Inr. .I Radiobon Oncology BKJ/. Phys, Vol. 22, pp. 68 1-684 Printed in the U.S.A. All n&s reserved.

Copyright

0360-3016/92 $5.0) + .Xl 0 1992 Pergamon Press

plc

??Session D: Bioreductive Mechanisms

CHARACTERIZATION

AMATO ANDREW

OF A CHO CELL LINE RESISTANT TO KILLING HYPOXIC CELL CYTOTOXIN SR 4233

J. GIACCIA,

PH.D.,

I. MINCHINTON, Department

PH.D.,

KIM

A. BIEDERMANN,

MARY

S. KOVACS,

PH.D., B.S.

LIANA

M. TOSTO,

AND J. MARTIN

BY THE

B.A.,

BROWN,

D. PHIL.

of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305

One approach to understanding the mechanism of selective hypoxic cell killing by the benzotriazine-di-N-oxide, SR 4233, is to characterize cell lines that exhibit increased resistance to killing by this drug. The Chinese Hamster Ovary cell line BL-10 was originally isolated on the basis of its hypersensitivity to killing by bleomycin. It is 2.7fold more resistant to hypoxic cell killing by SR 4233 than wild-type CHO on comparison of 4’s. However, both BL-10 and CHO possess the same sensitivity to killing by SR 4233 under aerobic conditions. We have excluded the explanation that differential metabolism of SR 4233 is responsible for its increased survival as both BL-10 and CHO produce the two-electron product SR 4317 at the same rate (3 nmoles/hr/106 cells). Analysis of free radical production, DNA double-strand break induction, and glutathione (GSH) levels suggested that the resistance of BL-10 to killing by SR 4233 might result from increased intracellular radical scavenger pathways. Using buthionine sulfoximine (BSO) to decrease cellular GSH levels, we found a marked increase in the sensitivity of BL-10 cells to SR 4233 killing under hypoxia, but a much smaller increase in the sensitivity of CHO cells. Taken together, these data imply that the high GSH levels in BL-10 cells is responsible for its resistance to SR 4233 cytotoxicity. BL-10, SR 4233, GSH, BSO.

ficient in DNA double-strand break repair, no human cell lines have been identified to be defective in this repair pathway. However, it has been found that tumor cell lines, especially from head and neck cancers, may vary in their fast kinetics of DNA double-strand break rejoining and this repair correlated with increased radioresistance (8). To further aid in our understanding of the cellular pathways that affect hypoxic cytotoxicy of SR 4233, we have characterized the CHO cell line BL- 10 ( 10) that was originally isolated on the basis of its hypersensitivity to killing by bleomycin. We found that BL- 10 was resistant to killing by SR 4233 under hypoxia, suggesting that a defect in the pathway causing bleomycin sensitivity gave rise to an increase in cellular resistance to SR 4233. Here we report that the resistance of BL- 10 to killing by SR 4233 under hypoxia is due largely to its increased intracellular GSH content, when compared to parental CHO cells.

INTRODUCTION

The ability to selectively kill hypoxic tumor cells would increase the efficacy of radiotherapy and chemotherapy. The benzotriazine di-N-oxide SR 4233 is a potent and selective hypoxic cytotoxin, the mechanism of which involves the production of free radicals that produce DNA double strand breaks (1, 3, 12). Recently, we undertook a study of human and rodent cell lines to determine what molecular mechanisms are responsible for the differential cellular cytotoxicity of SR 4233 under hypoxic conditions (2). These differences in sensitivities to killing by SR 4233 could, for the most part, be correlated with the level of bioreductive metabolism in repair proficient cells. Only the DNA double-strand break repair deficient cell lines XR- 1 (4) and V3 ( 11) deviated from this correlation. As SR 4233 will soon undergo clinical trials, we are interested in predicting which tumors will respond to the drug. From our previous data, we concluded that two important cellular predictors of cellular sensitivity to killing by SR 4233 are bioreductive metabolism and proficiency in DNA double-strand break repair. Although numerous rodent cell lines have been isolated that are de-

METHODS

AND

MATERIALS

Cell culture The bleomycin-sensitive cell line, BL- 10 was derived from CHO-K 1 cells following a single mutagen treatment

Presented at the Seventh International Conference on Chemical Modifiers of Cancer Treatment, Sheraton Sand Key Resort, Clearwater, FL, 2-5 February 199 1. Reprint requests to: Amato J. Giaccia, Ph.D.

work was supported by U.S.P.H.S. Grant CA 15201 from the National Cancer Institute, D,H,H.S. We would also like to thank Ms. Chiyoye Adachi for typing the manuscript. Accepted for publication 26 July 199 1.

