Metabolism of Ascites Tumor Cells

II. Inhibition of Respiration by Glycolyzable andNonglycolyzable Sugars Phosphorylated by

Hexokinase*

W. D. YUSHOK(Division of Cancer Biochemistry, Biochemical Research Foundation, Newark, Delaware)

SUMMARY

The effects of twelve monosaccharides on the oxygen uptake of Krebs-2 ascitescarcinoma cells were determined during prolonged incubation. Seven sugars wereshown to inhibit respiration. The incubation periods required to induce inhibitionwere inversely related to the maximum rate of phosphorylation of each sugar by tumorhexokinase. Of these inhibiting sugars, the nonglycolyzable were 2-deoxyglucose,glucosone, and mannoheptulose; and the glycolyzable were glucose, mannose, fructose,and glucosamine. Glucosone, a slowly phosphorylated sugar of highest affinity tohexokinase, controlled the degree of inhibition and its time of onset when added totumor cells in combination with 2-deoxyglucose or with each of the glycolyzablehexoses. The nonphosphorylated sugars, galactose, 3-o-methylglucose, L-sorbose,lyxose, and xylose, were found to be noninhibitory to respiration.

The effects of representative glucose analogs and ho-mologs on ascites tumor respiration have been investigated to ascertain the particular characteristics of sugarsassociated with respiratory inhibition. This is a part ofour studies concerned with the respiration-inhibitingsugars directed toward the elucidation of the intracellularchemical factors involved in the control of respiratory andphosphate metabolism of ascites tumor cells.

The inhibition of tumor respiration by glucose, nowknown as the Crabtree Effect, was first reported to occurin tumor slices by Crabtree (3) in 1929 and in ascitestumor cells by Kun, Talalay, and Williams-Ashman (9)and El'tsina and Seitz (4) in 1951. The Crabtree Effect

was found by Racker (14) and by Brin and McKee (2)to be elicited in ascites tumor cells by two other glycolyzable hexoses—fructose and mannose.

2-Deoxy-D-glucose (2DG), a nonglycolyzable glucoseanalog readily phosphorylated by tumor hexokinase (11),was first reported to inhibit ascites tumor respiration in1957 (22). In those preliminary experiments conducted inthis laboratory, glucosone was found to counteract respiratory inhibition by 2DG and glycolyzable sugars during short incubation periods (22). Further investigationshave been conducted on the metabolic effects of 2DG,glucosone, and glucose added separately and in combina-

* This work has been supported in part by Public HealthService research grant No. C-5118 from the National CancerInstitute.

Received for publication March 6, 1963.

tion and incubated for prolonged periods under differentconditions.

Twelve monosaccharides have been studied for theireffects on ascites tumor respiration. Seven sugars, eachphosphorylated by tumor hexokinase (11), have beenshown to inhibit oxygen uptake of Krebs-2 ascites carcinoma cells incubated in the presence of pyruvate. Fivesugars, which inhibit tumor fructolysis (21) but are notphosphorylated at a detectable rate by hexokinase, havebeen found to be noninhibitory to tumor respiration. Theresults of additional studies bearing on the intricatemechanisms of respiratory inhibition by glycolyzable andnonglycolyzable sugars and of concomitant changes inphosphate metabolism are presented in this series ofpapers.

MATERIALS AND METHODSKrebs-2 ascites carcinoma cells were propagated intra-

peritoneally in white Swiss mice. Krebs cells werewashed free of extracellular ascitic fluid and of most ofthe blood cells by suspending the cells 3 times in themedium to be used in the metabolic experiment, followedeach time by centrifugation at low speed for short periodsas previously described in detail (20). The packed volumeof Krebs cells was determined in each experiment bycentrifugation of the cell suspension in duplicate Bauer-Schenk tubes (20).

Krebs-Ringer phosphate buffer (18), modified to contain phosphate at a concentration of 12 mit, pH 7.5, and

187

on March 28, 2019. © 1964 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

1S8 Cancer Research Vol. 24, February 1964

no calcium, was prepared with glass-redistilled water andwas used in most of the experiments for suspension ofcells during both the washing and manometric procedures.In the experiments designed to determine the influence ofadded orthophosphate on oxygen uptake, the Krebs cellswere washed and suspended in a phosphate-free mediumof the following composition: 147 niM NaCI, 5.9 niMKC1, and 1.5 niM AIgS04 in glass-redistilled water. Thephosphate-free medium for suspension of cells in themain chambers of the Warburg vessels was buffered atpH 7.5 by 20 HIMsodium glycylglycinate and contained5 mM sodium pyruvate.

