Chemical enhancement of cisplatin cytotoxicity in a human ovarian and cervical cancer cell line

GYNECOLOGIC

ONCOLOGY

38, 315-322 (1990)

Chemical Enhancement of Cisplatin Cytotoxicity in a Human Ovarian and Cervical Cancer Cell Line GUY M. BoIKE,“.’ EDGAR Peru,‘? BERND-UWE SEWN,* HERVY E. AVERETTE,* TING-CHOA CHOU,$ MANUEI PENALVER,* DANIEL DONATO,* MICHAEL SCHIANO, * SUSAN G. HILSENBECK,~ AND JAMES PERRAS* *Division qf Gynecologic Oncology, University of Miami. Miami, Floridu 33101; tDepartment of Obstetrics and Gynecology, University oj Graz, Graz. A-8036 Austria, *Department of Pharmacology. Memorial Sloan Kettering Cancer Center, New York, New York 10021; and iDivision of Biostutistics, University of Miami School of Medicine, Miami, Florida 33101

Received December 2, 1989

While many advances have been made in the chemotherapy of gynecologic cancers, treatment failures remain a major clinical problem. A growing understanding of the mechanisms of tumor cell resistance to antineoplastic drugs provides a framework for the development of chemotherapy regimens containing agents capable of modulating tumor response. Using a short-term ATP bioluminescence assay we studied the ability of two methylxanthines (caffeine, pentoxifylline) and an inhibitor of ADP-ribosyl transferase (3-aminobenzamide) to enhance cisplatin cytotoxicity in gynecologic cancer cell lines. Our findings of significantly enhanced cisplatin-induced cytotoxicity with two different analysis techniques confirms the effectivenessof these agents. These results may have future clinical significance. 0 1990 Academic Press. Inc. INTRODUCTION

Cisplatin (DDP) is one of the most active antineoplastic agents against gynecologic cancers. While encouraging response rates have been observed in ovarian cancer patients, treatment failures are common, presumably due to development of cisplatin-resistant tumor populations [l-3]. In cervical cancer, cisplatin is the most active single agent studied; however, low response rates of short duration are common [4,5]. The mechanisms of tumor cell resistance to cisplatin are poorly understood but may involve decreased drug uptake, increased levels of intracellular thiols (e.g., glutathione, metallothioneins), decreased DNA platination, and enhanced tumor cell repair of cisplatin-damaged DNA. In experimental models of ovarian cancer, investigators have

reported modulation or enhancement of cisplatin’s antineoplastic effect by calmodulin antangonists [6], glutathione-depleting agents [7,8], and inhibition of DNA repair [9]. Few studies exist exploring modulation of cisplatin-induced cytotoxicity in human cervical cancer models. Conflicting reports exist regarding caffeine enhancement of cisplatin cytotoxicity in the HeLa cell line [IO, 111. In vitro studies have demonstrated modulation of radiation-induced killing by a variety of agents thought to interfere in DNA repair [ 12- 141. The potential utility of these compounds in modulating tumor cell sensitivity to cisplatin remains to be defined. Recent interest in the chemical modulation of cancer treatment [15-171 is based on the growing understanding of mechanisms involved in tumor cell resistance and the ability of various nontoxic compounds to sensitize or restore tumor cell susceptibility to standard cytotoxic therapy. With the slow introduction of new active cytotoxic drugs, it is likely that agents capable of modifying tumor cell response to standard therapy will assume increasing importance in cancer chemotherapy. MATERIALS

’ American Cancer Society Clinical Oncology Fellow. To whom all correspondence should be addressed at Department of Obstetrics and Gynecology, Division of Gynecologic Oncology (D-52), P. 0. Box 016960, Miami, FL 33101.

