Heterogeneous Methylation and Deletion Patterns of the INK4a/ARF Locus Within Prostate Carcinomas
2002; Elsevier BV; Volume: 160; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)62547-3
ISSN1525-2191
AutoresNoboru Konishi, Mitsutoshi Nakamura, Munehiro Kishi, M Nishimine, Eiwa Ishida, Keiji Shimada,
Tópico(s)Urological Disorders and Treatments
ResumoTo elucidate the role of p53/p16INK4a/RB1 pathways in prostate carcinogenesis, we analyzed the p14ARF, p16INK4a, RB1, p21Waf1, p27Kip1, PTEN, p73, p53, and MDM2 gene status of multiple areas within 16 histologically heterogeneous prostate carcinomas using methylation-specific polymerase chain reaction, differential polymerase chain reaction, and immunohistochemistry. All focal areas examined had Gleason scores ranging from 1 to 5. Methylation of either PTEN or p73 was undetected in any sample, whereas expression of MDM2 seemed to be an independent event within small foci of 4 of 16 tumors. Loss of p14ARF, p16INK4a, RB1, and p27Kip1 expression correlated with homozygous deletion or promoter hypermethylation. One carcinoma showed co-deletion of both p14ARF and p16INK4a in two of five areas examined; two areas within another tumor demonstrated concurrent hypermethylation of the promoter regions of the same genes. Focal hypermethylation of RB1, p21Waf1, and p27Kip1 was detected within two, two, and three tumors, respectively. These findings indicate that both genetic and epigenetic events occur independently in intratumor foci and further suggest hypermethylation-induced loss of gene function may be as critical as specific genetic mutations in prostate carcinogenesis. To elucidate the role of p53/p16INK4a/RB1 pathways in prostate carcinogenesis, we analyzed the p14ARF, p16INK4a, RB1, p21Waf1, p27Kip1, PTEN, p73, p53, and MDM2 gene status of multiple areas within 16 histologically heterogeneous prostate carcinomas using methylation-specific polymerase chain reaction, differential polymerase chain reaction, and immunohistochemistry. All focal areas examined had Gleason scores ranging from 1 to 5. Methylation of either PTEN or p73 was undetected in any sample, whereas expression of MDM2 seemed to be an independent event within small foci of 4 of 16 tumors. Loss of p14ARF, p16INK4a, RB1, and p27Kip1 expression correlated with homozygous deletion or promoter hypermethylation. One carcinoma showed co-deletion of both p14ARF and p16INK4a in two of five areas examined; two areas within another tumor demonstrated concurrent hypermethylation of the promoter regions of the same genes. Focal hypermethylation of RB1, p21Waf1, and p27Kip1 was detected within two, two, and three tumors, respectively. These findings indicate that both genetic and epigenetic events occur independently in intratumor foci and further suggest hypermethylation-induced loss of gene function may be as critical as specific genetic mutations in prostate carcinogenesis. In recent years, prostate cancer has shown an ∼3% annual increase worldwide. In American men this cancer is clinically diagnosed in 1 of every 11 men; one-third of those diagnosed will develop significant life-threatening disease, making it the second most lethal neoplasia.1Konishi N Cho M Yamamoto K Hiasa Y Genetic changes in prostate cancer.Pathol Int. 1997; 47: 735-747Crossref PubMed Scopus (17) Google Scholar The prostate cancer mortality rate in Japan has increased more than ten-fold throughout the last 3 decades, making it now the eighth leading cause of male cancer death. Epidemiological studies suggest increasing incidence might coincide with wider adoption of a Western lifestyle.2Watanabe M Nakayama T Shiraishi T Stemmermann GN Yatani R Comparative studies of prostate cancer in Japan versus the United States. A review.Urol Oncol. 