The rate of respiration of Krebs cells was determinedby the conventional Warburg technic (18) at 37.5°C.after a 10-minute pre-incubation period with air as thegas phase. Each sugar solution was tipped from theside-arm into the main chamber immediately after thepre-incubation period. The fluid volume of each Warburg vessel was 3.0 ml. Carbon dioxide was absorbed ineach vessel by 0.2 ml. 5 per cent KOH in the center wellcontaining a strip of Whatman No. 40 filter paper to increase the absorptive surface. The pH of some cell suspensions was determined at the end of the incubationperiod by means of a Beckman Model G pH meter.

The proportion of viable and dead cells in a populationof Krebs cells was determined by the trypan blue stainingtechnic (15). Precipitation of trypan blue from solutionby salts during storage, which can produce variablestaining results, was avoided by the preparation withglass-redistilled water and storage at 7°C.of a stock solu

tion of 0.125 per cent trypan blue (National Aniline).The working trypan blue solution was prepared justbefore use by mixing 4 parts trypan blue stock solutionwith 1 part Krebs-Ringer phosphate stock solution at aconcentration 5 times that of isotonic solution. A Krebscell suspension containing 10-40 ¿Jcells/ml was mixedwith an equal volume of buffered trypan blue solutionand was applied on a hemocytometer for counting undera microscope of the unstained live cells and the distinctlyblue dead cells. By this method no faintly blue stainedcells of questionable category were observed.

The sugars were of the D or natural configuration unlessindicated otherwise. Solutions of glucosone were usuallyneutralized with NaOH to a pH of 7.5. Generous samplesof mannoheptulose were supplied by N. K. Richtmyer ofthe National Institutes of Health. Galactose (PfanstiehlC. P.) was freed of fermentable contaminants by treatment with Baker's yeast and by recrystallization from 80per cent ethanol. The sources of the other sugars usedin this study were previously reported (21). Each of thesugars was dissolved in the same buffer medium that wasused to suspend Krebs cells in that particular experiment.Solutions of sodium pyruvate (Nutritional BiochemicalCorp.) were stored at 7°C.and were used within 2 days of

their preparation.

RESULTSInhibitory sugars.—Oxygen uptake of Krebs cells in

cubated in the presence of 5 m\i pyruvate1 was inhibited

1The rate of oxygen uptake of 2DG-treated Krebs cells incubated in the absence of substrate was significantly lower than that

TABLE 1INHIBITION OP OXYGEN UPTAKE OP KREBS CELLS BY SUGARS

SUGARNoneGlycolyzable

:GlucoseFructoseMannoseGlucosamineNongl

ycolyzable :2-DeoxyglucoseGlucosoneMannoheptuloseCONC.(nui)1010101010105215

10RELATIVE

RATEOFOXYGENUPTAKE*1st

br.1006468688664

959696101

1032dhr.1005957597759

81838988

913dhr.1005555577759

79788280

80FINALPH7.506.456.426.457.087.56

7.467.487.427.517.48PHOSPHO-

RYLATIONRATEt1002406160120

86

Krebs cells were preincubated for 10 minutes at 37.5°C. in

Krebs-Ringer phosphate buffer containing 15 Amóles pyruvatebefore sugar solution was tipped in from the side-arm of eachWarburg vessel. These typical data were obtained in four experiments in which an average of 92^1. of cells were suspended in3.0 ml. total fluid volume in each vessel.

* The average rates of oxygen uptake of the control cell suspen

sions containing pyruvate but no sugar were 81.9, 83.3, and 78.0¿mioles/ml cells during the 1st, 2d, and 3d hours of incubation,respectively.

t Maximal rate of phosphorylation of sugar by Krebs tumorhexokinase (11) relative to glucose as 100, with the exception ofthe relative rate for glucosamine which was reported by Sols andCrane (17) for brain hexokinase.

by seven sugars, as shown by representative data in Table1. Each of the three well known glycolyzable sugars,glucose, fructose, and mannose, exhibited the CrabtreeEffect to essentially the same degree during the entire3-hour incubation, beginning within the first 10-minuteperiod. Each produced approximately the same amountof acid, as indicated by the same pH of the medium at theend of the experiment. The presence of excess glucose atthe end of the 3-hour period of incubation was indicatedby the glucose oxidase method (10). Glucosamine hadless inhibiting effect on respiration and also less acidifyingeffect than the other glycolyzable sugars. The acid produced from freshly dissolved glucosamine was shown to belactic acid by the lactic dehydrogenase method of Hornand Bruns (5). The lactic acid formed from glucosamineby Krebs cells incubated aerobically without pyruvate fora 30-minute period was approximately one-half the amountformed from glucose.

