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Involvement of proliferating cell nuclear antigen in DNA repair after damage induced by genotoxic agents in human fibroblasts.

The association of the proliferating cell nuclear antigen (PCNA) to DNA synthesis sites during the process of DNA repair, was investigated in human diploid fibroblasts after treatment with different genotoxic agents. For this purpose, confluent cultures were treated with agents that form primarily DNA adducts, such as u.v.-C, 8-methoxypsoralen (8-MOP) and 4,4',6-trimethylangelicin (TMA), or with agents that induce strand breaks, such as X-rays or bleomycin (BLM). Chromatin-associated PCNA was detected with a monoclonal antibody and indirect immunofluorescence labelling. Quantitative analysis was performed by flow cytometry. Not all of the tested agents were able to activate the association of PCNA with chromatin. X-rays, u.v.-C and BLM induced a significant increase 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 associated PCNA, even at post-incubation repair times as long as 10 h. However, these drugs damaged DNA, as shown by the formation of micronucleated cells 48 h after treatment. The lack of PCNA activation was not due to an inhibition of the repair mechanism, since in TMA-treated fibroblasts, subsequent irradiation with u.v.-C induced an increase in PCNA 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.