Characterization of L5178Y Murine Lymphoblasts Resistant to Quinone Antitumor Agents¹

The exact contribution of the quinone group to the activity of quinone antitumor agents remains uncertain. Two L5178Y murine lymphoblastic cell lines resistant to the model quinone antitumor agent, hydrolyzed benzoquinone mustard, and one partial-revertant cell line were isolated and characterized. The antitumor activity of hydrolyzed benzoquinone mustard has been shown previously to be due to its ability to induce free radical mediated DNA strand breaks. Resistant cells were obtained by growing a cloned L5178Y parental cell line in media containing increasing concentrations of hydrolyzed benzoquinone mustard. L5178Y/HBM2 cells were selected from L5178Y cells growing in media containing 0.2 mm drug, while L5178Y/HBM10 cells were selected from cells growing in media containing 1.0 mm drug. The L5178Y/HBMR cells were obtained by growing L5178Y/HBM10 cells in media without hydrolyzed benzoquinone mustard. The resistant cell lines, L5178Y/HBM2 and L5178Y/HBM10, were 2.5- and 6-fold less sensitive, respectively, to hydrolyzed benzoquinone mustard compared to parental cells, and this was accompanied by a decrease in the formation of DNA single and double strand breaks by this drug. The partial-revertant cell line, L5178Y/HBMR was 2.9-fold less sensitive to hydrolyzed benzoquinone mustard compared to parental cells.

Drug uptake appeared to be lower in the resistant cells compared to parental cells. The resistant cells had a slightly elevated level of superoxide dismutase activity compared to parental cells, but there was no increase in the mRNA for superoxide dismutase nor any amplification of the gene for this enzyme. Intracellular catalase activities of the L5178Y/HBM2 and L5178Y/HBM10 cells were elevated by 1.25- and 2.6-fold, respectively, and the increased enzyme activity in the L5178Y/HBM10 cells appeared to result from a 3.6-fold increase in mRNA for this enzyme. Glutathione peroxidase activity was slightly elevated in L5178Y/HBM2 cells, but was unchanged in the other resistant cells. The L5178Y/HBM2 and L5178Y/HBM10 cells showed increased concentrations of glutathione and elevated levels of glutathione transferase activity. The resistant cell lines also had DT-diaphorase activity that was 3- and 24-fold higher in L5178Y/HBM2 and L5178Y/HBM10 cells, respectively, compared to sensitive cells. However, cytochrome P-450 reductase activity and the ratio of reduced to oxidized pyridine nucleotides was unchanged in the resistant cell lines.

The partial-revertant cell line, L5178Y/HBMR, showed approximately the same level of resistance to hydrolyzed benzoquinone mustard as the L5178Y/HBM2 cells. This lower degree of resistance compared to L5178Y/HBM10 cells was accompanied by a decrease in glutathione concentration to a level below that found in the parental cells and a decrease in DT-diaphorase activity to the level found in L5178Y/HBM2 cells. The catalase and glutathione transferase activities were only slightly decreased in the revertant cells compared to L5178Y/HBM10 cells.

These studies suggest that resistance to hydrolyzed benzoquinone mustard in L5178Y/HBM2 and L5178Y/HBM10 cells is multifactoral. Drug uptake may be decreased in the resistant cells. Elevated levels of DT-diaphorase may lead to preferential reduction of the hydrolyzed benzoquinone mustard quinone ring directly to the hydroquinone, thus decreasing the formation of the hydrolyzed benzoquinone mustard semiquinone and of oxygen free radicals and hydrogen peroxide. In addition, increased levels of glutathione, glutathione transferase activity, glutathione peroxidase, and catalase activity may also contribute to the resistance in these cells by inactivating free radicals and hydrogen peroxide generated by redox reactions of the quinone agent. These resistant cell lines should prove valuable in evaluating the role of the quinone group in the cytotoxic activity of quinone antitumor agents.