AND METHODS

Cell culture. The human cervical carcinoma cell line, ME-180, and the human ovarian cancer cell line, CAOV3, were obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in Eagle’s modified essential medium (EMEM) supplemented with 5% fetal calf serum (FCS), 100 pg/ml streptomycin, and 100 units/ml penicillin (GIBCO, Grand Island, NY) in a 5% CO2 atmosphere at 37°C. Cells were removed from tissue culture flasks by treatment with 0.25% trypsin + 0.02% EDTA for 10 min at 37°C.

315 0090-8258190 $1.50 Copyright 0 1990by Academic Press.Inc. All rights of reproductionin any form reserved.

316

BOIKE ET AL.

Chemicals. Caffeine, pentoxifylline, and 3-aminobenzamide were purchased from Sigma Chemical Company (St. Louis, MO). Cisplatin was generously provided by Bristol Laboratories (Evansville, IL). Caffeine, pentoxifylline, and cisplatin were freshly dissolved in water, filtered sterilized using a 0.2-pm acrodisc (Gellman Sciences, Ann Arbor, MI), and further diluted in medium. 3-Aminobenzamide was freshly dissolved in 95% ethanol, filter sterilized, and further diluted in medium prior to use. ATP Bioluminescence Studies. For ATP bioluminescence studies, 2 x lo4 cells were plated in 24-well tissue culture plates (Costar, Cambridge, MA) in a final volume of 1 ml. After overnight incubation, cells were exposed to cisplatin for 90 min at concentrations bracketing the reported peak plasma level of 2.5 pug/ml [18]. Following the addition of fresh medium, inhibitors were added from concentrated stocks for an additional 24-hr period. On the seventh day aftr cisplatin exposure, assays were terminated and ATP was extracted with the addition of 1 ml of 2% trichloroacetic acid (TCA) to each culture well. The media, cells, and TCA were gently mixed with a pipet and a 100-~.~1 aliquot from each well was transferred to a plastic tube (12 x 55 mm). The TCA extract was neutralized to pH 7.8 by the addition of an equal volume of 0.1 M Tris buffer, pH 9.0, and a 20-~1 aliquot from each sample was placed in a luminometer (United Technologies Packard, Downers Grove, IL). Following injection of 50 ~1 of luciferin-luciferase reagent (Picozyme, Los Alamos Diagnostics Inc., Los Alamos, NM) the luminescence was determined for 20 sec. For each assay, a standard curve relating ATP concentration to luminescence was performed. Data analysis and statistical evaluation. ATP bioluminescence values were converted to surviving fraction (fractional ATP) by dividing luminescence values of experimental groups by those of untreated control groups. All experiments were done in triplicate or quadruplicate and repeated two or three times. Results of representative experiments were analyzed with a two-way analysis of variance (DDP dose vs DDP + modulator combination). Analyses of log-transformed survival data were conducted using both standard F tests and Brown Forsythe statistics, which are an F test analog of an unequal variance t test. Group means were compared using contrasts from the standard analysis. The analyses were performed using SAS (PROC GLM) [19] and BMDP (Program 7D) [20]. The cisplatin concentration or dose necessary to produce 50% inhibition (I&) was calculated using the median effect plot of log(f,/f,) versus log D where f, = fraction unaffected = surviving fraction (SF), fa = fraction affected = (l-SF), and D = dose of cisplatin [2 1,221. A second group of experiments were designed and

A

?-

ATP STANDARD CURVE

,lS

.I4 LOG

4 ! 1 LOG

(+

-13

-i2

ATP(MOLE

COUNTED)

2

3

OF

CELLS

-11

1 4

COUNTED)

FIG. 1. (A) ATP standard curve. ATP standard was serially diluted in medium and treated with TCA and neutralizing buffer, and ATP bioluminescence determined on a 20-~1 aliquot (4 x 10-l* to 7.8 x IO-” mole). (B) ATP bioluminescence vs cell number. Cells were serially diluted in medium from I x IO6 to 5 x lo3 cells/ml. After preparation, ATP bioluminescence determinations were made on 2O/pl aliquots (2500 to 12,500 cells). Mean values of triplicate samples are shown. Standard deviations (SD) were <5% of means.

