(CANCER RESEARCH 46, 6105-6110, December, 1986]

Quantitative Analysis of Cellular Glutathione by Flow Cytometry UtilizingMonochlorobimane: Some Applications to Radiation and Drug Resistance inVitro and in Vivo1

Glenn C. Rice, Edward A. Bump, Dennis C. Shrieve, William Lee, and Mary Kovacs

Department of Radiology, Cancer Biology Research Laboratory, Stanford University, Stanford, California 94305 [G. C. R., M. KJ; Joint Center for Radiation Therapy,Harvard Medical School, Boston, Massachusetts 02115 [E. A. B.]; Department of Radiation Oncology, Radiation Oncology Research Lab, CED-200, University ofCalifornia, San Francisco, California 94143 [D. C. S]; and SRI International, Menlo Park, California 94025 [W. LJ

ABSTRACT

An assay using a bimane derivative has been developed to detectfree glutathione (GSH) in individual viable cells by flow cytometry.Monochlorobimane |ij7i-(ClCH2CH3)-l,5-diazabicycla|3.30]acta-3,6,-diene-2,8-dione], itself nonfluorescent, reacts with GSH to form a highlyfluorescent derivative. High pressure liquid chromatography analysisshowed that, using specific staining conditions, the only low molecularweight fluorescent derivative formed in Chinese hamster ovary cells wasthat formed with GSH. Very little reaction with protein sulfhydryls wasobserved. Rates of GSH depletion in Chinese hamster ovary cells exposedto diethylmaleate were essentially the same, whether measured by relativefluorescence intensity, by flow cytometry or by enzymatic assay oncellular extracts. This method was shown to be useful for measurementof GSH resynthesis, uptake, and depletion by prolonged hypoxia andmisonidazole treatment. Since measurements are made on individual cells,cell-to-cell variation and populational heterogeneity in GSH content arerevealed by flow cytometry. Although under most conditions In vitroGSH content is relatively homogeneous, under certain circumstances,such as release from hypoxia, heterogeneity in populational GSH levelswas observed. The significance of this heterogeneity is discussed in regardto the induction of gene amplification and drug resistance by transienthypoxia. Numerous subclones of Chinese hamster ovary cells selected bygrowth in Adriamycin or methotrexate-containing medium express elevated levels of GSH per cell. The method was extended to quantitate theGSH content of cells excised from EMT-6/SF mouse tumors that hadbeen treated in vivo with L-buthionine-S-Ä-sulfoximine, an inhibitor ofGSH synthesis. The Invariate analysis (forward angle light scatter versusmonochlorobimane fluorescence) of cells derived from these tumors gaveexcellent resolution of normal and tumor cells and demonstrated extensiveheterogeneity in the tumor cell population with respect to GSH contentper cell.

INTRODUCTION

Cellular GSH2 is a strong nucleophile and confers cellularprotection against damage produced from free radicals, oxi-dants, and electrophiles. Modulation of GSH concentrations inmammalian cells can influence cellular radiosensitivity (1-4),the cytotoxicity of several chemotherapeutic agents (5, 6), andcellular responses to hyperthermia (7, 8). One limitation ofcurrent thiol assays is an inability to detect populational heterogeneity. The development of fluorescent thiol probes andmethods involving flow cytometry offer the potential for analysis (and sorting) of subpopulations according to thiol content.Often only a small subpopulation is important in regard to a

Received 4/18/86; revised 8/8/86; accepted 8/14/86.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by NIH grants CA 40324, Ca 32827, CA42391, CA 38847, and Contract NOI-CM 17485 from the National CancerInstitute, Department of Health and Human Services.

2The abbreviations used are: GSH, glutathione; MBCL, monochlorobimane;

MBBR, monobromobimane; FCM, flow cytometry; FALS, forward angle lightscatter; CHO, Chinese hamster ovary; BSO, L-buthionine-S-Ä-sulfoximine;HPLC, high pressure liquid chromatography; PSH, protein sulfhydryl; DHFR,dihydrofolate reductase.

given end point (for instance, viable cells at decreased survivallevels).

