11
The Hexose Monophosphate Shunt in Glucose Catabolism in Ascites Tumor Cells CHARLESE. WENNER,JOHN H. HACKNEY,ANDFRANCISMOLITERNO (Department of Experimental Biology, Roswett Park Memorial Institute, Buffalo 3, N.Y.) In the neoplastia cell there are at least two major pathways for the utilization of carbohy drates—the Embden-Meyerhof glycolytic path way, which is quantitatively the more important (27), and the hexose monophosphate shunt (1, 17, 27). The present study was carried out to determine the physiological role of the hexose monophosphate shunt in ascites tumor cells and the factors which control its operation. Recent evidence has suggested that the hexose mono- phosphate shunt provides both intermediates and reducing capacity in the form of TPNH1 for reductive synthesis (cf. review in 13, 15, 18). This paper lends strong supporting evidence for the latter concept of the function of the hexose monophosphate shunt. The capacity of intact tu mor cells to produce TPNH can be demonstrated by the addition of artificial electron acceptors which markedly stimulate a TPN-dependent oxi dation of carbon-1 of glucose. The present study affirms the idea that the TPNH is utilized for reductive synthesis by a demonstration of an anaerobic oxidation of carbon-1 of glucose to car bon dioxide, which can be stimulated to its aerobic level by the addition of a physiological electron acceptor such as pyruvate. The effect of glucose concentration on COz formation via the hexosemonophosphate shunt was studied, since Racker (22) had found that high glucose concentrations stimulated carbon-1 oxidation by Ehrlich ascites cells. However, the most important rate-limiting step in the operation of this pathway that was found was the oxidation of TPNH. MATERIALS AND METHODS Tumors.—The mouse tumors used in this study are those described in Table 1. Source references and details of most of these tumors have been listed by Hauschka et al. (11, 12). 'Abbreviations used: DPN and TPN = oxidized diphos- pho- and triphosphopyridine nucleotide, respectively; DPNH and TPNH = reduced diphospho- and triphosphopyridine nucleotide, respectively; ATP = adenosine triphosphate; DNP = dinitrophenol; Q = ¿»liters/rag dry wt/hour. Received for publication June 5, 1958. Incubation of tissue.—The tumor cells were removed from the peritoneal cavity 7-10 days after implantation, at which time significant growth had occurred. The cell suspensions were centrifuged at 1000 X g for 10 minutes at 5° C., resus- pended in calcium-free Ringer phosphate (0.1 M, pH 7.4), and centrifuged again. The initial sediments of lymphoma and K2D ascites cells were resuspended free of the lower red blood cell pellet. Two or three repetitions removed the erythro- cyte contamination. Cell suspensions were adjusted to contain 200 mg. of packed cells/ml (approximately 25 mg. dry weight) and dispersed with a very loose-fitting Potter-Elvehjem ho- mogenizer which did not break the cells. For most experiments, 1.0 ml. was added to 2.0 ml. of calcium-free Ringer phosphate solution in a Warburg flask containing substrates and factors as required. Tissue slices were made with a Stadie-Riggs slicer. Approximately 200 mg. of cells (fresh weight) were then added to a final volume of 3.0 ml. of Ringer phosphate containing substrates and factors in Warburg vessels. For broken-cell preparations, the following methods were used: Solid tumors derived from hyperdiploid Ehrlich (EL) ascites cells were homogenized in a Potter-Elvehjem glass homogenizer. Ascites cells, after being washed with calcium- free Krebs-Ringer buffer, were homogenized in either of two ways. For the experiments reported in this paper, the cells were homogenized in the Servali Omni-Mixer at full speed at 3° C. for 3 minutes, at which time few intact cells remained. Since homogenization by this method breaks up the nuclei to a considerable degree, it was of interest to compare the properties of the soluble fraction obtained by a more gentle procedure. Similar results to those reported here were obtained with the soluble fraction obtained by a method in which a 75 per cent recovery of nuclei was obtained. This technic is a modification of the procedure of Lamanna and Malette as described in Methods and Enzymology for the rupture of yeast cell suspensions (3). The ascites cells were disintegrated through mechanical agitation in the presence of grade 10 glow beads (60-80 mesh, 200 m^ average diameter, obtained from Minnesota Mining and Manufacturing Co., Minneapolis). Eighteen to 20 gm. of glass beads were placed in the Servali Omni-Mixer with 10 ml. of a 10 per cent cell suspension in isotonic sucrose. The cells were then disrupted for a period of 2 minutes at a rotor speed of 5000 r.p.m., at which speed the rotor knife blades did not break up the cells in the absence of the beads. When the nuclei obtained by this procedure were examined under the phase microscope, very little cyto- plasmic contamination was present. Differential centrifugation of the homogenate was carried out in the following manner. The ascites cells, homogenized in 5 volumes of ice-cold calcium-free Krebs-Ringer solution, were centri fuged at 20,000 X g for 20 minutes at l°-3° C. in the Servali refrigerated centrifuge, and the 1105 Research. on January 15, 2020. © 1958 American Association for Cancer cancerres.aacrjournals.org Downloaded from

