[CANCER RESEARCH 42, 3934-3940, October 1982]0008-5472/82/0042-OOOOS02.00

Enhanced Survival of Adriamycin-treated Chinese Hamster Cells by2-Deoxy-D-glucose and 2,4-Dinitrophenol1

J. R. Colofiore, G. Ära,D. Berry, and J. A. Belli2

Department of Radiation Therapy and Joint Center for Radiation Therapy, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT

The purpose of this work was to determine the role ofmetabolic energy [adenosine triphosphate (ATP)] in the expression of cytotoxicity of the antitumor drug, Adriamycin (ADRM),in Chinese hamster fibroblasts. When ADRM-sensitive V79cells or an ADRM-resistant variant, 77A, were pretreated witheither 6 mM 2-deoxy-o-glucose or 0.2 rtiM 2,4-dinitrophenol for18 hr and then exposed to ADRM for 1 hr at 37°,we observed

a substantial increase in cellular survival. In the case of V79cells pretreated with deoxyglucose, as much as a 1000-foldincrease in survival was measured. The maximum dose ofADRM for V79 cells was 3 ^ig/ml, and for 77A cells it was 15fig/ml.

The effects of metabolic inhibitors on intracellular ATP content, ADRM uptake, mitotic indices, cellular multiplication, andplating efficiency were measured in untreated as well as ininhibitor-treated cells. The same concentrations of deoxyglu

cose (6 mw) and dinitrophenol (0.2 mM) that were used forcytotoxicity studies were also used in the biochemical studies.Under conditions of pretreatment with metabolic inhibitorswhich increased the survival of cells, a lowering of ATP contentand a concomitant slight enhancement of drug uptake wereobserved. Plating efficiency was not affected. We also haveobserved a partial inhibition of growth after pretreatment withmetabolic inhibitors as shown by lowered mitotic indices andby cell counts. We conclude that the increase in survival ofboth ADRM-sensitive and resistant cells is not due to decreased

intracellular accumulation of the drug. Flow microfluorimetricanalysis of V79 cells pretreated with deoxyglucose showed noreduction in the numbers of cells in the S phase of the cellcycle, the most sensitive compartment for ADRM, while pretreatment with dinitrophenol, on the other hand, significantlyreduced the number of cells in S phase. When inhibitors wereremoved, and the cells were rinsed, refed complete growthmedium, and then exposed to a single dose of drug (1.5 ftg/mlfor V79; 10 jug/ml for 77A) at various times up to 7 hr, cellularsurvival decreased while ATP content increased as a functionof time. We conclude that the effects of the metabolic inhibitorsare reversible and that cells slowly regain their sensitivity toADRM. As intracellular levels of ATP increase, sensitivity toADRM increases. Thus, metabolic energy may be important forcytotoxic damage by ADRM. However, the enhancement incellular survival may also be partly related to slower cell cycleprogression.

INTRODUCTION

ADRM3 is an anthracycline antibiotic which is currently one

Received March 12, 1982; accepted June 23, 1982.' This work was supported by NIH Grants RO1 CA 25333 and CA12662.2 To whom requests for reprints should be addressed. Present address:

Department of Radiation Therapy, The University of Texas Medical Branch,Galveston, Texas 77550.

3 The abbreviations used are: ADRM. Adriamycin (doxorubicin MCI); DG, 2-

of the most widely utilized antitumor agents (7, 20). Althoughselectively toxic to many types of cancer cells, ADRM is alsotoxic to normal cells. Moreover, treatment with ADRM leads tothe development of tumor cell resistance, a common consequence of using antitumor compounds. The molecular basis forunderstanding all the cytotoxic effects of ADRM is as yetincomplete, but a number of important findings have beensubstantiated.

ADRM binds to and intercalates into DMA (11, 22), resultingin inhibition of DMA replication and RNA transcription (30). Thequinone structure of ADRM may be reduced enzymatically,leading to the generation of free Superoxide radical ions. Suchfree radicals may then produce extensive strand scission ofDMA (6).