2000; 5: 274-283Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Advances in molecular biology have shown us an accumulation of genetic alterations in the step-wise process of tumorigenesis, but the actual genetic basis of the disease is not fully understood. Indeed, recent data have revealed there is actually little direct proof to support genetic mutation as the primary and/or only cause of prostate cancer. Epigenetic mechanisms, such as hypermethylation, are suspected of being more responsible in tumor progression.3Rennie PS Nelson CC Epigenetic mechanisms for progression of prostate cancer.Cancer Metastasis Rev. 1998; 17: 401-409Crossref PubMed Scopus (74) Google Scholar For example, the p16INK4a gene was reported to be mutated in one of three prostate cancer cell lines,4Liu Q Neuhausen S McClure M Frye C Weaver-Feldhaus J Gruis NA Eddington K Allalunis-Turner MJ Skolnick MH Fujimura FK Kamb A CDKN2 (MTS1) tumor suppressor gene mutations in human tumor cell lines.Oncogene. 1995; 10: 1061-1067PubMed Google Scholar but methylated in three of five others.5Herman JG Merlo A Mao L Lapidus RG Issa JP Davidson NE Sidransky D Baylin SB Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers.Cancer Res. 1995; 55: 4525-4530PubMed Google Scholar Both genetic and epigenetic alterations of the INK4a/ARF locus may provide impairment in both p14ARF/p53 and p16INK4a/RB1 pathways in the development and progression of prostate carcinomas. p14ARF confines MDM2 to a subsection of the nucleus and stabilizes intranuclear p53 protein by preventing its cytoplasmic transport,6Zhang Y Xiong Y Yarbrough WG ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways.Cell. 1998; 92: 725-734Abstract Full Text Full Text PDF PubMed Scopus (1393) Google Scholar, 7Pomerantz J Schreiber-Agus N Liegeois NJ Silverman A Alland L Chin L Potes J Chen K Orlow I Lee HW Cordon-Cardo C DePinho RA The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53.Cell. 1998; 92: 713-723Abstract Full Text Full Text PDF PubMed Scopus (1329) Google Scholar, 8Kamijo T Weber JD Zambetti G Zindy F Roussel MF Sherr CJ Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2.Proc Natl Acad Sci USA. 1998; 95: 8292-8297Crossref PubMed Scopus (785) Google Scholar, 9Stott FJ Bates S James MC McConnell BB Starborg M Brookes S Palmero I Ryan K Hara E Vousden KH Peters G The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2.EMBO J. 1998; 17: 5001-5014Crossref PubMed Scopus (1007) Google Scholar, 10Jones PA Cancer. Death and methylation.Nature. 2001; 409: 143-144Crossref Google Scholar indicating that p14ARF may act as an upstream regulator of p53 function.11Kamijo T Zindy F Roussel MF Quelle DE Downing JR Ashmun RA Grosveld G Sherr CJ Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF.Cell. 1997; 91: 649-659Abstract Full Text Full Text PDF PubMed Scopus (1380) Google Scholar Although homozygous deletion of p14ARF has been reported in ∼40% of glioblastomas,12Nakamura M Watanabe T Klangby U Asker C Wiman K Yonekawa Y Kleihues P Ohgaki H p14ARF deletion and methylation in genetic pathways to glioblastomas.Brain Pathol. 2001; 11: 159-168Crossref PubMed Scopus (208) Google Scholar the human p14ARF promoter has been shown to contain a CpG island that is also aberrantly methylated in gliomas, colorectal adenomas, and carcinomas,12Nakamura M Watanabe T Klangby U Asker C Wiman K Yonekawa Y Kleihues P Ohgaki H p14ARF deletion and methylation in genetic pathways to glioblastomas.Brain Pathol. 