Acknowledgements-This

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10,

SURVIVAL OF EL-10 AND CHO TO A 1 H TREATMENT OF SR 4233 UNDER HYPOXIA

.

METABOLISM

8-

.Oool ,

0

0 I

1

I

50

100

150

200

SR 4233 (PM)

Fig. 1. Survival of CHO and BL- IO to varying concentration of SR 4233 under hypoxia for lhr. The ratio of the slopes of BL10 and CHO fitted by liner regression analysis is 2.7.

(10). CHO and BL- 10 cells were cultured in Alpha MEM with 10% fetal calf serum. For survival experiments, equal numbers of exponentially growing cells were treated with SR 4233 under hypoxia in media without serum for 1 hr, washed, counted, and then plated in complete media to clone. Colonies with greater than 50 cells were counted. Cellular depletion of GSH was accomplished by incubating the cells with 50 PM buthionine sulfoximine (BSO) for 24 hr to decrease intracellular GSH content before treatment with SR 4233 under hypoxia. GSH was measured by HPLC as described by Minchinton (7).

TIME

(HR)

Fig. 2. Rate of the two electron reduction of SR 4233 to SR 43 I7 under hypoxia in CHO and BL- IO. For the substrate concentration of 200 FM SR 4233, the rate of metabolism for BLIO was 3.22 f 0.29 nmol SR 4317/106 survivors and for CHO was 2.9 I k 0.10 nmol SR 43 I7/ 1O6 survivors.

supplemented

scribed by Stamato and Denko (9) except that cells were exposed to SR 4233 for 1 hr in glass dishes before being cast into agarose plugs. The induction of DNA breaks is proportional to the fraction of DNA eluted from the gel (9) which was determined by the equation: Percentage

of DNA released =

Quantitation offree radical production with the ACAS 5 70 interactive laser cytometer Exponentially growing cells on glass coverslips were incubated with 5 PM 2,7,-dichlorofluorescin diacetate (DCFDA)* in media at 37°C for 15 min, and then treated with SR 4233 under hypoxia for 1 hr. These cells were then washed 4X with PBS containing 5 PM glucose and their fluorescence was quantitated using ACAS 570t equipped with an argon ion laser tuned to 488 nm with emission measured above 5 15 nm. This assay measures the oxidized form (DCF) of DCF-DA.

Induction of DNA double-strand breaks by pulsed-field gel electrophoresis Induction of DNA double-strand breaks was quantitated by pulsed-field gel electrophoresis (PFGE) as de-

* Molecular

Probes, Eugene, OR.

number

of cpm in lane

total cpm in lane + well I

SR 4233 metabolism studies Reduction of SR 4233 to SR 4311 was measured in CHO and BL- 10 cells as described by Baker et al (1). This technique takes advantage of the fact that SR 43 17 is approximately 64X more fluorescent at wavelength 4 16 nm than SR 4233. The results of this technique have been verified by HPLC analysis.

from gel x 100.

RESULTS The increased resistance to killing by SR 4233 under hypoxia of BL- 10 compared to parental CHO cells is shown in Figure 1. BL- 10 is 2.7 times more resistant than CHO to killing by SR 4233 under hypoxia by liner regression analysis of their slopes. To determine whether increased resistance of BL- 10 to killing by SR 4233 was due to decreased metabolism of the drug, we measured the rate of reduction under hypoxia of SR 4233 to the two electron product SR 43 17 for both cell lines. Under hypoxic conditions, SR 4233 is thought to be reduced to the 1-oxide product SR 43 17 by two single electron reductions. Figure 2 shows that both cell lines metabolize SR 4233 to a similar extent, excluding the possibility that the resistance of BL-10 to killing by SR 4233 results from decreased metabolism of drug. Since SR 43 17 is the two electron reduction product of SR 4233, the possibility exists that BL-10 cells could perform a two electron reduction in one step, thereby bypassing the toxic one electron free radical pathway. Therefore, we developed an assay to detect intracellular free radical formation by quantitating the oxidation of the dye 2’,7-dichlorofluorescin diacetate (DCF-DA) to its

t Meridian

instruments,

Place, MI.

