DNA cross-linking as an indicator of sensitivity and resistance of mouse L1210 leukemia to cis-diamminedichloroplatinum(II) and L-phenylalanine mustard.

The relationship between DNA cross-linking and cell killing by c;'s-diamminedichloroplatinum(ll) (c/s-DDP) and L-phenylalanine mustard (L-PAM) was studied in L1210 cell culture lines and in mice bearing sensitive and resistant lines of L1210 leukemia. A line of L1210 mouse leukemia cells was developed which is resistant to c/s-DDP in vitro. These cells, designated ZCR9, are cross-resistant to L-PAM. The effect of both drugs on the ZCR9 cells, compared to the parent L1210 K25 cells, was examined by DNA alkaline elution with and without the use of proteinase. The resistant line was similar to the normal line with regard to the kinetics of DNA cross-link formation and removal following treatment with c/s-DDP or L-PAM. For both drugs, maximum cross-linking occurred after 6 hr; this is pre sumed to represent the time required for conversion of drugDNA monoadducts to cross-links. In the resistant line, interstrand cross-linking by c/s-DDP or L-PAM and DNA-protein cross-linking by c/s-DDP were all reduced relative to the parent line. The interstrand cross-linking was reduced in approxi mately the same proportion as the cytotoxicity (in terms of dose modification factors). DNA-protein cross-linking by L-PAM, however, was similar in the two cell lines. The relationship between DNA cross-linking and cell killing by c/s-DDP and LPAM was also studied in mice bearing sensitive and resistant lines of L1210 leukemia. The cells were removed from un treated mice and tested in vitro for DNA cross-linking produced by the two drugs. Tumor sensitivity was assessed by comparing the survival of treated versus untreated mice which had been inoculated with the same cells used in cross-linking assays. A L1210 line which had been developed for resistance to c/sDDP exhibited marked reductions in both types of cross-linking by this drug when compared to its sensitive parent line. This line was not resistant to L-PAM and exhibited no significant depression in cross-linking by this drug. A second line, made resistant to L-PAM, showed marked reductions in L-PAM-induced cross-linking compared to its parent line. This line was cross-resistant to c/s-DDP but showed only a modest reduction in c/'s-DDP-induced cross-linking. Thus, in three of the four cell-drug comparisons, DNA cross-linking and in vivo cell killing were well correlated. The reason for the deviation of the fourth case was investigated in preliminary studies, but no definitive answer was obtained. The results suggest that DNA crosslinking correlates with tumor sensitivity to bifunctional agents. INTRODUCTION c/s-DDP,2 like the bifunctional nitrogen mustards, is capable of undergoing bifunctional addition reactions with DNA, pro ducing interstrand and intrastrand cross-links, as well as DNAprotein cross-links (6, 10, 11, 15-18). It is likely that one or more of these classes of bifunctional DNA lesions is responsible for the cytotoxicity and antitumor activity of these drugs. Previous work in our laboratory has utilized the alkaline elution technique to study DNA interstrand and DNA-protein cross-linking in mammalian cells (5, 9). DNA cross-linking, especially of the interstrand type, correlates with cytotoxicity in several different cell types (3, 15-17). The kinetics of cross link formation and removal following c/s-DDP treatment was found to be generally similar to that following treatment with LPAM (melphalan 15-17).3 After treatment with either drug, cross-linking increases for a few hr and then slowly decreases. These 2 chemically different types of agents therefore lend themselves to comparative studies aimed at elucidating the roles of various types of cross-links in producing the biological effects. In the current work, we have derived a resistant line of L1210 cells in culture and compared it with the parent line. The results suggested the possibility that tumor sensitivity to DNA crosslinking agents can be predicted by in vitro cross-linking meas urements. This hypothesis was then tested in 2 sensitive/ resistant pairs of L1210 tumor lines in mice. MATERIALS AND METHODS A cloned line (designated K25) of L1210 mouse leukemia cells was grown in RPMI Tissue Culture Medium 1630 (con taining 0.03% glutamine, freshly added) plus 20% heated fetal calf serum as described previously (18). A c/'s-DDP-resistant line (designated ZCR9) was derived as follows. L1210 K25 cells were treated with 10~" M methylnitrosourea for 1 hr. After recovery of exponential growth, the cells were treated with c/sDDP, and survivors were cloned in soft agar. A clone was selected, treated again with c/s-DDP, and recloned in soft agar. An additional methylnitrosourea treatment and recovery was followed by 5 cycles of c/s-DDP treatment and cloning. The colony survival levels following each treatment were 2.15 x 10~5 to 4.5 x 10~2. Ampuls of the resulting resistant line (ZCR9) were stored in liquid nitrogen. The cells were grown for 1To whom requests for reprints should be addressed, at Building 37, Room 5D17. NIH, Bethesda, Md. 20205. Received February 5, 1980; accepted November 3, 1980. 2 The abbreviations used are: c/s-DDP, c/s-diamminedichloroplatinum(ll); LPAM, L-phenylalanine mustard; RPMI, Roswell Park Memorial Institute. 3 W. E. Ross, L. A. Zwelling, and K. W. Kohn, unpublished observations. 640 CANCER RESEARCH VOL. 41 American Association for Cancer Research Copyright © 1981 on February 21, 2013 cancerres.aacrjournals.org Downloaded from DNA Cross-Linking and Tumor Sensitivity no more than 6 weeks before going back to a fresh ampul. During this time, resistance was maintained.