Both XPA and DNA polymerase eta are necessary for the repair of doxorubicin-induced DNA lesions.

Doxorubicin (DOX) is an important tumor chemotherapeutic agent, acting mainly by genotoxic action. This work focus on cell processes that help cell survival, after DOX-induced DNA damage. In fact, cells deficient for XPA or DNA polymerase eta (pol eta, XPV) proteins (involved in distinct DNA repair pathways) are highly DOX-sensitive. Moreover, LY294002, an inhibitor of PIKK kinases, showed a synergistic killing effect in cells deficient in these proteins, with a strong induction of G2/M cell cycle arrest. Taken together, these results indicate that XPA and pol eta proteins participate in cell resistance to DOX-treatment, and kinase inhibitors can selectively enhance its killing effects, probably reducing the cell ability to recover from breaks induced in DNA.

DNA damage induced by the anthracycline cosmomycin D in DNA repair-deficient cells.

PURPOSE: Anthracyclines have been widely used as antitumor agents, playing a crucial role in the successful treatment of many types of cancer, despite some side effects related to cardiotoxicity. New anthracyclines have been designed and tested, but the first ones discovered, doxorubicin and daunorubicin, continue to be the drugs of choice. Despite their extensive use in chemotherapy, little is known about the DNA repair mechanisms involved in the removal of lesions caused by anthracyclines. The anthracycline cosmomycin D is the main product isolated from Streptomyces olindensis, characterized by a peculiar pattern of glycosylation with two trisaccharide rings attached to the A ring of the tetrahydrotetracene. METHODS: We assessed the induction of apoptosis (Sub-G1) by cosmomycin D in nucleotide excision repair-deficient fibroblasts (XP-A and XP-C) as well as the levels of DNA damage (alkaline comet assay). RESULTS: Treatment of XP-A and XP-C cells with cosmomycin D resulted in apoptosis in a time-dependent manner, with highest apoptosis levels observed 96 h after treatment. The effects of cosmomycin D were equivalent to those obtained with doxorubicin. The broad caspase inhibitor Z-VAD-FMK strongly inhibited apoptosis in these cells, and DNA damage induced by cosmomycin D was confirmed by alkaline comet assay. CONCLUSIONS: Cosmomycin D induced time-dependent apoptosis in nucleotide excision repair-deficient fibroblasts. Despite similar apoptosis levels, cosmomycin D caused considerably lower levels of DNA damage compared to doxorubicin. This may be related to differences in structure between cosmomycin D and doxorubicin.

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 gammaH2AX 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 IIalpha 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.

Association of low repair efficiency with high hormone receptors expression and SOD activity in breast cancer patients.

OBJECTIVES: To evaluate the antioxidant status and repair capacity in breast cancer patients as well as the relationship between these parameters and expression of critical proteins in breast cancer tissue. DESIGN AND METHODS: Blood samples were obtained from 25 female breast cancer patients and 19 healthy women. The antioxidant status was determined by the concentration of thiobarbituric-reactive substances (TBARS) and activity of superoxide dismutase (SOD) and catalase (CAT). The basal DNA damage and repair capacity in lymphocytes were evaluated by comet assay. The expression of p53, c-erbB2, Ki-67, estrogen receptor (ER) and progesterone receptor (PR) in cancer tissue was detected by immunohistochemical staining. RESULTS: The breast cancer patients presented significantly elevated endogenous DNA damage in lymphocytes and lower susceptibility to DNA damage induced by H(2)O(2) when compared to the control group. There is a negative correlation between TBARS and sensitivity to peroxide induced DNA damage in patients. The percentage of residual damage after H(2)O(2) treatment followed by 3h of post-incubation is significantly higher in patients and also correlates positively with SOD activity, ER and PR expression and negatively with the basal DNA damage. CONCLUSIONS: Our results demonstrate low repair capacity in lymphocytes of breast cancer patients and suggest that the regulation of DNA repair is sensitive to cellular redox state and can be modulated by ER/PR status.