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[CANCER RESEARCH 52. 3515-3520. July I. I992| Modulation of the Cell Cycle-dependent Cytotoxicity of Adriamycin and 4Hydroperoxycyclophosphamide by Novobiocin, an Inhibitor of Mammalian Topoisomerase II1 Francis Y. F. Lee,2 Deborah J. Flannery, and Dietmar W. Siemann Tumor Biology Division, university of Rochester Cancer Center, Rochester, New York 14642 ABSTRACT MATERIALS AND METHODS Centrifugal elutriation was used to obtain synchronized cell populations in various cell cycle phases without prior growth-perturbing manipulation. Treatment of these subpopulations with novobiocin (NOVO), a putative inhibitor of the mammalian topoisomerase II enzyme, revealed a unique cell cycle phase-dependent cytotoxicity for this agent. At a concentration of 0.3 m\i, NOVO was cytotoxic only to a specific cell subpopulation in the ( ; i-S phase boundary. Cells in other cell cycle phases were completely unaffected. Additionally, S and (.M phase cells progressed through the cell cycle relatively unaffected by NOVO but were blocked at the d-S boundary. NOVO treatment protected tumor cells from Adriamycin (ADR)induced lethality but sensitized them to the toxic action of 4-hydroperoxycyclophosphamide, an alkylating agent. These opposing effects of NOVO were demonstrated in all of the four tumor cell lines investigated: A431 and HEp3 (derived from human squamous cell carcinomas); MLS, a human ovarian cancer cell line; and a Chinese hamster ovary cell line. The degree of protection against ADR was the greatest for S-phase cells, intermediate for cells in early (;, and M phases, and the least for late d cells. This cell cycle-dependent protection by NOVO, which is identical to the cell cycle-dependent cytotoxicity of ADR, was consistent with the idea that NOVO interfered directly with the cell-killing mechanism of ADR. In contrast, even though the cytotoxic activity of 4-hydroperoxycyclophosphamide exhibited significant cell cycle dependency, NOVO enhanced 4-hydroperoxycyclophosphamide lethality equally for all cell cycle phases. Cell Culturing. All cell lines were grown as monolayer cell cultures. Chinese hamster ovary cells were maintained in Ham's F-IO medium, HEp3 in Ham's F-12 medium, A431 and MLS in »-minimumessential INTRODUCTION There is considerable current interest in the role of Topo II' in determining the sensitivity of tumor cells to chemotherapeutic agents, notably the epipodophyllotoxins (1, 2), anthracyclines (3), and alkylating agents (4-6). It has been demonstrated in several studies that tumor cell lines that were resistant to DNA intercalators, whether acquired or innate, had decreased Topo II activity (7-9), while those resistant to alkylating agents had increased Topo II activity (1, 6). Clearly, these directly opposing relationships between Topo II activity and the antitumor efficacy of two of the most frequently used classes of anticancer agents have important implications for the optimal use of these agents in polychemotherapy. However, the precise role of Topo H-mediated processes in determining antitumor efficacy is still not well understood. Here we describe experi ments, using the putative Topo II inhibitor novobiocin, aiming at defining the possible role of Topo II in the cell cycledependent cytotoxicity of ADR and 4-OOH-CP. medium. All media were obtained from Gibco and were supplemented with 10% fetal calf serum, 5 ITIMglutamine, and 10 ITIM4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. Three-day-old exponentially growing cells were used in all experiments. Drugs and Drug Treatment. ADR was obtained from Adria Labora tory (Columbus, OH); 4-OOH-CP was kindly provided by Dr. R. Borch (University of Rochester, Rochester, NY) and Dr. P. Hilgard (AstaWerke AG, Bielefeld, Germany). NOVO was purchased from Sigma Chemical Co. Cells were generally treated with cytotoxic agents while still attached to Petri dishes. In some experiments, cells were treated in suspensions, at a concentration of 2-3 x IO5 cells/ml, following standard trypsinization procedures to obtain single