2001; 11: 159-168Crossref PubMed Scopus (208) Google Scholar, 13Esteller M Cordon-Cardo C Corn PG Meltzer SJ Pohar KS Watkins DN Capella G Peinado MA Matias-Guiu X Prat J Baylin SB Herman JG p14ARF silencing by promoter hypermethylation mediates abnormal intracellular localization of MDM2.Cancer Res. 2001; 61: 2816-2821PubMed Google Scholar, 14Esteller M Corn PG Baylin SB Herman JG A gene hypermethylation profile of human cancer.Cancer Res. 2001; 61: 3225-3229PubMed Google Scholar and in esophageal squamous cell carcinomas.15Xing EP Nie Y Song Y Yang GY Cai YC Wang LD Yang CS Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma.Clin Cancer Res. 1999; 5: 2704-2713PubMed Google Scholar However, there has been no study of silencing of the p14ARF by methylation/deletion specifically in prostate carcinomas. Inactivation of RB1 by mutation, deletion, and/or promoter hypermethylation has been reported as an alternative molecular mechanism to p16INK4a inactivation, CDK4 amplification, or CCND1 amplification/rearrangement in human tumors, including prostate carcinomas.16Konishi NHY Tsuzuki T Matsuda H Tao M Nakamura M Naitoh H Kitahori Y Shiraishi T Yatani R Shimazaki J Lin JC Detection of pRB, p16/CDKN2 and p15INK4B gene alterations with immunohistochemical studies in human prostate carcinomas.Int J Oncol. 1996; 8: 107-112PubMed Google Scholar Prostate cancer poses unique problems in terms of treatment and prognosis because of the frequent histological heterogeneity encountered. We previously reported on both known and unknown genetic changes within prostate carcinomas17Konishi N Hiasa Y Nakamura M Kitahori Y Matsubara K Nagai H Different patterns of DNA alterations detected by restriction landmark genomic scanning in heterogeneous prostate carcinomas.Am J Pathol. 1997; 150: 305-314PubMed Google Scholar that suggested that multicentric genetic events occur leading to tumor progression. We now additionally investigate the potential role of epigenetic, as well as genetic, mechanisms within the p53/p16INK4a/RB1 pathway in this series of prostate carcinomas. The 16 prostate carcinomas examined in this study were obtained from radical prostatectomies. No initial chemotherapy or hormonal treatments were instituted before tumor excision. A slice of whole prostate was fixed in 10% neutral-buffered formalin and embedded in paraffin, and the remainder of the tumors were frozen at −80°C for later DNA extraction. Consecutive sections were cut at 4 μm and mounted for immunohistochemical analyses and histopathological evaluation using conventional hematoxylin and eosin (H&E) staining; the H&E-stained sections also served as a guide for the immunohistochemical and DNA analyses. Three to five different foci from each tumor were selected based on representative morphology, size, and lack of contamination with normal prostatic tissues and histologically graded according to the Gleason system for prostate carcinoma.18Gleason DF Histologic grading of prostate cancer: a perspective.Hum Pathol. 1992; 23: 273-279Abstract Full Text PDF PubMed Scopus (834) Google Scholar DNA methylation patterns in the CpG islands of the p14ARF, p16INK4a, RB1, p21Waf1, p27Kip1, PTEN, and p73 genes were determined by methylation-specific PCR (MSP).19Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5215) Google Scholar Sodium bisulfite modification was performed using the CpGenome DNA Modification Kit (Intergen, Oxford, UK) according to the manufacturer's protocol with minor adjustments.12Nakamura M Watanabe T Klangby U Asker C Wiman K Yonekawa Y Kleihues P Ohgaki H p14ARF deletion and methylation in genetic pathways to glioblastomas.Brain Pathol. 