analyzed using the median effect analysis technique of Chou and Talalay [21,22]. This design calls for a doseresponse curve to cisplatin alone, modulator alone, and combinations of cisplatin and modulator in increasing doses at a fixed ratio. Survival data from these experiments were analyzed with the Dose-Effect Analysis with Microcomputers program (Elsevier-Biosoft, Cambridge, United Kingdom) [23]. RESULTS Characterization of the ATP bioluminescence assay. Figure 1A depicts the linear relationship between logtransformed luminescence and amount of ATP assayed. Serial dilutions from a frozen stock of an ATP standard (Picochec, Los Alamos Diagnostics Inc.) were assayed after treatment with TCA and neutralizing buffer as out-

MODULATION

OF CISPLATIN

ME-180

-

1 0

4

2

, 8

6

DAYS AFTER CISPLATIN EXPOSURE

FIG. 2. ATP levels vs time in ME-180 cells exposed to varying concentrations of cisplatin. ME-180 cells (2 X 104)were placed in 24well culture plates. Twenty-four hours later, cells were exposed to varying concentrations of cisplatin (DDP) for 90 min. Serial ATP determinations (0 to 7 days) were performed. Luminescence values were converted to moles of ATP per well using the ATP standard curve and graphed as the means f SD of quadruplicate determinations.

A

ME-180

-

DwMctE

-

DDP+PTXl

-

DDP + PTX 2.5

CYTOTOXICITY

317

lined under Materials and Methods. The linear regression coefficient (v’) was greater than 0.95 for all ATP standard curves performed. Figure 1 reveals that the ATP bioluminescence assay is highly sensitive, capable of quantitating less than 1 x lo-l4 mole of ATP. Under static conditions, a linear relationship between cell number and luminescence was noted for the ME-180 cell line (Fig. 1B) with a linear regression coefficient (r”) of 0.999. Under assay conditions, lo-20 tumor cells per 2Wgmlaliquot can be accurately quantified. Dynamic changes in ATP levels with time in ME-180 cells untreated or exposed to cisplatin are displayed in Fig. 2. ATP bioluminescence determinations on the initial cell suspension (20,000 cells/ml) revealed that ME-180 tumor cells contain approximately 6.2 x IO-” mole ATP/cell. Analysis of ATP levels with time in untreated ME-180 controls yields an ATP doubling time of less than 24 hr. A dose- and timedependent effect on ATP levels was noted following exposure to cisplatin. The cytotoxic effects of cisplatin appear to occur early after treatment, with decreases in ATP levels noted as early as 24 hr. At lower concentrations of cisplatin (1.25 and 2.5 pg/ml) cells subsequently resumed proliferation at a rate similar to that of untreated controls, but at higher doses (5.0 and 10.0 pg/ml) ATP levels persisted at or decreased below initial values. Survival curves generated from these data revealed very similar survival curves at 3, 5, and 7 days following exposure to cisplatin (curves not shown). An assay time of 7 days was chosen for all subsequent experiments. Chemical enhancement of cisplatin-induced cytotoxby icity. Modulation of cisplatin-induced cytotoxicity

B

CAOV-3

2.5

-

DDPMONE

-

DDP+PTXl

-

DDP + PTX 2.5

5.0

DDP DOSE (wa/rnt)

FIG. 3. Modulation of cisplatin cytotoxicity by pentoxifylline. Cells were exposed to cisplatin for 90 min followed by pentoxifylline (PTX, final concentration I .O or 2.5 m&f) for an additional 24 hr. Day 7 ATP survival data are graphed from means of quadruplicate samples. The SDS were less than 10% of the means.