2DG was found to inhibit respiration to essentially thesame extent as glucose but without altering the pH of the

of the endogenous respiration control during the 1st hour ofincubation but did not differ significantly from it during the 3dhour. Glucosone was found to stimulate endogenous respirationslightly throughout the incubation period, but the stimulatedendogenous rate was, nevertheless, substantially below that inthe presence of pyruvate (W. D. Yushok, unpublished data).

on March 28, 2019. © 1964 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

YusHOK—Metabolismof Asdtes Tumor Cells. II 189

medium during a 3-hour period (Table 1). Respiratoryinhibition by 2DG was detected in the initial 10-minuteperiod by the Warburg manometric technic and remainedwithin the same range during the entire 3-hour period.

Following the addition of glucosone or of manno-heptulose to the pyruvate-treated cells, inhibition ofrespiration was not significant during the 1st hour butbecame evident during further incubation. Neitherglucosone nor mannoheptulose was glycolyzed at a detectable rate as indicated by the insignificant change ofpH of the medium.

The nonglycolyzable sugars, galactose, 3-o-methyl-glucose, L-sorbose, xylose, and lyxose, were tested, eachat a concentration of 60 niM, along with the sugars listedin Table 1, but were found to have no effect on Krebs cellrespiration.

Lack of effect of sugars on cell viability.—The percentageof viable Krebs cells determined by the trypan blue methodat the end of a 3-hour period of aerobic incubation in thepresence of excess 2DG, glucosone, mannoheptulose,glucose, and without sugar was found to be 96, 94, 94, 92,and 91, respectively. Also in the presence of pyruvate,the viability of Krebs cells was not affected by treatmentwith these nonglycolyzable sugars.

Inhibitory effect with and without inorganic phosphate.—Inhibition of oxygen uptake by 2DG was greatest whenthe cells were incubated without added inorganic phos-

TABLE 2EFFECT OF PHOSPHATEON RESPIRATORYINHIBITION BY

GLUCOSEAND 2-UEOXYGLrcosE

TABLE 3INHIBITION BY GLDCOSONEOF THE CRABTREE EFFECT AND

GLYCOLYSISOP GLUCOSE,MANNOSE, AND FRUCTOSE

EXP.NO.123GLUCOSECONC.(nui)0015150000000000000002DGCONC.(mu)0000151500.50.51122022222PHOSPHATECONC.(mu)0404040020202002102040RELATIVE

RATEOFOXYGENUPTAKE1st

hr.1001007570547010064835570547310059727677792dhr.1001037370507010070845470526910050667069743dhr.100103757053721007382537551671004968707172FINAL

pn7.527.526.316.197.487.50

EXP.NO.45SUGAR cose,(mu)00Glucose

10Glucose10Mannose10Mannose10Fructose10Fructose1000Fructose

15Fructose15Fructose15Fructose15Fructose

15GLUCOSONECONC.(mu)050505050200.10.512RELATIVE

RATEOFOXYGENUPTAKE1st

hr.1009464906892689610096648494971002dhr.100835982598357831008955578190873dhr.10078558258855582100825252698582FINALPH7.607.486.457.286.457.306.427.507.567.426.166.306.887.337.42

Krebs cells were preincubated for 10 minutes with glycylgly-cine buffer and pyruvate in the main chamber of every Warburgvessel and with phosphate where indicated before hexose solutionwas tipped in from the side-arm at zero time. Each vessel contained 76.6 /ni. of cells in Exp. 1, 80.6 /il. in Exp. 2, and 85.3 /J.in Exp. 3 suspended in a total fluid volume of 3 ml.

