Interactions of Mitomycin C with Mammalian DMA Detected by Alkaline Elution1
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[CANCER RESEARCH 45, 3510-3516, August 1985] Interactions of Mitomycin C with Mammalian DMA Detected by Alkaline Elution1 Robert T. Dorr,2 G. Timothy Bowden, David S. Alberts, and James D. Liddil Departments ol Medicine [fÃ.T. D., D. S. A., J. D. L] and Radiology [G. T. B.J, Cancer Center Division, The University of Arizona, Tucson, Arizona 85724 ABSTRACT and by DNA renaturation studies using enhanced ethidium dye intercalation to indicate cross-linking (7). These studies used The antitumor antibiotic mitomycin C (MMC) was studied in bacterial or X-phage DNA and could not evaluate the dynamics vitro using L1210 leukemia and 8226 human myeloma cells. or specific type of DNA cross-linking produced by MMC due to Cytotoxicity was evaluated by colony formation in soft agar, and assay limitations. An alternate technique, DNA alkaline elution, DMA damage was analyzed using alkaline elution filter assays. has been useful in quantitating both the time course and dose- The purposes of these studies were: (a) to characterize the time response relationships of mammalian DNA cross-linking for a course of MMC-DNA damage; (b) to characterize the type of large number of alkylating agents. These include the bischloro- DNA damage [DNA-DNA interstrand cross-links (ISC), DNA- ethylamines, mechlorethamine (nitrogen mustard or HN2), and protein cross-links (DPC), single and double strand breaks melphalan (10), the platinum coordination compound cisplatin (SSBs, DSBs)]; and (c) to correlate this damage with cytotoxicity (11) and the chloroethylnitrosoureas (12). In addition to time and in vitro. Colony-forming assays showed the D0 value for 1 h dose studies, the alkaline elution technique can be modified to MMC to be 15.0 UM for L1210 cells and 17 nu for 8226 cells. differentiate DNA-DNA ISC, DPCs, and DNA SSBs produced by Alkaline elution studies showed that dose-dependent ISCs and a given drug treatment (13). In this report, we describe the rapid DPCs formed rapidly following MMC exposure. Removal of formation and slow elimination of DNA-DNA ISCs and DPCs cross-links was delayed, with only 50% repaired 32 h after produced by MMC in mammalian L1210 cell DNA as analyzed exposure. There was a good correlation between ISCs and by the alkaline elution technique. A brief preliminary description cytotoxicity in dose-response studies in each cell line. ISCs of these investigations has been reported previously (14). appeared to comprise most of the MMC-DNA lesions in both cell lines. No DNA SSBs or DSBs were observed following MMC MATERIALS AND METHODS exposure. Nuclei isolated from both cell lines and exposed to MMC produced less MMC alkylation than whole cells but, again, Cell Cultures. Murine L1210 leukemia cells were maintained and no strand breaks were evident. These results demonstrate that treated in suspension in RPMl 1640 medium supplemented with 10% MMC is principally an alkylating agent when used at pharmaco (vol/vol) fetal calf serum, 1% (v/v) penicillin (100 units/ml), and strepto logical (cytotoxic) concentrations In vitro. The lack of evidence mycin (100 ^g/ml) (all from Grand Island Biological Co.). The cells were for DNA strand breaks discounts a significant role for putative grown in 10% COz-90% air atmosphere at 37°C. Cells in logarithmic quinone-generated oxygen free radicals in the production of growth (doubling time of 12 h) were labeled in complete medium for 36 h by the addition of [2-14C]thymidine [0.1 /iCi/ml; 55 mCi/mmol (Research MMC cytotoxicity. Products International Corp., Mount Prospect, IL)]. Cells were "chased" in nonradioactive medium for an additional 12 h prior to drug treatments. INTRODUCTION Cells suspended in complete medium were exposed to MMC (Mutamy- cin; Bristol Laboratories, Syracuse, NY) diluted in phosphate-buffered MMC3 is a Sfreptomyces-derived antibiotic with anticancer saline (0.15 M NaCI, 0.71 mw KH2PO4, and 4.28 mw K2HPO4, pH 7.4) and maintained for 1 h at 37°C. After 1 h, drug was removed by activity in a variety of human tumors (1, 2). Early observations by Iyer and Szybalski (3, 4) documented the cross-linking of centrifugation twice at 800 x g into iced fresh complete medium. Iced cell suspensions (3 to 1 x 10^/ml) were irradiated with X-rays at 3 Gy/ complementary DNA strands as the molecular mechanism of its min using a 4-MeV linear accelerator (Linac 4; VarÃanAssociates, Palo cytotoxic action. Besides DNA alkylation, recent reports have Alto, CA). The human myeloma cell line RPMl 8226 (CCL155; American also suggested that MMC can produce DNA strand scission via Type Culture Collection, Rockville, MD) was treated