(CANCER RESEARCH 46, 3939-3944. August 1986] Cytotoxicity and DNA Lesions Produced by Mitomycin C and Porfiromycin in Hypoxie and Aerobic EMT6 and Chinese Hamster Ovary Cells1

Paula M. Fracasso2 and Alan C. Sartorelli3

Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, Ne»'Haven, Connecticut 06510

ABSTRACT cally resistant group within solid tumors; thus, hypoxic neo- plastic cells may be capable of proliferating and causing tumor Solid neoplasms may contain deficient or poorly functional vascular beds, a property that leads to the formation of hypoxic tumor cells, which regrowth after treatment that produces tumor regression. Our form a therapeutically resistant cell population within the tumor that is laboratory has suggested that hypoxic cells in solid tumors may difficult to eradicate by ionizing irradiation and most existing chemother- exist in an environment conducive to reductive processes, which apeutic agents. As an approach to the therapeutic attack of hypoxic cells, might be exploited by the use of chemotherapeutic agents that we have measured the cytotoxicity and DNA lesions produced by the become cytotoxic after reductive activation (5, 6). This class of bioreductive alkylating agents mitomycin C and porfiromycin, two struc drugs, which we have called bioreductive alkylating agents, turally similar antibiotics, in oxygen-deficient and aerobic cells. Mito consists of compounds which require metabolic reduction to mycin C and porfiromycin were preferentially cytotoxic to hypoxic EMT6 form species capable of alkylating critical cellular macromole- cells in culture, with porfiromycin producing a greater differential kill of cules. The expectation is that hypoxic tumor cells would readily hypoxic EMT6 cells relative to their oxygenated counterparts than did activate and be preferentially susceptible to drugs of this class. mitomycin C. Chinese hamster ovary cells were more resistant to these MC,4 an antineoplastic antibiotic in clinical use for the treat quinone antibiotics; although in this cell line, porfiromycin was signifi cantly more cytotoxic to hypoxic cells than to aerobic cells, and the ment of a variety of solid tumors (7,8) and the related antibiotic, degree of oxygénationdid not affect the toxicity of mitomycin C. PM, are prototypic bioreductive alkylating agents. Previous Alkaline elution methodology was utilized to study the formation of studies by our laboratory and by others have demonstrated that DNA single-strand breaks and DNA interstrand cross-links produced by a variety of cultured cell lines are more sensitive to the mito mitomycin C and porfiromycin in both EMT6 and Chinese hamster ovary- mycin antibiotics under hypoxic conditions than in the presence cells. A negligible quantity of DNA single-strand breaks and DNA of air (9-17). The mitomycin antibiotics are thought to exert interstrand cross-links were produced in hypoxic and aerobic Chinese their cytotoxic activity by the formation of interstrand cross hamster ovary cells by exposure to mitomycin C or porfiromycin, a links between complementary strands of DNA (18-32); these finding consistent with the considerably lower sensitivity of this cell line to these agents. In EMT6 tumor cells, no single-strand breaks appeared investigations have demonstrated that the highest degree of cross-linking occurs in DNAs of high guanine and cytosine to be produced by these antitumor antibiotics under both hypoxic and content (22, 24, 27); furthermore, the cross-linking of DNA by aerobic conditions; however, a significant number of DNA interstrand cross-links were formed in this cell line following drug treatment, with MC is enhanced as the pH of the environment is lowered, substantially more DNA interstrand cross-linking being produced under suggesting that preferential activation of this antibiotic could hypoxic conditions. Mitomycin C and porfiromycin caused the same occur in tumor cells which may tend to have a lower pH than amount of cross-linking under conditions of oxygen deficiency; however, normal cells (24, 33, 34). Of major significance is the evidence mitomycin C produced considerably more DNA cross-linking than did by several investigators (18, 20, 23, 35) that the rate of removal porfiromycin in oxygenated cells. DNA interstrand cross-links were of interstrand cross-links by mammalian cells is important to observed in hypoxic EMT6 cells throughout a 24-h period following the sensitivity of cells to MC. While the production of DNA removal of mitomycin C and porfiromycin, with a decrease in DNA interstrand cross-links is believed to be part of the mechanism interstrand cross-links observed at 24 h. An increase in DNA interstrand cross-links occurred in aerobic EMT6 cells treated with mitomycin C by which MC and PM exert their cytotoxicity, no direct evi and porfiromycin at 6 h after drug removal, with a decrease in these dence exists that these lesions are responsible for the antineo lesions being observed by 24 h, suggesting that the rate of formation of plastic activity of these antibiotics. The present report provides the cross-links may be slower and the removal of cross-links more rapid more extensive data on the toxicity of MC and PM to aerobic under aerobic conditions. These results were consistent with the degree and hypoxic EMT6 and CHO cells and, in addition, since these of cytotoxicity produced by these agents to hypoxic and aerobic EMT6 antibiotics are believed to exert their antineoplastic activity by cells. The findings support the conclusion that the formation and removal the formation of DNA interstrand cross-links, utilizes alkaline of DNA interstrand cross-links are important for the expression of elution methodology to assess the importance of these DNA cytotoxicity but are not the sole determinants of the toxicity produced by lesions to the cytotoxicity of these drugs under conditions of mitomycin C and porfiromycin under both hypoxia and aerobiasis. hypoxia and normal aeration.