2001; 11: 159-168Crossref PubMed Scopus (208) Google Scholar, 20Nakamura M Yonekawa Y Kleihues P Ohgaki H Promoter hypermethylation of the RB1 gene in glioblastomas.Lab Invest. 2001; 81: 77-82Crossref PubMed Scopus (150) Google Scholar The primer sequences for methylated and unmethylated PCR of p14ARF, p16INK4a, RB1, p21Waf1, p27Kip1, and p73 have been previously reported.19Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5215) Google Scholar, 21Esteller M Tortola S Toyota M Capella G Peinado MA Baylin SB Herman JG Hypermethylation-associated inactivation of p14ARF is independent of p16INK4a methylation and p53 mutational status.Cancer Res. 2000; 60: 129-133PubMed Google Scholar, 22Simpson DJ Hibberts NA McNicol AM Clayton RN Farrell WE Loss of pRb expression in pituitary adenomas is associated with methylation of the RB1 CpG island.Cancer Res. 2000; 60: 1211-1216PubMed Google Scholar, 23Corn PG Kuerbitz SJ van Noesel MM Esteller M Compitello N Baylin SB Herman JG Transcriptional silencing of the p73 gene in acute lymphoblastic leukemia and Burkitt's lymphoma is associated with 5′ CpG island methylation.Cancer Res. 1999; 59: 3352-3356PubMed Google Scholar The primer sequences for methylated and unmethylated PCR of PTEN are as follows: 5′-TTT TCGT TCG GCG CGG TTT CG-3′ (sense) and 5′-GCC GCG CCG AAA ACC CGA ACG-3′ (anti-sense) for the methylated reaction; 5′-TTG TTT GGT GTG GTT TTG TTT GTT T-3′ (sense) and 5′-ACC ACC ACA CCA AAA ACC CAA ACA-3′ (anti-sense) for the unmethylated reaction. MSP conditions for p14ARF, p16INK4a, RB1, p21Waf1, and p27Kip1were previously described.12Nakamura M Watanabe T Klangby U Asker C Wiman K Yonekawa Y Kleihues P Ohgaki H p14ARF deletion and methylation in genetic pathways to glioblastomas.Brain Pathol. 2001; 11: 159-168Crossref PubMed Scopus (208) Google Scholar, 20Nakamura M Yonekawa Y Kleihues P Ohgaki H Promoter hypermethylation of the RB1 gene in glioblastomas.Lab Invest. 2001; 81: 77-82Crossref PubMed Scopus (150) Google Scholar, 24Nakamura M Sakaki T Hashimoto H Nakase H Ishida E Shimada K Konishi N Frequent alterations of the p14ARF and p16INK4a genes in primary central nervous system lymphomas.Cancer Res. 2001; 61: 6335-6339PubMed Google Scholar The annealing temperature for both PTEN-methylated and -unmethylated reactions was 64°C, whereas that for both p73- methylated and -unmethylated reactions was 60°C. Amplified products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining. To assess homozygous deletion, we performed differential PCR with primers covering exon 1β of the p14ARFusing GAPDH as a reference. Differential PCR for homozygous deletion of p16INK4a exon 1α was performed using the β-actin gene as a reference. The primer sequences and PCR conditions were previously described.12Nakamura M Watanabe T Klangby U Asker C Wiman K Yonekawa Y Kleihues P Ohgaki H p14ARF deletion and methylation in genetic pathways to glioblastomas.Brain Pathol. 2001; 11: 159-168Crossref PubMed Scopus (208) Google Scholar, 15Xing EP Nie Y Song Y Yang GY Cai YC Wang LD Yang CS Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma.Clin Cancer Res. 1999; 5: 2704-2713PubMed Google Scholar The PCR product was analyzed on an 8% acrylamide gel. Gels were photographed using a DC290 Zoom digital camera (Eastman Kodak, Rochester, NY), and densitometry of the PCR fragments was performed using Kodak Digital Science ID Image Analysis Software (Version 3.5.2, Eastman-Kodak). Samples presenting <20% of the control signal were considered homozygously deleted.12Nakamura M Watanabe T Klangby U Asker C Wiman K Yonekawa Y Kleihues P Ohgaki H p14ARF deletion and methylation in genetic pathways to glioblastomas.Brain Pathol. 