REINITIATION OF DNA-SYNTHESIS AND CELL-DIVISION IN SENESCENT HUMAN FIBROBLASTS BY MICROINJECTION OF ANTI-P53 ANTIBODIES

In human fibroblasts, growth arrest at the end of the normal prolifera tive life span (induction of senescence) is dependent on the activity of the tumor suppressor protein p53, In contrast, once senescence has been established, it is generally accepted that reinitiation of DNA sy nthesis requires loss of multiple suppressor pathways, for example, by expression of Simian virus 40 (SV40) large T antigen, and that even t his will not induce complete cell cycle traverse. Here we have used mi croinjection of monoclonal antibodies to the N terminus of p53, PAb180 1 and DO-1, to reinvestigate the effect of blocking p53 function in se nescent human fibroblasts. Unexpectedly, we found that both antibodies induce senescent cells to reenter S phase almost as efficiently as SV 40, accompanied by a reversion to the ''young'' morphology. Furthermor e, this is followed by completion of the cell division cycle, as shown by the appearance of mitoses, and by a four-to fivefold increase in c ell number 9 days after injection. Immunofluorescence analysis showed that expression of the p53-inducible cyclin/kinase inhibitor p21(sdi1/ WAF1) was greatly diminished by targeting p53 with either PAb1801 or D O-1 but remained high and, moreover, still p53 dependent in cells expr essing SV40 T antigen, As previously observed for induction, the maint enance of fibroblast senescence therefore appears to be critically dep endent on functional p53, We suggest that the previous failure to obse rve this by using SV40 T-antigen mutants to target p53 was most probab ly due to incomplete abrogation of p53 function.