INVOLVEMENT OF PROLIFERATING CELL NUCLEAR ANTIGEN IN DNA-REPAIR AFTERDAMAGE-INDUCED BY GENOTOXIC AGENTS IN HUMAN FIBROBLASTS

The association of the proliferating cell nuclear antigen (PCNA) to DN A synthesis sites during the process of DNA repair, was investigated i n human diploid fibroblasts after treatment with different genotoxic a gents. For this purpose, confluent cultures were treated with agents t hat form primarily DNA adducts, such as u.v.-C, 8-methoxypsoralen (8-M OP) and 4,4',6-trimethylangelicin (TMA), or with agents that induce st rand breaks, such as X-rays or bleomycin (BLM). Chromatin-associated P CNA was detected with a monoclonal antibody and indirect immunofluores cence labelling. Quantitative analysis was performed by flow cytometry . Not all of the tested agents were able to activate the association o f PCNA with chromatin. X-rays, u.v.-C and BLM induced a significant in crease in PCNA complex as compared to the control samples. In contrast , 8-MOP and TMA did not show any detectable effect on the levels of as sociated PCNA, even at post-incubation repair times as long as 10 h. H owever, these drugs damaged DNA, as shown by the formation of micronuc leated cells 48 h after treatment. The lack of PCNA activation was not due to an inhibition of the repair mechanism, since in TMA-treated fi broblasts, subsequent irradiation with u.v.-C induced an increase in P CNA levels comparable to that found in cells treated with u.v.-C alone . These results indicate that PCNA is involved in DNA excision repair of genotoxic agents, but suggest that similar types of lesions may be repaired with alternative pathways not requiring PCNA.