Use of bimane derivatives as fluorescent labeling agents forcellular thiols was first developed by Kosower et al. (9). Durandand Olive (10) used MBBR as a fluorescent thiol reagent forflow cytometric studies. They did not, however, discriminatebetween staining of GSH and other nonprotein or proteinsulfhydryls. In the course of our studies, some of which arereported here, we found MBBR to be too reactive to allowspecific labeling of GSH in intact cells under easily controlledconditions. We therefore synthesized a less reactive and morespecific analogue, MBCL, and describe its properties and someapplications here.

MATERIALS AND METHODS

Cell Lines. Exponential Chinese hamster ovary cells (HA-1 subline)were grown as monolayers in Eagle's minimum essential medium

supplemented with 10% fetal calf serum. All cells were trypsinizedimmediately prior to analysis and stained in phosphate buffered salineat a concentration of 106/ml. EMT-6/SF tumors in BALB/c mice were

prepared as single cell suspensions as previously described (11). TheAA8 Chinese hamster ovary cell line was used for the hypoxia and drugresistance studies. Subclones resistant to growth in either ISO n\imethotrexate or 280 n\i Adriamycin were selected using cloning ringsand expanded for at least 5 passages in the presence of drug prior toGSH analysis. (Survival for an untreated population is approximately10~4for both Adriamycin and methotrexate.) The methotrexate resistant cells were selected in Ham's F12 medium lacking glycine, hypoxan-

thine, and thymidine supplemented with 10% dialyzed fetal calf serumand the Adriamycin resistant cells in o •minimal Eagle's medium sup

plemented with 10% fetal calf serum.Hypoxia. Hypoxia was induced by incubation in an atmosphere of

95% N2 and 5% CÜ2at 37°Cin nylon chambers as previously described

(12).Reagents. MBCL was synthesized from monobromobimane as de

scribed (13) by conversion to the trifluoroacetate, hydrolysis to thealcohol, and by reaction with lithium chloride in thionyl chloride.Monochlorobimane was dissolved in ethanol and stored at 0-5°Cin

the dark. BSO was obtained from Sigma Chemical and diethylmaleatewas obtained from Chemalog. Hoechst 33342 was obtained from Cal-biochem.

Flow Cytometry. A Coulter dual laser EPICS V was used for flowcytometry analysis. The cells were excited with 50 mW UV and meancellular fluorescence was calculated numerically using only the datafrom the largest peak. Cells (106/ml) were stained with 10 /IM MBCLat 20°Cfor 5 min prior to FCM analysis.

Biochemical Methods. HPLC analysis was performed on a Waters(Milford, MA) Chromatograph using a CIg bondapak column (Waters)and a fluorimeter (LCD; Minton, Riviera Beach, FL). Cells stainedwith MBCL were disrupted in 1% acetic acid in methanol with sonication and chromatographed as described.3 Peaks were identified by

comparison of retention times with GSH standards.For analysis of macromolecular MBCL or MBBR (Sigma) staining,

3E. A. Bump, G. C. Rice, E. K. Farnum, and W. W. Lee. Analysis of cellular

glutathione by flow cytometry and monochlorobimane, submitted for publication.

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QUANTITATIVE ANALYSIS OF CELLULAR GSH USING MBCL

cells were stained with either reagent and extracted as described.3Protein and GSH adducts were separated on a G-25 sephadex columnand bound bimane was determined fluorometrically. Fluorescence intensity was measured on a Perkin-Elmer (Model MPF-44B) fluorescence spectrophotometer, with excitation and emission settings of 395and 470 nm, respectively.

