Prevention of cisplatin-DNA adduct repair and potentiation of cisplatin-induced apoptosis in ovarian carcinoma cells by proteasome inhibitors

Histones H2A and H2B are known to be reversibly post-translationally modifi ed by ubiquitination. We previously observed in cultured tumor cells that p roteasome inhibition stabilizes polyubiquitinated proteins, depletes unconj ugated ubiquitin, and thereby promotes the deubiquitination of nucleosomal histones in chromatin. Provocative indirect evidence suggests that histone ubiquitination/deubiquitination cycles alter chromatin structure, which may limit accessibility of DNA repair proteins to damaged sites. In the presen t study, we focused on the relationship between the ubiquitination status o f histone H2A, the structure of chromatin, and the efficiency of nucleotide excision repair (NER) of cisplatin-DNA adducts in human ovarian carcinoma cells exposed to the antitumor drug cisplatin. Pretreating cells with the p roteasome inhibitor lactacystin (LC) or N-acetyl-leucyl-Leucyl-norleucinal (ALLnL) induced deubiquitination of ubiquitinated histone H2A (uH2A) and co ncomitantly promoted chromatin condensation, increased the extent of cispla tin-DNA adducts, and diminished NER-dependent repair of cisplatin-DNA lesio ns, compared with control cells treated with cisplatin alone. Both proteaso me inhibitors also prevented the increase in ERCC-1 mRNA expression that oc curs in cells exposed to cisplatin. Cells treated with the combination of A LLnL and cisplatin underwent apoptosis, as indicated by caspase-dependent p oly(ADP-ribose) polymerase (PARP) cleavage, more quickly than cells treated with tither agent alone. Additionally, the combination of ALLnL and cispla tin potently increased p53 levels in cell lysates and stimulated the bindin g of p53 to chromatin. Together, these observations suggest that proteasome inhibition may be exploited therapeutically for its potential to sensitize ovarian tumor cells to cisplatin. (C) 2000 Elsevier Science Inc.