INTRODUCTION MATERIALS AND METHODS Solid neoplasms are known to contain deficient vascular beds Cultured Cell Lines. EMT6 mouse mammary tumor cells (subline and areas of severe vascular insufficiency and as a result may EMT6-Rw supplied by Dr. Sara Rockwell, Department of Therapeutic develop regions containing hypoxic tumor cells (1-3). These Radiology, Yale University) and CHO cells (subline HA-1 supplied by malignant cells, which frequently constitute 5 to 30% of the Dr. Daniel S. Kapp. Department of Therapeutic Radiology, Yale Uni total viable tumor cell population (4), may form a therapeuti- versity) were grown at 37°Cin Waymouth's medium (Grand Island Biological Co., Grand Island, NY) supplemented with 15% fetal bovine serum (Grand Island Biological Co.) and antibiotics in a humidified Received 10/31/84; revised 3/7/86, 4/28/86; accepted 5/1/86. 1This research was supported in part by American Cancer Society Grant CH- atmosphere of 95% air/5% CO2. Stock cultures of EMT6 and CHO 211 and by USPHS Medical Scientist Training Program Grant GM-07205 to P. M. F. * The abbreviations used are: MC. mitomycin C; PM, porfiromycin; CHO, 2 Present address: Department of Internal Medicine. Beth Israel Hospital, Chinese hamster ovary cells; PBS, phosphate-buffered saline (0.2 g KC1. 0.2 g Boston, MA 02115. KH2P04, 8 g NaCI, and 1.15 g Na2PO4/liter, pH 7.4); SDS, sodium dodecyl 1To whom requests for reprints should be addressed. sulfate. 3939 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1986 American Association for Cancer Research. CYTOTOXICITY AND DNA LESIONS IN CULTURED CELLS cells were seeded in 25-cm2 plastic flasks at 5 to 10 x 10* and 1 to 2 x polyethylene Luer-lok syringe. These cells were then lysed on the filter 10* cells/ml of medium, respectively, with subculturing carried out with 5 ml of a lysis solution containing 2% SDS (99% purity; BDH every 3 to 4 days. Other characteristics of these cell lines have been Chemicals, Ltd., Poole, England), 0.025 M disodium EDTA (Fischer described in detail previously (36, 37). Scientific Co., Fair Lawn, NJ) and 0.1 M glycine (Bethesda Research Cell Survival Studies. To monitor cell survival, 2 x 10s EMT6 cells Laboratories, Inc., Gaithersburg, MD), pH 10.0, which was allowed to and 3 to 4 x 10s CHO cells/10 ml of Waymouth's medium supple flow through the filter by gravity. The lysate was deproteinized by mented with 15% fetal bovine serum, penicillin (100 units/ml), and placing 2 ml of 2% SDS, 0.025 M disodium EDTA, and 0.1 M glycine streptomycin (100 ni; nil), were seeded in glass milk dilution bottles, (pH 10.0) containing proteinase K (0.5 mg/ml; 20 mAnson units/mg; and cultures were allowed to grow for 3 days, at which time the cells E. Merck, Darmstadt, West Germany) on the filters in the upper were in mid-exponential growth. The medium was then removed and chamber of the filter holder. Following the addition of proteinase K, replaced with 5 ml of fresh medium. Bottles were sealed with sterile 40 ml of a solution containing tetrapropylammonium hydroxide (RSA rubber sleeve stoppers (Aldrich Chemical Co., Milwaukee, WI) and Corp., Elmsford, NY), 0.02 M EDTA, free acid (Sigma Chemical Co., fitted with 13-gauge needles for gas inflow and 18-gauge needles for St. Louis, MO), and 0.1% SDS (pH 12.1) was placed in the syringe. gas outflow; tubing connected to the outflow needles was immersed in Both solutions were pumped through the filter in the dark at a rate of water to allow visible monitoring of gas flow and to prevent back flow 0.035 ml/min, allowing the proteinase digestion time to be approxi of air into cultures. To produce hypoxia, cultures were gassed contin mately 2 h. Eluted fractions were collected at 3-h intervals for 15 h. uously for 3 h at 37°Cwith a humidified mixture of 95% N2/5% CO2 When all of the fractions were collected, they were processed as follows. (Presto Welding Service Centers, North Haven, CT). This incubation The eluting solution remaining in the funnel reservoir was gently poured technique produces full radiobiological hypoxia (oxygen content, 10 off and discarded. The solution remaining in the pump tubing and filter ppm) after 2 h of exposure to 95% N2/5% CO2. Normally aerated holder was pumped at maximum speed into an empty scintillation vial. cultures were incubated in a humidified atmosphere of 95% air/5% The filter was removed and placed in a scintillation vial containing 0.4 CO2 (Presto Welding Service Centers). At this time, appropriate dilu ml of l N HC1. Subsequently, the filter was heated at 60'C for 1 h to tions of either MC or PM (the generous gifts of Dr. T. W. Doyle, depurinate the DNA. After heating the filter, it was cooled to room Bristol Laboratories, Syracuse, NY) dissolved in ethanol (final concen temperature and 2.5 ml of 0.4 N NaOH (which converts the apurinic tration of ethanol = 0.35%) were added directly to cultures without sites to strand breaks) was added for 1 additional h. Finally, 10 ml of breaking the hypoxia, by injecting a small volume of drug through the 0.4 N NaOH was added to the funnel to flush the filter holder and rubber sleeve. After exposure to each drug for l h under hypoxia or air, pump tubing and a 2.5ml aliquot of this solution was assayed