cells. Cells were suspended in complete »-minimumessential medium in a type 1vial at 37°Cas described by Whillans and Rauth (10) and were continuously gassed with 95% air-5% CO;. For both treatment procedures, a 10-iil aliquot of a stock solution of the appropriate drug was given per ml of medium. Clonogenic Cell Survival Assay. At the end of drug exposure, cells were washed and single cell suspensions were obtained. The cell number was counted and a variable number of cells were plated to determine survival by clonogenic assay. At an appropriate interval following plating, the exact length of time being cell line dependent, dishes were stained with crystal violet. Colonies containing more than 50 cells were counted to determine cell survival. Centrifugal Elutriation. Cells in various cell cycle phases were isolated by centrifugal elutriation. which separates cells according to size and density (11). Briefly, single cell suspensions of tumor cells were elu triated in ice-cold complete media containing 10% fetal calf serum. During the elutriation, the reservoir, rotor, and collection flasks were kept at 4°C.After 10*cells were loaded into a Beckman JE-6 elutriator rotor, subfractions were collected at decreasing rotor speeds. Cell volume and number were determined with a Coulter Channelyzer. Since most tumor cells are aneuploid. flow cytometry was used as a second measure of tumor cell purity. Cells were fixed with 70% methanol and stained with mithramycin or propidium iodide, and the fluorescence intensity was quantilated by a Coulter EPICS V flow cytometer. Glutathione Assay by High Performance Liquid Chromatograph). Cellular GSH content was analyzed by the high performance liquid chromatography technique described previously (11). Briefly, tumor cells (2-5 x IO5) were washed with phosphate-buffered saline, and centrifuged at 400 x g for 10 min at 4°C,and the cell pellet was stored at -70°C before analysis. Thawed cell pellets and frozen-solid tumor cubes (1 mm') were homogenized with 200 n\ or 20 volumes (w/v), respectively, of a 20 ITIM5-sulfosalicylate solution and centrifuged in an Eppendorf microcentrifuge for 40 s. The supernatant was derivatized Received 1/2/91 ; accepted 4/21/92. using the fluorescent reagent monobromobimane. The fluorescent GSH The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in conjugate was eluted in a Waters Radial-PAK reversed-phase bonded accordance with 18 U.S.C. Section 1734 solely to indicate this fact. octadecylsilane (CIK)cartridge column (8 mm inside diameter) using 1This work is supported by NIH Grant CA-36858 and BRSG Sub isocratic conditions consisting of a mobile phase of 23% acetonitrile in S7RR05403-27. 2To whom requests for reprints should be addressed, at Experimental Thera 40 ITIMammonium phosphate buffer, pH 7.2, containing 5 mM tetrapeutics Department, Pharmaceutical Research Institute. Bristol-Myers Squibb butylammonium hydroxide, and a flow rate of 3 ml/min. Fluorescence Co., 5 Research Parkway, P. O. Box 5100, Wallingford. CT 06492-7660. of the effluent was detected at 410 nm with excitation at 340 nm. GSH "The abbreviations used are: Topo II, topoisomerase 11; NOVO, novobiocin; concentration was determined from peak height with reference to a ADR, Adriamycin; GSH. glutathione; 4-OOH-CP, 4-hydroperoxycyclophos phamide. linear calibration curve constructed using synthetic GSH standards. 3515 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1992 American Association for Cancer Research. NOVOBIOCIN MODULATION OF ANTICANCER DRUG ACTION Typical performance characteristics of this assay are: lower limit of detections, 8.0 pmol on column; coefficient of variation, 4-8%; accu racy, 92-109%. RESULTS Cell Cycle Selective Cytotoxicky of NOVO NOVO exhibits pronounced cell cycle phase selectivity in its toxic action. Following a 4-h treatment with 0.3 HIM NOVO, lethality was observed only for a small population of cells in the G]-S boundary of the cell cycle (Fig. 1). For this particular subpopulation, clbnogenic cell survival was reduced to ~30% of control. Cells in other parts of the cell cycle were completely unaffected. 