RESULTS AND DISCUSSION

Specificity. CHO cells were stained with 20 MMMBCL for10 min, lysed, and processed for HPLC analysis of low molecular weight fluorescent derivatives. Previous data3 indicated

that 20 MMresulted in maximal labeling kinetics in CHO cells(reaction rate for GSH and MBCL = 0.23 M/s"1 as measured

by HPLC). The only low molecular weight fluorescent productformed under these conditions was the GSH derivative (Fig. 1).At no time was there any indication of reagent conjugation toany other thiol species. This extraordinary specificity of MBCLfor GSH in CHO cells (as well as in a wide variety of mouseand human cell lines)3 is due to the enzyme GSH 5-transferase,

which catalyzes the conjugation of GSH with MBCL, forminga fluorescent adduct that is retained within the cell. An apparentKm of approximately 10 MMfor MBCL and GSH in the presenceof rat liver GSH 5-transferase has been measured, which is inagreement with the concentration dependence of GSH labelingin CHO cells3 and in agreement with the Kmfor MBCL reported

by Hulbert and Yakuba (14).Since 20 MMMBCL results in maximal labeling kinetics in

CHO cells, we chose this concentration to examine the kineticsof GSH-MBCL derivatization using HPLC. Fig. 2A illustratesthat derivatization was complete after a 4-min exposure ofCHO cells to 20 MMMBCL at 25"C. These data were obtained

by GSH peak area integration of the HPLC tracing. These dataare similar to reaction kinetics of CHO cells as measured byflow cytometry (Fig. 2B). In this experiment, cells were stainedwith MBCL and the relative amount of fluorescence per cell

M

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Fig. 1. HPLC tracings for intact CHO cells first stained with 20 /IM MBCLfor 10 min before lysing and processing for HPLC. The only low molecularweight thiol peak observed from the lysates (A) comigrated with the GSH standard(A).

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Fig. 2. Time dependence of MBCL (mBO)-GSH conjugation in CHO cellsas measured by HPLC (A) or flow cytometry (B). Cells were stained with MBCLas described and immediately centrifuged, extracted with 1% acetic acid inmethanol, and GSH assayed by HPLC. //, increase in cellular fluorescence asmeasured by flow cytometry as a function of time following addition of MBCL.

was quantitated by FCM. The reaction was essentially completeby 4 min in accord with Fig. 2A. We have since extended thesedata to other cell lines including 10T-1/2, EMT-6/SF, and ahuman fibrosarcoma cell line. We have found maximal labelingkinetics to be 20 MMfor all cell lines except EMT-6/SF, whichis maximal at approximate 40 MM(data not shown).

Although results indicated that the only low molecular weightthiol labeled with MBCL was GSH (Fig. 1), we were interestedin possible conjugation of MBCL with cellular macromolecules(protein —SH, phosphates, carboxylates, etc.) Separation onG-25 sephadex indicated that under previously described labeling conditions very little binding to cellular protein sulfhydrylswas observed (Fig. 3). By 30 min only 2.2% of the totalfluorescence could be attributed to PSH fluorescence. This iscontrasted to monobromobimane which has been used by otherinvestigators (10) for thiol analysis. The data in Fig. 3 indicatethat MBBR reacts quite readily with cellular PSH, such that by10 min, 15% of the total cellular fluorescence is attributable toPSH. This is undoubtedly due to the increased rate of reaction(by an order of magnitude) of MBBR as compared to MBCLwith sulfhydryls.3 Therefore, we conclude that total cellularMBCL-associated fluorescence accurately reflects the GSHcontent of the cell under these staining conditions.

Applications to the Measurement of GSH Depletion, Transport, and Resynthesis in Vitro. In Fig. 4A, CHO cells weredepleted of GSH by treatment with 10 MMdiethylmaleate. Cells

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QUANTITATIVE ANALYSIS OF CELLULAR GSH USING MBCL

M(9

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Fig. 3. Reaction of MBCL (mBCI) and a similar analogue, monobromobimane(mBBr) with protein sulfhydryls following addition of 20 iOA bimane to intactcells. Note the much lower reactivity of MBCL than MBBR to cellular PSH.

were harvested at various times and GSH analyzed either byFCM or by the enzymatic assay of Tietze (15).The relative GSHlevels as measured by FCM or by enzymatic assay were in closeagreement over 2 orders of magnitude. The FCM methodologyis useful not only for GSH depletion studies but also for kineticstudies of GSH uptake (Fig. 4B) and GSH resynthesis afterdepletion with diethylmaleate (Fig. 4Q. The FCM methodology is rapid, simple, and highly sensitive (changes to 2% ofcontrol can be differentiated from background with as little as40 mW laser excitation).