2001; 11: 159-168Crossref PubMed Scopus (208) Google Scholar, 15Xing EP Nie Y Song Y Yang GY Cai YC Wang LD Yang CS Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma.Clin Cancer Res. 1999; 5: 2704-2713PubMed Google Scholar, 24Nakamura M Sakaki T Hashimoto H Nakase H Ishida E Shimada K Konishi N Frequent alterations of the p14ARF and p16INK4a genes in primary central nervous system lymphomas.Cancer Res. 2001; 61: 6335-6339PubMed Google Scholar LOH assays were performed using the Genetic Analyzer 310 (PE Biosystems, Norwalk, CT) capillary electrophoresis system. The marker D13S153 lies in intron 2 of the RB1 gene.25Toguchida J McGee TL Paterson JC Eagle JR Tucker S Yandell DW Dryja TP Complete genomic sequence of the human retinoblastoma susceptibility gene.Genomics. 1993; 17: 535-543Crossref PubMed Scopus (136) Google Scholar PCR was performed for 28 cycles with 58°C annealing temperature. The analysis was performed using the Gene Scan Program (PE Biosystems) following the manufacturer's protocol. The p53 mutations in tumors 1 to 9 have been previously reported.26Konishi N Hiasa Y Matsuda H Tao M Tsuzuki T Hayashi I Kitahori Y Shiraishi T Yatani R Shimazaki J Lin JC Intratumor cellular heterogeneity and alterations in ras oncogene and p53 tumor suppressor gene in human prostate carcinoma.Am J Pathol. 1995; 147: 1112-1122PubMed Google Scholar For tumors10 to 16, PCR amplification of exons 5 to 9 of p53, single-strand conformation polymorphism analysis, and DNA sequencing was performed according to these published conditions.26Konishi N Hiasa Y Matsuda H Tao M Tsuzuki T Hayashi I Kitahori Y Shiraishi T Yatani R Shimazaki J Lin JC Intratumor cellular heterogeneity and alterations in ras oncogene and p53 tumor suppressor gene in human prostate carcinoma.Am J Pathol. 1995; 147: 1112-1122PubMed Google Scholar The expression of each gene was assessed immunohistochemically using a polyclonal anti-human antibody to p14ARF (FL-132, SC1661; Santa Cruz Biochemicals, Santa Cruz, CA) and monoclonal antibodies to p16INK4a (F-12, SC1661; Santa Cruz Biochemicals), pRB (clone G3-245; Pharmingen, San Diego, CA), p21Waf1 (F-5, SC-6246; Santa Cruz Biochemicals), p27Kip1 (clone 57; Transduction Laboratories, Lexington, KY), and MDM2 (clone IF2; Oncogene Res. Products, Boston, MA). After deparaffinization, sections were heated for 5 minutes in 10 mmol/L of sodium citrate buffer (pH 6.0) in a pressure cooker. The sections were then incubated overnight at 4°C with a 1:500 dilution of antibodies to p14ARF, pRB, and p27Kip1, a dilution of 1:1000 for p16INK4a, and a dilution of 1:100 for p21Waf1 and MDM2. The reactions were visualized using a Histofine SAB-PO kit and diaminobenzidine as the chromogen (Nichirei, Tokyo, Japan) with hematoxylin counterstaining. For immunostaining, the reactivity was recorded as (−) when <5% of cancer cells were positive, according to the previous study.24Nakamura M Sakaki T Hashimoto H Nakase H Ishida E Shimada K Konishi N Frequent alterations of the p14ARF and p16INK4a genes in primary central nervous system lymphomas.Cancer Res. 2001; 61: 6335-6339PubMed Google Scholar As shown in Table 1, of the 16 prostate adenocarcinomas evaluated, simultaneous homozygous deletion of the p14ARF and p16INK4a gene was detected by differential PCR in only one case (tumor 3, Figure 1). Similarly, hypermethylation of the promoter regions of both p14ARF and p16INK4a also occurred in a single carcinoma (tumor 8, Figure 2). Concurrent deletion or methylation of p14ARF and p16INK4a was detected in two of five foci within individual tumor (tumors 3 and 8, respectively). The two foci showing deletions