The current investigation is directed toward probing thecytotoxic mechanism of ADRM. Since the transport of the drug,either cytoplasmic or nuclear, the production of Superoxideradicals, or the binding of drug to DMA may require metabolicenergy (ATP), we have examined the role of metabolic inhibitionin the cytotoxicity of ADRM. The glucose analog, DG, as wellas the uncoupler of oxidative phosphorylation, DNP, wereutilized to block cellular energy production. The effects ofthese metabolic inhibitors on the survival response to ADRMand drug uptake in V79 cells and its ADRM-resistant variant,77A, were therefore investigated.

Although DG and DNP both decrease levels of intracellularATP, the mechanisms for doing so are dissimilar. DG is ananalog of glucose and mannose, is phosphorylated by ATP,and blocks glucose entry. Metabolically, the action of DG onanaerobic glycolysis is to block the formation of fructose 6-phosphate with a concomitant loss of an ATP-generating step.That DG lowers metabolic energy has been well documented(2, 14). DNP also depletes metabolic energy in cells, but itdoes so by a completely different mechanism, namely, byuncoupling electron transport from oxidative phosphorylation(16).

The present investigation uses metabolic inhibitors to examine the role of ATP in cytotoxic damage produced by ADRM.Other mechanisms by which metabolic inhibitors might changecellular sensitivity to ADRM, in particular by induction of specific membrane protein synthesis or by facilitation of DNArepair, are also discussed.

MATERIALS AND METHODS

Chemicals. ADRM (doxorubicin HCI) was obtained from Adria Laboratories, Inc., Columbus, Ohio, and reconstituted in PSA to a concentration of 2000 /¿g/ml.DG, DNP, ATP, and firefly lantern extract, wereobtained from Sigma Chemical Company, St. Louis, Mo.

deoxy-D-glucose; DNP, 2,4-dinitrophenol; PSA, Puck's Saline A; aMEM, a modification of Eagle's minimal essential medium supplemented with penicillin, strep

tomycin, and calf serum; FMF, flow microfluorlmetry; Ml, mitotic index.

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Increased Survival to ADRM by Metabolic Inhibitors

Cells. Chinese hamster fetal lung fibroblasts (clone V79-182) were

used as the parental cell line. Cell culture methods have been describedin detail elsewhere (5). An ADRM-resistant variant, 77A, was derivedfrom V79-182 (4) by growing the cells in low levels of ADRM (0.05 /ig/ml) for 77 weeks. Thereafter, the cells were grown in drug-free medium.

Cytotoxicity Studies. To determine the ADRM dose response, cellswere dispersed from Retri dish surfaces with 0.05% trypsin:EDTA inPSA, counted, and diluted in oMEM, with or without metabolic inhibitors. Cells were plated into 100-mm Retri dishes (Falcon Plastics,

Oxnard, Calif.) in numbers sufficient to produce approximately 200colonies at all survival levels. All ADRM solutions were kept in the darkand at 4°.Stock dilutions were made in complete growth medium eitherwith or without metabolic inhibitors. After 18 hr at 37° in complete

medium (o-glucose concentration, 5.5 mM), either with or without DG

(6.0 mW), or complete medium with or without DNP (0.22 row), cellswere exposed to appropriate ADRM concentrations, rinsed with PSA,and refed complete growth medium («MEM) for colony formation. Alldrug exposures were for 1 hr at 37°. Immediately after refeeding, cellswere incubated at 37° for 7 to 10 days. At this time, colonies were

stained with a 2% méthylèneblue solution and counted, correctionswere made for plating efficiency, and surviving fractions were determined. The survival data for each experiment were derived from thesame starting cell suspension. Three replicate dishes were used foreach survival point. Each inhibitor was tested in at least 3 separateexperiments.

ADRM Uptake. ADRM exhibits specific fluorescence which enablesaccurate measurement of intracellular accumulation of fluorescentspecies. The excitation and emission wavelengths used were 485 nmand 590 nm, respectively. Following a 1-hr exposure to ADRM, after

18 hr of preincubation in metabolic inhibitors, dishes were rinsed oncewith PSA and harvested with 5 ml of 0.05% trypsin:EDTA (Ca2*- andMg2Mree). An aliquot of the cell suspension was counted, and 4 ml

were spun at 900 x g for 5 min. The supernatant was then discarded.Cells were lysed by addition of 5 ml of water at 4°and vigorously

pipeted. Five ml of n-butyl alcohol were then added, vortex-mixed for1 min, and finally centrifuged at 900 x g for 1 hr at 20°.Three ml of

the butyl alcohol layer were pipeted into plastic cuvets, and the fluorescence was then measured in a Perkin-Elmer MPF-4 fluorimeter.