similarly but with a quinone-generated oxygen free radicals (5-7). The contribution modification to increase the [14C]thymidine labeling time to 60 h to of oxygen free radical effects to MMC cytotoxicity is not estab accommodate the slower tumor doubling time of approximately 32 h. lished. Indeed, DNA cross-linking appears to correlate well with Nuclear isolation was performed according to the method of Ross er MMC cytotoxicity in terms of drug doses (8) and the onset of al. (15). Phase microscopy was used to confirm the presence of nuclei effects (9). in the final preparation. The cross-linking effects of MMC have been studied using A standard double layer soft-agar plating system was used to evaluate MMC activity against exponentially growing L-1210 and 8226 cells in DNA thermal denaturation and buoyant density techniques (3) vitro (16). MMC (Mutamycin) was added to cell suspensions for 1 h at 1 Supported in part by Grants CA 23074, CA 17094, CA 26972, and CA 17343 37°C. from the National Cancer Institute, NIH, Department of Health and Human Services, Following drug exposure, cells were washed twice by centrifugation Bethesda, MD 20205. (800 x g) and plated at 4 x 104 cells/ml (L1210) or 2 x 10s cells/ml Presented in part at the Seventy-fifth Annual Meeting of the American Associa (8226) into 35-mm Petri dishes containing 0.3% agar in complete me tion for Cancer Research, May 10,1984, Toronto, Canada (14). 2To whom requests for reprints should be addressed, at Cancer Center, Arizona dium. Triplicate plates were incubated under 10% CO2 and 90% hu Health Sciences Center, 1501 N. Campbell Avenue, Tucson, A2 85724. midified air to facilitate colony formation. Colonies of >60 pm size were 'The abbreviations used are: MMC, mitomycin C; ISC, ¡nterstrand DNA-DNA cross-links; DPC, DNA-protein cross-links; SSB, single-strand breaks; DSB, double- counted 5 to 7 days after plating (L1210) or 10 to 14 days after plating (8226) by an automated image analysis system |FAS-ll Omincon; Bausch strand breaks; SDS, sodium dodecyl sulfate. Received 12/14/84; revised 4/24/85; accepted 4/26/85. and Lomb, Rochester, NY (17)]. CANCER RESEARCH VOL. 45 AUGUST 1985 3510 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1985 American Association for Cancer Research. MMC CYTOTOXICITY AND DNA CROSS-LINKING Alkaline DNA Elution. The technical and theoretical aspects of this to-one-terminus model of Ross ef al. (23): procedure have been reviewed in detail elsewhere (13). Briefly, 1.0 x 106 L1210 or 0.5 x 106 8226 cells were exposed to drug, washed by PC=KI- r)-1-(1 (C) centrifugation in cold medium, irradiated on ice, and loaded gently into 50-ml filter stacks housing a 25-mm, 2-/¿mpore size Polyvinylchloride where Po is the frequency of DNA single strand breaks produced by 30 Gy and r and r0 are the fractions of DNA eluting in the slow elution phase filter (Type BS; Millipore Corp., San Francisco, CA). The cells were washed with ice-cold phosphate-buffered saline and lysed immediately in the presence or absence of drug, respectively. The degree to which r exceeds r0 is a measure of DPCs. DNA SSB and DSBs induced by MMC on the filters with 5 ml of pH 10.0 detergent solution in the presence or were also quantitated as Gy-equivalents, defined as the dose of radiation absence of proteinase k (0.5 mg/ml). Assays performed to detect total DNA cross-linking used a lysis solution of 2 M NaCI, 0.04 M disodium which produces an equivalent number of DNA strand breaks in control EDTA, 0.2% AMauroylsarcosine, and a pH 12.1 elution solution of tetra- experiments with radiation alone (17). propylammonium hydroxide (Eastman Organic Chemicals, Rochester, NY). Assays performed to detect DNA-DNA ISC were performed with a RESULTS pH 10.0 lysis solution of proteinase k (0.5 mg/ml) (E. Merck Co., Darmstadt, Germany; Type XI fungal from Tritirachium alba 10-20-m The survival of L1210 cells following a 1-h exposure to various Anson units/mg of protein) 2% SDS, 0.1 M glycine, 0.02 M disodium doses of MMC was determined by cloning in soft agar (Chart 1). EDTA, and a pH 12.1 elution solution of 1% SDS/0.02 M EDTA/tetrapro- The quasithreshold dose (DQ) for MMC against exponentially plyammonium hydroxide. For DPC assays, the AMauroylsarcosine lysis and non-SDS-containing elution solutions were used. In this latter assay, growing L1210 cells was approximately 5.0 ¿/M/h.The mean lethal dose (D0) was approximately 15 ^M) (5 ¿<g/ml).DNAcross- cells were irradiated to a total of 30 Gy to produce very short DNA strands which are retained on the PVC filters only if the DNA strand is linking in L1210 cells exposed to MMC was assessed by 3 covalently linked to a large protein (18). To assess whether DNA SSBs alkaline elution techniques.