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.
Jarrard
D F
DF
Department of Toxicology, University of Wisconsin Comprehensive Cancer Center and Medical School, Madison 53792, USA. jarrard@surgery.wisc.edu
Sarkar
S
S
Shi
Y
Y
Yeager
T R
TR
Magrane
G
G
Kinoshita
H
H
Nassif
N
N
Meisner
L
L
Newton
M A
MA
Waldman
F M
FM
Reznikoff
C A
CA
D000368:Aged; D016922:Cellular Senescence; D002869:Chromosome Aberrations; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D019175:DNA Methylation; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D017353:Gene Deletion; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009362:Neoplasm Metastasis; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism; D050725:Papillomavirus E7 Proteins; D011467:Prostate / Q000378:metabolism* / Q000473:pathology; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D012097:Repressor Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C493956:CDKN1A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; C059731:oncogene protein E7, Human papillomavirus type 16