In order to establish the relationship between fluorescence measuredin the butyl alcohol layer and the ADRM concentration in the presenceof cell lysate, a standard curve was constructed. Cells that had not

been exposed to ADRM were harvested and treated as describedabove. Known amounts of ADRM were added at the H2O (4°)step. A

standard curve was constructed as fluorescence versus ADRM concentration. To determine the uptake for experimental points, fluorescence was measured and ADRM concentration was determined bymeans of the appropriate standard curve.

ATP Determination. ATP was measured using a modification of aluciferase method (9). Cells were plated in 100-mm dishes and rinsed

once with cold PSA. Intracellular ATP was extracted by placing 0.6 Nperchloric acid onto adherent cells for 30 min. The acid extract wasthen neutralized with cold potassium hydroxide.

An aliquot of neutralized cell extract was placed in a scintillation vialcontaining glycylglycine buffer (50 mM), sodium arsenale (0.1 M), andMgSCvt (40 mM) at pH 7.4. Diluted firefly extract was then added, andthe vial was counted in a Beckman LS 250 liquid scintillation counter.Counts accumulating during 10 sec were compared to standardsprepared from commercial ATP (Sigma).

FMF. Cells grown in 150-mm culture dishes were treated withHoechst indicator dye 33342 with 5% Triton X-100. The resulting cell

suspension was analyzed in a flow cytometer built at the Sidney FarberCancer Institute (26). Percentages of cells in d and G2 + M, respectively, were estimated by doubling the numbers of cells found in the lefthalf of the G, peak and the right half of the G2 + M peak. The numbersof cells in S phase were then obtained by subtraction.

Ml. To determine the number of mitotic figures, cells were removedfrom 150-mm dishes by trypsinization. Following centrifugation, cells

were suspended in hypotonie KCI (0.075 M) for 10 to 15 min and fixedin methanol glacial acetic acid (3:1). After fixation, cells were placedon glass slides and stained with Giemsa. The number of mitotic cellswas then scored.

RESULTS

Survival Studies. The survival responses of asynchronouspopulations of V79 or 77A cells to ADRM were typically bi-phasic. A survival curve obtained with ADRM-sensitive V79cells after an 18-hr preincubation in full growth medium, with

or without DG, is shown in Chart 1^. The maximum dose ofADRM in the survival experiments for V79 was 3 ftg/ml. Cellspretreated with DG exhibited a 3-log increase in survival at the

1.0

Chart 1. ADRM survival response for V79(A) and for 77A (B) cells. For both V79 and77A, cells were incubated in aMEM with orwithout DG for 18 hr before and during theexposure to ADRM (1 hr at 37°). Ñ. average

number of cells per colony at the time of drugexposure; PE, plating efficiency; bars. S.E.

01t-

001 -

0001 -

00001 -

00000130

B.77A•aMEM

PÕ--66% N=229

o= aMEM« SmMOG

PE= 60% N'1.94

0.001 r

00001

AORIAMYCIN (»g/ml )

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J. P. Colofiore et al.

Chart 2. ADRM survival response for V79W and for 77A (8) cells. For both V79 and77A, cells were incubated in aMEM with orwithout DNP for 18 hr before and during theexposure to ADRM (1 hr at 37°).Ñ. average

number of cells per colony at the time of drugexposure; PE, plating efficiency: bars. S.E.

001 -

0001-

0 0001 r

00000105 1.0 15 2.0 25 30

1.0

0.1

0.01

0.001

0.0001

B, 77A

*0.2mMCNP%

N=1.90

10 15

highest dose of ADRM used.Chart 1ßshows the survival curve obtained when ADRM-

resistant 77A cells were exposed to ADRM after an 18-hr

preincubation in full growth medium, with or without DG. ForDG-treated 77A cells, a 2-log increase in cellular survival was

observed at the highest dose of ADRM. For 77A cells, themaximum dose of ADRM was 15 fig/ml.