Effects of Hypoxia and Misonidazoie on GSH levels. TheFCM method was used to detect depletion of cellular GSH inCHO cells by prolonged hypoxia or by treatment with theradiosensitizer misonidazole under hypoxic conditions. Cellswere exposed for various durations to either 0, 0.5 or 5.0 mMmisonidazole in either air or hypoxia. No change in fluorescencewas observed for cells incubated with or without misonidazolefor up to 18 h (Fig. 5/Õ).However, hypoxia alone decreasedGSH levels by approximately 35% after 4 h (Fig. 5B), similarto results obtained in EMT-6/SF cells (16). Addition of misonidazole to the hypoxic culture rapidly reduced GSH levels andthe effect was concentration dependent. The populational distribution of GSH content was remarkably homogenous, evenafter treatment with hypoxia or misonidazole. Fig. 6 illustratesFCM distributions of control CHO cells and cells treated witheither 18 h of hypoxia (Fig. 6A) or incubated with 4 h ofhypoxia simultaneously with 5 mM misonidazole (Fig. (ill). Forinstance, in control populations a 5-fold difference in fluorescence intensity on the average separated the lower 1% to theupper 1% of the distribution.

Applications to Gene Amplification and Drug Resistance. Theonly heterogeneity we have observed in populational GSH levelsin vitro was after release from chronic hypoxia. When CHOcells were placed in hypoxia for 24 h and released for 12 hbefore trypsinizing and analyzing GSH content by FCM, 2distinct populations of differing GSH content were evident (Fig.7). (The multiple smaller peaks of lower GSH content are deadcells.) When these cells were analyzed simultaneously withFALS, we note that the subpopulation of elevated GSH contentwas also of increased size (Fig. 7). It has previously been shownthat transient hypoxia can induce increased amplification ofseveral genes including the DHFR (12) and p-glycoproteingenes.4 Amplification of the DHFR gene results in cellularresistance to methotrexate (17) and amplification of the p-glycoprotein gene is associated with the multidrug cross-resistance phenotype ( 18). It was further shown that hypoxia induces

4 G. C. Rice, C. A. Hoy, V. Ling, and R. T. Schimke. Amplification of thedihydrofolate reducíaseand p-glycoprotein genes by hypoxia in Chinese hamsterfibroblasts, submitted for publication.

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Fig. 4. Measurement of cellular GSH depletion, uptake, and resynthesis. A,kinetics of GSH depletion in CHO cells by addition of 200 «IMdiethylmaleate.Replicates were assayed either by enzymatic assay of Tietze (15) (•)or by flowcytometry with MBCL (•).Control is untreated cells. II. GSH uptake kineticsusing the flow cytometry-MBCL method. Cells were first depleted of GSH by a2-h treatment with 200 MMdiethylmaleate before readdition of 3 HIMGSH to themedium. C, kinetics of GSH resynthesis following diethylmaleate depletion. Cellswere rinsed and returned to 37*C with (•)or without (•)10 (/\i BSO to inhibit

resynthesis.

S-phase DNA overreplication, such that the treated S-phasecells have >4 C DNA content and increased forward angle lightscatter (12). In fact, it is from this subpopulation of increasedsize (and as shown in Fig. Hi. GSH content) from which ariseall of the amplified cells (12).