CHO

cell

line resistant to killing by

SR

4233 0

A.

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so8

600

1

60 -

-0

600

400

10

0

200

so0

200

O SR4233 (PM)

0 0

200 PM SR 4233 TREATMENT

Fig. 3. Production of free radicals in CHO and BL- 10 cells after a I hr treatment with SR 4233 under hypoxia. Average fluorescence for at least 200 cells is plotted. Background fluorescence

is the same for both cells without SR 4233 treatment.

fluorescent product dichlorofluorescein (DCF) (Biedermann, K. A., Graham, R. and Brown, J. M. Quantitation of free radical production by SR4233 in individual cells. Presented at 38th Annual Meeting of Radiation Research Society, New Orleans, LA, April 7-12, 1990.). Using this assay, production of free radicals in BL- 10 cells was 50% of that of CHO after a 1 hr treatment with 200 PM SR 4233 (Figure 3). This decrease in free radicals formed after SR 4233 treatment in BL-10 cells correlated with a 40% decrease in DNA double-strand break induction in BL10 cells compared to CHO cells for a 1 hr treatment with SR 4233 (Figure 4). This decrease in free radical production by BL- 10 could be interpreted either as metabolic bypass of the one electron reduction product for the two electron reduction product, or an increase in intracellular free radical scavengers that react with the radical species before it can oxidize DCF-DA or produce DNA breaks. The latter of these two possibilities is the more likely in view of the fact that HPLC measurements of GSH cellular content showed that BL-IO cells possessed 2.5 times the amount of GSH as do CHO cells (BL-lO= 15 fmol/cell, CHO= 6 fmol/cell). To prove that this increase in GSH correlated with the resistance of BL-10 cells to SR 4233 cytotoxicity, we depleted the GSH levels in both CHO and BL-10 cells with a 24 hr incubation with 50 PM BSO and then tested the sensitivity of both cell lines to SR 4233 for 1 hr under hypoxic conditions. We found that by depleting GSH in both cell lines to less than 5% of the untreated controls, the sensitivity of the BL- 10 cells was increased dramatically, whereas that of the CHO cells was increased to a much lesser extent (Figure 5).

Fig. 4. SR 4233 induction of DNA double strand breaks as analyzed by pulsed field gel electrophoresis. The percentage of DNA released from the well for BL-IO and CHO is graphically rep-

resented. ble-strand break repair (5). We recently have found that BL-10’s bleomycin hypersensitivity is due to a lack of alpha glutathione S-transferase (GST) actiivity (Giaccia, A. J., Lewis, A. D., Denko, N. C., Cholon, A., Evans, J. W., Waldren, C. A., Stamato, T. D., and Brown, J. M. Genetic mapping the CHO variant BL-10’s hypersensitivity to bleomycin killing to a lack ofglutathione S-transferase alpha activity. Cancer Research in press 199 1.). Normally, GSTs possess two binding sites, one for GSH and another for xenobiotics or peroxidation products (6). Thus, a possible explanation for the increased intracellular GSH content of BL- 10, would be that BL- 10 has a defective GSH binding site in its non-functional alpha GST protein that causes an increased intracellular GSH content. We believed that this increased level of GSH accounts for BL-10’s resistance to SR 4233. Indeed, when intracellular GSH levels in BL-10 cells were depleted, its sensitivity to killing by SR 4233 was dramatically increased. This is to be contrasted with the data for CHO cells that showed considerably less effect of GSH depletion. Since BL- 10 possesses the same rate of SR 4233 metabolism as CHO, then GSH must react with SR 4233 free radicals before they can cause DNA damage. EFFECTOF

10-b 0

DISCUSSION BL-10 is hypersensitive to killing by bleomycin (10). Unlike XR-1 and V3 cells, it does not possess sensitivity to ionizing radiation (lo), and is proficient in DNA dou-

GSH DEPLETION

BY 50pM BSO ON THE SENSlTlVlTV

OF BL-10 AND CHO TO KILLING

.

1. 20

"'.I. 40

60

BY SR 4233

00

100

CONC.SR4233(pM)

Fig. 5. Survival of CHO and BL-10 cells to a 1 hr treatment of SR 4233 under hypoxia after 24 hr pre-incubation with 50 &I BSO to deplete GSH levels.

