Oldenburg J, Begg A C, van Vugt M J, Ruevekamp M, Schornagel J H, Pinedo H M, Los G
Division of Experimental Therapy, The Netherlands Cancer Institute, Amsterdam.
Cancer Res. 1994 Jan 15;54(2):487-93.
Cisplatin resistance was developed in sublines of the CC531 rat colon adenocarcinoma cell line by continued low level drug exposure. Two relatively stable lines were obtained (RL2 and RL4) which were 6- and 20-fold more resistant to cisplatin. In addition, a subline more sensitive than the parent line by a factor of 2 (RLS) was obtained by subculture from a treated tumor. Mechanisms of resistance to cisplatin were investigated in these four lines, with the aim of determining the relative contributions of different resistance mechanisms at various resistance levels. Drug accumulation linearly decreased with increasing drug resistance. A 20-fold resistance was associated with only a 5-fold decrease in accumulation, suggesting that other resistance mechanisms may be involved in the total degree of resistance. Intracellular glutathione, measured fluorometrically, also increased with increasing resistance, varying by a factor of 4 between the most and least resistant lines. Reduction of glutathione levels by buthionine sulfoximine to parent line levels increased sensitivity but the cells remained considerably more resistant than parent cells. Resistant lines cultured in the absence of drug became progressively more sensitive, without accompanying changes in total glutathione levels. DNA-drug adducts, the presumed toxic lesion, were measured immunocytochemically. Initial levels decreased with increasing platinum resistance, although not proportional to resistance (factor of 5 decrease for 20-fold resistance). Drug dose ratios for equal initial adducts were similar to dose ratios for equal drug accumulation, implying that intracellular concentrations solely determine DNA adduction and that differences in glutathione level had little influence on the proportion of drug which eventually formed adducts. After 48 h, a better correlation between remaining adducts and resistance was found (factor 12 less adducts for 20-fold resistance). This implies that repair of adducts was important in determining survival. These data indicate that decreased drug accumulation played a proportionally greater role in the moderately resistant cell line and that adduct repair played a progressively greater role in the highly resistant cell line.
