Interactions of Mitomycin C with Mammalian DMA Detected by Alkaline Elution1

[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 37Â°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 37Â°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 37Â°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, Â¡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)].

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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-/Â¿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 Â¿/M/h.The mean lethal dose (D0) was approximately 15 ^M) (5 Â¿

Relative elution = log r0 - log r (A)

where ra and r are the relative retentions of DNA on the filter after 12 h of elution in the absence and presence of X-rays, respectively. At a constant level of X-rays, the ratio of the relative elution with X-rays only 0.1 to the relative elution with X-rays and drug treatment will represent the (Ã¬) 10 (5) - (io) <ÃŒ5) factor by which X-ray sensitivity has been decreased by the drug. This MITOMYCIN C CONCENTRATION 1 HR. uM (uo/ml) is taken to represent the cross-link factor and has been shown to be Chart 1. Dose-survival effects of exponentially growing L1210 murine leukemia independent of the X-ray dose used to elicit the effect (22): cells (â€¢)or 8226 cells (O) exposed to MMC. All drug exposures were for 1 h at 37Â°C. Vertical bars indicate one standard error for 3 separate experiments. The Relative elution (X-rays only) quasithreshold dose, 0, (equal to the intercept of the exponential portion of the Cross-link factor = (B) Relative elution (X-rays plus drug treatment) survival curve with the abscissa) was approximately 5.0 *IM for L1210 cells and 12.5 /IM for 8226 cells. The mean lethal dose, D0 (equal to the concentration required to reduce the survival colony-forming units by 63% on the exponential The DPCs lesions were converted to Gy-equivalents using the bound- part of the survival curve) was 15 ^ M for L1210 cells and 17 M for 8226 cells.

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Table 1 apparent that MMC cross-links form rapidly and are slowly Mitomycin C-induced DNA cross-links removed over a 32-h observation period. There was no signifi DNA cant reduction in cross-linking until 17 h after MMC exposure. MMC expo cross-link fac cross-link fac (Gy- tor'1.14 To determine the presence of DNA-DNA ISCs following MMC, sure(jjM/h)1.0 (Grays)6.0 torÂ«1.64 equivalents)0.76 a group of elutions were performed using a 1-h drug treatment 30.0 and subsequent cell lysis with proteinase K. This procedure 5.010.0 6.0 4.32 1.65 removes most of the DNA-associated proteins and DPCs; thus, 30.0 4.27 any subsequent [14C]DNA retention on the filters is assumed to 6.0 5.71 2.19 30.0 5.26 represent DNA-DNA interstrand cross-linking (13). 20.0X-rays 6.0 8.96DNA-DNA 2.95DPC Chart 3 shows the dose-response data for these assays. It 30.0Total 12.10 " Cross-link factors were determined using Relative Elution Equations A and B was again apparent that DNA-DNA interstrand cross-linking be described in "Materials and Methods." Results represent the mean of triplicate comes significant only after a MMC exposure of 15.0 ftw/h (5.0 (total cross-links)or duplicate experiments (ISC and DPC assays). " DNA-proteincross-links were quantitated as Gy-equivalentsusing the bound- /Â¿g/ml/h)and that a dose-response relationship exists up to 60 to-one-terminusmodelof Ross et al. (23).This is detailedin EquationC in "Materials tiÂ»(or 20 /ig/ml/h). However, at equal MMC exposures, DNA- and Methods." DNA cross-link factors were about one-half of those obtained in the total cross-link assays (Table 1). Chart 4 shows the dose- response relationship for DNA-protein cross-links after MMC concentrations greater than 15 M are achieved (Table 1, Column 5). The Diphasic elution evident in Chart 4 is typical for the DPC type of assay in which the latter slow phase is assumed to be

Oio 3.5 12.0 170 24O 320 TIME AFTER MITOMYCIN EXPOSURE (HOURS) Chart 2. Kinetics of formation and disappearance of total DNA cross-linking assayed by alkalineelution in L1210 cells exposed to 15 ^M MMC at 37Â°Cfor1 h. Cells were washed free of drug and postincubated in complete medium at 37Â°C for up to 32 h prior to alkaline DNA elution performed without proteinase K. In these assays, DNA cross-linking includes both ISCs and DPCs. DNA cross-link factors were calculated using the percentage of [â„¢C]DNAremainingon the filter after 12 h of elution as described in "Materials and Methods." Light protection of cells and DNA was maintained throughout the experimental runs. Values depicted are the mean of 2 experiments.