Chart 2A shows the survival curve obtained when V79 cellswere exposed to ADRM after an 18-hr preincubation in full

growth medium, with or without DNP. At the level of DNP used,a 2-log increase in cell survival was observed for pretreated

V79 cells at the maximum dose of ADRM (3 /¿g/ml).Chart 26 illustrates the survival curve obtained when ADRM-

resistant 77A cells were exposed to ADRM after an 18-hr

preincubation, with or without DNP. For 77A cells pretreatedwith DNP, there was a 1-log increase in survival at the highest

dose of ADRM (15 jug/ml). In the survival experiments forADRM, for both V79 and 77A cells, the doses of each metabolicinhibitor were chosen for their ability to lower intracellular ATPwithout alteration of plating efficiency. DG at concentrations of7 rriM or higher produced a gradual loss of plating efficiency.For DNP, at levels of 0.25 mw and beyond, plating efficiencyfell significantly.

ATP, Plating Efficiency, and Cell Multiplication. That theintracellular ATP content of V79 and 77A cells is lowered byan 18-hr preincubation with metabolic inhibitors, either DG or

DNP, is shown in Table 1. Prior treatment with DG loweredintracellular ATP for V79 cells by a factor of 3. In the case ofDNP, ATP content was less affected for V79, being lowered bya factor of 1.5.

For 77A cells pretreated with DG, ATP was depleted by afactor of 2. Prior incubation of 77A cells in DNP, however,lowered the content of ATP by a factor of 1.5. The effects ofplating efficiency and cell multiplication following an 18-hr prior

incubation for both cell lines in either metabolic inhibitor compared to control are also shown in Table 1. Cell multiplicationwas measured by calculating the average number of cells percolony (Ñ) immediately before the cells were treated with

ADRIAMYCIN(»g/ml)

Table 1Plating efficiency, cell multiplication (Ñ), and ATP content of V79 and 77A cells

pretreated with metabolic inhibitors or untreated control (aMEM)

CelllineV79V7977A77APretreatmentcondi

tion (18hr)aMEM

(control)aMEM +DG«MEM

(control)«MEM+DNPaMEM

(control)aMEM +DGaMEM

(control)aMEM + DNPCell

multiplication(/9)a1.78

1.362.12

1.832.29

1.942.18

1.90Plating

efficiency(%)67±4b63

±472

±472 ±466

±760±266

±562 ±3ATP

(nmol/mgprotein)7.8

±1.2C

2.6 ±0.56.3

±0.35.4 ±0.26.4

±0.43.7 ±0.58.6

±0.27.2 ±0.3

N, average number of cells per colony at the time cells were treated withADRM.

6 Mean ±S.E.c Average ±S.E. of duplicate.

ADRM. Pretreatment with either metabolic inhibitor did notaffect plating efficiencies but lowered cellular multiplication forboth cell lines after an 18-hr incubation.

ADRM Uptake Studies. In order to determine if the enhancedsurvival to ADRM might be due to any differences in uptake ofADRM in metabolic inhibitor-treated V79 or 77A cells, we havemeasured ADRM uptake using a fluorimetrie assay. Drug uptake experiments were performed, and intracellular ADRM wasmeasured at doses of ADRM comparable to those used in thesurvival experiments. Since uptake of ADRM is cell densitydependent, comparable densities of cells were used in inhibitor-treated versus untreated (control) cultures. That pretreatment with DG or DNP does not lower uptake of ADRM is shownin Chart 3A for V79 cells and in Chart 36 for 77A cells. On thecontrary, treatment with metabolic inhibitors resulted in a slightenhancement of drug uptake. Because the ring structure ofDNP might possibly increase fluorescence, we constructedstandard curves for uptake of ADRM in the presence of DNP.We observed no spurious fluorescence under these conditionsand therefore have ruled out enhanced fluorescence of DNPitself as the reason for any increased uptake in the presence

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Increased Survival to ADRM by Metabolic Inhibitors

A,V79

D <3MEM(Control)W/, i'-- '•'•••'•"

I aMEM»0.2mMDNP

Chart 3. ADRM uptake in V79 (A) and 77A(B) cells. All cells received a 1-hr exposure toADRM at 37°.Cells were grown in aMEM with

or without inhibitor pretreatment.

B.77A

Table 2

Ml for V79 cells at Time Oat 6 and 20 hr after removal of DG or DNP: data from2 separate experiments.

Cells were pretreated for 18 hr with metabolic inhibitors, rinsed with PSA,refed complete medium, and prepared for Ml after 0, 6, or 20 hr. Five hundredcells were counted in each experiment.