4 hr 4 hr C O •¿ <—t *j 1 ü 11 CO 24 5-, i-i hr 24 hr 24 hr 00 C CO G2M 0.1 2500 2000 3000 Cell volume 3500 (/urn ) Fig. 1. Cell cycle-specific cytotoxicity of NOVO. Monolayer cultures of MLS cells in exponential growth were treated with 0.3 IHM NOVO or phosphatebuffered saline for 4 h. Single cell suspensions were then prepared for centrifugal elutriation. Cell cycle phase distribution was determined by flow cytometry following DNA labeling with propidium iodide. Fig. 3. Effects of two different concentrations of NOVO (0.3 and 1.8 m\i) on the cell cycle transit of S and ( ;. M cells. A mixture of S and G2M cells obtained by centrifugal elutriation were exposed to NOVO at time 0. DNA content was determined by flow cytometry analysis of propidium iodide-labeled cells at the indicated time. Effects of NOVO on Cell Cycle Progression c 'S IDO E 80 CJ O ü 8X10 7X105 M S 0) O 6X10 5 5X10 O 0) O tt 3X10 10 Time 20 30 (hr) Fluorescence Intensity Fig. 2. Effects of NOVO (0.3 m.\t) on the cell cycle progression and growth of the human ovarian tumor cell line MLS. Purified G, cells were plated at time 0 in complete medium with or without NOVO. At various times after, triplicate plates of cells were trypsinized, cell number was counted, and DNA content was determined by flow cytometry. A, percentage of G, cells remaining at various times after plating (initial percentage of d cells, 89%). The major effect of NOVO (•)was the blockage of G, to S phase transition as compared with controls (O). As a result, cell division was inhibited (B). Cani D, DNA histograms of initially purified G, cells at various times following plating with or without NOVO, respectively. NOVO at a concentration of 0.3 HIM inhibited the cell cycle progression from G, to S phase (Fig. 2). Fig. 2, A and C, shows that under normal conditions the number of G, cells in a Gì phase-enriched cell population declined progressively with time as individual cells transited the cell cycle. At 16 h, the number of G, cells reached a minimum (31%), while 44 and 25% of the cells were in S and G^M phase, respectively. With Novo, the G, to S transition was blocked (Fig. 2, A and D). At 16 h, 73% of the cells remained in the late G, phase of the cell cycle while 17 and 10% were in S and G:M phases, respectively. The effects of NOVO on cell cycle progression are confirmed by cell growth measurement (Fig. 2Ä).Under control conditions the number of cells roughly doubled 30 h following the plating of G, cells. With NOVO in the growth medium, however, no increase in cell number was apparent. The effects of NOVO (0.3 HIM) on cell cycle progression appeared to be specific for cells in the G,-S transition phase. Cells in S and G:M phases progressed normally through the cell cycle at similar rates as cells under control conditions (Fig. 3). However, at very high NOVO concentration (1.8 HIM),cell cycle progression was blocked at the G:M phase (Fig. 3). Cell Cycle-dependent Cytotoxicity of ADR and 4-OOH-CP ADR. The four different cell lines investigated in this study all exhibited similar patterns of cell cycle phase-dependent responses to ADR (Figs. 4A and 5Ä).Cells in the G, phase of the cell cycle were always the most resistant, whereas S and 3516 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1992 American Association for Cancer Research. NOVOBIOriN MODULATION OF ANTICANCER DRUG ACTION NOVO after 4-OOH-CP was removed produced no sensitization effect (Fig. 6Ä). «,•<<sç£> •¿'" >-... C 0u Effects of NOVO on Glutathione Metabolism - À S"AÀ Gi oCi_Dr/l0.01 Because GSH is known to be a critical determinant of the cytotoxicity of 4-OOH-CP (12-14), we have carried out studies on the effects of NOVO on the status of GSH metabolism using the MLS tumor cell line. As depicted in Fig. 8/Õ,NOVO treatment alone did not affect the GSH level of cells in all phases of the cell cycle. These results show that the enhance ment of 4-OOH-CP cytotoxicity by NOVO was not simply due to a direct effect on the level of GSH per se. However, the sensitizing action of NOVO may