TY - JOUR
T1 - Effect of the anti-neoplastic drug doxorubicin on XPD-mutated DNA repair-deficient human cells
AU - Saffi, Jenifer
AU - Agnoletto, Mateus H.
AU - Guecheva, Temenouga N.
AU - Batista, Luís F.Z.
AU - Carvalho, Helotonio
AU - Henriques, João A.P.
AU - Stary, Anne
AU - Menck, Carlos F.M.
AU - Sarasin, Alain
N1 - Funding Information:
The authors acknowledge Danièle Pham for technical assistance. J. Saffi was supported by a post-doctoral fellowship from CAPES (Brasília, Brazil). This research was founded by CNPq (Brasília, Brazil), FAPERGS-PPSUS 2006 (Porto Alegre, Brazil), USP-COFECUB (Sao Paulo, Brazil and Paris, France) and FAPESP (São Paulo, Brazil). Grants to A. Sarasin have been given by the “Ligue Contre le Cancer” (Paris, France) and the “Agence Nationale de la Recherche, ANR:MUTATRANSCI” (Paris, France).
PY - 2010/1/2
Y1 - 2010/1/2
N2 - 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 γH2AX 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 IIα 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.
AB - 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 γH2AX 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 IIα 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.
KW - Cancer
KW - Doxorubicin
KW - Nucleotide excision repair
KW - Topoisomerase II
KW - Xeroderma pigmentosum group D
UR - http://www.scopus.com/inward/record.url?scp=72949107121&partnerID=8YFLogxK
U2 - 10.1016/j.dnarep.2009.10.003
DO - 10.1016/j.dnarep.2009.10.003
M3 - Article
C2 - 19926538
AN - SCOPUS:72949107121
SN - 1568-7864
VL - 9
SP - 40
EP - 47
JO - DNA Repair
JF - DNA Repair
IS - 1
ER -