PretreatmentconditionsaMEM

(control)aMEMH-DGaMEM

+DNPaMEM

+DGaMEM-DNPaMEM

+DGaMEM+ DNPTime

after removal ofinhibitors(hr)0620MlExperiment14.41.42.02.03.04.03.6(%)Experiment24.01.62.42.22.64.24.4

of inhibitor.Ml. As a result of metabolic inhibition after pretreatment with

DG or DNP, we have measured a decrease in cell multiplicationfor V79 by calculation of Ñ values and also by cell counts. Wetherefore wished to examine any effect of metabolic inhibitorson the growth of cells by measurement of the numbers ofmitotic cells under our standard conditions of pretreatment withDG or DNP and in untreated control cultures (aMEM).

These data are presented in Table 2. V79 cells were culturedfor 18 hr, with or without metabolic inhibitors. Five hundredcells were scored for mitotic figures for each inhibitor in 2separate experiments and in aMEM (control) alone. The number of cells in mitosis is expressed as the percentage of a totalof 500 cells for each of the 2 inhibitors. Inhibitor-treated cellsshowed a reduction in the number of mitotic figures. Thenumber of mitotic cells decreased from 4% in the control(aMEM) to between 1 and 2% in DG and to 2% in DNP. Since,after pretreatment with metabolic inhibitors there were fewercells in mitosis, measurement of Ml has verified the partialgrowth inhibition that we have observed by calculation of Ñvalues (cell multiplication) and by cell counts.

Reversibility of Metabolic Inhibition. In order to determineif the effects of the metabolic inhibitors were reversible, wehave measured the survival fractions and the levels of intracel-lular ATP with time after removal of DG or DNP for both cell

EXTRACELLULAR ADRM (ug/ml)

0.01 -

0.0001 ;-

A.V79aMEM(Conlroll

aMEM•»6mM DG

802

HOURS

Chart 4. Survival fractions and ATP levels of V79 cells with time after removalof metabolic inhibitors. Cells were pretreated for 18 hr in aMEM alone or inaMEM plus inhibitors. At Time 0, cells were rinsed once with PSA, and then refedaMEM. Survival fractions and ATP levels were assayed with time after removal ofinhibitors. For each point, cells were exposed to a single dose of ADRM (1.5 /ig/ml) for 1 hr at 37°.A and C, survival fraction and ATP levels, respectively, for

cells pretreated with DG; 6 and 0, survival fraction and ATP levels, respectively,for cells pretreated with DNP. Each value for survival is the average of triplicates.Each value for ATP is the average of duplicates.

lines. These experiments were performed using a single doseof ADRM, 1.5 ¿ig/ml for V79 and 10.0 jug/ml for 77A cells.Both the survival fractions and levels of ATP were measured atincrements up to 7 hr after removal of the inhibitors. Thesurvival fractions and levels of ATP measured as a function oftime after removal of inhibitors are shown in Chart 4 for V79and in Chart 5 for 77A. For V79 or 77A cells pretreated withDG or DNP, survival began to decrease gradually at first andthen more substantially after 7 hr. For both cell lines, survivalfractions approached control values after 7 hr. We have ob-

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8 02468

HOURSChart 5. Survival fractions and ATP levels of 77A cells with time after removal

of metabolic inhibitors. Cells were pretreated for 18 hr in aMEM alone or inaMEM and inhibitors. At Time 0, cells were rinsed once with PSA and then refedaMEM. Survival fractions and ATP levels were assayed with time after removal ofinhibitors. For each point, cells were exposed to a single dose of ADRM (10 /ig/ml) for 1 hr at 37°.A and C, survival fraction and ATP levels, respectively, for

cells pretreated with DG; ßand D, survival fraction and ATP levels, respectively,for cells pretreated with DNP. Each value for survival is the average of triplicates.Each value for ATP is the average for duplicates.

served that levels of intracellular ATP increased with time afterremoval of the metabolic inhibitors and reached control levelsat 7 hr after removal of DG or DNP. Since survival fractionsdecreased and ATP levels increased with time after metabolicinhibitors were removed, we conclude that the effects of metabolic inhibition are slowly reversible.