nevertheless involve GSH. This possibility was suggested by the observation that NOVO enhanced the GSH depletion induced by 4-OOH-CP. Previous dose-response studies where tumor cells were incubated with various concentrations of 4-OOH-CP have shown that deple tion of GSH occurred only with cytotoxic concentration of 4OOH-CP. Significant depletion invariably indicates a break down of the cellular defense mechanism which protects cells from damage by the toxic metabolites of 4-OOH-CP (14). Additional data suggested that in treated cells GSH was lost through conjugation reaction with the GSH-reactive toxic me tabolites of 4-OOH-CP, acrolein and phosphoramide mustard. The present finding, shown in Fig. 8Ä,that cells treated with s 0.001 - A1 A 0.0001 -.*..,_*\ , <=,, s 800 ,C,M , o, , 1800 s A ,C2M , 2800 3800 4800 Celi volume (um^) i o B '. 0.1 •¿, " 0.01- J_j_^i 500 1500 -C 2500 u. i wu ].^*,/* 3500 Cell volume I, Oo Fig. 4. /4, cell cycle-dependent cytotoxicity of ADR on the survival of clonogenic cells of three cell lines. O, CHO; A, A43I; •¿, HEp3. Cells were treated with 0.5 ¿ig/mlADR for 3 h. B, cell cycle-dependent cytotoxicity of 4-OOH-CP on the survival of clonogenic cells of three cell lines. O, CHO; A, A431;T, MLS. Cells were treated with 10, 50, and 25 MM4-OOH-CP for 3 h, respectively. \Q/Y°\à '°\3 •¿Â¿ ;^0 ' •¿| 0.010-D(0.002-0A 10 G^M phase cells were invariably more sensitive. 4-OOH-CP. A radically different pattern of cell cycle-de pendent responses was observed for 4-OOH-CP. For this cytotoxic agent, cells in the early G, phase of the cell cycle were always the most sensitive to the cytotoxicity of 4-OOH-CP. As cells traversed the cell cycle from G, through S to G2M their sensitivities to 4-OOH-CP became progressively reduced (Fig. 45). Effects of NOVO on the Cell Cycle-dependentCytotoxicities of ADR and 4-OOH-CP ADR. NOVO protected exponentially growing cells (both Chinese hamster ovary and A431) against the cytotoxicity of ADR (Fig. 5A). The protective efficacy of NOVO was concen tration dependent; significant protection was observed at a concentration of 0.01 HIM.In addition, the protective efficacy of NOVO was cell cycle dependent; cells in S phase were protected to a greater degree than cells in G, phase (Fig. 5B). 4-OOH-CP. NOVO enhanced the cytotoxicity of 4-OOHCP (Fig. 6A). The sensitizing activity of NOVO was dose dependent; its effectiveness increased with increasing NOVO concentration, from 0.01 to 0.3 IHM(Fig. dB). However, in contrast to its effects on ADR, the enhancement by NOVO of cellular 4-OOH-CP chemosensitivity was not cell cycle depend ent (Fig. 7). The timing of NOVO treatment also appeared to be critical for its activity. Simultaneous presence of NOVO and 4-OOH-CP was essential for interaction; treatment of cells with 0.2 1.[Novobiocin] 0.4 0.6 0.8 (mM) C 0.1 - o 4_> o IS t-, OD C 0.01 - 2000 4000 3000 Cell volume (/um Fig. 5. . I. dose-dependent protection of two cancer cell lines against ADR cytotoxicity by NOVO. O, A431; »,HEp3. Cells were incubated with NOVO (0.3 HIM) for 1 h initially followed by the coincubation with ADR (0.5 ng/ml) and NOVO (0.3 mM) for an additional 3 h. Bars, SD. B, cell cycle-dependent protection by NOVO (0.3 m.M)against the cytotoxic action of ADR in the MLS cancer cell line. Cells were treated for 3 h with 0.25 fig/ml ADR. Single cell suspensions were then prepared for centrifugal elutriation. O, ADR alone; •¿, ADR plus NOVO. Bars. SD. 3517 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1992 American Association for Cancer Research. NOVOBIOCIN MODULATION OF ANTICANCER DRUG ACTION both NOVO and 4-OOH-CP exhibited greater degrees of GSH depletion than cells treated with 4-OOH-CP alone suggests that NOVO may act through a mechanism which resulted in an increase in the level of toxic 4-OOH-CP metabolites. The precise mechanism by which this may occur is not yet known. DISCUSSION The problem of acquired drug resistance to antineoplastic agents by cancer cells is currently attracting a good deal of research interest. For the DNA intercalator class of antineo plastic agents, a number of biochemical mechanisms of acquired resistance have been identified (for a review see Ref. 13). However, the existence of innately resistant cell subpopulations within the solid tumor mass may also be an important cause of treatment failure in patients receiving first-line chemotherapy. A good case in point is the innate resistance of nonproliferating cells to Topo II-interactive antineoplastic agents (e.g., Adria- 10 0 10 20 30 40 50 [4-OOH-CP] 1E-1 1 15- |E-2J GO t) "o e I 10 - 1e-40.1 Novobiocm 0.2 conc. 0.3 c o o (mM) Fig. 6. A, effects of NOVO on the cytotoxicity of 4-OOH-CP in the MLS ovarian cancer cell line. Cells were incubated with NOVO (0.3 mM) for l h followed by coincubation with various concentrations of 4-OOH-CP for an additional 3 h. NOVO enhanced the cytotoxicity of 4-OOH-CP by a factor of 2.1 at the 10% survival level. Bars. SD. B, effects of incubation with NOVO (0.3 mM), simultaneously with or following 4-OOH-CP exposure, on the cytotoxicity of the alkylating agent. The MLS human ovarian cancer cells were treated either simultaneously with 4-OOH-CP and NOVO for 4 h (•),or in two stages: first with 4-OOH-CP for 4 h, washed and then incubated with NOVO for an additional 4 h (A). Bars, SD. {--Õ 1.0 -. 5 - 1 00 U 1500 2000 2500 Cell volume B (/um 3 3500 ) 100 - DC co Ãœ ï-NoYOblocm (lona 3000 80 - 13 13 •¿ (•H O 4-OOH-CP llana ï 0 •¿l-< 1-1 O v> 0.01 - 0 10 20 30 40 50 [4-OOH-CP] 0.001 1800 2400 3000 3600 Cell Volume (pm3) Fig. 1. Effects of NOVO on the cell cycle-dependent cytotoxicity of 4-OOHCP in the MLS ovarian cancer cell line. Cells were treated with NOVO (0.3 mM) for 1 h followed by coincubation with 25 JIM4-OOH-CP for another 3 h. Single cell suspensions were then prepared for centrifugal elutriation. Bars, SD. Fig. 8. A, effects of NOVO on the GSH content of MLS human ovarian cancer cells in different phases of the cell cycle. Exponentially growing cells were treated with NOVO (0.3 mM) for 4 h prior to separation into the various cell cycle phases by centrifugal elutriation. Bars, SD. B, NOVO enhanced the GSH contentdepleting effects of 4-OOH-CP. MLS cells were incubated with NOVO (0.3 m\i) for 1 h followed by coincubation with various concentrations of 4-OOH-CP for another 3 h. Bars, SD. 3518 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1992 American Association for Cancer Research. NOVOBIOCIN MODULATION OF ANTICANCER DRl'Ci ACTION mycin, etoposide). Several recent studies have associated the "resistance" of nonproliferating cells to Topo II-interactive antineoplastic agents with reduced Topo II activity in these cells (7, 9,15). Similarly, the greater sensitivity of cells in active DNA synthesis (S phase) has been correlated with increased Topo II activity in this cell cycle phase (9, 16). The present results obtained using a variety of different tumor models clearly support this hypothesis. An important feature of the present investigation is the use of countercurrent centrifugal elutriation to obtain cell popula tions in different cell cycle phases. This technique has two advantages: (a) cells in different cell cycle phases can be ob tained simultaneously from the same exponentially growing cell populations; (/») prior manipulation of growth conditions is not required, thus avoiding complications resulting from growth perturbation (11). The characteristic cell cycle-dependent cytotoxicity of ADR demonstrated for the four different cell lines in this study is in agreement with previously published data (17, 18). Cells in S phase as a rule showed the greatest and G, cells the least sensitivity to ADR (Figs. 4A and 5B). The findings that NOVO abolished the cell cycle-dependent cytotoxicity of ADR (Fig. 5B) are consistent with the hypothesis that the cytotoxicity of ADR is partly caused by DNA strand breakages resulting from the stabilization of DNA "cleavable complexes" (19, 20). According to this hypothesis the greater sensitivity of proliferating cells to ADR, as compared to stationary phase cells, is a consequence of the higher Topo II activities in actively dividing cells (9, 15). The present findings suggest that NOVO may abrogate the Topo Il-mediated cell cycle-dependent cyto toxicity of ADR. This interpretation is in agreement with other studies using DNA intercalators other than ADR (9, 15, 21). It should be noted, however, that the work of Smith and Bell (22) suggested that at least part of the effects of NOVO may be attributed to a reduction in cellular accumulation of ADR. The effects of NOVO on 4-OOH-CP cytotoxicity have not been previously reported, although a number of studies have investigated its effects on other alkylating agents (4, 5). In this study we showed that NOVO augmented the cytotoxicity of 4OOH-CP in exponentially growing MLS human ovarian cancer cells by a dose enhancement factor of 2.1 at the isoeffect of 1 log cell kill (Fig. 6/1). Unlike its effects on ADR, however, the enhancement of 4-OOH-CP cytotoxicity appeared not to be cell cycle dependent (Fig. 7). In addition, 4-OOH-CP treatment alone did not exhibit the same cell cycle-dependent cytotoxicity as that described above for ADR (cf. Fig. 4, A and B). Further more, for the two cell lines investigated thus far, namely A431 and MLS, significant differences in sensitivity to 4-OOH-CP were not observed for exponential versus stationary cells (results not shown). These findings argue against the direct involvement of Topo II perse in the mechanism of alkylating agent toxicity enhancement by NOVO. Other known effects of NOVO may play important roles in this respect. NOVO has been shown to inhibit replicative and repair DNA syntheses (23), affect mitochondrial structure and ATP metabolism (24), and impair DNA and RNA polymerases activity (25). The observation in this study, that NOVO treatment immediately following a 3-h ex posure to 4-OOH-CP did not enhance the activity of the cytotoxic agent (Fig. 6Ä),suggests that NOVO probably effected a process(es) which occurs concurrently with 4-OOH-CP expo sure. We have obtained preliminary results showing that com bined 4-OOH-CP and NOVO caused a greater degree of glutathione depletion than 4-OOH-CP alone. In previous studies we have established that glutathione depletion is an effective indicator of cytotoxicity because it accurately reflects the amount of alkylating toxic species formed from the parent 4OOH-CP (14, 26). Therefore, the greater depletion of GSH by combined NOVO and 4-OOH-CP may indicate that the for mation of the toxic phosphoramide mustard and acrolein from the parent 4-OOH-CP was enhanced by NOVO. However, the mechanism by which this occurs is not known at present. There is strong evidence that NOVO impairs Topo II activity by inhibiting the ATPase portion of this enzyme (27, 28). The present study shows that treatment of tumor cells with NOVO at concentrations that can inhibit cell cycle progression (from G, to S phase), protect against ADR cytotoxicity, and produce synergistic cell killing with 4-OOH-CP did not in fact cause significant cytotoxicity. It appears therefore that NOVO is comparatively nontoxic for mammalian cells. It should be noted that at extremely high concentrations (>500 n\\) NOVO has been reported to be toxic to mammalian cells (5, 22) and to cause the accumulation of cells in G?M phase of the cell cycle (22, 29). However, as shown in this study, NOVO was effective in protecting against ADR cytotoxicity at the concentration of 50 UMor less (Fig. 5Ä);additionally, Utsumi et al. (21) found that 100 ¿¿M NOVO can abrogate the cytotoxicity of the DNA intercalator 4'-(9-accidinylamino)methanesulfon-w-anisidide. Thus, assuming that the above protective effects of NOVO were the result of its inhibition of Topo II, it is likely that the toxic action of NOVO at high concentrations is not a direct conse quence of Topo II inhibition. The only toxicity that we observed with low NOVO exposure (0.3 m\i for 4 h), as revealed in the cell fractionation experiments, was uniquely cell cycle phase specific and therefore would not otherwise be detected in an unfractionated cell population. Only cells in the Gi-S boundary (which usually constitute less than 5% of