Effects of gemcitabine on cis-platinum-DNA adduct formation and repair in a panel of gemcitabine and cisplatin-sensitive or -resistant human ovarian cancer cell lines.
Gemcitabine (dFdC) can increase the sensitivity of both cisplatin (CDDP)-sensitive and -resistant cell lines. It has been postulated that both formation and repair of platinum-(Pt)-DNA adducts are related to these effects. Therefore, we investigated the effects of dFdC on the formation and repair of Pt-DNA adducts in the human ovarian cancer cell line, A2780, and its CDDP- or dFdC-resistant variants, ADDP and AG6000, which have a different expression of various repair enzymes. Cells were exposed for 1 h to CDDP alone or combined with dFdC in IC50 concentrations, followed by a 1-h exposure to thiourea and, subsequently, by a drug-free period of 1, 3 or 23 h (i.e. 2, 4 or 24 h after CDPP +/- dFdC removal). Pt-DNA adducts were quantified with 32P-post-labeling. The gene expression of the repair enzymes, XPA and XRCC1, was the same in all 3 cell lines but ERCC1, ERCC3 and XPC were 2-6 times higher in AG6000 compared to A2780 cells. In contrast, both ERCC1 and ERCC3 were 10- and 1.5-fold lower in ADDP cells compared to A2780. The mismatch enzyme, MLH1, was lower in ADDP cells. At equally toxic CDDP concentrations, all cell lines formed comparable peak levels of total Pt-DNA adducts (36-48 fmol/microg DNA). However, the time at which peak levels were reached showed large variation. The repair of the adducts was very efficient in the resistant cell lines whereas, in A2780 cells, plateau levels were retained until 24 h after CDDP exposure. In A2780 cells, dFdC shifted the adduct peaks from 4 h to directly after CDDP exposure and increased peak levels by >3.9-fold. dFdC also enhanced the repair of adducts by >1.7-fold and increased the Pt-GG:Pt-AG ratio compared to CDDP alone by >1.4-fold. Overall, dFdC decreased the area under the Pt-DNA adduct-time curve (AUA0-25 h) in A2780 cells by 2.7-fold. In ADDP cells, dFdC shifted the adduct peaks from 2 to 4 h and increased them by >2.2-fold. dFdC also increased the Pt-GG:Pt-AG ratio during the repair process by 1.4-fold. Overall, dFdC increased the AUA0-25 h in ADDP cells by 1.7-fold. In AG6000 cells, dFdC increased the Pt-GG:Pt-AG ratio by 1.6-fold directly after exposure but did not clearly affect the AUA0-25 h. In conclusion, dFdC can affect both Pt-DNA adduct formation and repair, depending on the initial sensitivity of the cells.
Peters
Godefridus J
GJ
Department of Medical Oncology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands. gj.peters@vumc.nl
Van Moorsel
Catharina J A
CJ
Lakerveld
Bianca
B
Smid
Kees
K
Noordhuis
Paul
P
Comijn
Elizabeth C
EC
Weaver
Dennis
D
Willey
James C
JC
Voorn
Daphne
D
Van der Vijgh
Wim J F
WJ
Pinedo
Herbert M
HM
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002945:Cisplatin / Q000494:pharmacology; D018736:DNA Adducts; D004260:DNA Repair / Q000187:drug effects*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D005260:Female; D006801:Humans; D010051:Ovarian Neoplasms / Q000473:pathology*; D014407:Tumor Cells, Cultured; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D018736:DNA Adducts; D003841:Deoxycytidine; D002945:Cisplatin; D000093542:Gemcitabine