Cell cycle checkpoint abrogator UCN-01 inhibits DNA repair: association with attenuation of the interaction of XPA and ERCC1 nucleotide excision repair proteins.
UCN-01, an anticancer agent currently in Phase I clinical trials, has been found to potentiate the cytotoxicity of cisplatin (CDDP). Because mammalian cells remove CDDP-induced DNA adducts through the nucleotide excision repair (NER) pathway, we determined the effects of UCN-01 on NER by measuring its effects on the interaction of the repair factors XPA and ERCC1 and the phosphorylation/dephosphorylation of the repair proteins. The repair activity, as measured by an in vitro repair synthesis assay and an in vivo host-cell reactivation assay using A549 cells, was significantly reduced. Although expression of XPA and ERCC1 proteins was elevated in cells exposed to UCN-01, the treatment resulted in a decreased ERCC1 level in the Triton X-100-insoluble fraction of cell lysates. A pull-down assay using the MBP-XPA fusion protein showed a significant reduction in the binding of ERCC1 to XPA in nuclear extracts from UCN-01-treated cells compared with untreated cells, suggesting that UCN-01 reduced the XPA-ERCC1 interaction. Consistent with these data, lower repair incision activity was found in the cell extracts from UCN-01-treated cells. In vitro phosphorylation revealed that UCN-01 had no effect on the phosphorylation/dephosphorylation status of either XPA or ERCC1; however, UCN-01 caused dephosphorylation of an unidentified XPA-bound protein with an apparent molecular mass of 52 kDa. Taken together, these data demonstrate the NER-inhibitory action of UCN-01, which is associated with the inhibition of the XPA-ERCC1 interaction by UCN-01 and with the effect of UCN-01 on the phosphorylation/dephosphorylation of an XPA-bound, 52-kDa protein, the identity of which remains to be determined.
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Division of Pathology and Laboratory Medicine, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
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D000470:Alkaloids / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002289:Carcinoma, Non-Small-Cell Lung; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D004260:DNA Repair / Q000187:drug effects*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D019311:Staurosporine / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured; D014983:Xeroderma Pigmentosum; D051760:Xeroderma Pigmentosum Group A Protein
D000470:Alkaloids; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D011506:Proteins; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; C054852:7-hydroxystaurosporine; C071447:ERCC1 protein, human; D004720:Endonucleases; D019311:Staurosporine