation of two p16 gene transcripts con-taining a novel sequence in exon 1 (E1b) [29]. A veryrecent report by Herman et al. [30] has now shown

that several prostate cancer cell lines (DuPro-1, PC-3,and TSU-PR1) do not express p16 due to methylation.While Herman et al. did not detect p16 expression inDuPro-1 and PC-3 cell lines by RT-PCR assay, wehave clearly detected p16 mRNA expression in Du-Pro-1 and PC-3 cell lines on Northern blot using fulllength p16 cDNA probe (Fig. 2). Whether we havedetected alternate transcript form (E1b) of p16 [29] onour Northern analysis needs to be further clarified.

Fig. 3. PCR-SSCP (A–E) and DNA sequence analysis (F) of p16protein coding region in human placental (HP) and prostate cancercell line DNAs. A: p16 exon 1 [DU 145 (1), HP (2), LNCaP (3),PC-3 (4), DuPro-1 (5), and 1LN (6)]. The reduced band intensitiesin lanes 5 and 6 are due to less DNA present during PCR reaction.B, C, and D represent overlapping regions of p16 exon 2 [B: DU145 (1), LNCaP (2), PC-3 (3), DuPro-1 (4), and 1LN (5); C: HP (1),DU 145 (2), LNCaP (3), PC-3 (4), DuPro-1 (5), and 1LN (6); D: HP(1), DU 145 (2), LNCaP (3), PC-3 (4), DuPro-1 (5), and 1LN (6)]

and p16 exon 3 [E: 1LN (1), DU 145 (2), LNCaP (3), PC-3 (4), andDuPro-1 (5)]. Mobility shifts as indicated by arrows were observedin exon 2 overlapping fragments of DU 145 cell line DNA shownhere in B and C. Cloning and DNA sequencing of the SSCP variantbands exhibited the same G to A alteration in codon 84 as shownin F. The SSCP variant bands in exon 3 of DU 145 cell line DNAshown by arrows in E were cloned and sequenced, revealed C/Gpolymorphism in 38 untranslated region.

TABLE I. Detection of the p16 Gene Mutations in Prostate Cancer Cells/Tissues*

Source Location Nucleotide change Amino acid change

DU 145 Exon 2; codon 84 GAC to TAC Asp to Tyr

DU 145 Nucleotide 494 in non-coding AAACCTCGGGAAACTT toC 329 sequences 38 to exon 3 AAACCTCCGGAAACTTC 330

*C 329, C 330 DNA of tumor and normal regions, respectively, from a radical prostatectomy specimen.

192 Gaddipati et al.

More importantly in situ-based p16 mRNA expres-sion in primary prostate cancer specimens shouldprovide relevant information concerning the status ofp16 expression in prostate cancer. Since a reciprocalrelationship between CDK4 gene amplification/over-expression was reported in cell lines with wild typep16 gene [12,13], we have also investigated the statusof CDK4 in prostate cancer cells showing no p16 al-terations. However, CDK4 expression was not foundto be significantly elevated in the cell lines exhibitingwild type p16.

Although we did not analyze metastatic prostatecancer clinical specimens in this report, the detectionof a p16 mutation in one of five cell lines derived frommetastatic prostate cancer suggests a low rate of p16mutation in metastatic disease. However, a cell cul-ture origin of p16 mutation in DU 145 cell line cannotbe ruled out. Unlike p16, four of five metastatic can-cer cell lines analyzed here are reported to have p53mutations, and there appears to be a good correlationin terms of recent findings showing frequent p53 mu-tations [16,31,32] in metastatic prostate cancer speci-mens.

In summary, this study demonstrates that the in-activation of the tumor suppressor gene p16 via mu-tations in the protein coding sequence is not a com-mon mechanism in prostate cancer. In the light ofrecent developments concerning p16 inactivation bymethylation or by deletions scored by new microsat-ellite markers [25], additional studies of p16 alter-ations employing in situ hybridization is needed toclearly establish the role of p16 in prostate cancer.

ACKNOWLEDGMENTS

We thank Mike Gandolph for technical assistance.This research was supported by the Center for Pros-tate Disease Research, a program of the Henry M.Jackson Foundation for the Advancement of MilitaryMedicine, 1401 Rockville Pike, Suite 600, Rockville,MD 20852-3007.

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