Ku70 and Rad51 vary in their importance for the repair of doxorubicin- versus etoposide-induced DNA damage.
For DNA targeting anticancer drugs, cellular DNA repair mechanisms may cause resistance and hamper the therapeutic outcome. DNA damage induced by topoisomerase IIα inhibitors like etoposide and anthracyclines, which are a mainstay of cancer therapy, is also repaired in many cell types, but the impact and precise mechanisms of this repair are still obscure. To investigate the DNA damage response of human adenocarcinoma HT29-cells to doxorubicin and to compare the involvement of Ku70 and Rad51 in the repair of doxorubicin- versus etoposide-induced DNA damage, we assessed cell cycle distribution and cell death, DNA damage, proteins relevant for repair by homologous recombination and non-homologous end-joining, and clonogenicity following exposure to doxorubicin at clinically achievable concentrations. Also, we assessed changes in the repair kinetics after siRNA-mediated attenuation of Ku70 or Rad51 expression. We found that exposure to doxorubicin for 24 h induced a substantial amount of DNA damage that was largely repaired when doxorubicin was removed and the cells were maintained in drug-free medium. Nevertheless, a pronounced G(2)/M arrest occurred at times when repair was maximal. This was followed by a distinct increase in cell death and loss of clonogenicity. In this regard, responses to doxorubicin and etoposide were similar. However, distinct differences in the repair process following doxorubicin versus etoposide were seen in concentration dependency, time-course and requirement of Ku70 and Rad51 proteins. In spite of the shared molecular target of doxorubicin and etoposide, DNA lesions induced by these compounds are repaired differently.
Clinical Pharmacy, Institute of Pharmacy, Hamburg University, Bundesstrasse 45, Hamburg, Germany. i.schonn@online.de
Ilona
I
Schonn
Jana
J
Hennesen
Dorothee C
DC
Dartsch
D034961:Antigens, Nuclear / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D003114:Colony-Forming Units Assay; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004317:Doxorubicin / Q000494:pharmacology*; D005047:Etoposide / Q000494:pharmacology*; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D019073:HT29 Cells; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D000072200:Ku Autoantigen; D034741:RNA, Small Interfering / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism*; D014162:Transfection
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D005047:Etoposide; D004317:Doxorubicin; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen