Activity of Quinone Alkylating Agents in Quinone-Resistant Cells1

(CANCER RESEARCH 50, 2872-2876, May 15, 1990] Activity of Quinone Alkylating Agents in Quinone-resistant Cells1

Asher Begleiter2 and Marsha K. Leith

Departments of Internal Medicine [A. B., M. K. LJ and Pharmacology and Therapeutics [A. B.J, University of Manitoba, and Manitoba Institute of Cell Biology [A. B., M. K. LJ, 100 Olivia Street, Winnipeg, Manitoba R3E OV9, Canada

ABSTRACT ating activity of these agents. Studies have shown that the alkylating activity of MMC is dependent on reduction of the The role of the quinone group in the antitumor activity of quinone quinone group (2, 5-9). Similarly, AZQ was shown to produce alkylating agents, such as mitomycin C and 2,5-diaziridinyl-3,5- DNA cross-links only after reduction (4, 10). bis(carboethoxyamino)-l,4-benzoquinone, is still uncertain. The quinone group may contribute to antitumor activity by inducing DNA strand We have previously studied the activity of model quinone breaks through the formation of free radicals and/or by influencing the alkylating agents in LSI78Y lymphoblasts. BM and BDM alkylating activity of the quinone alkylators. The cytotoxic activity and showed significantly greater potency in L5178Y cells compared DNA damage produced by the model quinone alkylating agents, benzo- with the nonquinone alkylating agent, AM (11, 12). The qui quinone mustard and benzoquinone dimustard, were compared in LSI78Y none agents induced both DNA strand breaks and DNA-DNA murine lymphoblasts sensitive and resistant to the model quinone anti- cross-links in L5178Y cells, and both their cytotoxic activity tumor agent, hydrolyzed benzoquinone mustard. The resistant cell lines, and strand break activity could be partially inhibited by the L5178Y/HBM2 and L5178Y/HBM10, have increased concentrations of protective enzyme, catalase (11-13). Studies with these model glutathione and elevated catalase, Superoxide dismutase, glutathione .V- compounds in L5178Y cells resistant to alkylating agents also transferase, and DT-diaphorase activity. L5178Y/HBM2 and L5178Y/ indicated a significant role for DNA-DNA cross-linking in the HBM10 cells were 7.4- and 8.5-fold less sensitive to benzoquinone mustard and 1.7- and 4.3-fold less sensitive to benzoquinone dimustard, cytotoxic activity of BM and BDM (14). respectively, compared with sensitive cells, but showed no resistance to We have isolated and characterized LSI78Y cell lines resist the non-quinone alkylating agent, aniline mustard. The formation of DNA ant to the nonalkylating quinone model compound, HBM (15). double strand breaks by benzoquinone mustard was reduced by 2- and 8- Resistance in these cell lines appeared to be multifactorial. fold in L5178Y/HBM2 and L5178Y/HBM10 cells, respectively, while L5178Y/HBM2 cells, which were 2.5-fold resistant to HBM, double strand break formation by benzoquinone dimustard was reduced showed reduced drug uptake, had slightly elevated levels of only in the L5178Y/HBM10 cells. The number of DNA-DNA cross Superoxide dismutase and catalase activity, a 2-fold increase in links produced by benzoquinone mustard was 3- and 6-fold lower, and intracellular glutathione, and a 3-fold increase in GST and DT- the number produced by benzoquinone dimustard was 35% and 2-fold diaphorase activity compared with L5178Y sensitive cells. lower in L5178Y/HBM2 and L5178Y/HBM10 cells, respectively, com L5178Y/HBM10 cells, which were 6-fold resistant to HBM pared with L5178Y parental cells. In contrast, cross-linking by aniline mustard was unchanged in sensitive and resistant cells. Dicoumarol, an compared with sensitive cells, showed reduced drug uptake and inhibitor of DT-diaphorase, increased the cytotoxic activity of both similar increases in Superoxide dismutase activity and glutathi benzoquinone mustard and benzoquinone dimustard in L5178Y/HBM10 one levels. However, the L5178Y/HBM10 cells also had a 3- cells. This study provides evidence that elevated DT-diaphorase activity fold increase in catalase activity, an 11-fold increase in GST in the resistant cells contributes to resistance to benzoquinone mustard activity, and a 24-fold increase in DT-diaphorase activity. HBM and benzoquinone dimustard, possibly by decreasing the formation of the produced significantly reduced levels of DNA strand breaks in semiquinone intermediates of these agents. The altered reduction of the the resistant cells. quinone groups in the resistant cells may be responsible for the decreased In the present study we have examined the activity of the DNA-DNA cross-linking and lowered induction of DNA strand breaks model quinone alkylating agents, BM and BDM, in the qui- by the quinone alkylating agents. These findings demonstrate that the none-resistant cell lines in order to evaluate the role of the quinone group can modulate the activity of quinone alkylating agents. The study also suggests that the semiquinone intermediates of benzoqui quinone group in the cytotoxic activity of these agents. none mustard and benzoquinone dimustard may be the active alkylating species of these two agents. MATERIALS AND METHODS Materials. Benzoquinone mustard [di(2'-chloroethyl)amino-l,4-ben- INTRODUCTION zoquinone), benzoquinone dimustard |2,5-bis[di(2'-chloroethyl)ami- Antitumor agents such as MMC,1 AZQ, and triaziquone no]-l,4-benzoquinone|, aniline mustard [A',A'-di(2'-chloroethyl)ani- contain both a quinone ring and alkylating groups in their line], and hydrolyzed benzoquinone mustard (di(2'-hydroxymethyl) structure. It has been suggested that the quinone group may amino-1,4-benzoquinone] were prepared as described previously (11, play a role in the cytotoxic activity of these agents. Reduction- 13). Fischer's medium and horse serum were obtained from Grand Island Biological Co., Grand Island, NY. ['4C)Thymidine (specific oxidation reactions of the quinone group may lead to the activity, 50 mCi/mmol) and ['Hjthymidine (specific activity, 50 to 80 formation of oxygen free radicals which can induce DNA dam Ci/mmol) were obtained from Du Pont-New England Nuclear, Boston. age (1), and this has been observed with MMC (2, 3) and AZQ (4). Additionally, the quinone group may influence the alkyl- MA. Proteinase K was from E. Merck, Darmstadt, Federal Republic of Germany; tetrapropylammonium hydroxide was from Eastman Ko Received 4/26/88; revised 12/27/89. dak Co., Rochester, NY; polycarbonate filters (0.8 ^m and 2.0 Mâ„¢) The costs of publication of this article were defrayed in part by the payment were from Nucleopore Corp., Pleasanton, CA; and dicoumarol was of page charges. This article must therefore be hereby marked advertisement in from Sigma Chemical Co., St. Louis, MO. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by the Medical Research Council of Canada. Cell Lines. The L5178Y, L5178Y/HBM2, and L5178Y/HBM10 cell 2To whom requests for reprints should be addressed, at Manitoba Institute of lines used in this study have been described previously (15). L5178Y cells were grown in Fischer's medium containing 12% horse serum, Cell Biology, 100 Olivia Street, Winnipeg. Manitoba R3E OV9. Canada. 3The abbreviations used are: MMC, mitomycin C; AZQ, 2.5-diaziridinyl-3,6- L5178Y/HBM2 cells were grown in Fischer's medium containing 12% bis(carboethoxyamino)-l,4-benzoquinone: BM, benzoquinone mustard; BDM, horse serum and 0.2 mM HBM, and L5178Y/HBM10 cells were grown benzoquinone dimustard; AM. aniline mustard; HBM, hydrolyzed benzoquinone in Fischer's medium containing 12% horse serum and 1.0 mM HBM. mustard; GST. glutathione 5-transferase; DIO,concentration of drug reducing the surviving cell fraction to 0.1. Cytotoxicity Assays. L5178Y parental or resistant cells were incu- 2872

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1990 American Association for Cancer Research. QUINONE ALKYLATORS IN QUINONE-RESISTANT CELLS bated with different concentrations of the model antitumor agents for treated with 30 nM BM or 3 nM BDM in the presence or l h at 37Â°Cin medium containing horse serum. Cytotoxic activity in absence of 100 pM dicoumarol. Although there was a slight the parental and resistant cell lines was determined by a soft agar increase in the cytotoxicity of both BM and BDM in the cloning assay, as described previously (11, 16). Cloning efficiencies presence of dicoumarol, this effect was not statistically signifi ranged from 35 to 66% for L5178Y parental cells, from 51 to 90% for cant. The concentration of BM and BDM used in these studies L5178Y/HBM2 cells, and from 37 to 80% for L5178Y/HBM10 cells. A linear regression analysis of each concentration-survival curve was produced similar levels of cell kill in LSI78Y and L5178Y/ obtained, and the D,0 was derived from the negative reciprocal of the HBM 10 cells in the absence of dicoumarol. regression slope as previously described (17). The degree of resistance Induction of DNA Double Strand Breaks by Quinone Model of the L5178Y/HBM2 and L5178Y/HBM10 cells was determined Compounds in L5178Y Sensitive and Resistant Cells. L5178Y from the ratio of the Â£>,ovaluein the resistant cells to the D,a in the sensitive and resistant cells were incubated with various concen parental cells. The cytotoxicity of each agent in the sensitive and trations of HBM, BM, or BDM at 37Â°Cfor 1 h. The number resistant cell lines was compared statistically by a i test comparing the of DNA double strand breaks induced in the L5178Y, L5178Y/ significance of the differences of the slopes of the concentration-survival HBM2, and L5178Y/HBM10 cells was determined by elution curves. assay (Fig. 3). The induction of double strand breaks by HBM For inhibition studies with dicoumarol, L5178Y or L5178Y/HBM10 was reduced by 2-fold in L5178Y/HBM2 cells and by 30-fold cells were pretreated with or without 100 MMdicoumarol for 15 min. in L5178Y/HBM10 cells compared with the level found in the L5178Y cells were then treated with either 30 nM BM or 3 MMBDM, while L5178Y/HBM10 cells were treated with either 0.5 MMBM or 10 sensitive cells, and the latter effect was statistically significant MMBDM at 37Â°Cfor 1 h. Cytotoxicity was determined by clonogenic (P < 0.01). With BM, the level of DNA double strand breaks assay. The results were analyzed statistically by a two-tailed / test was reduced by 2-fold and 8-fold in L5178Y/HBM2 and comparing the significance of the difference of the mean surviving cell L5178Y/HBM10 cells, respectively, compared with parental fraction in the absence or presence of dicoumarol. The concentration cells, and again the latter result was statistically significant. of dicoumarol used in this study was not toxic to the cells. However, DNA double strand break formation by BDM was Determination of DNA Double Strand Breaks. Cells labeled with [14C]-thymidine were treated with various concentrations of HBM, BM, not significantly different in L5178Y and L5178Y/HBM2 cells, or BDM for l h at 37Â°Cin medium containing horse serum. DNA but was significantly reduced by only 40% to minimum detect able levels in L5178Y/HBM10 cells (P < 0.01). double strand breaks were measured using an elution assay as described Production of DNA-DNA Cross-Links by Model Alkylating previously (13, 18, 19). The level of DNA strand breaks was calculated from the elution profiles and was expressed as rad equivalents (dose of Agents in LSI78Y Sensitive and Resistant Cells. LSI78Y sen radiation inducing an equivalent number of breaks) as determined from sitive and resistant cells were treated with various concentra calibration curves. A linear regression analysis of each concentration- tions of BM, BDM, or AM at 37"C for 1 h, and the number of activity curve was obtained, and the strand-breaking activity of each DNA-DNA cross-links produced in the L5178Y, L5178Y/ agent in the sensitive and resistant cell lines was compared statistically HBM2, and L5178Y/HBM10 cells was determined by elution by a t test comparing the significance of the difference of the slopes of assay (Fig. 4). The production of cross-links by BM was reduced the concentration-activity curves. by 3-fold and 6-fold in L5178Y/HBM2 and L5178Y/HBM10 Determination of DNA-DNA Cross-Linking. Cells labeled with [14CJ- cells, respectively, compared with sensitive cells, while cross thymidine were treated with various concentrations of BM, BDM, or AM for l h at 37Â°Cin medium containing horse serum. DNA-DNA link formation by BDM was 35% and 2-fold lower in these cross-links were measured using an elution assay as described previously cells, respectively, compared with L5178Y cells. These effects (13. 20). The level of cross-linking was calculated as described by Kohn were statistically significant (P< 0.01). In contrast, the number et al. (20). A linear regression analysis of each concentration-activity of cross-links produced by AM in sensitive and resistant cells cune was obtained, and the cross-linking activity of each agent in the was not significantly different. sensitive and resistant cell lines was compared statistically by a I test comparing the significance of the difference of the slopes of the con centration-activity curves. DISCUSSION The quinone ring may play an important role in the cytotoxic activity of a number of antitumor agents. Reduction-oxidation RESULTS reactions of the quinone group can lead to the formation of Cytotoxicity of Model Compounds in LSI78Y Sensitive and oxygen free radicals and active oxygen species (1) which may Resistant Cells. The cytotoxic activities of the model antitumor contribute to the antitumor activity (3, 21) and cardiotoxicity agents, HBM, BM. BDM, and AM, in parental L5178Y cells of the anthracyclines (22-25) and to the antitumor action of and in the resistant cells, L5178Y/HBM2 and L5178Y/ MMC (2, 3), AZQ (4), and streptonigrin (26). In addition, the HBM 10, were determined by a soft agar cloning assay after quinone group may influence the alkylating activity of alkylat- treatment of cells with drug for l h at 37Â°C(Fig. 1). A compar ing quiÃ±ones,such as MMC (2, 5-9) and AZQ (4, 10), which ison of the D10values (Table 1) shows that the L5178Y/HBM2 are activated by reduction of the quinone. In previous studies and L5178Y/HBM10 cells were 2.5- and 6.2-fold resistant to with model compounds we have shown that the model quinone HBM, 7.4- and 8.5-fold resistant to BM, and 1.7- and 4.3-fold alkylating agents, BM and BDM, show increased cytotoxicity resistant to BDM, respectively, compared with parental and cross-linking activity in L5178Y lymphoblasts compared L5178Y cells. In contrast, the resistant cells showed no cross- with a nonquinone alkylating agent, AM, and increased cyto resistance to the nonquinone model agent, AM. toxicity and DNA double strand break formation compared Effect of Dicoumarol on the Cytotoxicity of Model Compounds with HBM (12). Additional work in alkylator-resistant cells in L5178Y Sensitive and Resistant Cells. L5178Y/HBM10cells indicated an important role for cross-linking in the cytotoxic were treated with 0.5 //M BM or 10 ^M BDM in the presence activity of BM and BDM and suggested that the enhanced or absence of 100 ^M dicoumarol. The presence of dicoumarol DNA strand break formation by BM and BDM may be due to significantly increased the cytotoxicity of both BM and BDM the ability of these agents to generate free radicals close to their in L5178Y/HBM10 cells (Fig. 2), but the level of resistance to target by binding to DNA (14). these agents was only partially reversed. L5178Y cells were We have isolated and characterized L5178Y cell lines resist- 2873

Fig. 1. Concentration-survival curves for LSI78Y parental and resistant cells treated with model antitumor agents. LSI78V parental cells (â€¢),L5178V/HBM2 cells (â€¢).or L5178Y/ HBM10 cells (A) were incubated at 37Â°Cfor 1 h with HUM, BM, BDM, or AM at the concen trations shown. The surviving cell fraction was determined by a clonogenic assay as described in the text and is plotted against the concentra tion of drug used. Points, mean of 5 to 7 quad ruplicate determinations: bars. SE. On occasion, the confidence intervals are too small to be shown. io- 0 5 10 0 0.25 0.5 0 10 20 0 15 [HYDROLYZEDBENZOOUINONE [BENZOOUINONE BENZOOUINONE ANILINEMUSTARD] MUSTARD](mm) MUSTARD](Â¿IM) DIMUSTARD](,uM)

Table I Cytotoxic activity of model antitumor agents in 7.5778Ysensitive and resistant cells L5178Y, L5178Y/HBM2, or L5178Y/HBM10 cells were incubated with different concentrations of the model antitumor agents at 37'C for 1 h, and the surviving cell fraction was determined by a clonogenic assay as described in the text. A linear regression analysis of each concentration-survival curve in Fig. 1 was obtained, and the D\0 (mean Â±SE) was derived from the negative reciprocal of the slope of the regression line. The cytotoxic activity of each model agent in the resistant cells was compared with its cytotoxic activity in the sensitive cells by a 2-lailed t test, comparing the significance of the difference in the slopes of the linear regression lines from the concentration-survival plots. cells(0,0. cells(0,0, 10cells(0,o, agentHydrolyzedModel antitumor M)0.56 M)1.37Â±0.06x M)3.48 benzoquinone mustard Â±0.08 X IO'3 10~w Â±0.18 X 10-M Benzoquinone mustard 0.19 Â±0.01 x IO'7 1.37 Â±0.14 x IO"7" 1.58 Â±0.07 x IO-7" 1.83Â±0.09x IO'6 3.14 Â±0.12 x 10-M Benzoquinone dimustard 7.86 Â±1.31 x 10-" 1.20Â±0.08x IO'5L5178Y/HBM21.34Â±0.36x 10-'*L5178Y/HBM IO'5**P< Aniline mustardL5178Y 1.22 Â±0.18 x 0.001. * P = not significant.

HBM 10 cells are 6-fold resistant to this nonalkylating quinone compared with parental cells. These cells may be resistant to CONTROL HBM because of reduced induction of DNA strand breaks

DICOUMAROL resulting from decreased formation and/or increased inactivation of free radicals. Decreased formation of free radicals may be due to reduced drug uptake or increased drug inactivation -1 and extrusion resulting from elevated levels of glutathione and 10 GST activity (27). In addition, elevated levels of DT-diaphorase may decrease the formation of the semiquinone radical of HBM, and thus oxygen free radicals, by converting the quinone agent directly to the hydroquinone (28). DNA strand breaks may be further reduced in these cells by increased catalase and Superoxide dismutase activity and elevated glutathione that can -2 inactivate free radicals and hydrogen peroxide that are formed. 10 The present study provides evidence that the quinone group plays an important role in the cytotoxic activity of quinone alkylating agents. The quinone-resistant cell lines showed cross- resistance to the quinone alkylating agents, BM and BDM, but not to the nonquinone alkylating agent, AM. Since the L5178Y/HBM2 and L5178Y/HBM10 cells did not demon -3 10 strate any resistance to AM despite having elevated concentra BENZOOUINONE BENZOQUINONE tions of glutathione and GST activity, it would appear that this MUSTARD DIMUSTARD detoxification mechanism did not play a major role in resistance Fig. 2. Effect of dicoumarol on cytotoxic activity of BM and BDM in L5I78Y'/ to alkylating agents in these cells. HBM10 cells. L5178Y/HBM10 cells were preincubated with or without 100 Ã•/M dicoumarol for 15 min at 37'C and then were incubated with either 0.5 ii\t BM Resistance mechanisms which can reduce the formation of or 10 iim BDM for 1 h. The surviving cell fractions were determined by a free radicals or inactivate these reactive species may contribute clonogenic assay as described in the text and represent the mean of 4 to 11 to the resistance to BM and BDM. These include decreased determinations. Bars, SE. For each quinone agent the results were evaluated statistically by 2-tailed r tests comparing the significance of the differences of the drug uptake; elevated GST, catalase, and Superoxide dismutase means of cells treated with or without dicoumarol. activity; and increased levels of glutathione. This suggestion was supported by the finding that the formation of DNA double ant to the model quinone antitumor agent, HBM (15). The strand breaks by BM and BDM was reduced in L5178Y/ cytotoxic activity of HBM appears to be due to the formation HBM 10 cells. However, the induction of double strand breaks of free radical-generated DNA strand breaks (11, 13). L5178Y/ by these agents is not significantly reduced in L5I78Y/HBM2 HBM2 cells are 2.5-fold resistant to HBM. while L5178Y/ cells, and the reduced formation of double strand breaks did 2874

2000

Fig. 3. DNA double strand breaks induced in L5178Y parental and resistant cells by 1500 model quinone agents. L5178Y (â€¢).LSI78V/ HBM2 (â€¢).orL5178Y/HBM10 (A) cells were incubated at 37Â°Cfor l h with the concentra tions of HBM, BM, or BDM shown and were assayed for DNA double strand breaks using 1000 elution assays as described in the text. Double strand breaks are presented as rad equivalents (dose of radiation producing an equivalent number of breaks) and represent the mean of 3 to 11 determinations. Bars, SE. For each quinone agent, the results were evaluated sta 500 tistically by ( tests comparing the significance of the differences of the slopes of the concen tration-activity curves in the resistant cells to the slope in the parental cells. 4 0 2 4 75 150 [HYDROLYZED BENZOQUINONE [BENZOQUINONE [BENZOQUINONE MUSTARD] (mM) MUSTARD] (Â¡M) DIMUSTARD] (/4I)

600

Fig. 4. DNA-DNA cross-linking produced by model alkylating agents in L5178Y parental and resistant cells. L5178Y (â€¢).L5178Y/ 400 HBM2 (â€¢).orL5178Y/HBM10 (A) cells were incubated at 37Â°Cfor l h with the concentra tions of BM, BDM. or AM shown and were a: Ã¬ assayed for DNA-DNA cross-linking using o elution assays as described in the text. The level of cross-linking is presented as rad equiv alents and represents the mean of 3 to 7 deter 200 minations. Bars, SE. For each alkylating agent, the results were evaluated statistically by t tests comparing the significance of the differences of the slopes of the concentration-activity curves in the resistant cells to the slope in the parental cells.

O 100 200 O 0.1 0.2 0.3 O 20 40 [ANILINE MUSTARD] (jM) [BENZOQUINONE [BENZOQUINONE MUSTARD](pU) DIMUSTARD] (jM) not correspond closely with the reduced sensitivity of the cells fold elevated in L5178Y/HBM2 and L5178Y/HBM10 cells to the antitumor agents. Thus, other mechanisms must also (15), respectively, compared with parental cells. The importance contribute to resistance to BM and BDM in these cells. of DT-diaphorase in the mechanisms of resistance to the model There was a significant decrease in the production of DNA- quinone agents in these cells was further supported by the DNA cross-links by BM and BDM in the L5178Y/HBM2 and ability of dicoumarol, an inhibitor of DT-diaphorase activity, L5178Y/HBM10 cells, but cross-linking by AM was unchanged to increase the cytotoxic activity of BM and BDM in the in these cells compared with parental LSI78Y cells. This find resistant cells. The elevated DT-diaphorase activity in the re ing suggests that the decreased alkylating activity of the quinone sistant cells may result in increased reduction of BM and BDM agents was not due to a direct effect on the nitrogen mustard directly to their hydroquinone forms without formation of alkylating group. It has been shown previously that reduction semiquinone intermediates. These findings suggest that the of the quinone group is required to activate the alkylating semiquinone intermediates, obtained by one electron reduction activity of bioreductive alkylating agents, such as MMC and of BM and BDM, may be responsible for the cytotoxic activity AZQ (2, 8-10). In a similar manner, reduction of the quinone of these antitumor agents. If semiquinones are the active alkyl group may enhance the cross-linking activity of BM and BDM. ating species of BM and BDM and are responsible for the Thus, mechanisms of resistance effecting reduction of the qui production of the DNA strand breaks by induction of oxygen none group may be responsible for the reduced cross-linking free radicals, then elevated levels of DT-diaphorase activity in observed in the L5178Y/HBM2 and L5178Y/HBM10 cells. the resistant cell lines could decrease the formation of the However, cytochrome P-450 reducÃase activity, which is re semiquinones and could, at least in part, account for the reduced sponsible for the one electron reduction of quinone groups (29), levels of cross-linking, double strand break formation, and was unchanged in the resistant cells (15). In contrast, DT- cytotoxicity of the quinone alkylating agents in these cells. diaphorase activity, which produces a two electron reduction of These findings contrast with results obtained with other the quinone directly to the hydroquinone (28), was 3- and 24- quinone alkylating agents. We have found that MMC produced 2875

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1990 American Association for Cancer Research. QUINONE ALKYLATORS IN QUINONE-RESISTANT CELLS increased cytotoxicity and DNA cross-linking in L5178Y/ 7. Dorr. R. T.. Bowden. G. T.. Alberts. D. S.. and Liddil, J. D. Interaction of mitomycin C with mammalian DNA detected by alkaline elution. Cancer HBM10 cells compared with parental L5178Y cells. Both of Res., 45: 3510-3516. 1985. these activities in L5178Y/HBM10 cells were decreased by the 8. Pan. S-S.. Iracki. T.. and Bachur. N. R. DNA alkylation by enzyme-activated mitomycin C. Mol. Pharmacol., 29: 622-628. 1986. addition of dicoumarol (30). Similarly, Siegel et al. (31) have 9. Toniasz, M.. Chowdary. D.. Lipman. R.. Shinotakahara. S., Veiro. D.. shown that AZQ was more cytotoxic to HT-29 human colon Walker. V.. and Verdine. G. L. 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Pharmacol., 34: 2629-2636. 1985. groups catalyzed by DT-diaphorase, may contribute to the 13. Begleiter. A., and Blair. G. W. Quinone induced DNA damage and its alkylating and cytotoxic activity of MMC and AZQ. relationship to antitumor activity. Cancer Res., 44: 78-84. 1984. 14. Begleiter. A. The contribution of alkylation to the activity of quinone anti- In summary, we have shown that L5178Y lymphoblasts tumor agents. Can. J. Physiol. Pharmacol., 64: 581-585. 1986. resistant to the model quinone antitumor agent, HBM, are 15. Begleiter. A., Leith. M. K.. McClarty, G.. Beenken. S.. Goldenberg, G. J., cross-resistant to the quinone alkylating agents, BM and BDM, and Wright. J. A. Characterization of LSI 78V murine lymphoblasts resistant to quinone antitumor agents. Cancer Res., 48: 1727-1735, 1988. but not to the nonquinone alkylating agent, AM. Elevated levels 16. Chu. M. Y., and Fischer. G. A. 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Asher Begleiter and Marsha K. Leith

Cancer Res 1990;50:2872-2876.

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