A further surprising finding regarding GSH content and drugresistance is shown in Fig. 8. Numerous CHO clones wereisolated that were resistant to continuous growth in 150 UMmethotrexate, or 280 UM Adriamycin. These clones were isolated by cloning rings, expanded, and analyzed for GSH content. Of 19 clones analyzed that were resistant to continuoustreatment with Adriamycin, all were found to have elevatedGSI? content, up to 3.3-fold greater than control. Of the 21methotrexate resistant clones analyzed, all were found to have

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QUANTITATIVE ANALYSIS OF CELLULAR GSH USING MBCL

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treated with 0, O.S. or 5 m\i misonidazole in full medium for the indicatedduration before analysis by flow cytometry. Data are averages of at least duplicatesamples with all SDs <10%. B, same as A except that the cells were placed in anatmosphere of 95% N, and 5% CO2.

elevated GSH content. These clones ranged from 1.1- to 2-foldgreater than control. We found no relationship between averagecell size and GSH content among the various succiones. Thus,these differences likely represent changes in cellular GSH concentration rather than simply increased cell size. Additionalwork regarding the relationship between amplification of theDHFR and p-glycoprotein genes to amplification of the GSH5-transferase gene and elevated GSH levels will be publishedelsewhere.5 These data, however, point to the potential impor

tance elevated GSH levels may play in drug resistance to twounrelated agents.

Applications in Vivo. Fig. 9A shows a GSH distribution foran EMT-6/SF tumor excised, dispersed into a single cell suspension, and stained with MBCL. Bivariate analysis simultaneously measuring FALS and MBCL fluorescence of the samesample demonstrated extensive heterogeneity in GSH contentand the presence of 2 major subpopulations with differing GSHcontent (Fig. 9B): a small subpopulation (low FALS) of lowGSH content and a large subpopulation (high FALS) of higherGSH content. In the hope of discriminating normal cells fromtumor cells a replicate of the tumor was stained with Hoechst

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and cells treated with 18 h hypoxia (H). B, control and cells treated with S.OHIMmisonidazole (A/) under hypoxia. Note the lack of heterogeneity in populationalthiol content for both control and treated samples.

33342. This dye binds to DNA, and as is shown in Fig. 9E,differentiates the diploid normal cells from the heterodiploidtumor cells. Simultaneous measurement of Hoechst 33342fluorescence and FALS clearly shows that the normal diploidcells were almost all smaller (lower FALS) than the heterodiploid tumor cells (Fig. 9F). Integrating the small number ofnormal cells that had an elevated FALS signal (Fig. 9F, inset)indicated that on the average, only 6-8% of the large cells wereactually normal cell contaminants; the remainder were tumorcells. Thus, the subpopulation consisting of elevated FALS andGSH in Fig. 9B can be assumed to consist predominantly oftumor cells. The tumor cells were found to have approximately3.4-fold more GSH than the normal cells.

GSH distributions for EMT-6/SF tumors treated with BSO,5 mg/kg in vivo 12 and 24 h prior to excision show a largedecrease (to a level 24.4% of untreated) in the tumor (7)subpopulation's fluorescence (Fig. 9, C and D). Although this

is difficult to see with the univariate distribution (due to theoverlap with the normal cells) it is quite clearly resolved in theDivariate distribution. This decrease is consistent with enzymatic measurements made under similar treatment conditions.6

Note that the normal (N in Fig. 9, B and D) cells (low FALS)were depleted to a much less extent (to a level 61% of untreated)than the tumor cells with in vivo BSO administration. Thus, a

9G. C. Rice. Manuscript in preparation. 6 D. C. Shrieve, unpublished observations.

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QUANTITATIVE ANALYSIS OF CELLULAR GSH USING MBCL

Fig. 7. Effects of transient hypoxia on cellular heterogeneity of GSH levels. A, univariateMBCL (mBCl) distribution for cells treatedfor 24 h in chronic hypoxia followed by releasein air for 12 h. Note the 2 major subpopula-tions of altered GSH content. B, simultaneousmeasurement of MBCL fluorescence and forward angle light scatter (F. A. L. S.) of thesame sample as in A. Note that the subpopulation of elevated GSH content is also of elevated size. Rei., relative.

