Abstract
DNA damage can lead to either DNA repair with cell survival or to apoptotic cell death. Although the biochemical processes underlying DNA repair and apoptosis have been extensively studied, the mechanisms by which cells determine whether the damage will be repaired or the apoptotic pathway will be activated is largely unknown. We have studied the role of nucleotide excision repair (NER) in cisplatin DNA damage-induced apoptotic cell death using both normal human fibroblasts and NER-defective xeroderma pigmentosum (XP) XPA and XPG cells. The caspase-3 activation experiment demonstrated a greatly increased casapse-3 activation in the NER-defective cells following cisplatin treatment. The flow cytometry experiment revealed an altered cell cycle arrest pattern of the NER-defective cells following cisplatin treatment. The results obtained from the Western blot experiment showed that NER defects resulted in enhanced CHK1 phosphorylation and p21 induction after cisplatin treatment. The cisplatin treatment-induced ATM phosphorylation, however, was attenuated in NER-defective cells. The results obtained from our immunoprecipitation experiment further demonstrated that the ATM protein interacted with the TFIIH basal transcription factor and the XPG protein of the NER pathway. It also showed that a functional XPC protein was required for the association of the ATM protein to genomic DNA. These results suggest that the NER process may prevent the cisplatin treatment-induced apoptosis by activating the ATM protein, and that the presence of the XPC protein is essential for recruiting the ATM protein to the DNA template.
Highlights
Many anticancer drugs are targeted to the genomic DNA of cancer cells to generate DNA damage and block DNA replication and/or gene transcription, resulting in cell cycle arrest and apoptotic cell death
Using both normal human fibroblasts (NF) and Nucleotide excision repair (NER)-defective xeroderma pigmentosum group A (XPA) and XPG cells, we demonstrated that the NER defects led to an altered cell cycle arrest and an increased apoptotic cell death after cisplatin treatment
We further demonstrated that the ataxiatelangiectasia mutated (ATM) protein was phosphorylated in cisplatin-treated NF cells, whereas this cisplatin treatment-induced ATM phosphorylation was attenuated in XPA and XPG cells
Summary
Many anticancer drugs are targeted to the genomic DNA of cancer cells to generate DNA damage and block DNA replication and/or gene transcription, resulting in cell cycle arrest and apoptotic cell death (apoptosis). Studying the DNA damage-mediated signaling process in these NER-defective cells will provide important insights into the mechanism of DNA repair in preventing DNA damage-induced apoptosis. These results suggest that the NER process may prevent cisplatin treatment-induced apoptosis by activating the ATM pathway, which in turn, enhances cell cycle arrest to allow for DNA repair to take place. The DNA damage recognition and binding of XPC protein helps recruit the ATM protein to the DNA template These results provide an important insight into the mechanism by which the NER pathway coordinates with other cellular processes including cell cycle arrest and apoptosis in determining the fate of damaged cells
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