EVIDENCE THAT TRANSCRIPTIONAL ACTIVATION BY P53 PLAYS A DIRECT ROLE IN THE INDUCTION OF CELLULAR SENESCENCE

Wild-type p53 is necessary for the growth arrest of human diploid fibr oblasts (HDF) (and many other cell types) at the end of their prolifer ative lifespan. Although p53 may actively mediate senescence, possibly in response to telomere erosion, it is however equally possible that it is merely a permissive factor required for response to some other i nducer. To address this question, we have generated stable transfectan ts of early passage HDF, represented here by clone LacZ21, in which ex pression of a beta-galactosidase reporter construct reflects p53 trans activation activity. During continuous passage, the proportion of beta -gal positive LacZ21 cells remained below 2% for 25 population doublin gs (pd), first became significantly increased after 29 pd, and thereaf ter increased rapidly, reaching a maximum of 88% in fully-senescent ce lls (32 pd), which exceeded the response observed following an optimum dose (20 J/m(2)) of u.v. radiation. Correspondingly, the proportion o f cells incorporating bromodeoxyuridine (BrdU) (initially 45-50%) bega n to fall at 29 pd and thereafter dropped rapidly to below 1% by pd 32 . There was therefore a near-perfect reciprocal relationship between r eporter construct expression and DNA synthesis as cells approached sen escence. Furthermore, a dominant-negative p53 mutant (introduced by re troviral transduction) rescued LacZ21 cells from senescence and genera ted colonies with extended lifespan in which beta-gal expression was t otally abolished. These data, although not excluding the need for othe r p53 functions, strongly suggest that p53-mediated transactivation of growth regulatory genes is a direct trigger, rather than a permissive factor, for cellular senescence.