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Fig. 8. Correlation of drug resistance and elevated GSH levels. CHO cloneswere selected for continuous growth in 280 n\i Adriamycin (A) and ISO n\imethotrexate (B). GSH levels were then screened by the MBCL-flow cytometrymethod. Shown are GSH levels as a percentage of control, with the control levelsshown as ±1 SD. All clones screened had elevated levels of GSH. There was nocorrelation between increased cell size and increased GSH content between clones(data not shown).

therapeutic gain has been achieved in effect; i.e., prior to BSOtreatment, tumor cells had 3.4-fold more GSH per cell, whileafter BSO treatment, tumor cells had only 1.5 times the GSHcontent per cell as did the surrounding normal cells. Further,i/i vivo applications will be published elsewhere.7 However, these

data point to the power and utility of measurement of GSH ata single cell level to assess tumor GSH heterogeneity as well asthe ability to combine such analysis with other parameters (suchas FALS) to discriminate multiple cell types.

In conclusion, we have demonstrated that a bimane derivative, monocholorobimane, possesses high specificity to GSH inlive mammalian cells. Analysis of fluorescence by flow cytometry is a simple, rapid, and sensitive measure of relative GSHcontent per cell. If desired, these values can be converted toabsolute levels if control values for GSH per cell are known.We show that this new methodology is useful for measuringeither GSH depletion, uptake, or resynthesis following depletion. Also, depletion due to physical manipulation (hypoxia) or

7 D. C. Shrieve and G. C. Rice. Manuscript in preparation.

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Fig. 9. In vivo applications of the MBCL (mfiCV)-flow cytometry technique.A, MBCL distribution for an EMT-6 tumor excised and immediately stained withMBCL. lì,same distribution with simultaneous measurement of FALS. Similarly,C and l>depict univariate and bivariate distributions, but tumors were first treatedin vivo with 2 injections of BSO (5 mg/kg) 12 and 24 h before excision andstaining. J. control levels of GSH for either the large subpopulation [predominantly composed of tumor (T) cells] and the small subpopulation [normal (N)cells] as well as the average levels for these 2 subpopulations observed followingin vivo BSO treatment. E and /. Hoechst 33342 (Ho342) distributions as aunivariate or bivariate (simultaneous with FALS). Note that nearly all of theheterodiploid cells (i.e., tumor cells) are large (elevated FALS) while nearly all ofthe diploid cells (normal) are small. The small contamination of normal cells inthe high FALS subpopulation (inset) was estimated on the average to containapproximately 8% of the total cells with high FALS. Therefore, the small andlarge subpopulations in A-D can be assumed to contain mostly all normal andtumor cells, respectively.

by drug (misonidazole) is rapidly quantitated. Although GSHlevels are generally homogenous in cell cultures in vitro, undercertain circumstances heterogeneity can be induced, such asfollowing release from hypoxia. This heterogeneity is unresolv-able using standard methods of GSH analysis and may lead tonew insights into understanding the role elevated GSH may

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QUANTITATIVE ANALYSIS OF CELLULAR GSH USING MBCL

play in drug resistance. Analysis of heterogeneity was shown tobe particularly useful in in vivo tumor applications whereby theGSH content of normal and tumor subpopulations can beseparated by using multiparameter analysis.

ACKNOWLEDGMENTS

The authors thank Dr. Martin Brown for a critical review of thismanuscript.

REFERENCES

1. Bump, E. A., Yu, N. Y., and Brown, J. M. Radiosensitization of hypoxictumor cells by depletion of intracellular glutathione. Science (Wash. DC),2/7:544-545, 1982.

2. Shrieve, D. C, Denekamp, J., and Michinton, A. I. Effects of glutathionedepletion by buthionine sulfoximine on radiosensitization by oxygen andmisondidazole in vitro. Radiât.Res., 102: 283-294, 1985

3. Clark, E. P., Epp, E. R., Biaglow, J. E., Morse-Gaudio, M., and Zachgo, E.Glutathione depletion, radiosensitization, misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine. Radiât.Res.,98:370-380, 1984.