for its cells were washed twice with 5 ml of sterile PBS, and treated with content of radioactivity. A 2-ml aliquot of lysis solution was obtained 0.05% trypsin (Grand Island Biological Co.) in PBS for 10 min. Cells and water was added to this fraction and to the fractions containing were collected by centrifugation and after appropriate dilutions were 0.4 N NaOH to give a final volume of 6 ml. All fractions, except the plated in replicate dishes and allowed to form colonies for 10 to 14 fraction which contained the filter, were mixed with 10 ml Aquassure days. Colonies were stained with Gram's crystal violet (Fischer Scien (New England Nuclear, Boston, MA) with 0.7% glacial acetic acid. The tific Co., Fairlawn, NJ) and counted. Both hypoxic and aerobic vehicle fraction containing the filter was mixed with 5 ml Aquassure with 0.7% (0.35% ethanol) controls were included in each experiment; the surviv glacial acetic acid. Samples were counted in a Beckman LS 7500 ing fractions for hypoxic cultures were calculated using the hypoxic scintillation counter. controls. DNA interstrand cross-linking indices were computed using the Assay of Single-Strand Breaks and DNA Interstrand Cross-Links by formula Alkaline Elution. EMT6 and CHO cells were seeded in glass milk dilution bottles at a density of 1 to 2 x 10s cells/10 ml of Waymouth's Cross-link index = - 1 medium supplemented with 15% fetal bovine serum and anti biotics. After a 24-h incubation period, sample cells were labeled with where r0 and r were the fractions of the [3H]- and 1'4C]DNA remaining [2-'4CJthymidine (0.02 ^Ci/ml; 55 mCi/mmol; Amersham Corp., Ar lington Heights, IL) and reference cells were labeled with [methyl-3H]- on the filter after approximately 10 h of elution (38, 39). thymidine (0.05 jiCi/ml; 2 Ci/mmol; Amersham Corp.). The radioac tive medium was removed after 24 h and replaced with fresh medium RESULTS until the start of experiments. This procedure enabled the labeled material to be incorporated into high molecular weight DNA. Alkaline Cell Survival Studies. Measurements of the surviving fraction elution technology has been described in detail (36, 37). Briefly, EMT6 of hypoxic and aerobic vehicle-treated controls demonstrated a or CHO sample cells prelabeled with [14C]thymidine were treated with slight cytotoxicity with both cell lines after exposure to hypoxia; 2 ¿IMMCor PM dissolved in ethanol for l h under hypoxia or air. At thus, the surviving fraction of the hypoxic vehicle-treated con this time, the drug-containing medium was removed, the cell monolayer trol cells was 0.472 and 0.534, whereas the aerobic vehicle- was washed with ice-cold Hanks' balanced salt solution containing treated control survivals were 0.738 and 0.794 for EMT6 and glucose but no calcium or magnesium (Grand Island Biological Co.), CHO cells, respectively. The survival curves for aerobic and and cells were either not irradiated or irradiated with 300 rads of X- hypoxic cells treated with various concentrations of MC are irradiation (250 kV; 15 mA; 2 mm Al filter; dose rate, 153 rads/min; Siemans Stabilipan) in 5 ml of Hanks' balanced salt solution to measure shown in Figs. 1 and 2. The findings with EMT6 cells treated single-strand breaks and DNA interstrand cross-links, respectively. with MC agreed with those reported by our laboratory previ Untreated EMT6 reference cells prelabeled with [3H]thymidine were ously (9, 11, 12, 14, 15); thus, the survival curves for EMT6 also washed and irradiated with 300 rads when used in the assays to cells exposed to various concentrations of MC showed that measure single-strand breaks and DNA interstrand cross-links. These hypoxic cells were significantly more sensitive to these anti reference cells provided DNA within each experiment with consistent biotics than their aerobic counterparts (Fig. 1). In contrast, elution kinetics which were not influenced by the elution rate of the CHO cells, which were considerably less sensitive than EMT6 experimental [MC]DNA. After X-irradiation, the cells were collected cells to this antibiotic, demonstrated little difference in their from the milk dilution bottles using a T-flask scraper (BélicoGlass, sensitivity to MC under aerobic and hypoxic conditions (Fig. Inc., Vineland, NJ), 5 ml of ice-cold medium was added to each bottle, 2). The differential toxicity of PM to hypoxic versus aerobic and the cells were dispersed by gentle pipetting. Approximately 2.5 x 10s MC-labeled sample cells and a similar number of 3H-labeled refer EMT6 and CHO cells was significantly greater than that of ence cells were mixed, diluted in ice-cold PBS, and collected on a 0.8- MC (Figs. 3 and 4). The findings indicate that PM and MC nm pore size, 2-mm diameter, polycarbonate filter (Nuclepore Corp., were almost equitoxic to hypoxic EMT6 cells, whereas PM was Pleasanton, CA), which was placed on a 25-mm polyethylene filter considerably less cytotoxic than MC to aerobic cells. For CHO holder (Swinnex; Millipore Corp., Bedford, MA) connected to a 50-ml cells, the results indicated significantly greater hypoxic cell 3940 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1986 American Association for Cancer Research. CYTOTOXICITY AND DNA LESIONS IN CULTURED CELLS

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0.0001 1.0 2.0 3.0 4.0 Mitomycin C (/¿M) 0001 Fig. 1. Survival of hypoxic and aerobic EMT6 cells after exposure to MC. 1.0 20 30 40 Exponentially growing cells were exposed to varying concentrations of drug for Porfiromycin I li under conditions of normal aeration or hypoxia, and cytotoxicity was estimated by the ability of cells to form colonies as described in "Materials and Fig. 3. Survival of hypoxic and aerobic EMT6 cells after exposure to PM. Methods." Data are expressed as a fraction of control survival of aerobic (O) or Exponentially growing cells were exposed to varying concentrations of drug for hypoxic (•)cellsand represent mean and SE (bars) of three experiments. l h under conditions of normal aeration or hypoxia, and cytotoxicity was estimated by the ability of cells to form colonies as described in "Materials and Methods." Data are expressed as a fraction of control survival of aerobic (O) or 1.0 hypoxic (•)cellsand represent mean and SE (bars) of three experiments.

1.0 u O

(fi O.I IO 20 30 4.0 01 Mitomycin C Fig. 2. Survival of hypoxic and aerobic CHO cells after exposure to MC. Exponentially growing cells were exposed to varying concentrations of drug for l h under conditions of normal aeration or hypoxia, and cytotoxicity was estimated by the ability of cells to form colonies as described in "Materials and Methods." Data are expressed as a fraction of control survival of aerobic (O) or hypoxic (•)cellsand represent mean and SE (bars) of three experiments. cytotoxicity than aerobic cell cytotoxicity at 2 and 4 /¿MPM 001 (Fig. 4); however,this increase in hypoxic CHO cell cytotoxicity 1.0 2.0 3.0 4.0 relative to that of oxygenated cells produced by PM was sub Porfiromycin stantially less than the differential cytotoxicity seen with EMT6 Fig. 4. Survival of hypoxic and aerobic CHO cells after exposure to PM. Exponentially growing cells were exposed to varying concentrations of drug for cells treated with this antibiotic. l h under conditions of normal aeration or hypoxia, and cytotoxicity was Formation of Single-Strand Breaks and DNA Interstrand estimated by the ability of cells to form colonies as described in "Materials and Cross-Links Produced by Antibiotic Treatment. Experiments Methods." Data are expressed as a fraction of control survival of aerobic (O) or using both hypoxic and aerobic EMT6 cells treated with 2 /¿M hypoxic (•)cellsand represent mean and SE (bars) of three experiments. MC or PM for l h demonstrated differences dependent upon the degree of oxygénation;thus,although the alkaline elution linking than those exposed to PM, which is also consistent with profiles of aerobic and hypoxic EMT6 cells treated with either the degree of cytotoxicity produced by these drugs in air. MC or PM for l h demonstrated no DNA single-strand breaks In the CHO cell line, neither MC nor PM caused detectable under either hypoxic or aerobic conditions, this cell line exhib single-strand breaks under aerobic and hypoxic conditions (Fig. ited DNA interstrand cross-links, with substantially more DNA 6), a finding identical to that observed with EMT6 cells; how interstrand cross-links occurring under hypoxic conditions than ever, contrary to the results obtained with the EMT6 carcinoma, with oxygénation(Fig.5). Both antibiotics produced a compa negligible DNA interstrand cross-linking was produced by rable amount of cross-linking under hypoxic conditions, a find either antibiotic under hypoxic or aerobic conditions. These ing consistent with the degree of cytotoxicity produced by these findings are consistent with studies which indicated that these agents (Figs. 1 and 3). Aerobic EMT6 cells treated with MC antibiotics caused considerably less toxicity to CHO cells. demonstrated a slightly greater amount of interstrand cross- Measurement of the formation and removal of single-strand 3941

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1986 American Association for Cancer Research. CYTOTOXICITY AND DNA LESIONS IN CULTURED CELLS

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< 0806 04 0.2 0806 04 0.2 Fraction of 3H-Lobeled DNA Retained on Filter 12 24 Time After Mitomycm C Removal (hr) Fig. 5. Alkaline elution profiles of hypoxic and aerobic EMT6 cells following Fig. 7. DNA interstrand cross-link index in EMT6 cells after MC treatment. treatment with MC or PM. Exponentially growing cells were exposed to 2 (/\i MC or PM for I h under conditions of normal aeration or hypoxia. Subsequently, Exponentially growing cells were treated with 2 fiM MC for I h under conditions the drug-containing medium was removed, and the cells were irradiated with 0 or of normal aeration or hypoxia. Subsequently, the drug-containing medium was 300 rads of X-irradiation and assayed for single-strand breaks or DNA interstrand removed, and the cells were irradiated with 300 rads and assayed for DNA cross-links by the alkaline elution assay described in "Materials and Methods." interstrand cross-linking by the alkaline elution assay, as described in "Materials and Methods." The DNA interstrand cross-linking index was determined using Alkaline elution profiles for the measurement of single-strand breaks and DNA interstrand cross-links are shown by open and closed symbols, respectively. O, * the formula control cells: D, •.MC-treated cells; A, A, PM-treated cells. Cross-link index - l 1.0 0.8 where >,,and r were the fractions of control and drug-treated DNA remaining on —°-o_o the filter after approximately 10 h of elution (38, 39). 03,hypoxic EMT6 cells; _.. I È 0.6 HypomC aerobic EMT6 cells. Data represent mean and SE (bars) of three experiments. û

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008 u•oc 0.806 0.4 0.2 0.80.6 04 02 Froction of 3H-Lobeled DMA Retained on Filter o1 Fig. 6. Alkaline elution profiles of hypoxic and aerobic CHO cells following 004"e treatment with MC or PM. Exponentially growing cells were exposed to 2 /IM t—i° MC or PM for 1 h under conditions of normal aeration or hypoxia. Subsequently, the drug-containing medium was removed, and the cells were irradiated with 0 or 300 rads of X-irradiation and assayed for single strand breaks or DNA interstrand 0-TiÕ1 cross-links, respectively, by the alkaline elution assay described in "Materials and Methods." Alkaline elution profiles for the measurement of single-strand breaks O 6 \2 24 and DNA interstrand cross-links are shown by the open and closed symbols, Time After Porfiromycin Removal (hr) respectively. O, •,control cells; D, •MC-treated cells; A, A, PM-treated cells. Fig. 8. DNA interstrand cross-link index in EMT6 cells after PM treatment. Exponentially growing cells were treated with 2 t¡MPMfor I h under conditions of normal aeration or hypoxia and processed as described in Fig. 7. Data represent breaks and DNA interstrand cross-links in EMT6 cells as a mean and SE (bars) of three experiments. There were significantly more DNA function of time following exposure to 2 UM MC or PM for 1 interstrand cross-links immediately following PM treatment (P < 0.05), 6 (P < 0.03), and 12 (P < 0.05) h after PM removal in hypoxic (O) than in aerobic (D) h and then removal of antibiotics by washing, in a manner EMT6 cells. analogous to cell survival studies, demonstrated that DNA interstrand cross-links were produced by these agents in hypoxic h after drug removal. In contrast, significantly more DNA EMT6 cells which were maintained over the 24-h postincuba- interstrand cross-links were observed in hypoxic EMT6 than tion period used, with approximately an equivalent amount of aerobic EMT6 cells at 0, 6, and 12 h after removal of PM. cross-linking occurring over the 0 to 12-h period and fewer cross-links at 24 h (Figs. 7 and 8). No single-strand breaks DISCUSSION could be detected in EMT6 cells under either hypoxia or air throughout the 0 to 24-h period after drug removal. Few de Previous work by our laboratory has demonstrated that MC tectable DNA interstrand cross-links were present immediately and PM are more toxic to hypoxic EMT6 cells than to their after MC and PM treatments, respectively, in aerobic EMT6 aerobic counterparts; MC was shown to be essentially equitoxic cells; however, an increase in DNA interstrand cross-links to hypoxic and aerobic CHO cells in culture (9, 11, 14-16). occurred at 6 h postincubation for both antibiotics, with the The results of the present study extend these findings on the number of cross-links decreasing during the 12- to 24-h period cytotoxicity of MC and PM and confirm the earlier investiga following drug exposure. Considerably more DNA interstrand tions which demonstrate that these agents are preferentially cross-links were observed immediately following MC treatment cytotoxic to hypoxic EMT6 cells as compared to their oxygen of hypoxic EMT6 cells. There were, however, no significant ated counterparts and that MC has slight and no preferential differences between hypoxic and aerobic EMT6 cells in the toxicity that is dependent upon the degree of oxygénationin amount of DNA interstrand cross-links occurring 6,12, and 24 CHO cells. This latter finding is in agreement with the report 3942

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1986 American Association for Cancer Research. CYTOTOXICITY AND DNA LESIONS IN CULTURED CELLS of Rauth et al. (13) who demonstrated that MC (l Mg/ml) was suggest a relationship between the cytotoxicity of these drugs only slightly toxic to hypoxic and aerobic CHO cells. Measure and the formation of DNA interstrand cross-links. Thus, in ment of the toxicity of PM to hypoxic and aerobic cultured both cell lines, single-strand breaks were not detected: however, cells yielded results different from those of MC. PM produced the DNA interstrand cross-links present in antibiotic-treated relatively great hypoxic cell cytotoxicity to EMT6 carcinoma EMT6 cells may have masked any single-strand breaks which cells but exhibited considerably less aerobic toxicity than that may have occurred during or after drug treatments by retention caused by MC. CHO cells treated with PM were also more of the DNA on the filter. Furthermore, even though MC is susceptible under conditions of hypoxia. These findings agree known to form hydrogen peroxide and hydroxyl radicals after with earlier studies which demonstrated that PM was an active reductive activation (52-54) and these radicals may cause sin anticancer drug but with lower potency and less toxicity to gle-strand breaks, Bradley and Erickson (55) have reported normal tissue than MC ( 11,40). That the results may be related rapid repair rates for single-strand breaks induced by hydrogen to differences in the susceptibilities of the two antibiotics to peroxide in V79 cells. MC was more toxic to hypoxic EMT6 reductive activation was suggested by the findings of Patrick et cells than to corresponding aerobic cells and more DNA inter al. (41) that hydrogénation of the MC related material mito- strand cross-linking was observed under hypoxic conditions mycin B over a platinum catalyst at atmospheric pressure in than under aerobic conditions immediately following drug treat dimethylformamide produced an aziridinomitosene, whereas ment. A similar pattern was also observed in PM-treated EMT6 hydrogénationof PM under the same conditions gave no such cells. Furthermore, the aerobic toxicity that occurs with MC in product. Since both of these antibiotics have identical redox EMT6 cells is greater than that produced by PM, and the potentials, it is not clear why differences should exist between degree of DNA cross-linking under these conditions corre these agents in their susceptibility to reduction. sponds with these results. Early studies have demonstrated cross-linking of bacterial Both MC and PM produced DNA interstrand cross-links in DNA in situ and cross-linking of purified bacterial DNA in the EMT6 cells that persisted over a 24-h period following drug presence of cell lysates in vitro after exposure to MC (21, 26). removal. Interestingly, maximum formation of DNA inter Iyer and Szybalski (22, 27) have demonstrated that either strand cross-links in aerobic EMT6 cells occurred several hours chemical or enzymatic reduction of MC and PM was essential after drug removal with both MC and PM, whereas under for cross-linking of bacterial DNA in vitro, and an increase in hypoxic conditions, maximum cross-linking of DNA was ob cross-linking occurred when bacterial DNA of high guanine- served at the time of drug removal. With MC, the amount of cytosine content was used. These investigators extended their interstrand cross-linking observed in aerobic EMT6 cells was findings to include studies on the production of cross-links in comparable to that occurring in hypoxic cells at 6 and 12 h DNA using a cultured human cell line (26, 27). An amount of after drug removal, indicating that the rate of formation of DNA cross-linking in mammalian cells in vitro and in vivo interstrand cross-links in EMT6 cells is slower under air. Sub comparable to that obtained with bacterial DNA in vitro and in stantially fewer DNA interstrand cross-links were observed at vivo was not obtained, even at relatively high concentrations of 24 h after MC or PM removal in hypoxic and aerobic EMT6 MC, unless purified DNA which had been deproteinized was cells. These results may indicate that removal of cross-links is used. [14C]PM was used to prove covalent binding of drug to taking place in these cells by repair processes, although it is bacterial DNA (28). Other investigators have confirmed the possible that cell division occurred and that the decrease in the covalent binding of [14C]PM to DNA from other bacterial number of cross-links reflects the fact that the original popu sources, and additionally, demonstrated covalent binding of lation of cells was diluted at 24 h after drug exposure. Although ['HJMC to DNA (42-44). Using synthetic polynucleotides, a relationship between DNA interstrand cross-links and cyto both [I4C]PM and [3H]PM were found to bind to guanine toxicity to the mitomycin antibiotics appears to exist, other polynucleotides to a greater extent than to other polynucleo factors also must be considered; thus, it is possible that cross- tides (42, 45). The preference of MC for the guanine-cytosine linking of other cellular macromolecules, including cell mem base pair in DNA cross-linking was confirmed directly by Lown brane components, structural proteins, and enzymes may occur et al. (24) using an ethidium bromide fluorescence assay. Fuji- at highly sensitive sites in these molecules and lead to the wara (18) and Fujiwara and Tatsumi (20) observed DNA-DNA differential cytotoxicity observed in hypoxia and normal aera interstrand cross-links with MC-treated normal human fibro- tion. blasts using alkaline sucrose sedimentation, hydroxyapatite In conclusion, the studies presented in this report demon chromatography, and S,-nucIease digestion methodologies, strate a relationship between the cytotoxicity of the mitomycin while Fornace and Little (46) and Foranee et al. (47) observed antibiotics in hypoxic and aerobic cell lines and the degree of DNA-DNA and DNA-protein cross-links with MC-treated hu DNA interstrand cross-linking; thus, porfiromycin is more man fibroblasts using alkaline elution methodology. In recent selective for hypoxic cells than is MC, both with respect to its years, several investigators have been able to identify MC capacity to kill cells and to cross-link DNA. CHO cells are adducts after chemical or enzymatic reduction of the drug in relatively resistant to the cytotoxic effects of MC and PM the presence of nucleotides, calf thymus DNA, and rat liver compared to EMT6 cells, and relatively few DNA interstrand DNA (48-51). While alkylation of DNA and the production of cross-links were produced in CHO cells by these agents. In DNA-DNA interstrand cross-links are believed to be part of EMT6 cells, however, which are considerably more sensitive to the mechanism by which MC and PM exert their anaerobic the mitomycin antibiotics, particularly under hypoxic condi cytotoxicities, no direct evidence exists that these lesions are tions, these drugs produced a much greater degree of DNA responsible for the antineoplastic activity of these antibiotics. interstrand cross-linking, although the finding that the total The alkaline elution technique developed by Kohn et al. (38, number of DNA cross-links increases with time in oxygenated 39) was utilized to study the relationship of DNA single-strand EMT6 cells to the maximum level found in hypoxic cells breaks and DNA interstrand cross-links produced by MC and indicates that factors such as repair of these lesions may be of PM to the toxicity of these antibiotics to hypoxic and aerobic importance in the differential sensitivity of hypoxic and aerated EMT6 and CHO cells. The results of the alkaline elution studies cells to these agents. Further studies are required to clarify the 3943