were Gleason grades 5 and 4, whereas those with the co-methylation were grades 3 and 4. Hypermethylation of p16INK4a exon 2 was found in all areas examined in each of 11 tumors (69%, Table 1).Table 1Heterogeneous Alterations of Multiple Genes in Prostate Cancerp14ARFp16INK4aRB1p21Waf1p27Kip1PTENp73p53MDM2ID No.Gleason scoreIHC*IHC, immunohistochemistry.Methyl or del†p14ARF methylation was detected in promoter regions, and deletion in exon 1β.IHCMethyl or del‡p16INK4a methylation was detected in promoter regions and deletion in exon 1α.Methyl§p16INK4a methylation was detected in exon 2.IHCLOH at D6S153MethylIHCMethylIHCMethylMethylMethylIHCMutationIHC1 13+−+−Methyl−NI¶NI, noninformative.Methyl−Methyl+−−−++− 24+−+−Methyl−NIMethyl−Methyl+−−−++− 33+−+−Methyl−NIMethyl−−+−−−++− 44+−+−Methyl−NIMethyl−Methyl+−−−++− 53+−+−Methyl−NI−−Methyl+−−−−−−2 11+−+−Methyl−LOH∥LOH, loss of heterozygosity.−−−+−−−−−− 22+−+−Methyl+LOH−−−+−−−−−− 32+−+−Methyl−LOH−−−+−−−−−− 43+−+−Methyl−LOH−−−+−−−−−− 52+−+−Methyl−LOH−−−+−−−−−−3 15−del−delMethyl+RET**RET, retention of heterozygosity.−+−−Methyl−−−−− 24−del−delMethyl+RET−+−−Methyl−−−−− 35−−−−Methyl+RET−+−−Methyl−−−−− 45+−+−Methyl+RET−+−+Methyl−−−−− 54−−−−Methyl+RET−+−−Methyl−−−−+4 15+−+−Methyl+RET−+−+−−−−−− 24+−+−Methyl+RET−+−+−−−−−− 34+−+−Methyl+RET−+−+−−−−−− 44+−+−Methyl+RET−+−+−−−−−− 53+−+−Methyl−n.a.n.a., not available.−+−+−−−−−−5 14+−+−Methyl−n.a.−−−+−−−−−− 24+−+−Methyl−n.a.−−−+−−−−−− 35+−+−Methyl−RET−−−−−−−−−− 45+−+−Methyl−RET−+−+−−−−−− 55+−+−Methyl+RET−+−+−−−−−+6 13+−+−Methyl+RET−−−+−−−−−− 24+−+−Methyl+RET−+−+−−−−−− 34+−+−Methyl+LOH−+−+−−−−−− 43+−+−Methyl−LOH−+−−−−−−−− 54+−+−Methyl+LOH−+−+−−−−−−7 14−−−−−+n.a.−+−+−−−++− 23+−−−−+RET−+−+−−−++− 33+−−−−+RET−+−+−−−++− 43+−−−−+RET−+−+−−−−−− 53+−+−−+RET−+−+−−−−−−8 14+−+−Methyl+RET−−Methyl−Methyl−−−−− 23−Methyl−MethylMethyl+RET−−Methyl−−−−−−− 34−Methyl−MethylMethyl+RET−−−−Methyl−−−−− 45+−+−Methyl+RET−−−−Methyl−−−−− 54+−+−Methyl+RET−−−−Methyl−−−−−9 12+−+−−−NI¶NI, noninformative.−+−−−−−−−− 23+−+−−−NI−+−−−−−−−− 34+−+−−−NI−+−+−−−−−− 44+−+−−+NI−+−+−−−−−−10 12+−+−Methyl+NI−+−+−−−−−− 23+−+−Methyl+NI−+−+−−−−−− 33+−+−Methyl+n.a.n.a., not available.−+−+−−−−−− 44+−+−Methyl+n.a.−+−+−−−−−+ 54+−+−Methyl+NI−+−+−−−−−−11 15+−+−−+RET**RET, retention of heterozygosity.−+−−−−−−−− 25+−+−−+RET−+−−−−−−−− 33+−+−−+n.a.−−−+−−−−−− 44+−+−−+LOH∥LOH, loss of heterozygosity.−−−−−−−−−− 54+−+−−+LOH−−−−−−−−−−12 14+−+−−+RET−+−+−−−−−− 25+−+−−+RET−+−+−−−−−− 35+−+−−+RET−+−+−−−−−− 45+−+−−+RET−+−+−−−−−−13 13+−+−Methyl+RET−+−−Methyl−−−−− 25+−+−Methyl−RET−−−−−−−−−− 35+−+−Methyl−RET−−−−Methyl−−−−− 44+−+−Methyl−RET−−−−Methyl−−−−− 53+−+−Methyl−RET−−−−Methyl−−−−−14 13+−+−Methyl+RETMethyl−−+−−−−−− 23+−+−Methyl−RETMethyl−−+−−−−−− 34+−+−Methyl−RETMethyl+−+−−−−−−15 15+−+−Methyl+RET−−−+−−−−−− 24+−+−Methyl+RET−−−−−−−−−− 34+−+−Methyl+RET−−−−−−−−−− 45+−+−Methyl+RET−−−−−−−−−− 54+−+−Methyl+RET−+−−−−−−−−16 14+−+−−+NI−+−−−−−−−+ 24+−+−−+NI−+−+−−−−−− 33+−+−−+NI−−−+−−−−−−* IHC, immunohistochemistry.† p14ARF methylation was detected in promoter regions, and deletion in exon 1β.‡ p16INK4a methylation was detected in promoter regions and deletion in exon 1α.§ p16INK4a methylation was detected in exon 2.¶ NI, noninformative.∥ LOH, loss of heterozygosity.** RET, retention of heterozygosity.