p16/pRb pathway alterations are required for bypassing senescence in human prostate epithelial cells.

The cell cycle regulatory genes p16/CDKN2 and RB are frequently deleted in prostate cancers. In this study, we examined the role of alterations in p16 and pRb during growth, senescence, and immortalization in vitro of human prostate epithelial cells (HPECs). HPECs are established from normal prostate tissues and cultured on collagen-coated dishes. Our results show that p16 is reproducibly elevated at senescence in HPECs. HPECs are immortalized using human papilloma virus 16 E6 and/or E7 as molecular tools to inactivate p53 and/or pRb, respectively. Immortalization occurs infrequently in this system and only after a latent period during which additional genetic/epigenetic changes are thought to occur. Notably, all of the E6-immortalized HPEC lines but none of the E7 lines show inactivation of p16/CDKN2 (by deletion, methylation, or mutation) in association with immortalization. In contrast, E7 lines, in which pRb function is abrogated by E7 binding, retain the high levels of p16 observed at senescence. Thus, all lines show either a p16 or pRb inactivation. Analysis of six independent lines from metastatic prostate cancers reveals a similar loss of either p16 or pRb. Comparative genomic hybridization of HPECs shows that gains of chromosomes 5q, 8q, and 20 are nonrandomly associated with bypassing senescence (probability = 0.95). These results suggest that high levels of the cyclin-dependent kinase inhibitor p16 mediate senescence G1 arrest in HPECs and that bypassing this block by a p16/pRb pathway alteration is required for immortalization in vitro and possibly tumorigenesis in vivo. Our results further indicate that inactivation of the p16/pRb pathway alone is not sufficient to immortalize HPECs and that additional genetic alterations are required for this process.

[1]  T. Kiyono,et al.  Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells , 1998, Nature.

[2]  P. Carroll,et al.  Genetic alterations in primary bladder cancers and their metastases. , 1998, Cancer research.

[3]  J W Gray,et al.  Positional cloning of ZNF217 and NABC1: genes amplified at 20q13.2 and overexpressed in breast carcinoma. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[4]  M A Newton,et al.  On the statistical analysis of allelic-loss data. , 1998, Statistics in medicine.

[5]  G. Hannon,et al.  Myc activates telomerase. , 1998, Genes & development.

[6]  P. Jones,et al.  The role of DNA methylation in expression of the p19/p16 locus in human bladder cancer cell lines. , 1998, Cancer research.

[7]  M. Newton,et al.  Overcoming cellular senescence in human cancer pathogenesis. , 1998, Genes & development.

[8]  M. Droller.  Telomerase activity: a biomarker of cell proliferation, not malignant transformation. , 1998, The Journal of urology.

[9]  Takafumi Nishizaki,et al.  Genetic alterations in primary breast cancers and their metastases: Direct comparison using modified comparative genomic hybridization , 1997, Genes, chromosomes & cancer.

[10]  H. Frierson,et al.  p53, bcl-2 and retinoblastoma proteins as long-term prognostic markers in localized carcinoma of the prostate. , 1997, The Journal of urology.

[11]  J. Herman,et al.  Deletional, mutational, and methylation analyses of CDKN2 (p16/MTS1) in primary and metastatic prostate cancer , 1997, Genes, chromosomes & cancer.

[12]  A. Levine.  p53, the Cellular Gatekeeper for Growth and Division , 1997, Cell.

[13]  G. Hannon,et al.  Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[14]  A. Klein-Szanto,et al.  Expression of p21 is not required for senescence of human fibroblasts. , 1996, Cancer research.

[15]  R. Blelloch,et al.  A molecular genetic model of human bladder cancer pathogenesis. , 1996, Seminars in oncology.

[16]  D. Schaid,et al.  Allelic imbalance and microsatellite instability in prostatic adenocarcinoma. , 1996, Cancer research.

[17]  J. Brooks,et al.  An uncertain role for p53 gene alterations in human prostate cancers. , 1996, Cancer research.

[18]  O. Pereira-smith,et al.  Replicative Senescence: Implications for in Vivo Aging and Tumor Suppression , 1996, Science.

[19]  C. Reznikoff,et al.  Elevated p16 at senescence and loss of p16 at immortalization in human papillomavirus 16 E6, but not E7, transformed human uroepithelial cells. , 1996, Cancer research.

[20]  P. Carroll,et al.  Genetic alterations in untreated metastases and androgen-independent prostate cancer detected by comparative genomic hybridization and allelotyping. , 1996, Cancer research.

[21]  L. Liotta,et al.  Analysis of 99 microdissected prostate carcinomas reveals a high frequency of allelic loss on chromosome 8p12-21. , 1996, Cancer research.

[22]  G. Peters,et al.  Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence , 1996, Molecular and cellular biology.

[23]  M. Ittmann,et al.  Alterations of the retinoblastoma gene in clinically localized, stage B prostate adenocarcinomas. , 1996, Human pathology.

[24]  D. Louis,et al.  CDKN2/p16 or RB alterations occur in the majority of glioblastomas and are inversely correlated. , 1996, Cancer research.