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1986 American Association for Cancer Research. CYTOTOXICITY AND DNA LESIONS IN CULTURED CELLS role of repair in the differential sensitivity of oxygenated and to antitumor antibiotics. V. Reaction of mitomycin C with DNA examined by ethidium fluorescence assay. Can. J. Biochem., 54:110-119, 1976. hypoxic cells to the mitomycins as well as to ascertain the 25. Matsumoto, I., and Lark, K. G. Altered DNA isolated from cells treated with reason for the relatively great cytotoxicity of MC and PM mitomycin C. Exp. Cell Res., 32: 192-196, 1963. towards hypoxic EMT6 cells compared to the relative resistance 26. Szybalski, W. Chemical reactivity of chromosomal DNA as related to mu- tagenicity: studies with human cell lines. Cold Spring Harbor Symp. Quant. of CHO cells. It is conceivable that differences in factors such Biol. 29: 151-159, 1964. as uptake, activation, and/or subsequent reaction with DNA 27. Szybalski, W., and Iyer, V. N. Crosslinking of DNA by enzymatically or chemically activated mitomycins and porfiromycins, bifunctionally ''alkylat are responsible for the different sensitivities of the two cell lines ing" antibiotics. Fed. Proc., 23: 946-957, 1964. to these agents. 28. Szybalski, W., and Iyer, V. N. Binding of CM-labeled mitomycin or porfiro mycin to nucleic acids. Microb. Genet. Bull., 21: 16-17, 1964. 29. Tomasz, M. H2O2 generation during the redox cycle of mitomycin C and ACKNOWLEDGMENTS DNA-bound mitomycin C. Chem.-Biol. Interact. 13:89-97, 1976. 30. Tomasz, M., Lipman, R., Snyder, J. K., and Nakanishi, K. Full structure of The authors would like to thank Drs. Neil W. Gibson and Leonard a mitomycin C dinucleoside phosphate adduci. Use of differential FT-IR C. Erickson for invaluable discussions during the course of this work. spectroscopy in microscale structural studies. J. Am. Chem. Soc., 705:2059- 2063, 1983. 31. Topal, M. D., and Baker, M. S. 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Cytotoxicity and DNA Lesions Produced by Mitomycin C and Porfiromycin in Hypoxic and Aerobic EMT6 and Chinese Hamster Ovary Cells

Paula M. Fracasso and Alan C. Sartorelli

Cancer Res 1986;46:3939-3944.

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