15 UM and up to 60 /IM. significant total DNA cross-linking was produced (proportional to the concentration of drug used). Additional experiments with this total cross-linking technique were aimed at determining the time course of cross-link formation and removal following a MMC exposure. The loss of cross-links QI over time is believed to represent enzymatic repair of drug- 8 IO I2 I4 I6 IS 20 22 24 induced DNA damage (10). For these assays, L1210 cells were HOURS OF ELUTION washed free of MMC after a 1-h 15 Â¿Â¿Mexposureand were Chart 3. Effects of MMC and radiation on DNA-DNA ISCs in L1210 cells. Cells were exposed to MMC 3-60 ^M for 1 h at 37Â°C,Â¡radiatedon ice with 6.0 Gy X- allowed to incubate in fresh medium for varying times prior to rays, and lysed with a proteinase K-containing solution. Alkaline elution was then irradiation, lysis, and alkaline DNA elution. Six post-MMC time performed over 17 h using the 1% SDS pH 12.1 solution. The retention of [14C]DNAon the filters is seen to increase proportionally to the concentration of periods were evaluated: 1.0, 3.5, 12, 17, 24, and 32 h. Chart 2 MMC, indicating that dose-dependent ISCs are produced following a 1-h drug demonstrates the dynamics of cross-linking formation and re exposure. MMC (15 MM)alone(without X-rays)did not increasethe elution of DNA moval over time following MMC exposure. It is immediately under alkalineconditions.

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amount of DNA from the 8226 control cells (about 16%) was not

-O CONTROL NO X-RAYS retained in the absence of cell irradiation. MMC 19/M In the 8226 cell line, MMC cross-linked DNA in a manner similar to that with the L1210 cells. There was again no evidence of MMC-induced SSBs with intact human 8226 cells exposed up to 60 fiM of MMC (data not shown). Thus, in both human and murine tumor cell lines, unirradiated cellular DNA did not elute more rapidly when exposed to MMC. Double Strand Breaks. Recent experiments with functionally similar benzoquinone mustards have shown that quinone-generated DNA SSBs can be masked by alkylating effects in DNA filter assays (25). However, DSBs were still apparent when the DNA-filter assays used a neutral elution technique. Because of the possibility that MMC-induced DNA alkylation similarly masks JOSy ' MMC SSBs, a series of MMC-DSB assays were performed with L1210 and 8226 cells. Chart 5 shows the negative results of these assays. MMC at 1-h exposures up to 300 ^M (20 times the D0 value for cytotoxicity) did not produce enhanced DNA elution. The sensitivity of DSB detection in these assays was slightly 30 Syt MMC 30

Chart 4. Effects of MMC on DPCs in L1210 cells exposed to MMC for 1 h and obtained with 8226 human myeloma cell nuclei. Control DNA postirradiated on ice with 30 Gy of X-rays. The cells were lysed with an N- was retained to 70 to 90% for assays with and without proteinase lauroylsarcosine solution, pH 10.0, and DNA was eluted over a 17-h period using K in the lysis solution, respectively. Chart 6 shows the results of a pH 12.1 tetrapropylammonium hydroxide solution. The Diphasic elution pattern observed is typical for DPCs detected using this assay technique. A MMC dose- these assays which demonstrated significant DNA cross-linking response pattern is evident for DPCs in this cell line. by MMC in nuclei. However, there appeared to a quantitatively less MMC cross-linking in nuclei compared to whole cells (Charts dependent on DNA-protein adducts. 2 and 3). In the absence of post-MMC exposure radiation, 8226 None of the 3 cross-linking assays demonstrated rapid DNA elution in intact L1210 cells exposed to MMC in the absence of X-irradiation. Thus, there was no evidence of direct MMC-induced SSBs in these cross-linking assays using MMC exposures >5 /Â¿M/h.Separate experiments to directly evaluate MMC SSBs were performed using 1 h MMC exposure up to 30 /Â¿M.Inthis assay, cells are not irradiated following MMC exposure, and a proteinase Â«-containing lysis solution is used to remove any DPCs. In intact L1210 cells, there was no evidence of DNA SSBs at any MMC exposure up to 30 MM/", a dose lethal to about 95% of colony-forming L1210 cells (Chart 1). Effects on Human Myeloma Cells. In order to confirm these results in human cells, the multiple myeloma cell line 8226 (24) was evaluated similarly for cytotoxicity and for ISCs and SSBs following MMC. For these cells, a dose-response relationship was initially established for MMC cytotoxicity using the soft-agar colony-forming assay. The survival of colony-forming units was Â°'o' i ' Ã• similar to that with L-1210 cells (Chart 1). These 2 cell lines were HOURS OF ELUTION also compared on the basis of the effect of X-irradiation on total Charts. Effects of MMC on DSBs from L1210 cells (A) or 8226 cells (fl). The elutions are performed at pH 9.6 following a 1-h proteinase K lysis. Control cells DNA retention on PVC filters. The L1210 cells were slightly more on ice were exposed to X-ray doses from 1 to 10 krads (x), prior to elution. MMC- responsive to X-rays than were the 8226 cells. However, a larger treated cells were not irradiated prior to elution (30 Â«Â¿M,â€¢;300 AIM,A).

