Alkylating Agent Resistance among Four Human Melanoma Cell Lines Selected for Levels π-transferaseS Cross-Resistance and Glutathione-Updated Version

A panel of four cell sublines, each selected for resistance to a different antineoplastic agent, has been developed from a human malignant mela noma cell line G3361. Following repeated exposure to escalating doses of the drug of interest, cloned sublines were developed that are 9-fold resistant to cisplatin (G3361/CP), 11-fold resistant to 4-hydroxyperoxycyclophosphamide (4-HC) (G3361/HC), 4-fold resistant to carmustine (BCNU) (G3361/BCNU), and 4-fold resistant to melphalan (G3361/ PAM). The cross-resistance of each resistant cell line was determined for cisplatin, BCNU, 4-HC, melphalan, carboplatin, nitrogen mustard, and Adriaimdn. In general, the alkylating agent-resistant cell lines were specifically resistant to the drug used for selection with the exception of the G3361/CP line, which was > 10-fold resistant to the cisplatin analogue carboplatin, 4-fold resistant to 4-HC, and slightly (1.5-fold) resistant to melphalan, and the G3361/BCNU line, which was slightly (1.8-fold) resistant to melphalan. Collateral sensitivity of the G3361/CP, G3361/ PAM, and G3361/4HC lines to killing by BCNU was also observed. Glutathione-5-transferase activity was elevated in each of the alkyl ating agent-resistant cell lines by 3to 5-fold with chlorodinitrobenzene substrate. On Western blotting, the glutathione-S-transferase-ir (GSTT) isoenzyme protein was elevated in the resistant cells by 3to 5-fold. A complementary DNA (pTS4-10) coding for GST-ir has been cloned from a Xgtll library, sequenced, and used as a probe to determine the relative levels of GST-ir mRNA in the alkylating agent-resistant cell lines. GST-ir mRNA levels were elevated (8to 15-fold) in the resistant cell lines, indicating that the GST-ir increases were mediated through an increase in mRNA levels. GST-ir elevations are a frequent event in cells selected for alkylating agent resistance, and in some cases, of multiple drug resistance. However, the lack of cross-resistance among cell lines selected for resistance to different alkylating agents, all of which have elevated GST-ir levels, indicates that increased levels of GST-ir cannot be the predominate mechanism for resistance to the tested drugs in these cell lines.