Conditions of incubation of Krebs cells were the same as inTable 1. Each Warburg vessel contained 73.3 *tl. of cells in Exp.4 and 102.6 n\. in Exp. 5.

phate (Table 2). 2DG at a concentration of 15 mM inhibited Krebs cell respiration 46-50 per cent in the absenceof phosphate and 28-30 per cent in the presence of 4 mMphosphate (Exp. 1). The 2DG inhibition was also partlyreversed by 2 mM phosphate when 2DG was at levels forboth maximal inhibition (1 and 2 HIM)and submaximalinhibition (0.5 HIM)as shown in Exp. 2. The degree ofreversal of 2DG inhibition by 10^0 HIMphosphate wasonly slightly greater than that by 2 mM phosphate (Exp.3).

As shown by the data in Table 1 (Exp. 1), glucose, incontrast to 2DG, inhibited oxygen uptake to the sameextent both in the absence and in the presence of 4 mMinorganic phosphate.

Control of respiration inhibition by glucosone.—Theresults presented in Table 3 show that during the 1sthour of incubation 5 mM glucosone inhibited pyruvate-supported respiration 6 per cent and reduced the CrabtreeEffect of glucose, mannose, and fructose to 10, 8, and 4per cent, respectively. During the 2d and 3d hours ofincubation, the combinations of glucosone with eachglycolyzable sugar inhibited oxygen uptake to the sameextent as glucosone alone. Acid formation from eachglycolyzable sugar was strongly suppressed by 5 mMglucosone (Exp. 4).

Glucosone at concentrations of 1 and 2 mM reversedthe Crabtree Effect of fructose to the same level of inhibition as by glucosone alone (Exp. 5). When its initialconcentration was 0.5 mM, glucosone fully suppressed theCrabtree Effect of fructose during the 1st hour, but itseffectiveness decreased during further incubation and therespiratory inhibition during the 3d hour approachedthat of fructose. Glucosone initially at 0.1 mM partlyblocked the respiratory inhibition by fructose during the

on March 28, 2019. © 1964 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

190 Cancer Research Vol. 24, February 1964

TABLE 4EFFECTOF GLUCOSONEOF RESPIRATORYINHIBITION

BY2-DEOXYGLUCOSE

EXP.NO.67GLUCOSONECONC.(ox)0550552DGCONC.(mu)1010015150INHIBITION

OF OXYGENUPTAKE(PERCENT)1st

hr.353627832dhr.4118173419193dhr.411622361918

Conditions of incubation of Krebs cells were the same as inTable 1.

1st hour, but the oxygen uptake subsequently decreasedto the inhibited rate of the fructose control. The pHvalues of Exp. 5 indicated that glucosone at 2 HIMcompletely inhibited acid formation from fructose throughoutthe 3 hours of aerobic incubation. Lower concentrationsof glucosone permitted a decrease of pH, which was inversely related to the initial concentration of glucosone.

Glucosone in combination with 2DG was found to havethe same effect on tumor respiration as glucosone alone(Table 4). Glucosone at 5 IBM controlled the rate ofoxygen uptake in the presence of 2DG at levels of 10 HIM(Exp. 6) and of 15 muí(Exp. 7).

Capacity for A TP synthesis by respiration and glucolysis.—The theoretical capacity for adenosinetriphosphate(ATP) synthesis from adenosinediphosphate (ADP) byKrebs cells incubated in the presence of pyruvate, glucose,and 2DG was calculated from averaged data on respiration presented in this paper and on glucolysis reportedin the preceding paper of this series (20) and is presentedin Table 5. Respiring Krebs cells incubated in thepresence of pyruvate as sole substrate were shown to havethe capacity to synthesize 450 jumólesATP/ml cells/hour,when the calculations were based on the assumption ofmaximum efficiency of oxidative phosphorylation in theliving cells with the formation of 6 moles ATP from ADPper mole oxygen consumed ( 13). In the presence of excess2DG, the theoretical ATP formation by the inhibitedrespiratory system was lowered to 300 /¿moles. The ATPavailable to the reactive phosphate pool was further reduced by the quantity used for phosphorylation of 2DG,which is reported in the following paper (12) to be 37/¿molesin the 1st hour of incubation, to give a net synthesis of 263 /¿moles. In the presence of glucose, thecalculated theoretical ATP formation by the inhibitedrespiration was 303 /¿moles. The decreases in the •respiratory ATP formed was compensated for by the 173/¿molesATP synthesized by aerobic glucolysis. The netATP synthesized by the combination of both respirationand glucolysis, 476 /¿moles,was nearly the same as thatsynthesized by pyruvate-supported respiration in theabsence of glucose. The ATP formed by anaerobic glucolysis was calculated to be only 11 per cent lower than thequantity formed by pyruvate-maintained respiration.