fixed concentrations of pentoxifylline (1 or 2.5 mM) are shown in Fig. 3. Pentoxifylline enhanced cisplatin cytotoxicity in both the human cervical cancer cell line ME-180 (Fig. 3A) and the human ovarian cancer cell line CAOV-3 (Fig. 3B). Increasing concentrations of pentoxifylline, while relatively nontoxic to both cell lines, produced a concentration-dependent enhancement of cisplatin cytotoxicity. The modulation of IC,,‘s and surviving fraction at the clinically achievable peak plasma concentration for cisplatin (2.5 pg/ml) is shown in Table 1. Postcisplatin treatment with pentoxifylline reduces the I& dose of cisplatin for both cell lines, resulting in 1.3- to 3.4-fold enhancement of cytotoxicity. At the cisplatin peak plasma concentration (PPC) of 2.5 pgjml 1181, pentoxifylline produces up to a 19.5-fold enhancement of cytotoxicity (Table 1). Modulation of cisplatin-induced cytotoxicity by fixed concentrations of caffeine (1 or 2.5 mm is depicted in Fig. 4. Caffeine enhanced cisplatin cytotoxicity in both ME-180 (Figure 4A) and CAOV-3 (Figure 4B) cell lines in a magnitude similar to that of pentoxifylline. The en-

318

BOIKE ET AL.

TABLE 1 Summary of Cytotoxicity Data Cell line

Drug combination

ME-180

ME-180 CAOV-3

DDP DDP DDP DDP DDP DDP DDP DDP DDP DDP DDP DDP

Enhancement of I&

Enhancement at PPC (2.5 pg/ml)

2.53 2.01 0.74

1.30 3.40

1.80 19.50

1.60 0.95

1.60 2.70

3.60 17.40

1.32

1.83

0.91

2.65

4.10 11.10 -

IGO” b.dml DW

alone + PTX 1 mM + PTX 2.5 mM + CAF 1 mM + CAF 2.5 mM + 3-AMB 5 mM + CAF 1 mM + 3-AMB 5 mM alone + PTX 1 mM + PTX 2.5 mM + CAF 1 mM + CAF 2.5 mM

1.54 0.61

2.52 2.90 2.52 2.90

0.53 0.61 0.53

8.25 15.00 7.02

11.00

” Concentration of cisplatin required to cause 50% decrease in surviving fraction (e.g., SF = 0.5).

-wpMCNE -

DDP+CAFl

-

DDP + CAF 2.5

hancement in I&, values and observed cytotoxicity at the cisplatin PPC of 2.5 pg/ml are shown in Table 1. Enhancement of cisplatin cytotoxicity by 5 mM 3-aminobenzamide (3-AMB) and the combination of 1 mM caffeine plus 5 mM 3-AMB is represented graphically in Fig. 5. While 3-AMB enhanced cytotoxicity, the combination of caffeine plus 3-AMB produced an even greater potentiation of cell kill, as demonstrated by the 2.65-fold enhancement in I(& for DDP + CAF + 3AMB versus 1.83-fold for DDP + CAF (Table 1). Statistical analysis of the above data with a two-way ANOVA are shown in Table 2. Fixed effects were assumed. Within-cell effects varied greatly, and a log transform was only partially successful in stabilizing the vari-

2.5 DDP DOSE

B

(kO/ml)

ME-180

CAOV-3

-

DOPKONE

-

DDP+CAFl

-

DDP + CAF 2.5

.

f-

I---

DDPALCNE DDP+SAMB DDP + CAFL-AMB

DDP DOSE

@g/ml)

2.5 DDP DOSE (pg/ml)

FIG. 4. Modulation of cisplatin-induced cytotoxicity by caffeine. Following cisplatin exposure (90 min), caffeine (CAF, 1 or 2.5 rnM) was added for an additional 24 hr. Day 7 ATP survival data are shown as means of quadruplicate samples with SDS < 10% of the means.

FIG. 5. Modulation of cisplatin-induced cytotoxicity by 3-aminobenzamide with or without caffeine. Following cisplatin exposure (90 min), 3-aminobenzamide (3-AMB, 5 mM) or caffeine (1 mM) and 3aminobenzamide (5 mM) were added for 24 hr. Day 7 ATP survival data are shown as means of triplicate samples with SDS < 10% of the means.