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Mutant rodent cell lines have proved to be powerful genetic tools that aid in our understanding of complex genetic pathways. Analysis of the CHO mutants XR-1, V3 and BL-10 have added to our understanding of the molecular mechanisms responsible for SR 4233 cytotoxicity. However, these cell lines were originally isolated on their sensitivity to ionizing radiation and bleomycin. Although it is tempting to test other variant rodent cell lines for SR 4233 sensitivity, it would be considerably more useful to determine which cellular enzymes are responsible for its bioactivation under hypoxia by isolating CHO mu-

Volume 22. Number 4, 1992

tants based on their sensitivity or resistance to killing by SR 4233. These studies are planned. So far, we have identified three predictors for cellular sensitivity to killing by SR 4233 under hypoxia in cell culture: cellular metabolism of SR 4233, proficiency in DNA double-strand break repair, and intracellular radical scavengers. In viva, a fourth predictor of sensitivity to killing by SR 4233 would be the degree and/or extent of tumor hypoxia. Taken together, these predictors will be important in determining which tumors should or should not be given SR 4233 treatment.

REFERENCES 1. Baker, M. A.; Zeman, E. M.; Hirst, V. K.; Brown, J. M. Metabolism of SR 4233 by Chinese hamster ovary cells:

basis of selective hypoxic cytotoxicity. Cancer Res. 48:59475952; 1988. 2. Biedermann, K. A.; Wang, J.; Graham, R. P.; Brown, J. M.

3.

4.

5.

6.

7.

SR 4233 cytotoxicity and metabolism in DNA repair-competent and repair-deficient cell cultures. Br. J. Cancer 63: 35% 362; 1991. Brown, J. M.; Lemmon, M. J. SR 4233: A tumor specific radiosensitizer active in fractionated radiation regimes. Radiother. Oncol. 20: I5 l- 156; 199 I. Giaccia, A. J.; Weinstein. R.; Stamato, T. D. Cell cycledependent repair of double-strand DNA breaks in a gammaray-sensitive Chinese hamster ovary cell. Somat. Cell. Genet. 11:485-491; 1985. Giaccia, A. J.; Shieh, J.; Cholon, A.; Brown, J. M. Biochemical evidence for two different mechanisms for bleomycin induced cell killing. Mutat. Res. (In press). Mannervik, B.; Danielson, U. H. Glutathione transferasesstructure and catalytic activity. CRC Biochem. 23:283-337: 1988. Minchinton, A. I. Measurements of glutathione and other

8.

9.

10.

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12.

thiols in cells and tissues: a simplified procedure based on the HPLC separation of monobromobimane derivatives of thiols. Int. J. Radiat. Oncol. Biol. Phys. 10: 1503- 1506; 1984. Schwartz, J. L.; Mustafi, R.; Beckett, M. A.; Weichselbaum, R. R. Prediction of the radiation sensitivity of human squamous cell carcinoma cells using DNA filter elution. Radiat. Res. 123:1-6; 1990. Stamato, T. D.; Denko, N. Asymmetric field inversion gel electrophoresis: a new method for detecting DNA double strand breaks in mammalian cells. Radiat. Res. 121: 196205; 1990. Stamato, T. D.; Peters, B.: Patil, P.; Denko, N.; Weinstein, R.: Giaccia, A. Isolation and characterization ofbleomycinsensitive Chinese hamster ovary cells. Cancer Res. 47: I5881592; 1987. Whitmore, G. F.; Varghese, A. J.: Gulyas, S. Cell cycle responses of two X-ray sensitive mutants defective in DNA repair. Int. J. Radiat. Biol. 56:657-665; 1989. Zeman. E. M.; Brown, J. M.; Lemmon, M. J.; Hirst, V. K.; Lee, W. W. SR 4233: a new bioreductive agent with high selective toxicity for hypoxic mammalian cells. Int. J. Radiat. Oncol. Biol. Phys. 12: 1239- 1242; 1986.

Characterization of a CHO cell line resistant to killing by the hypoxic cell cytotoxin SR 4233.

One approach to understanding the mechanism of selective hypoxic cell killing by the benzotriazine-di-N-oxide, SR 4233, is to characterize cell lines ...
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