DISCUSSIONThe seven sugars which inhibited pyruvate-supported

oxygen uptake of Krebs cells have in common the propertyof phosphorylation by hexokinase (11, 17). The time required for the development of significant respiratoryinhibition by each sugar appears to be related to itsmaximum rate of phosphorylation (Table 1), and eachsugar has the additional property of a significant affinityto hexokinase. The rapidly phosphorylated sugars, suchas the glycolyzable ones and 2DG, induced full respiratoryinhibition within the first 10-minute period after addition.The slowly phosphorylated sugars, glucosone and manno-heptulose, produced significant respiratory inhibition afterthe 1st hour of incubation.

Although the five sugars which are not phosphorylatedby hexokinase inhibit anaerobic fructolysis (21), theyhave been found to be noninhibitory to Krebs cell respiration. Even though galactose at high concentrations wasreported to be phosphorylated at a low but detectablemaximum rate by tumor hexokinase (11), its high Michaelis constant of 175 HIMprecluded an effective rate ofphosphorylation at the lower but still substantial concentration. According to Racker (14) oxygen uptake ofEhrlich cells was not affected by galactose or by threecarbohydrates, ribose, sucrose, and glycerol, which areinert in the hexokinase reaction. Significant affinity tohexokinase without phosphorylation, which is characteristic of lyxose (11, 17) and xylose (17), is apparently notrelated to respiratory inhibition.

Each of the well known glycolyzable sugars, glucose,mannose, and fructose, inhibited Krebs cell respirationto the same extent; this is in agreement with the resultsreported for Ehrlich ascites cells (2, 14). The equalinhibitory rates of respiration and the identical rates

TABLE 5EFFECTOF2-DEOXYGLucosBANDGLUCOSEONTHETHEORETICAL

MAXIMALCAPACITYFORATP SYNTHESISFROMADPBYKREBS CELLS

SUGAR(10mu)None2-Deoxyglucose

GlucosePYRUVATE(mM)555ATP

FORMATION*GJMOLES/MLCELLS/HR)Respiration450300

303Glucolysis00173ÃŽNet450263|476

* Calculations were based on averaged data for glucolysis (20)and oxygen uptake of Krebs cells and on the theoretical formationof 6 moles ATP from ADP per mole oxygen consumed and on thenet synthesis by glucolysis of 1 mole ATP from ADP per molelactate produced (13).

t During the 1st hour of incubation with 2DG, 37 ¿¿molesATPwere used for the phosphorylation of 2DG as shown in the subsequent paper (12)and were subtracted from the calculated quantityof ATP synthesized by the inhibited respiration to give the netvalue.

ÎUnder anaerobic conditions, glucolysis (20) is capable ofsynthesizing over 400 /¿molesATP/ml cells/hr when the efficiencyof phosphorylation is assumed to be the same as under aerobicconditions.

on March 28, 2019. © 1964 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

YTJSHOK-—Metabolismof Ascites Tumor Cells. II 191

(20) of aerobic glycolysis of the three hexoses in Krebscells suggest a relationship between the metabolic processes. Since the glycolytic rates of the three hexosesare less than one-tenth of the potential rates of theirphosphorylation by Krebs cell hexokinase (11), thisenzyme activity does not limit the metabolic rate.

Glucosamine has been shown to have less inhibitoryeffect on Krebs cell respiration than the other glycolyz-able sugars and to be converted to lactic acid by Krebscells incubated under aerobic conditions at a lower ratethan the other glycolyzable sugars. The latter phenomenon was shown by Scholefield (16) to occur in Ehrlich cells. Since glucosamine is phosphorylated readilyby hexokinase, Krebs cells apparently have the necessaryenzymes to convert glucosamine-6-phosphate at a limitedrate to one of the intermediary metabolites of glycolysis.