Since we have observed a partial growth inhibition in V79cells pretreated with DG or DNP by measurement of Ml, weexamined the effects on Ml with time after removal of theinhibitors. After an 18-hr pretreatment with DG or DNP, cells

were rinsed with PSA and refed full growth medium. At 6 hrand again at 20 hr after removal of the inhibitors, Mis weremeasured. These data are shown in Table 2 for V79 cells. Asis seen, the numbers of mitotic cells increased at 6 hr afterremoval of inhibitors and reached control values (approximately4%) after 20 hr. These data indicate that the effects on mitosisafter metabolic inhibition by DG or DNP in V79 cells are slowlyreversible and reach control levels after 24 hr. In summary,after removal of metabolic inhibitors, we have observed thatsurvival decreased, ATP content increased, and the number ofcells in mitosis returned to control values. We therefore conclude that both cell lines gradually regain their sensitivity toADRM.

MF. The cytotoxicity of ADRM is cell cycle specific, sincecells in S phase are especially sensitive to the drug. Therefore,we examined whether cells were accumulating in any cell cyclecompartment as a result of pretreatment with either DG or DNP.We performed flow microfluorometric analyses on V79 cells,both inhibitor-pretreated and untreated (control) cells. Repre

sentative effects of inhibitor pretreatment on cell cycle distributions are shown for V79 cells in Chart 6. For FMF, cells werepretreated with inhibitors for 18 hr but were not given ADRM.Representative DNA histograms are shown for V79 cells: inaMEM, control (no inhibitor) in Chart 6A; in «MEMplus DG inChart 68; and in aMEM plus DNP in Chart 6C. The left peakrepresents cells in Gìor G0; troughs represent cells in S phase,the most sensitive compartment; while the right peak represents cells in G? and M.

The FMF tracings for V79 cells pretreated with either metabolic inhibitor were not superimposable with the pattern forcontrol cells in aMEM alone. Since the same number of cellswas used, the apparent discrepancies in shapes of the curveswere attributed to differences in calibration of the flow cytom-eter, resulting in variations in the total number of channelsopen.

Table 3 compares the effects of DG or DNP pretreatment oncell cycle distributions for V79 cells as determined by FMF.The number for each cell cycle compartment shown in percentages is a reflection of the integrated area under each peak(see Chart 6). For survival responses, the number of cells in Sphase is the most important in that cells are most sensitive toADRM. After DG pretreatment, the number of cells in S phase

DNA CONTENT

Chart 6. Flow microfluorimetric analyses of cell cycle distributions for V79: A,aMEM (control); B. aMEM plus 6 mM DG; C, aMEM plus 0.2 mM DNP. DNAcontent corresponds to Gìphase (/eft peak), S phase ((roughs), and G2 + Mphases (righi peak).

Table 3

Effect of DG or DNP on cell cycle distributions for V79 by flow microfluorimetricanalysis

Average of 2 separate experiments. Values for each replicate experiment werewithin 10% of the mean.

% of cellsinPretreatment

conditionsaMEM

(control)aMEM+ 6.0 mwDGaMEM-1-0.2 mM DNPG,455465S313121Gj

+M241514

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has not been reduced; whereas after DNP treatment, the number has been lowered. For both metabolic inhibitors, the number of cells in G2 and M phases of the cell cycle has also beenreduced. Although cells in mitosis are also sensitive to ADRM,the number of cells in M in an asynchronous population of V79cells was only 4%, and thus it represented a small percentageof the population of cells. Since G2 is not an ADRM-sensitive

cell cycle compartment, we think that the reduction in thenumber of cells in G2 is not of major significance in the interpretation of our survival data.

DISCUSSION

An inhibitor of anaerobic glycolysis, DG, or an uncoupler ofoxidative phosphorylation, DNP, both substantially increasedthe survival of V79 or 77A cells to ADRM. In fact, in somecases, cells pretreated with inhibitors showed up to a 3-log

decrease in kill at the highest dose of ADRM used. The initialslopes of the survival curves for cells treated with metabolicinhibitors were very different compared to control, which wouldaccount for large differences in the surviving fractions seen athigher concentrations of ADRM. Under the conditions of pretreatment with metabolic inhibitors (6 mw DG and 0.2 mwDNP), levels of intracellular ATP were lowered and cellulargrowth was partially inhibited, which resulted in lower mitoticindices. However, neither plating efficiency nor drug uptakewas lowered. In fact, the enhanced survival to ADRM occurreddespite an increase in uptake of ADRM in inhibitor-pretreated