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was similar to that seen after melphalan, another alkylating agent which can produce profound and delayed myelotoxocity in vivo (28). This suggests that alkaline DNA elution assays might be useful as an in vitro preclinical tool to detect compounds which will produce more severe hematological toxicities in clinical trials. Cross-linking studies using proteinase K treatments demon strated that roughly one-half of the total MMC-induced DNA adducts were due to DNA-DNA interstrand cross-links. This confirms in a mammalian system the earlier findings of Iyer and Szybalski (3) and Lown (7), who have described DNA cross- linking in bacterial DNAs examined with heat-denaturation, su crose sedimentation, or ethidium dye fluorescent-enhancement techniques. The hypothesis that MMC produces multiple DNA adducts was also tested using DPC assays. MMC did cause dose- dependent DPCs which became significant only after MMC ex posures 15 M/h. It should be recalled, however, that some DPCs can be formed by large doses of ionizing radiation alone (29,30). In addition to MMC, a number of other bifunctional alkylating agents also produce DPCs. These include mechlorethamine (31), cyclophosphamide (32), cisplatin (11), and the nitrosoureas (33). In addition to DNA cross-linking, other in vitro studies have

4 6 8 10 suggested that MMC may cause strand breaks following the HOURS OF ELUTION generation of oxygen free radicals from cyclic reduction of the Chart 6. Effects of MMC on DNA-DNA ISC in nuclei isolated from 8226 human quinone moiety (1, 5, 7). In the current study, DNA elutions myeloma cells. Alkaline elution was performed over 17 h without (open symbols) performed without postirradiation demonstrated no apparent and with (closed symbols) proteinase K. MMC 15 Â»IMincreased DMA retention on MMC-induced SSB. This result was the same in both cell lines the filter in postirradiated nuclei (O). In the absence of radiation, neither 15 nu (â€¢) nor 30 >Â¡M(A) MMC increased DNA elution in these assays performed with and in isolated nuclei. This suggests that MMC is primarily an proteinase K to detect drug-induced SSBs alkylating agent if drug concentrations are used within the phar macological range. In addition, a previous study from our labo DNA was highly retained on the filters, indicating the presence ratory showed that a MMC-sensitive human breast cancer cell of MMC-induced cross-links in human nuclei. There was no line, MCF-7 adenocarcinoma, was not protected from MMC evidence of MMC-augmented rapid DNA elution (strand break cytotoxicity by coincubation with 10 mw concentrations of the age) in nuclei from this cell line, even following high MMC free radical scavengers dimethyl sulfoxide and mannitol (34). exposures of up to 30 jiM/h. Lown (7) described MMC-induced single strand nicks in covalently closed circular supercoiled PM2 DNA. The addition of biological free radical scavengers, catatase and Superoxide dis- DISCUSSION mutase, reduced MMC-induced strand breaks in this system. These DNA alkaline elution experiments demonstrate that Tomasz ef al. (35) observed that H2O2 production was involved MMC rapidly cross-links mammalian DNA in a dose-dependent during the redox cycling of both free MMC and drug which was fashion. There is a good correlation between the dose of MMC covalently bound to DNA. Doroshow (6) also demonstrated that required for cytotoxicity and that producing significant DNA MMC may enhance Superoxide and hydrogen peroxide formation cross-linking. The threshold for both effects in murine and human in rat sarcosomal preparations. In this report, neither dimethyl cells appears to be in the range of 3 UM (1.0 g/ml/h). The D0 sulfoxide nor catatase decreased Superoxide formation, mea value of 5.0 /Â¿g/mlfor L1210 cell cytotoxicity in the current study sured by the reduction of acetylated cytochrome C. In these in compares to a D0 value of 1.0 /tg/ml in cultured LoVo human vitro systems, a variety of MMC concentrations were used. Some colon cancer cells (26). Both cytotoxicity and DNA cross-linking concentrations far exceeded the pharmacologically attainable increased proportionately with increasing MMC exposures up to peak MMC level in humans, which is in the range of 5.0 UM (36). 60 pM. This level of DNA cross-linking by MMC was similar to This may explain the difference between these prior studies and that reported by Fornace and Little (22). Time-course cross- the current results in which MMC exposure was limited to those linking studies with L1210 cells showed that MMC-induced DNA which were cytotoxic in the cell lines studied. cross-links are not removed for up to 24 h following drug Early studies of the mechanism of MMC cytotoxicity noted exposure and that only about 50% removal occurs 32 h after that DNA cross-linking did not readily occur with a purified DNA drug exposure. This indicates that MMC-induced DNA lesions system unless the drug was exposed to a cell lysate (4). In are rather stable, and this may explain some of the profound addition to cell lysates, microsomal enzymes, and NADPH (17, toxicity of this drug toward high DNA turnover in normal tissues 37), sodium hydrosulfite or borohydride, acidic pH (38), Clostrid- such as the bone marrow (27). Ross ef al. (10) have suggested ium diaphorase, xanthine oxidase (39), and electrochemical re that the persistence of DNA lesions after exposure to alkylating duction (40) can produce MMC alkylating activity. The current drugs may be a prime determinant of ultimate cytotoxicity. In the results with isolated nuclei demonstrate that sufficient MMC current study, the delayed removal or repair of MMC cross-links reduction can occur without cytosol to produce significant alkyl-

