Investigation of resistance to DNA cross-linking agents in 9L cell lines with different sensitivities to chloroethylnitrosoureas

The 9L-2, 9L-7, and 9L-8 cell lines, derived from the 9L in vivo rat brain tumor, were treated with nitrosoureas that can alkylate and cross-link DNA and carbamoylate intracellular molecules to various extents. Compared to 9L cells, 9L-2 cells were very resistant to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, and to 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-deoxyglucopyranose. The sensitivity of 9L-7 and 9L-8 cell lines to these drugs was intermediate between 9L and 9L-2. Treatment of 9L, 9L-2, 9L-7, and 9L-8 cell lines with 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea produced approximately the same level of cell kill. Compared to 9L cells, 9L-2 cells are 10-fold more resistant to the cytotoxic effects, 34-fold more resistant to the induction of sister chromatid exchanges, and have 40\% fewer DNA interstrand cross-links caused by treatment with 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea . In contrast, treatment of 9L and 9L-2 cells with 1-ethylnitrosourea produced approximately the same level of cell kill and induction of sister chromatid exchanges. Our results suggest that the resistance of 9L-2, 9L-7, and 9L-8 cells is related to DNA cross-linking and not to alkylation or carbamoylation. We studied the effects of other agents that form DNA cross-links with structures different from those formed by treatment with chloroethylnitrosoureas (CENUs) in 9L and 9L-2 cells. In contrast to results obtained with CENUs, 9L-2 cells were 2-fold more sensitive to the cytotoxic effects, 2-fold more sensitive to the induction of sister chromatid exchanges, and had 3-fold more cross-links formed than 9L cells treated with nitrogen mustard. However, the amount of cell kill, number of sister chromatid exchanges induced, and the DNA cross-linking were the same for 9L and 9L-2 cells treated with cis-diamminedichlorplatinum(II). Our results indicate that cellular resistance to CENUs is highly specific and that the mechanism of resistance does not allow cross-resistance with other DNA cross-linking agents. These and other results suggest that when DNA repair processes mediate cellular resistance to CENUs, other cross-linking agents will not be cross-resistant unless they form alkylation products that are affected by repair processes that mediate resistance to CENUs

Prediction of the relative in vitro sensitivity of 9L rat brain tumor cells to nitrosoureas by the sister chromatid exchange assay

In an earlier study we showed that there is a good correlation between sister chromatid exchange induction and cell kill in 9L cells treated with certain nitrosoureas. In the study reported here, we treated four 9L cell lines that have different sensitivities to chloroethylnitrosoureas with 1,3-bis (2-chloroethyl)-1-nitrosourea, chlorozotocin, and ethylnitrosourea and determined the number of sister chromatid exchanges induced. Cell lines that were most sensitive to the drugs with respect to cell kill were also most sensitive to induction of sister chromatid exchanges for a given drug, and the assay based on sister chromatid exchange is therefore predictive of the relative sensitivity of these cells to the drugs used