4. Malaise, E. P. Reduced oxygen enhancement of the radiosensitivity of glu-tathione-deficient fibroblasts. Radiât.Res., 95:486-494, 1983.

5. Babson, J. R., Abell, N.S., and Reed, D. J. The protective role of glutathione-redox cycle against Adriamycin mediated toxicity in isolated hepatocytes.Hindu'm. Pharmacol., 31: 2299-2304, 1981.

6. Roizin-Towle, L., Hall, E. J., Flynn, M., Biaglow. J. E., and Varnes, M. E.Enhanced cytotoxicity of melphalan by prolonged exposure to nitroimida-

zoles: the role of endogenous thiols. Int. J. Radiât.Oncol. Biol. Phys., 8:757-760,1982.

7. Mitchell, J. B., Russo, A., Kinsella, T. J., and Glatstein, E. Glutathioneelevation during thermotolerance induction and thermosensitization by glutathione depletion. Cancer Res., 43: 987-991, 1983.

8. Shrieve, D. C., Li, G. C., Astromoff, A., and Harris, J. W. Cellular glutathione, thermal sensitivity and thermotolerance in Chinese hamster fibroblastsand their heat resistant variants. Cancer Res., 46: 1684-1687, 1986.

9. Kosower, N. S., Kosower, E. M., Newton, G. L., and Ranney, A. M. Bimanefluorescent labels: labeling of normal human red blood cells under physiological conditions. Proc. Nati. Acad. Sci. USA, 76: 3382-3386, 1979.

10. Durand, R. E., and Olive, P. E. Flow cytometry techniques for studyingcellular thiols. Radiât.Res., 95:456-470, 1983.

11. Ono, K., and Shrieve, D. C. Enhancement of EMT6/SF tumor cell killingby mitomycin C and by cyclophosphamide following in vivo administrationof buthionine sulfoximine. Int. J. Radial. Oncol. Biol. Phys., in press, 1986.

12. Rice, G. C., Hoy, C. A., and Schimke, R. T. Transient hypoxia inducesamplification of the dihydrofolate reductase gene. Proc. Nati. Acad. Sci.USA, S3; 5978-5982, 1986.

13. Kosower, F. M., Pazhenchevsky, B., Dodvik, H., Kanety, H., Faust, D., andBimanes, G. Reactive halogen derivatives of syn and anf/'-l,5,diazabicyclo[3.3.0] octa-dienones (9,10-dioxabimanes). J. Org. Chem., 46: 1666-1673,1981.

14. Hulbert, P. B., and Yakuba, S. I. Monobromobimane, a fluorometric assayfor glutathione-5-transferase. J. Pharm. Pharmacol. 35: 384-386, 1983.

15. Tietze, F. Enzymatic method for quantitative determination of nanogramamounts of total and oxidized glutathione: applications to mammalian bloodand other tissues. Anal. Bipchem. 27: 502-522, 1969.

16. Shrieve, D. C., and Harris, J. W. The in vitro sensitivity of chronicallyhypoxic EMT6/SF cells to x-radiation and hypoxic cell radiosensitizers. Int.J. Radiât.Biol. Relat. Stud. Phys. Chem. Med., 48:127-138, 1985.

17. Alt, F. W., Kellems, R. E., Benino, J. R., and Schimke, R. T. Selectivemultiplication of dihydrofolate reductase genes in methotrexate resistantvariants of cultured murine cells. J. Biol. Chem., 253:1357-1370, 1978.

18. Riordan, J. R., Deuchars, K., Kartner, N., Alan, N., Trent, J., and Ling, V.Amplification of p-glycoprotein genes in multidrug resistant mammalian celllines. Nature (Lond.), 316:817-819, 1985.

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1986;46:6105-6110. Cancer Res   Glenn C. Rice, Edward A. Bump, Dennis C. Shrieve, et al.  

in Vivo and in Vitroand Drug Resistance Utilizing Monochlorobimane: Some Applications to Radiation Quantitative Analysis of Cellular Glutathione by Flow Cytometry

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