The inhibition of Krebs cell respiration by 2DG wasstrongest in the absence of added phosphate in the medium and was partly reversed by the addition of inorganicphosphate similar to that reported by Ibsen, Coe, andMcKee (8) to occur in Ehrlich cells. Since 2DG-treatedascites tumor cells do not acidify the medium, the effectof added phosphate on 2DG-inhibited respiration isapparently not due to increased buffering of the medium.

In the presence of glucosone, the glycolyzable sugarsand 2DG were ineffective for respiratory inhibition.Since glucosone has the highest affinity for tumor hexokinase of any of the sugars investigated but is very slowlyphosphorylated, it can effectively block the phosphorylation of the other sugars. This is illustrated by the factthat glucosone at low concentration suppresses the Crab-tree Effect of fructose, which has the lowest affinity amongthe glycolyzable sugars. The cessation of respiratoryrate control by glucosone at very low concentration in thepresence of excess fructose may be the result of the complete conversion of glucosone to its phosphorylated form.If this explanation is correct, glucosone-6-phosphate,unlike glucose-6-phosphate, does not inhibit tumor hexokinase, although attempts to obtain conclusive proof ofthis interaction with purified tumor hexokinase were notsuccessful (11). Since glucosone has been shown to stimulate slightly the endogenous respiration, it is possiblethat glucosone or its phosphorylated product is oxidizedby Krebs cells at a very slow rate.

The capacity of glucosone to reverse the CrabtreeEffect rules out the possibility discussed by Bloch-Fran-kenthal and Weinhouse (1) that high concentrations ofglucose per se may exert a direct inhibitory action on oneof the steps of electron transport. However, the effectsof glucosone decidedly indicate that phosphorylation byhexokinase is the primary reaction necessary for respiratory inhibition by both glycolyzable and nonglycolyzablesugars.

The capacity for ATP synthesis from ADP in Krebscells by respiration in the presence of pyruvate is similarto that by the combination of respiration and glucolysisand is only slightly higher than that by anaerobic glucolysis (Table 5). Therefore, the maintenance of a uniform rate of ATP synthesis appears to be a commonfeature of both the Pasteur Effect and its reciprocal, theCrabtree Effect. Since an ATP-depleting agent, such

as 2DG, does not produce energy for cell metabolismeither aerobically or anaerobically, it is not expected toalter the metabolic equilibrium in the same manner asan ATP-producing agent, such as glucose, and cannot beunambiguously associated with the inverse of the PasteurEffect. The Crabtree Effect is, indeed, the reciprocal ofthe Pasteur Effect when it is limited to the effect of glycolyzable sugars, which have one common mechanismof action, but not when its definition is broadened toinclude nonglycolyzable sugars as in Ibsen's review (8).

Although the Krebs ascites tumor has the theoreticalcapacity to synthesize from ADP over 450 jumólesATP/ml cells/hour by respiration alone or by the combinationof respiration and glycolysis, its ATP content is approximately 3 jumoles/ml cells (12), less than 1 per cent of thetotal ATP that can be formed in 1 hour. Therefore, therate of ATP utilization by enzymatic reactions in Krebscells maintained alive in vitro approaches the rate of ATPsynthesis. The cellular ATP concentration is apparentlydependent on the balance between the enzymatic reactions for synthesis and degradation of ATP. Furtherinvestigations of ATP-depleting sugars should helpelucidate the metabolic pathways and control mechanisms involved in ATP metabolism of cancer cells.

ACKNOWLEDGMENTS

2DG and glucosone were prepared in this laboratory, 2DG byF. B. Cramer and glucosone by Marie T. Hudson and Gladys E.Woodward (6).

REFERENCES

1. BLOCH-FHANKENTHAL,L., ANDWEINHOUSE, S. Metabolism ofNeoplastic Tissue. XII. Effect of Glucose Concentration onRespiration and Glycolysis of Ascites Tumor Cells. CancerRes., 17:1082-90, 1957.

2. BRIN, M., ANDMcKEE, R. W. The Inhibition of Respiration byGlucose, Fructose, and Mannose in the Ehrlich Mouse AscitesTumor. Cancer Res., 16:364-68, 1956.

3. CRABTREE, H. G. Observations on the Carbohydrate Metabolism of Tumours. Biochem. J., 23:536-45, 1929.

4. EL'TSINA, N. V., ANDSEITZ, I. F. Respiratory and Glycolytic

Phosphorylation in Tumor Cells. Doklady Akad. NaukS.S.S.R., 77:653-56, 1951.

