REGULATION OF P16(CDKN2) EXPRESSION AND ITS IMPLICATIONS FOR CELL IMMORTALIZATION AND SENESCENCE

p16(CDKN2) specifically binds to and inhibits the cyclin-dependent kin ases CDK4 and CDK6, which function as regulators of cell cycle progres sion in G(1) by contributing to the phosphorylation of the retinoblast oma protein (pRB). Human cell lines lacking functional pRB contain hig h levels of p16 RNA and protein, suggesting a negative feedback loop b y which pRB might regulate p16 expression in late G(1). By a combinati on of nuclear run-on assays and promoter analyses in human fibroblasts expressing a temperature-sensitive simian virus 40 T antigen, we show that p16 transcription is affected by the status of pRB and define a region in the p16 promoter that is required for this response. However , the effect is not sufficient to account for the differences in p16 R NA levels between pRB-positive and -negative cells. Moreover, p16 RNA is extremely stable, and the levels do not change appreciably during t he cell cycle. Primary human fibroblasts express very low levels of p1 6, but the RNA and protein accumulate in late-passage, senescent cells . The apparent overexpression of p16 in pRB-negative cell lines is the refore caused by at least two factors: loss of repression by pRB and a n increase in the number of population doublings.