†† n.a., not available. Open table in a new tab Figure 2A: Methylation-specific PCR of CpG islands in the p14ARF and p16INK4a promoters in prostate carcinomas. In foci 7-5, 8-1, and 8-4, only unmethylated DNA (U) is apparent. In case 8, p14ARF and p16INK4a methylation (M) was restricted to foci (8-2 and 8-3) lacking p14ARF and p16INK4a immunoreactivity. N.C., Normal control DNA from a normal blood; P.C., positive control for methylated DNA; U, PCR product amplified by unmethylated-specific primers; M, PCR product amplified by methylated-specific primers. B: Methylation-specific PCR for RB1, p21Waf1, and p27Kip1. Hypermethylation of RB1 and/or p21Waf1 promoters was found in four of five foci in only one tumor (tumor no.1). Hypermethylation of p27Kip1 was demonstrated in four foci in one tumor (tumor no. 8).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Hypermethylation of the RB1, p21Waf1, or p27Kip1 promoter was detected in two, two, and three tumors, respectively (Table 1, Figure 2). In general, methylation occurred in most, but not all, large foci within these few tumors and seemed to be independent events, ie, methylation of both the RB1 and p21Waf1promoters was detected in three of five foci in tumor 1 and concurrent methylation of p21Waf1 and p27Kip1 was detected in one of five foci in tumor 8. Methylated and unmethylated control DNAs showed the expected fragment sizes of 163 bp for RB1, 89 bp and 118 bp for p21Waf1, and 195 bp and 212 bp for p27Kip1 (Figure 2). Methylation of the PTEN and p73 promoters was not detected in any tumor examined. For RB1, 3 of 12 informative cases had allelic loss in more than two foci. In case 2, LOH was detected in all foci within the tumor. All 13 tumors with p14ARF expression showed a normal p14ARF gene status. Three tumors showed loss of p14ARF and p16INK4a expression (Table 1). Loss of p14ARF/p16INK4a expression seemed to correlate to homozygous co-deletion or promoter co-methylation. In one case (tumor 8), three foci showed expression of both p14ARF and p16INK4a, but the neoplastic cells within two other areas lacked expression of p14ARF/p16INK4a while showing promoter hypermethylation in both (Table 1, Figure 3); exon 2 of p16INK4a was also methylated in these two immunonegative foci. Two foci (3-3 and 3-5) in tumor 3 and focus 7-1 in tumor 7 showed loss of both p14ARF and p16INK4a expression without simultaneous deletion or methylation in the genes. Another anomaly of tumor 7 was that four of the five individual foci examined were immunonegative for expression of p16INK4a without further alteration in that gene. Nuclear immunoreactivity to p14ARF and p16INK4a was observed in normal prostate tissues. Negative immunoreactivity to pRB was evident in a majority of areas within six tumors and five tumors were primarily negative to p27Kip1. Of these samples, tumor 13 showed loss of expression of both genes, whereas no correlation was seen in the remainder of cases. Within this subset, two lesions (tumors 1 and 14) showed promoter hypermethylation of RB1 concurrent with immunonegativity and three tumors (tumors 3, 8, and 13) showed the same phenomenon with p27Kip1. Loss of protein expression tended to correlate with RB1 methylation in six of eight foci (75%) examined within tumors 1 and 14. Thirty-four of 42 foci without LOH showed RB1 expression, but six foci without RB1 methylation showed loss of the expression. In the three lesions with overall loss of p27Kip1expression, methylation was similarly detected in 12 of 15 foci (80%). However, as can be seen from Table 1, focal loss of protein expression without any other detected aberrations in the promoter regions of the genes discussed occurred far more frequently. A large fraction of prostate carcinomas (10 of 16 cases, 33 of 46 foci) revealed some loss of p21Waf1 expression, but p21Waf1 concurrent hypermethylation was detected in only six immunonegative foci among two tumors (tumors 1 and 8). Five tumors showed collective negative immunoreactivity to both pRB and p21Waf1, and four tumors expressed neither p21Waf1 nor p27Kip1. MDM2 protein was overexpressed in only one focus in each of four tumors and did not seem to correlate with any other gene alterations. In our previous examination of these prostate tumors, we detected p53 mutations in seven foci between two tumors;26Konishi N Hiasa Y Matsuda H Tao M Tsuzuki T Hayashi I Kitahori Y Shiraishi T Yatani R Shimazaki J Lin JC Intratumor cellular heterogeneity and alterations in ras oncogene and p53 tumor suppressor gene in human prostate carcinoma.Am J Pathol. 