CHARACTERIZATION OF RESISTANCE MECHANISMS TO CIS-DIAMINEDICHLOROPLATINUM(II) IN 3 SUBLINES OF THE CC531 COLON ADENOCARCINOMA CELL-LINE IN-VITRO

Cisplatin resistance was developed in sublines of the CC531 rat colon adenocarcinoma cell line by continued low level drug exposure. Two rel atively stable lines were obtained (RL2 and RL4) which were 6- and 20- fold more resistant to cisplatin. In addition, a subline more sensitiv e than the parent line by a factor of 2 (RLS) was obtained by subcultu re from a treated tumor. Mechanisms of resistance to cisplatin were in vestigated in these four lines, with the aim of determining the relati ve contributions of different resistance mechanisms at various resista nce levels. Drug accumulation linearly decreased with increasing drug resistance. A 20-fold resistance was associated with only a 5-fold dec rease in accumulation, suggesting that other resistance mechanisms may be involved in the total degree of resistance. Intracellular glutathi one, measured fluorometrically, also increased with increasing resista nce, varying by a factor of 4 between the most and least resistant lin es. Reduction of glutathione levels by buthionine sulfoximine to paren t line levels increased sensitivity but the cells remained considerabl y more resistant than parent cells. Resistant lines cultured in the ab sence of drug became progressively more sensitive, without accompanyin g changes in total glutathione levels. DNA-drug adducts, the presumed toxic lesion, were measured immunocytochemically. Initial levels decre ased with increasing platinum resistance, although not proportional to resistance (factor of 5 decrease for 20-fold resistance). Drug dose r atios for equal initial adducts were similar to dose ratios for equal drug accumulation, implying that intracellular concentrations solely d etermine DNA adduction and that differences in glutathione level had l ittle influence on the proportion of drug which eventually formed addu cts. After 48 h, a better correlation between remaining adducts and re sistance was found (factor 12 less adducts for 20-fold resistance). Th is implies that repair of adducts was important in determining surviva l. These data indicate that decreased drug accumulation played a propo rtionally greater role in the moderately resistant cell line and that adduct repair played a progressively greater role in the highly resist ant cell line.