MODULATION

OF CISPLATIN

antes. The remaining, moderately significant heteroscedasticity was primarily associated with the dose factor. The analysis results from the F tests and BrownForsythe statistics were nearly identical, and only the standard F test results are reported. Results revealed a significant effect of DDP dose (P < O.OOOl),DDP + all modulators (P < O.OOOl),and interaction of the two (P < 0.0001) for both cell lines. Comparison of DDP alone with each of the experimental groups individually was also highly significant (P < 0.0001). This finding was confirmed by analysis with the Student-Neuman-Keuls test. The second group of experiments examined the interaction of cisplatin with pentoxifylline and caffeine using the median effect analysis technique [21-231. This approach allows definitive determination of the nature of drug interaction over multiple levels of tumor cell kill. In each experiment, survival curves were generated for cisplatin alone, modulator alone (pentoxifylline or cafA 1.5

x

1

ME-180 CISPIATIN + PENTOXIFYLLINE

E z

ANTAGONISM ------------e-w-----

1.0

AD,,,T,“,S,J SYNERGISM

0’ F

I

_.- 0.0 ,

012

0:4

FRACTION

B 1.5

1

0.0-I 0.0

016 AFFECTED

010

110

(Fa)

CAOV-3 CISPLATIN + PENTOXIWNE

0.2 FRACTION

0.4

0.6 AFFECTED

0.8

1.0

(Fe)

FIG. 6. Median effect analysis of cisplatin and pentoxifylline. Following cisplatin exposure (90 min), cells were treated with pentoxifylline for 24 hr according to median effect design with increasing concentrations at a fixed ratio. Day 7 ATP survival data were analyzed using the classical isobologram equations to determine combination indices as a function of tumor cell kill (e.g., Fa = fraction affected).

319

CYTOTOXICITY

feine), and increasing concentrations of cisplatin + modulator in a fixed ratio (1.25 pg/ml: 1 mM). Assays were done in quadruplicate and performed three times. Figure 6 depicts a scatter plot of combination index (CI) versus tumor cell kill (fraction affected) for cisplatin and pentoxifylline. The combination index was calculated from the classical isobologram equations using actual data from all three experiments for each cell line. A CI > 1 indicates antagonism, a CI = 1 indicates additivism, and a CI < 1 synergism. Parameters derived from median effect analysis are summarized in Table 3. Mean combination indices at a level of 50% cell kill (CI 50) of 0.5 to 0.7 indicate a substantial synergistic enhancement of cisplatin cytotoxicity. A regression coefficient of greater than 0.9 indicates that each modulator produced a linear median effect plot with cisplatin and followed the basic mass-action principle. The slopes of the median effect plots for all groups were between 2 and 3, indicating sigmoidicity of the dose-response curves and highlighting the appropriateness of this analysis technique. DISCUSSION The ATP bioluminescence assay is a short-term, nonclonogenic assay that has been used to accurately quantify viable tumor cell number and in vitro tumor growth 124,251. Investigators studying the cytotoxic effects of chemotherapy with the ATP assay have reported excellent correlation with dye exclusion methods [26,27], [jH]thymidine incorporation ob27], and clonogenic stem cell assays [27-291. This chemosensitivity assay has been utilized to screen “fresh” ovarian cancer specimens against antineoplastic agents and appears to have potential predictive ability [30,31]. Our findings of 6.2 x lo-” mole of ATP/ME-180 cell is in the range 1 x lOpI4 to 6 x IO-l5 mole/cell reported for other cancer cell lines [27], and the observed ATP doubling time of ~24 hr correlates well with the cell doubling time of 16-24 hr originally reported for the ME-180 cell line [32]. Many investigators have previously reported that methylxanthines (e.g., caffeine, theobromine, theophylline) are capable of enhancing the cytotoxic effects of alkylating agents, in both in vitro and in vivo tumor models [11, 33-371. Our findings of significant caffeine enhancement of cisplatin-induced cytotoxicity against the human cervical cell line ME-180 and the human ovarian cell line CAOV-3 correlate well with the published literature on caffeine modulation of alkylating agent activity. Various mechanisms for this enhancement have been proposed including inhibition of postreplication repair of DNA, inhibition of poly(ADP-ribose)polymerase, and cell cycle effects with induction of mitosis in damaged G2 cells prior to completion of DNA repair [ 11,33,35,38]. Caffeine has not been used clinically with

320

BOIKE ET AL.