the whole population of cells) appeared to be killed by NOVO (Fig. 1). These results suggest that the NOVO treatment may be deleterious to survival only for cells the DNA of which was in the process of being unwound in preparation for DNA synthesis. In addition, NOVO prevented the cell cycle transition from G, to S phase (Fig. 2). Presumably, cells were blocked in the G,-S boundary at a point immediately before the unwinding of the supercoiled DNA. Additional studies concerning the exact position in the G]-S boundary at which cells were blocked, relative to those produced by hydroxyurea and nutrient deprivation, are in prog ress in this laboratory. In conclusion, we have demonstrated that NOVO can abolish the cell cycle-dependent cytotoxicity of ADR and enhance the toxicity of 4-OOH-CP in a variety of tumor cell lines. Prelim inary evidence suggests that these two effects of NOVO may be mediated by different mechanisms. Further investigations into the effects of NOVO on glutathione metabolism, drug uptake and accumulation, and DNA repair may provide important information into the mode of action of this clinically safe agent. This may ultimately aid in the design of combined regimens of NOVO and cytotoxic alkylating agents for the treatment of cancer. ACKNOWLEDGMENTS The authors wish to thank the Cell Separation Facility of the Uni versity of Rochester Cancer Center for technical support; Dr. R. Borch and Dr. P. Hilgard for the supply of 4-hydroperoxycyclophosphamide; and B. King for excellent technical assistance. 3519 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1992 American Association for Cancer Research. NOVOBIOCIN MODULATION OF ANTICANCER DRUG ACTION REFERENCES 1. Glisson, II . Sullivan, I !.. Gupta, R.. and Ross, W. Mediation of multi-drug resistance in a Chinese hamster ovary cell line by a mutant type II topoisomerase. NCI Monog., 4: 89-93, 1987. 2. Beck, W. T., Danks, M. K., Yallowich, J. C. Zamora, J. M., and Cirtain, M. C. Different mechanisms of multiple drug resistance in two human leukemic cell lines. In: P. Woolley and K. Tew. (eds.). Mechanisms of Drug Resistance in Neoplastic Cells, p. 212. San Diego, CA: Academic Press, 1988. 3. Robson, C. N., Hoban. P. R., Harris. A. L., and Hickson, I. D. Crosssensitivity to topoisomerase II inhibitors in cytotoxic drug-hypersensitive Chinese hamster ovary cell lines. Cancer Res., 47: 1560-1565, 1987. 4. Eder, J. P., Teicher, B. A.. Holden, S. A., Cathcart, K. N. S., Schnipper. L. E., and Frei, E., III. Effect of novobiocin on the antitumor activity and tumor cell and bone marrow survivals of three alkylating agents. Cancer Res., 49: 595-598, 1989. 5. Eder, J. P., Teicher, B. A., Holden, S. A., Senator, L., Cathcart, K., and Schnipper, L. E. Ability of four potential topoisomerase II inhibitors to enhance the cytotoxicity of cu-diamminedichloroplatinum(II) in Chinese hamster ovary cells and in an epipodophyllotoxin-resistant subline. Cancer Chemother. Pharmacol., 26: 423-425, 1990. 6. Tan, K. B., Mattern. M. R., Boyce. R. A., and Schein, P. S. Elevated topoisomerase II activity in nitrogen mustard-resistant cell lines. Proc. Nati. Acad. Sci. USA, 84: 7668, 1987. 7. Sullivan, D. M., Latham, M. D., and Ross, W. E. Proliferation-dependent topoisomerase II content as a determinant of antineoplastic drug action in human, mouse, and Chinese hamster ovary cells. Cancer Res., 47: 39733979, 1987. 8. Pommier, Y., Kerrigan, D., Schwartz, R. E., Swack, J. A., and McCurdy, A. Altered DNA topoisomerase II activity in Chinese hamster cells resistant to topoisomerase II inhibitors. Cancer Res.. 46: 3075-3081. 1986. 9. Markovits, J., Pommier, Y., Kerrigan, D., Covey, J. M., Tilchen, E. J., and Kohn, K. W. Topoisomerase Il-mediated DNA breaks and cytotoxicity in relation to cell proliferation and the cell cycle in NIH 3T3 fibroblasts and L1210 leukemia cells. Cancer Res., 47: 2050-2055, 1987. 10. Whillans, D. W., and Rauth. A. M. An experimental and analytical study of oxygen depletion in stirred cell suspensions. Radiât.Res., 84: 97. 1980. 