Effect of the anti-neoplastic drug doxorubicin on XPD-mutated DNA repair-deficient human cells

Doxorubicin (DOX), a member of the anthracycline group, is a widely used drug in cancer therapy. The mechanisms of DOX action include topoisomerase II-poisoning, free radical release, DNA adducts and interstrand cross-link (ICL) formation. Nucleotide excision repair(NER) is involved in the removal of helix-distorting lesions and chemical adducts, however, little is known about the response of NER-deficient cell lines to anti-tumoral drugs like DOX. Wild type and XPD-mutated cells, harbouring mutations in different regions of this gene and leading to XP-D, XP/CS or TTD diseases, were treated with this drug and analyzed for cell cycle arrest and DNA damage by comet assay. The formation of DSBs was also investigated by determination of gamma H2AX foci. Our results indicate that all three NER-deficient cell lines tested are more sensitive to DOX treatment, when compared to wild type cells or XP cells complemented by the wild type XPD cDNA, suggesting that NER is involved in the removal of DOX-induced lesions. The cell cycle analysis showed the characteristic G2 arrest in repair-proficient MRC5 cell line after DOX treatment, whereas the repair-deficient cell lines presented significant increase in sub-G1 fraction. The NER-deficient cell lines do not show different patterns of DNA damage formation as assayed by comet assay and phosphorylated H2AX foci formation. Knock-down of topoisomerase II alpha with siRNA leads to increased survival in both MRC5 and XP cells, however, XP cell line still remained significantly more sensitive to the treatment by DOX. Our study suggests that the enhanced sensitivity is due to DOX-induced DNA damage that is subject to NER, as we observed decreased unscheduled DNA synthesis in XP-deficient cells upon DOX treatment. Furthermore, the complementation of the XPD-function abolished the observed sensitivity at lower DOX concentrations, suggesting that the XPD helicase activity is involved in the repair of DOX-induced lesions. (C) 2009 Elsevier B.V. All rights reserved.CAPES (Brasilia, Brazil)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq (Brasilia, Brazil)FAPERGS-PPSUS 2006 (Porto Alegre, Brazil)Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS)USP-COFECUB (Sao Paulo, Brazil and Paris, France)Comité Français d´Evaluation de la Coopération Universitaire avec le Brésil (COFECUB)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP (Sao Paulo, Brazil)Ligue Contre le Cancer (Paris, France)Ligue Contre le Cancer (Paris, France)Agence Nationale de la Recherche de la France (ANR)Agence Nationale de la Recherche, ANR:MUTATRANSCI (Paris, France