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1985 American Association for Cancer Research. MMC CYTOTOXICITY AND DNA CROSS-LINKING ation of mammalian DNA. REFERENCES Molecular mechanisms of MMC alkylation appear to involve 1. Crooke, S. T. and Bradner, W. T. Mitomycin C: a review. Cancer Treat. Rev., an initial noncovalent, possibly intercalative association with DNA 3:121-139,1976. (41). In addition, the N7-position of guanine is apparently not 2. Godfrey, T. E. and Wilbur, D. W. Clinicalexperience with mitomycin C in large alkylated by MMC (41, 42). The Opposition of guanine was infrequent doses. Cancer (Phila.),29:1647-1652,1972. 3. Iyer, V. N. and Szybalski, W. A molecular mechanism of mitomycin action: originally favored by Iyer and Szybalski (3), and more recent linking of complementary DNA strands. Proc. Nati. Acad. Sci. USA, 50; 355- studies have confirmed MMC attack at the Opposition (43, 44) 362,1963. 4. Iyer, V. N. and Szybalski, W. Mitomycins and profiromycin: chemical mecha over alkylation at the N-2 position of guanine. nism of activation and cross-linkingof DNA. Science (Wash. DC), 745: 55-58, Since MMC is definitely a potent DNA alkylator, this could 1964. potentially mask the detection of simultaneously produced DNA- 5. Bachur, N. R., Gordon, S. L, and Gee, M. V. A general mechanism for microsomal activation of quinone anticancer agents to free radicals. Cancer strand breaks in the current alkaline elution assays. Kohn ef al. Res., 38: 1745-1750,1978. (13) have considered the problem of detecting mixtures of DNA 6. Doroshow, J. H. Mitomycin C-Â«nhancedSuperoxideand hydrogen peroxide formation in rat heart. J. Pharmacol.Toxico!., 218: 206-211,1981. strand breaks and cross-links in these assays. If a drug produces 7. Lown, J. W. The molecular mechanismof antitumor action of the mitomycins. a combination of both lesions, there should be a reduced elution In: S. K. Carter and S. T. Crooke (eds.),Mitomycin C: Current Status and New rate in irradiated cells, with an increased elution rate in unirra- Developments,pp. 5-26. New York: Academic Press, Inc., 1979. 8. Akhtar, M. H., Begletier,A., Johnson, D., Lown, J. W., McLaughlin, L. W., and diated cells. This phenomenon was not observed in any of the Sim, S. K. Studies related to antitumor antibiotics Part VI. Correlation of current MMC SSB elution assays. However, Begleiter and Blair covalent cross-linking of DNA by bifunctional aziridinoquinones with their antineoplastic activity. Can. J. Chem., 53: 2891-2905, 1975. (25) have recently observed quinone-induced DSBs in L5178Y 9. Wakaki, S. Recent advances in research on antitumor mitomycins. Cancer lymphoblasts exposed to benzoquinone mustard, a compound Chemother. Rep., 13: 79-86, 1961. 10. Ross, W. E., Ewig, R. A. G., and Kohn, K. W. Differences between melphalan which has functional groups similar to MMC. Of interest, this and nitrogen mustard in the formation and removalof DNA cross-links.Cancer alkylating quinone-containing compound induced no SSBs de Res., 38:1502-1506,1978. tectable by alkaline DNA elution assays. This was apparently 11. Zwelling, L. A., Anderson, T., and Kohn, K. W. DNA-protein and DNA interstrand cross-linking by c/s- and frans-platinum(ll)diamminedichlondein L1210 due to cross-link-induced masking of up to 6 Gy-equivalents of mouse leukemiacells and relation to cytotoxicity. Cancer Res., 39: 365-369, SSBs. However, the drug did produce DNA-DSBs in the same 1979. 