cells. Several investigators have shown that ADRM uptake wasdue to passive diffusion, whereas efflux of the drug was anATP-requiring, active, outward transport mechanism (10, 18).ADRM-resistant cells have been found to possess a markedly

enhanced efflux mechanism which rendered cells highly resistant to the cytostatic and cytotoxic effects of ADRM (17). Ifdrug efflux is a process of active outward transport, requiringATP, inhibitor-treated cells with lower ATP levels should retain

more drug. This may explain why we observed enhancementof drug uptake in metabolically inhibited V79 and 77A cells, ascompared to control.

We have also shown that the effects of metabolic inhibitionin both cell lines were slowly reversible. Once the metabolicinhibitors were removed, the surviving fractions decreased,while levels of ATP and Ml increased with time. Thus, afterremoval of metabolic inhibitors, both cell lines gradually regained their sensitivity to ADRM, approaching that of control.We have demonstrated further that the enhancement in survivalto ADRM after pretreatment with metabolic inhibitors is a general phenomenon in that we observe it in both ADRM-sensitiveas well as in ADRM-resistant cells. Although DG and DNP

exhibit differing modes of action, both metabolic inhibitorsshare a common primary effect, which is to change the levelsof metabolic energy (ATP) of cells. Therefore, we conclude thatthe physiological, i.e., the metabolic, state of the cells is important for the process of cytotoxicity, and that the increase insurvival to ADRM in cells treated with metabolic inhibitors isnot due to decreased drug uptake.

Cell cycle effects of ADRM have been well studied (3, 8).Cells in mitosis and early S phase were shown to be mostsensitive to the drug, whereas G,. G2, and late-S-phase cellswere shown to be least sensitive. For DG-pretreated V79 cells,FMF showed no reduction in the number of cells in S phase.

However, for DNP-pretreated cells, FMF showed fewer cells in

S phase, and thus we cannot disregard the possibility that theincrease in survival which we have observed after DNP pretreatment might be due, at least in part, to altered cell cycledistributions.

Metabolic inhibitors such as DG or DNP have additionaleffects on cells besides lowering levels of metabolic energy.These effects may be caused by decreased levels of energy ormay be unrelated to it. For example, in HeLa cells, DNP hasbeen shown to reduce the number of growing points in replicating DNA (12). Moreover, DNP has also been shown to effecta change in membrane potential which resulted in alteration ofintracellular pH (23). In addition to its effects on cellular energy,DG has been shown to alter patterns of glycosylation (21) incells and to be incorporated into cellular glycolipids and gly-coproteins (28, 29). Both DG and DNP have been shown toinduce specific membrane protein synthesis in several mammalian cell lines in response to a number of stressful conditions.For example, the induction of such stress proteins has beenreported for cells exposed to chemical agents such as aminoacid analogs (19), inhibitors of either glycosylation (24, 27), oroxidative phosphorylation (25); by heat shock (1 ), by deciliation(13), or by release from anoxia (15). Induction of cellularproteins in response to metabolic stress resulting from pretreatment with inhibitors may be related to the enhancement insurvival to ADRM. Furthermore, both DG and DNP may act toslow down scheduled DNA synthesis as well as the biosynthesisof other cellular macromolecules.

In summary, the enhancement in survival to ADRM which weobserved when V79 or 77A cells were pretreated with DG orDNP could be a result of ATP depletion. On the other hand,additional biochemical changes either related or unrelated toATP depletion could also contribute to the increased survivalto ADRM after metabolic inhibitor pretreatment. Specific membrane protein induction, changes in glycosylation patterns, oralteration in the rate of DNA synthesis following partial growthinhibition allowing time for repair are currently being investigated as possible mechanisms by which metabolic inhibitorsact to increase cell survival to ADRM.

ACKNOWLEDGMENTS

We thank Dr. Howard M. Shapiro for help with flow cytometry.

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3940 CANCER RESEARCH VOL. 42

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1982;42:3934-3940. Cancer Res   J. R. Colofiore, G. Ara, D. Berry, et al.   Cells by 2-Deoxy-d-glucose and 2,4-DinitrophenolEnhanced Survival of Adriamycin-treated Chinese Hamster

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