TABLE 2 Summary of Statistical Analyses Cell line

Group analyzed

ME-180

DDP + PTX, CAF

ME-180

DDP + CAF, 3-AMB

CAOV-3

DDP + PTX, CAF

Source of Variation

Degrees of freedom

F value

P value

DDP dose (A) DDP + modulator (II) Interaction (A x B) DDP dose (A) DDP + modulator (B) Interaction (A x B) DDP dose (A) DDP + modulator (B) Interaction (A x B)

2 4 8 3 2 6 3 4 I2

393.50 60.16 14.01 426.09 219.34 32.33 1610.22 210.53 17.40

0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001

chemotherapy due to the inability to achieve the necessary plasma concentrations without untoward side effects. Caffeine levels necessary to enhance chemotherapy tumor cell lethality are approximately 20-fold higher than maximally tolerated blood levels [ 11,39-4 13. Pentoxifylline (Trental, Hoechst-Roussel Pharmaceuticals) is an oxohexyl-substituted analog of theobromine clinically used for the treatment of intermittent claudication [42]. Fingert et al. initially reported enhanced in vitro cytotoxicity of thiotepa and nitrogen mustard against a human bladder cancer cell line when pentoxifylline was added after exposure to the antineoplastic agents [38]. In a subsequent report they demonstrated enhanced thiotepa cytotoxicity against human bladder and breast cancer cell lines, both in vitro and in vivo (murine subrenal capsule assay) [43]. Of additional significance was the finding that pentoxifylline appeared to selectively decrease the recovery capacity of cancer cells versus normal cells, producing an enhanced therapeutic response without increased host tissue toxicity. Pharmacokinetic studies of pentoxifylline in humans demonstrate peak plasma concentrations between 1102 and 1348 rig/ml [44,45]. As a potential chemotherapy modifier, pentoxifylline exhibits the most favorable pharmacokinetic profile of the methylxanthines studied. Our findings of significant enhancement of cisplatin cytotoxicity against both an ovarian and cervical cancer cell line

highlight the need for extended in vitro chemosensitivity testing of this combination against “fresh” gynecologic tumor samples and for controlled clinical trials. The findings presented here demonstrating a significant statistical interaction between cisplatin drug dose and enhanced cytotoxicity with pentoxifylline (Table 2) may have particular relevance to both cervical and ovarian cancer. The implication of this finding is that higher concentrations of cisplatin with pentoxifylline will augment enhancement of tumor kill. In cervical cancer, increased response rates (without increased survival) have been observed with higher doses of cisplatin (100 mg/m*) [4]. In ovarian cancer, very high levels of cisplatin can be achieved with intraperitoneal administration [46]. This is, to our knowledge, the first report of enhanced cisplatin cytotoxicity by pentoxifylline. The synthesis of large amounts of poly(ADP-ribose) by ADP-ribosyltransferase (ADPRT) is a common response to DNA damage, such as that caused by alkylating agents [34]. Several inhibitors of ADPRT, including theobromine, caffeine, and 3-aminobenzamide (3-AMB), prevent synthesis of poly(ADP-ribose) in response to DNA damage produced by alkylating agents. 3-AMB is the most specific inhibitor and is approximately 4 to 55 times more potent than theobromine or caffeine, respectively [34]. The primary effect of this inhibition with regard to alkylating agents appears to be inhibition of

TABLE 3 Summary of Median Effect Analysis Data

Cell line ME-180 CAOV-3 ” Mean k SD.