11. Lee, F. Y. F., Siemann. D. W.. Allalunis-Turner, M. J.. and Keng, P. C. Glutathione contents in human and rodent tumor cells in various phases of the cell cycle. Cancer Res., 48: 3661-3665, 1988. 12. Crook, T. R.. Souhami. R. L., Whyman, G. D.. and Mclean, A. E. M. Glutathione depletion as a determinant of sensitivity of human leukemia cells to cyclophosphamide. Cancer Res., 46: 5035-5038, 1986. 13. Moscow, J., and Cowan, K. Multidrug resistance. J. Nati. Cancer Inst., 80: 14-20, 1987. 14. Lee, F. Y. F., Planner), D. J., and Siemann, D. W. Prediction of tumor sensitivity to 4-hydroperoxycyclophosphamide by a glutathione-targeted as say. Br. J. Cancer, 63: 217-222, 1991. 15. Zwelling, L. A.. Estey, E., Silberman, L., Doyle, S., and Hittelman, W. Effect of cell proliferation and chromatin conformation on intercalator-induced. protein-associated DNA cleavage in human brain tumor cells and human fibroblasts. Cancer Res., 47: 251-257, 1987. 16. Dillenay, L. E., Denstman. S. C.. and Williams, J. R. Cell cycle dependence of sister chromatid exchange induction by DNA topoisomerase II inhibitors in Chinese hamster V79 cells. Cancer Res., 47: 206-209, 1987. 17. Barranco, S. C. Review of the survival and cell kinetics effects of Adriamycin (NSC-123127) on mammalian cells. Cancer Chemother. Rep., 6: 147-152, 1975. 18. Kim, S. II and Kim, J. H. Lethal effect of Adriamycin on the division cycle of HeLa cells. Cancer Res., 32: 323-325, 1972. 19. Tewey, K. M., Rowe, T. C., Yang, L.. Halligan, B. D., and Liu, L. F. Adriamycin-induced DNA damage mediated by mammalian DNA topoisom erase II. Science (Washington DC). 226: 466-468. 1984. 20. Zwelling. L. A., Kerrigan, D.. and Michaels, S. Cytotoxicity and DNA strand breaks by 5-iminodaunorubicin in mouse leukemia 11210 cells: comparison with Adriamycin and 4'-(9-acridinylamino)methanesulfon-m-anisidide. Can cer Res., 42: 2687-2691, 1982. 21. I isiimi. H., Shibuya, M. L., Kosaka, T., Buddenbaum, W. E., and Elkind, M. M. Abrogation by novobiocin of cytotoxicity due lo thètopoisomerase II inhibitor amsacrine in Chinese hamster cells. Cancer Res., SO: 2577-2581, 1990. 22. Smith, P. J., and Bell, S. M. A DNA topoisomerase H-independent route for novobiocin-mediated resistance to DNA binding agents. Cancer Chemother. Pharmacol., 26: 257-262, 1990. 23. Mattern, M. R., and Scudiero, D. A. Dependence of mammalian DNA synthesis on DNA supercoiling. III. Characterization of the inhibition of replicative and repair-type DNA synthesis by novobiocin and nalidixic acid. Biochim. Biophys. Acta, 653: 248-258, 1981. 24. Downes, C. S., Ord, M. J., Mulligan, A. M., Collins. A. R. S., and Johnson, R. T. Novobiocin inhibition of DNA excision repair may occur through effects on mitochondria! structure and ATP metabolism, not on repair topoisomerases. Carcinogenesis (Lond.), 6: 1343-1352, 1985. 25. Edenberg, H. Novobiocin inhibition of simian virus 40 replication. Nature (Lond.), 286: 529-531. 1980. 26. Lee, F. Y. F. Glutathione diminishes the antitumor activity of 4-hydroperox ycyclophosphamide by stabilizing its spontaneous breakdown to alkylating metabolites. Br. J. Cancer. 63: 45-50, 1991. 27. Wang, J. C. Recent studies of DNA topoisomerases. Biochim. Biophys. Acta, 909: 1-9, 1987. 28. Geliert, M., O'Dea, M. H.. Itah, T.. and Tomizawa, J. Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc. Nati. Acad. Sci. USA, 73: 3872-3878, 1976. 29. Rowley, R., and Kort. L. Novobiocin, nalidixic acid, etoposide, and 4'-(9acridinylamino)methanesulfon-m-anisidide effects on G3 and mitotic Chinese hamster ovary cell progression. Cancer Res., 49:4752-4757, 1989. 3520 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1992 American Association for Cancer Research. Modulation of the Cell Cycle-dependent Cytotoxicity of Adriamycin and 4-Hydroperoxycyclophosphamide by Novobiocin, an Inhibitor of Mammalian Topoisomerase II Francis Y. F. Lee, Deborah J. Flannery and Dietmar W. Siemann Cancer Res 1992;52:3515-3520. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/52/13/3515 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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