INVOLVEMENT OF THE PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) IN DNA-REPAIR INDUCED BY ALKYLATING-AGENTS AND OXIDATIVE DAMAGE IN HUMAN FIBROBLASTS

The involvement of the proliferating cell nuclear antigen (PCNA) in th e process of DNA repair induced by alkylating agents or by oxidative d amage was investigated in human quiescent fibroblasts by immunofluores cence and flow cytometry, Transition from soluble to the DNA-bound for m of PCNA, was taken as the parameter to determine its involvement in repair DNA synthesis. Treatment with the alkylating agents methylmetha ne sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine resulted in the rapid and dose-dependent increase in the nuclear binding of PCNA, Simi lar results were obtained with compounds such as hydrogen peroxide or tert-butyl hydroperoxide, which are known to induce oxidative DNA dama ge. Tert-butyl hydroperoxide may also generate malondialdehyde through a reaction of lipid peroxidation, This mutagenic and carcinogenic pro duct has been previously shown to form adducts with DNA, Therefore, th e possibility that tert-butyl hydroperoxide could induce DNA damage th rough this pathway was investigated by incubating cells directly in th e presence of malondialdehyde, Such treatment resulted in an increase in immunofluorescence associated with nuclear-bound PCNA, The ability of oxidative and alkylating agents to induce the nuclear binding of PC NA was also assessed in proliferating cells. In these conditions, trea tment with hydrogen peroxide or methylmethane sulfonate, resulted in a n increase in nuclear-bound PCNA in the G1 and in the G2 + M compartme nts, but not in S phase. At longer times after treatment, PCNA immunos taining was reduced to basal levels, while an increase in nuclear bind ing of p2l(waf1/cip1) protein was found in concomitance with cell-cycl e arrest. These results indicate that agents inducing DNA base alterat ions in vivo, promote the nuclear binding of PCNA, These lines of evid ence support the role of a PCNA-dependent reaction in the base excisio n repair system.