12. Erickson, L. C., Bradley, M. O., Kohn, K. W. Strand breaks in DNAfrom normal neutral elution assay used in the current study. At a concentra and transformed human cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea. tion of 60 nw, benzoquinone mustard produced about 5 Gy- Cancer Res., 37: 3744-3750,1977. equivalents of DSBs. This is very near the resolution limit of this 13. Kohn, K. W., Ewig, R. A. G., Erickson, L. C., and Zwelling, L. A. Measurement of strand breaks and cross-links by alkalineelution. in: E. C. Freidberg and P. assay. Similar MMC-induced DSBs were not seen in the current C. Hanwalt(eds.),DNA Repair:A Laboratory Manualof ResearchProcedures, studies, even following cell treatments with MMC concentrations Vol. 1, Part B, pp. 379-401. New York: Marcel Dekker, Inc., 1981. 14. Dorr, R.T., Bowden, G. T., Alberts, D. S., and Liddil.J. Molecularpharmacology of 0.3 to 300 M/h. of mitomycin C (MMC) and a polar metabolite(PM) on mammalianDNA. Proc. The new clinical agent diaziquone (diaziridinylbenzo quinone) Am. Assoc. Cancer Res., 25: 291,1984. also possesses quinone and alkylating aziridine moieties similar 15. Ross, W., Wozniak, A., Smallwood, S., and Yalowich, J. C. DNA damage by VP-16: mechanism and relationship to cytotoxicity. In: B. F. Issell, F. M. to MMC. Recent alkaline elution studies of diaziquone demon Muggia, and S. K. Carter (eds.), Etoposide (VP-16):Current Status and New strated a varying mixture of ISC DPC and SSB in different human Developments.New York: Academic Press, Inc., 1984. 16. Salmon, S. E., Hamburger, A. W., Soehnlen, B., Duire, B. G. M., Alberts, D. (fibroblast, embryo, colon) and murine (L1210) cell lines exposed S., and Moon, T. E. Quantitation of differentialsensitivity of humantumor stem to the drug. However, cell killing only correlated with ISC and cells to anticancerdrugs. N. Engl. J. Med., 298:1321-1327,1978. not with DNA strand scission (45). Thus, SSB following AZQ 17. Salmon, S. E., Young, L., Lebowitz, J., Thomson, S., Einspahr, J., Tong, T., and Moon, T. E. Evaluation of an automated image analysis system for were negligible in the HT-29 colon carcinoma, yet this cell line counting human tumor colonies. Int. J. Cell Cloning, 2:142-160,1984. was significantly more sensitive to the drugs cytotoxic action in 18. Kohn, K. W. and Ewig, R. A. G. DNA-proteincross-linking by frans-platinum(ll) diamminedichloridein mammaliancells, a new method of analysis. Biochim. vitro. And HT-29 cells demonstrated the largest degree of ISC Biophys. Acta, 562: 32-40,1979. at all AZQ concentrations. 19. Bradley, M. D. and Kohn, K. W. X-ray DNA double strand break production Our MMC results demonstrate that, at pharmacological drug and repair in mammalian cells as measured by neutral filter elution. Prog. NucleicAcid Res., 1: 793-804,1979. levels, MMC predominantly alkylates mammalian DNA without 20. Fornace, A. J. and Kohn, K. W. DNA-protein cross-linking by ultraviolet causing discernable DNA strand breakage. The drug causes radiation in normal human and xeroderma pigmentosum fibroblasts. Biochim. dose-dependent DNA-DNA and DPC cross-links in murine and Biophys. Acta, 435, 95-103,1976. 21. Kohn, K. W. and Grimek-Ewig,R. A. Alkalineelution analysis,a new approach human tumor cell lines and in isolated nuclei. 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Robert T. Dorr, G. Timothy Bowden, David S. Alberts, et al.

Cancer Res 1985;45:3510-3516.

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