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The formation and repair of cisplatin-DNA adducts in wild-type and cisplatin-resistant L1210 cells : comparison of immunocytochemical determination with detection in isolated DNA

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Author: Blommaert, F.A. · Floot, B.G.J. · Dijk-Knijnenburg, H.C.M. van · Berends, F. · Baan, R.A. · Schornagel, J.H. · Engelse, L. den · Fichtinger-Schepman, A.M.J.
Type:article
Date:1998
Institution: Centraal Instituut voor Voedingsonderzoek TNO
Source:Chemico-Biological Interactions, 108, 209-225
Identifier: 40055
doi: DOI:10.1016/S0009-2797(97)00108-7
Keywords: Nutrition · Cisplatin · DNA adducts · Drug resistance · Enzyme-linked immunosorbent assay · Immunocytochemistry · antiserum · cisplatin · dna · animal cell · article · colony formation · dna adduct · dna content · dna damage · dna synthesis · drug dna binding · drug resistance · drug sensitivity · enzyme linked immunosorbent assay · growth inhibition · immunocytochemistry · mouse · nonhuman · Animals · Antineoplastic Agents · Bicarbonates · Cell Division · Cisplatin · DNA Adducts · DNA Repair · DNA, Neoplasm · Drug Resistance, Neoplasm · Enzyme-Linked Immunosorbent Assay · Immunohistochemistry · Leukemia L1210 · Mice · Sensitivity and Specificity · Thiourea · Animalia · Rodentia

Abstract

We have studied the formation and repair of cisplatin-DNA adducts in wild-type mouse leukemia L1210/0 cells and in the sublines L1210/2 and L1210/5, which differ in cisplatin sensitivity. In a colony-formation assay these sublines were 9- and 22-fold more resistant compared to L1210/0, respectively. Cisplatin-induced DNA modification was studied at the cellular level by immunocytochemistry with antiserum NKI-A59 raised against cisplatintreated DNA. Levels of nuclear staining immediately after a 1-h treatment were similar to those seen after a 24-h post-incubation in drug- free medium. Clear differences in DNA platination were found between the cell lines: immediately after exposure, L1210/2 and L1210/5 showed only 32 and 14%, respectively, of the nuclear staining observed in L1210/0, and 48 and 13% after 24 h. In these experiments, adduct-specific nuclear staining was quantified as the area under the adduct versus concentration curves (AUC). The formation and repair in these cell lines of the bifunctional adducts cis- Pt(NH3)2d(pGpP)2 (Pt-GG), cis-Pt(NH3)2d(pApG) (Pt-AG) and cis- Pt(NH3)2(dGMP)2 (G-Pt-G) were studied with an enzyme-linked immunosorbent assay (ELISA). No relation between repair and resistance was observed. The results suggest that differences in induced DNA platination levels, rather than in repair, are responsible - at least in part - for the differences in cisplatin resistance. A mechanism such as an increased tolerance of the resistant cells to platinum-DNA damage may also be involved.