Drug combination DDP DDP DDP DDP

+ + + +

PTX CAF PTX CAF

Combination ratio (dml : mM) 1.25: 1 1.25: 1 1.25:l 1.25: 1

Combination index 50 (SF = 0.5) 0.565 0.792 0.628 0.747

5 t 2 k

0.073” 0.101 0.331 0.256

Regression coefficient 0.984 0.992 0.990 0.991

+ 0.011” k 0.013 r+ 0.014 2 0.012

Number of experiments 3 3 3 3

MODULATION

OF CISPLATIN

DNA excision repair, DNA strand rejoining, and enhanced cytotoxicity [34]. We report that post-treatment with 3-AMB significantly enhanced cisplatin cytoxicity in ME-180 cells and the combination of 3-AMB and caffeine further potentiated this effect. The use of multiple inhibitors of the DNA repair process to modulate the cytotoxic effect of chemotherapy requires further study, but these findings suggests a major role for DNA repair processes in tumor cell resistance to cisplatin. We are currently investigating the cell cycle perturbations induced by the above combinations of cisplatin and modulators with flow cytometry. While many advances have been made in the chemotherapy of gynecologic cancers, treatment failures continue to be a major problem. With the slow introduction of new, active cytotoxic agents into the clinic, it becomes increasingly important to pursue new treatment strategies. The growing understanding of tumor cell resistance to standard cytotoxic chemotherapy provides an opportunity to employ agents which circumvent this phenomenon. Results of our study suggest that nontoxic modulators of cisplatin cytotoxicity, such as pentoxifylline, may be ideally suited for clinical application. Much additional work is needed to establish the potential utility of this novel form of adjuvant therapy. REFERENCES 1. Goldie, J. H., and Coldman, A. J. The genetic origin of drug resistance in neoplasms: Implications for systemic therapy, Cancer Res. 44, 3643-3653 (1984).

2. Young, R. C. Ovarian cancer treatment: Progress or paralysis? Semin. Oncol. 11, 327-329 (1984). 3. Ozols, R. F.. and Young, R. C. Chemotherapy of ovarian cancer, Semin. Oncol. 11, 251-263 (1984). 4. Bonomi, P., Blessing, J. A., Stehman, F. B., DiSaia, P. J., Walton, L.. and Major, F. Randomized trial of three cisplatin dose schedules in squamous-cell carcinoma of the cervix: A Gynecologic Oncology Group study. J. C/in. Oncol. 3, 1079-1085 (1985). 5. Alberts, D. S., Kronmal. R., Baker, L. H., Stock-Novack, D. L., Surwit, E. A., Boutselis, J. G., and Hannigan, E. V. Phase II randomized trial of cisplatin chemotherapy regimens in the treatment of recurrent or metastatic squamous cell cancer of the cervix: A Southwest Oncology Group study, J. Clin. Oncol. 5, 1791-1795 (1987). 6. Kikuchi, Y., Oomori, K., Kizawa, I., Hirata, J., Kita, T., Miyauchi, M., and Kato, K. Enhancement of antineoplastic effects of cisplatin by calmodulin antagonists in nude mice bearing human ovarian carcinoma, Cancer Res. 47, 6459-6461 (1987). 7. Hamilton, T. C., Winkler, M. A., Louie, K. G., Batist, G., Behrens, B., Tsuruo, T., Grotzingenk, K., McKay, W., Young, R. C., and Ozols, R. F. Augmentation of adriamycin, melphalan and cisplatin cytotoxicity in drug resistant and sensitive human ovarian cancer cell lines by buthionine sulfoximine mediated glutathione depletion, Biochem. Pharmacol. 34, 2583-2586 (1985). 8. Andrews, P. A., Murphy, M. P., and Howell, S. B. Differential potentiation of alkylating and platinating agent cytotoxicity in hu-