5. HORN, H. D., AND BRUNS, F. H. Quantitative Bestimmungvon L(+)-Milchsäure mit Milchsäuredehydrogenase. Biochim.Biophys. Acta, 21:378-80, 1956.

6. HUDSON,M. T., ANDWOODWARD,G. E. Glucosone. I. Preparation and Properties. J. Franklin Inst., 264:61-68, 1957.

7. IBSEN, K. H. The Crabtree Effect: a Review. Cancer Res., 21:829-41, 1961.

8. IBSEN, K. H.; COE, E. L.; ANDMcREE, R. W. Some FactorsInfluencing Respiration and Glycolysis in Ehrlich AscitesTumor Cells. Cancer Res., 20:1399-1407, 1960.

9. RUN, E.; TALALAY,P.; ANDWILLIAMS-ASHMAN,H. G. Studieson the Ehrlich Ascites Tumor. I. The Enzymic and MetabolicActivities of the Ascites Cells and the Ascitic Plasma. CancerRes., ll:855-«3, 1951.

10. McCoMB, R. B., ANDYUSHOK,W. D. Colorimetrie Estimationof D-Glucose and 2-Deoxy-n-glucose with Glucose Oxidase. J.Franklin Inst., 265:417-22, 1958.

11. . Properties of Particulate Hexokinase of the Krebs-2Ascites Tumor. Biochim. Biophys. Acta, 34:515-26,1959.

12. . Metabolism of Ascites Tumor Cells. III. Effect of2-Deoxyglucose Phosphorylation on Phosphorus Metabolism.Cancer Res., 24:193-97, 1964.

13. PARDEE,A. B. Free Energy and Metabolism. In: D. M. GREEN-BERG (ed.), Chemical Pathways of Metabolism, Vol. 1, Chap.1. New York: Academic Press, 1954.

on March 28, 2019. © 1964 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

192 Cancer Research Vol. 24, February 1964

14. RACKER, E. Carbohydrate Metabolism in Ascites TumorCells. Ann. N. Y. Acad. Sci., 63:1017-21, 1956.

15. RICHTER, M. N., ANDMACUOWELI,, E. C. Studies on MouseLeukemia. VII. The Relation of Cell Death to the Potency ofInoculated Cell Suspensions. J. Exp. Med., 57:1-20, 1933.

16. SCHOLEFIELD,P. G. Studies on Fatty Acid Oxidation. VI.The Effects of Fatty Acids on the Metabolism of EhrlichAscites Carcinoma Cells. Cancer Res., 18:1026-32, 1958.

17. SOLS, A., AND CRANE, R. K. Substrate Specificity of BrainHexokinase. J. Biol. Chem., 210:581-95, 1954.

18. UMBHEIT,W. W.; BURRIS, R. H.; ANDSTAUFFER,J. F. Mano-metric Techniques. Minneapolis: Burgess Publishing Co.,1957.

19. Wu, R., AND RACKER, E. Regulatory Mechanisms in Carbohydrate Metabolism. IV. Pasteur Effect and Crabtree Effectin Ascites Tumor Cells. J. Biol. Chem., 234:1036-41, 1959.

20. YUSHOK,W. D. Metabolism of Ascites Tumor Cells. I. Rate ofGlycolysis and Competitive Utilization of Fructose, Mannose,and Glucose. Cancer Res., 19:104-11, 1959.

21. . Inhibition of Glucolysis and Fructolysis of Krebs-2Ascites Carcinoma Cells by Chemical Agents. Cancer Res.(Suppl.), 18: pp. 379-89, 1958.

22. YUSHOK, W. D., AND BATT, W. G. Influence of D-Glucosoneand 2-Deoxy-D-glucose on Respiration and the CrabtreeEffect. Abstr. Am. Chem. Soc. pp. 23c-24c, 1957.

on March 28, 2019. © 1964 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

1964;24:187-192. Cancer Res   W. D. Yushok  by Hexokinaseby Glycolyzable and Nonglycolyzable Sugars Phosphorylated Metabolism of Ascites Tumor Cells: II. Inhibition of Respiration

  Updated version

  http://cancerres.aacrjournals.org/content/24/2_Part_1/187

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/24/2_Part_1/187To request permission to re-use all or part of this article, use this link

on March 28, 2019. © 1964 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from