Investigation of Resistance to DMA Cross-Linking Agents in 9L Cell Lines with Different Sensitivities to Chloroethylnitrosoureas1

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(frans-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-amino2-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 crosslinking 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 c/s-diamminedichloroplatinum(ll). 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. 1Supported in part by NIH Grants CA-13525 and CA-31882 and the Phi Beta Psi Sorority. 2To whom requests for reprints should be addressed, at the Brain Tumor Research Center, 783 HSW, University of California, San Francisco, CA 94143. "Recipient of Teacher Investigator Award 1K-07NS 00604 NSPA from the National Institute of Neurological Communicative Disorders and Stroke. Received 12/14/84; revised 4/23/85; accepted 5/7/85. INTRODUCTION Certain CENUs4 are hydrolyzed intracellularly to reactive inter mediates that alkylate DNA, form DNA interstrand cross-links, and carbamoylate cellular proteins. Because all of these reac tions may cause cellular cytotoxicity, but with different efficien cies, it is necessary to establish which of these events is of primary importance to cell killing in sensitive cells and which processes are modified by cells resistant to the cytotoxic effects of these agents. Therefore, in this study we treated 9L cells and lines derived from the in vivo 9L rat brain tumor by BCNU treatment {1) with CENUs and other nitrosoureas with different capacities to alkylate DNA, cross-link DNA, and carbamoylate intracellular molecules (see Table 1). We measured cell kill, SCE induction, and DNA cross-link formation in order to obtain a better understanding of the molecular mechanisms of cell killing and cellular resistance to CENUs. Preliminary investigations have shown that 9L-2 cells are resistant to the cytotoxic effects of BCNU and to the induction of SCEs compared to 9L-cells treated with the same concentra tion of BCNU (2). However, from these studies we could not determine if the observed cellular resistance was specific for CENUs, or whether 9L-2 cells were also resistant to other DNAcross-linking agents. Schabel ef al. (3, 4) have performed exten sive studies on resistance and patterns of cross-resistance in L1210 and P388 cell lines. Schabel's results showed that L1210 and P388 cells resistant to BCNU were also resistant to other CENUs but retained the sensitivity of the parent line to phenylalanine mustard, cyclophosphamide, and c/s-Pt (3, 4). Erickson ef al. (5) have shown that human tumor cells resistant to BCNU are not cross-resistant to c/s-Pt. Tew and Wang (6) have reported that Walker 256 rat carcinoma cells highly resistant to HN2 and related derivatives are not cross-resistant with CENUs. These results suggest that cellular resistance to DNA cross-linking agents is highly specific. The experiments reported here were performed to determine whether 9L-2 cells resistant to BCNU are cross-resistant to other DNA cross-linking agents. MATERIALS AND METHODS Cell Lines. The 9L rat gliosarcoma is a well-established cell line (7). Sublines that are resistant to BCNU were produced by the following procedure. Fisher 344 rats bearing the intracerebral 9L tumor were treated with a single dose of BCNU, either 13.3 or 26.7 mg/kg. Twentyfour h after the dose was administered, rats were sacrificed, and tumors 4The abbreviations used are: CENU, chloroethylnitrosourea; BCNU, 1,3-bis(2chloroethylH -nitrosourea; ENU, W-ethylnitrosourea; CHLZ, 2-[3-<2-chloroethyl)-3nitrosoureido]-D-glucopyranose; BHCNU, 1,3-bis(frans-4-hydroxycyclohexyl)-1-ni trosourea; ACNU, 3-{4-amino-2-methyl-5-pyrimidinyl)methyl-1 -(2-chloroethyl)-nitrosourea; HN2, nitrogen mustard; c/s-Pt, c/s-diamminedichloroplatinum(ll); CFE, col ony-forming efficiency; SCE, sister chromatid exchange. CANCER RESEARCH VOL. 45 AUGUST 1985