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man ovarian carcinoma cells by glutathione depletion, Cancer Res. 45, 6250-6253 (1985). 9. Masuda, H., Ozols, R. F., Lai, G. M., Fojo, A., Rothenberg, M., and Hamilton, T. C. Increased DNA repair as a mechanism of acquired resistance to cis-diamminedichloroplatinum(II) in human ovarian cancer cell lines, Cancer Res. 48, 5713-5716 (1988). 10. Fraval, H. N. A., and Roberts, J. J. Effects of cis-platinum(H) diamminedichloride on survival and the rate of DNA synthesis in synchronously growing HeLa cells in the absence and presence of caffeine, Chem.-Biol. Interact. 23, I It-1 I9 (1978). Il. Blyfield, J. E., Murnane, J., Ward, J. F., Calabro-Jones, P.. Lynch, M., and Kulhanian, F. Mice, men, mustards, and methylated xanthines: The role of caffeine and related drugs in the sensitization of human tumours to alkylating agents, Brif. J. Cancer 43, 669-683 (1981). 12. Busse, P. M.. Bose, S. K., Jones, R. W., and Tolmach, L. J. The action of caffeine on X-irradiated HeLa cells. II. Synergistic lethality, Rudiat. Res. 71, 666-677 (1977). 13. Ward, J. F., Joner, E. L., and Blakely, W. F. Effects on inhibitors of DNA strand break repair on HeLa cell radiosensitivity, Cancer Res. 44, 59-63 (1984). 14. Kelland, L. R., and Steel, G. G., Modification of radiation doserate sparing effects in a human carcinoma of the cervix cell line by inhibitors of DNA repair, Int. J. Radiat. Biol. 54, 229-244 (1988). 15. Coleman, C. N., Bump, E. A., and Kramer, R. A. Chemical modifiers of cancer treatment, J. C/in. Oncol. 6, 709-733 (1988). 16. Chabner, B. A. The oncologic end game, J. C/in. Oncol. 4, 625638 (1986). 17. Berger, N. A. Cancer chemotherapy: New strategies for success, J. Clin. invest. 78, 1131-l I35 (1986). 18. Alberts. D. S., and Chen, H. S. G. Tabular summary of pharmacokinetic parameters relevant to in vitro drug testing. in Cloning of human tumor stem cells (S. Salmon, Ed.), Alan R. Liss, New York, pp. 351-359, (1980). 19. SAS aser’s guide: Statistics, Version 5 ed. SAS Carey, NC (1985). 20. Dixon. W. J. (ed.). BMDP statistical software manual. Univ. of California Press, Berkeley (1988). 21. Chou, T. C., and Talaly, P. Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 22, 27-55 (1984). 22. Chou. T. C., and Talalay. P. Analysis of combined drug effects: A new look at a very old problem, Trends Pharmacol. Sci. 4,450455 (1983). 23. Chou. T. C., and Chou, J. Nonlinear mass-law regression of doseeffect relationships by using computer simulation, Pharmacologist 29, 162-169 (1987). 24. Garewal, H. S., Ahmann, F. R., and Celniker, A. The ATP assay: Anticancer drug effects on malignant cell growth, Proc. Amer. Assoc. Cancer Res. 26, l317A (1985). 25. Garewal, H. S., Ahmann, F. R., and Woo, L. ATP levels provide a useful assay for the quantitation of growth and drug effects in malignant cells, C&n. Res. 32, 415A (1984). 26. Kuzmits, R., Aiginger, P., Miiller, M. M., Steurer, G., and Linkesch, W. Assessment of the sensitivity of leukaemic cells to cytoxic drugs by bioluminescence measurement of ATP in cultured cells. Clin. Sci. 71, 81-88 (1986). 27. Kangas, L., Gronroos, M., and Neiminen, A. L. Bioluminescence of cellular ATP: A new method for evaluating cytotoxic agents in vitro, Med. Biol. 62, 338-343 (1984).

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