thiourea and ammonium-bicarbonate

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 cisplatin-treated 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(pGpG) (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 plantinum-DNA damage may also be involved.

Topics: 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

Thiourea and NH4HCO3 are widely used to block the conversion of Pt-DNA monoadducts to diadducts prior to the enzymatic digestion of DNA and subsequent analysis of the relative proportion of the different types of Pt-DNA adducts. Our data show that NH4HCO3 (100 mM, 18 h, 25 degrees C) is much less effective than thiourea (10 mM, 10 min, 25 degrees C) at blocking monoadducts, apparently because considerable monoadduct-to-diadduct conversion occurs during the incubation of platinated DNA with NH4HCO3. Under these incubation conditions, neither NH4HCO3 nor thiourea treatment causes significant diadduct-to-monoadduct conversion. At 25 degrees C, thiourea causes no significant removal of either ethylenediamine(en)- or diaminocyclohexane(dach)-Pt monoadducts. However, at 37 degrees C, both en-Pt and dach-Pt monoadducts are selectively removed. Pt-DNA diadducts are stable to 10 mM thiourea at either temperature. These data suggest that previous experiments using NH4HCO3-blocked DNA are likely to have underestimated Pt-DNA monoadducts and to have overestimated diadducts. As a consequence, such studies are likely to produce inaccurate estimates for the repair of individual adducts. The data also show that although thiourea treatment is suitable for blocking Pt-DNA monoadducts under the conditions generally used (10 mM, 10 min, 25 degrees C), it can selectively remove Pt-DNA monoadducts at higher temperatures.

Topics: Bicarbonates; Chromatography, High Pressure Liquid; Cisplatin; DNA; DNA Adducts; Thiourea