1995; 147: 1112-1122PubMed Google Scholar these seven foci also showed expression of p53 protein with immunostaining. In this study, we confirmed the previous results and, additionally, found no significant association between p53 status and alterations in p14ARF or overexpression of MDM2. Within the multistep process of carcinogenesis, clonal populations arising within tumors may undergo separate individual genetic and epigenetic changes leading to aggressive growth advantages. The 16 prostate carcinomas analyzed in the current study demonstrated multiple genetic/epigenetic patterns; several foci showed promoter methylation or deletions in promoter regions that were not detected in other areas within the same tumor. Methylation of DNA is important in the genetic regulation of mammalian cells. CpG islands are GC-rich areas of the genome corresponding to the promoter regions of genes and are associated with transcriptional activity. The methylation status of these islands has been shown to be involved with inactivation of tumor suppressor genes. Recent available evidence would suggest that changes in gene expression through epigenetic mechanisms may be responsible for tumor progression in prostate cancer.3Rennie PS Nelson CC Epigenetic mechanisms for progression of prostate cancer.Cancer Metastasis Rev. 1998; 17: 401-409Crossref PubMed Scopus (74) Google Scholar Methylation silencing of p14ARF and p16INK4a is frequent in some types of tumors, including gliomas, colon, and esophageal carcinomas;12Nakamura M Watanabe T Klangby U Asker C Wiman K Yonekawa Y Kleihues P Ohgaki H p14ARF deletion and methylation in genetic pathways to glioblastomas.Brain Pathol. 2001; 11: 159-168Crossref PubMed Scopus (208) Google Scholar, 15Xing EP Nie Y Song Y Yang GY Cai YC Wang LD Yang CS Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma.Clin Cancer Res. 1999; 5: 2704-2713PubMed Google Scholar, 21Esteller M Tortola S Toyota M Capella G Peinado MA Baylin SB Herman JG Hypermethylation-associated inactivation of p14ARF is independent of p16INK4a methylation and p53 mutational status.Cancer Res. 2000; 60: 129-133PubMed Google Scholar however, methylation silencing of p14ARF is extremely rare in lymphomas13Esteller M Cordon-Cardo C Corn PG Meltzer SJ Pohar KS Watkins DN Capella G Peinado MA Matias-Guiu X Prat J Baylin SB Herman JG p14ARF silencing by promoter hypermethylation mediates abnormal intracellular localization of MDM2.Cancer Res. 2001; 61: 2816-2821PubMed Google Scholar, 14Esteller M Corn PG Baylin SB Herman JG A gene hypermethylation profile of human cancer.Cancer Res. 2001; 61: 3225-3229PubMed Google Scholar, 24Nakamura M Sakaki T Hashimoto H Nakase H Ishida E Shimada K Konishi N Frequent alterations of the p14ARF and p16INK4a genes in primary central nervous system lymphomas.Cancer Res. 2001; 61: 6335-6339PubMed Google Scholar and in pancreatic and hepatic carcinomas.13Esteller M Cordon-Cardo C Corn PG Meltzer SJ Pohar KS Watkins DN Capella G Peinado MA Matias-Guiu X Prat J Baylin SB Herman JG p14ARF silencing by promoter hypermethylatio
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