[25]  M. Skolnick,et al.  CDKN2 (MTS1) tumor suppressor gene mutations in human tumor cell lines. , 1995, Oncogene.

[26]  James Brugarolas,et al.  Radiation-induced cell cycle arrest compromised by p21 deficiency , 1995, Nature.

[27]  C Roskelley,et al.  A biomarker that identifies senescent human cells in culture and in aging skin in vivo. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[28]  P. Atadja,et al.  Increased activity of p53 in senescing fibroblasts. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[29]  J. Herman,et al.  5′ CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers , 1995, Nature Medicine.

[30]  J. Bartek,et al.  Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16 , 1995, Nature.

[31]  J. R. Smith,et al.  Mutant p53 rescues human diploid cells from senescence without inhibiting the induction of SDI1/WAF1. , 1995, Cancer research.

[32]  K. Vousden.  Regulation of the cell cycle by viral oncoproteins. , 1995, Seminars in cancer biology.

[33]  T. Ide,et al.  Increase in expression level of p21sdi1/cip1/waf1 with increasing division age in both normal and SV40-transformed human fibroblasts. , 1995, Oncogene.

[34]  G. Peters,et al.  Lack of cyclin D‐Cdk complexes in Rb‐negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene product. , 1995, The EMBO journal.

[35]  J. Brooks,et al.  Allelic loss of the retinoblastoma gene in primary human prostatic adenocarcinomas , 1995, The Prostate.

[36]  A. Okamoto,et al.  IS-12 Mutation and altered expression of P16^ in human cancer. , 1995 .

[37]  A. Okamoto,et al.  Mutations and altered expression of p16INK4 in human cancer. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[38]  M. Newton,et al.  Long-term genome stability and minimal genotypic and phenotypic alterations in HPV16 E7-, but not E6-, immortalized human uroepithelial cells. , 1994, Genes & development.

[39]  R. Hruban,et al.  Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma , 1994, Nature Genetics.

[40]  W. Clark,et al.  Germline p16 mutations in familial melanoma , 1994, Nature Genetics.

[41]  D. Sidransky,et al.  Rates of p16 (MTS1) mutations in primary tumors with 9p loss. , 1994, Science.

[42]  C. Cordon-Cardo,et al.  Immunohistochemical determination of p53 protein nuclear accumulation in prostatic adenocarcinoma. , 1994, The Journal of urology.

[43]  M. Skolnick,et al.  A cell cycle regulator potentially involved in genesis of many tumor types. , 1994, Science.

[44]  J Piper,et al.  Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[45]  F. Kaye,et al.  Absence of p16INK4 protein is restricted to the subset of lung cancer lines that retains wildtype RB. , 1994, Oncogene.

[46]  Goberdhan P Dimri,et al.  Molecular and cell biology of replicative senescence. , 1994, Cold Spring Harbor symposia on quantitative biology.

[47]  T. Tong,et al.  Cancer statistics, 1994 , 1994, CA: a cancer journal for clinicians.

[48]  U. Stein,et al.  Multidirectional differentiation in the normal, hyperplastic, and neoplastic human prostate: simultaneous demonstration of cell-specific epithelial markers. , 1994, Human pathology.

[49]  G. Hannon,et al.  A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4 , 1993, Nature.

[50]  C. Kao,et al.  Role of SV40 T antigen binding to pRB and p53 in multistep transformation in vitro of human uroepithelial cells. , 1993, Carcinogenesis.

[51]  J. Shay,et al.  E6 of human papillomavirus type 16 can overcome the M1 stage of immortalization in human mammary epithelial cells but not in human fibroblasts. , 1993, Oncogene.

[52]  J. Schalken,et al.  Colocalization of basal and luminal cell-type cytokeratins in human prostate cancer. , 1992, Cancer research.

[53]  W. Isaacs,et al.  Expression of the cellular adhesion molecule E-cadherin is reduced or absent in high-grade prostate cancer. , 1992, Cancer research.

[54]  R. Pignolo,et al.  Changes in gene expression during senescence in culture , 1992, Experimental Gerontology.

[55]  W. Isaacs,et al.  Wild-type p53 suppresses growth of human prostate cancer cells containing mutant p53 alleles. , 1991, Cancer research.

[56]  J. Shay,et al.  A role for both RB and p53 in the regulation of human cellular senescence. , 1991, Experimental cell research.

[57]  E. Hara,et al.  Cooperative effect of antisense-Rb and antisense-p53 oligomers on the extension of life span in human diploid fibroblasts, TIG-1. , 1991, Biochemical and biophysical research communications.

[58]  G. Demers,et al.  The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells , 1991, Journal of virology.

[59]  W. Lee,et al.  Promoter deletion and loss of retinoblastoma gene expression in human prostate carcinoma. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[60]  G. Stein,et al.  Failure to phosphorylate the retinoblastoma gene product in senescent human fibroblasts. , 1990, Science.

[61]  R. Newbold,et al.  Induction of immortality is an early event in malignant transformation of mammalian cells by carcinogens , 1982, Nature.

[62]  L. Hayflick.  THE LIMITED IN VITRO LIFETIME OF HUMAN DIPLOID CELL STRAINS. , 1965, Experimental cell research.