The Hexose Monophosphate Shunt in Glucose Catabolism in ...cancerres.aacrjournals.org/content/canres/18/9/1105.full.pdf · The Hexose Monophosphate Shunt in Glucose Catabolism in

  • Upload
    others

  • View
    14

  • Download
    0

Embed Size (px)

Citation preview

Page 1: The Hexose Monophosphate Shunt in Glucose Catabolism in ...cancerres.aacrjournals.org/content/canres/18/9/1105.full.pdf · The Hexose Monophosphate Shunt in Glucose Catabolism in

The Hexose Monophosphate Shunt in GlucoseCatabolism in Ascites Tumor Cells

CHARLESE. WENNER, JOHN H. HACKNEY,ANDFRANCISMOLITERNO

(Department of Experimental Biology, Roswett Park Memorial Institute, Buffalo 3, N.Y.)

In the neoplastia cell there are at least twomajor pathways for the utilization of carbohydrates—the Embden-Meyerhof glycolytic pathway, which is quantitatively the more important(27), and the hexose monophosphate shunt (1,17, 27). The present study was carried out todetermine the physiological role of the hexosemonophosphate shunt in ascites tumor cells andthe factors which control its operation. Recentevidence has suggested that the hexose mono-phosphate shunt provides both intermediates andreducing capacity in the form of TPNH1 forreductive synthesis (cf. review in 13, 15, 18).This paper lends strong supporting evidence forthe latter concept of the function of the hexosemonophosphate shunt. The capacity of intact tumor cells to produce TPNH can be demonstratedby the addition of artificial electron acceptorswhich markedly stimulate a TPN-dependent oxidation of carbon-1 of glucose. The present studyaffirms the idea that the TPNH is utilized forreductive synthesis by a demonstration of ananaerobic oxidation of carbon-1 of glucose to carbon dioxide, which can be stimulated to its aerobiclevel by the addition of a physiological electronacceptor such as pyruvate.

The effect of glucose concentration on COzformation via the hexosemonophosphate shuntwas studied, since Racker (22) had found thathigh glucose concentrations stimulated carbon-1oxidation by Ehrlich ascites cells. However, themost important rate-limiting step in the operationof this pathway that was found was the oxidationof TPNH.

MATERIALS AND METHODSTumors.—The mouse tumors used in this study are those

described in Table 1. Source references and details of mostof these tumors have been listed by Hauschka et al. (11, 12).

'Abbreviations used: DPN and TPN = oxidized diphos-pho- and triphosphopyridine nucleotide, respectively; DPNHand TPNH = reduced diphospho- and triphosphopyridinenucleotide, respectively; ATP = adenosine triphosphate; DNP= dinitrophenol; Q = ¿»liters/ragdry wt/hour.

Received for publication June 5, 1958.

Incubation of tissue.—Thetumor cells were removed fromthe peritoneal cavity 7-10 days after implantation, at whichtime significant growth had occurred. The cell suspensionswere centrifuged at 1000 X g for 10 minutes at 5°C., resus-pended in calcium-free Ringer phosphate (0.1 M, pH 7.4),and centrifuged again. The initial sediments of lymphoma andK2D ascites cells were resuspended free of the lower redblood cell pellet. Two or three repetitions removed the erythro-cyte contamination. Cell suspensions were adjusted to contain200 mg. of packed cells/ml (approximately 25 mg. dry weight)and dispersed with a very loose-fitting Potter-Elvehjem ho-mogenizer which did not break the cells. For most experiments,1.0 ml. was added to 2.0 ml. of calcium-free Ringer phosphatesolution in a Warburg flask containing substrates and factorsas required. Tissue slices were made with a Stadie-Riggsslicer. Approximately 200 mg. of cells (fresh weight) werethen added to a final volume of 3.0 ml. of Ringer phosphatecontaining substrates and factors in Warburg vessels.

For broken-cell preparations, the following methods wereused: Solid tumors derived from hyperdiploid Ehrlich (EL)ascites cells were homogenized in a Potter-Elvehjem glasshomogenizer. Ascites cells, after being washed with calcium-free Krebs-Ringer buffer, were homogenized in either of twoways. For the experiments reported in this paper, the cellswere homogenized in the Servali Omni-Mixer at full speedat 3°C. for 3 minutes, at which time few intact cells remained.

Since homogenization by this method breaks up the nucleito a considerable degree, it was of interest to compare theproperties of the soluble fraction obtained by a more gentleprocedure. Similar results to those reported here were obtainedwith the soluble fraction obtained by a method in whicha 75 per cent recovery of nuclei was obtained. This technicis a modification of the procedure of Lamanna and Maletteas described in Methods and Enzymology for the ruptureof yeast cell suspensions (3). The ascites cells were disintegratedthrough mechanical agitation in the presence of grade 10glow beads (60-80 mesh, 200 m^ average diameter, obtainedfrom Minnesota Mining and Manufacturing Co., Minneapolis).Eighteen to 20 gm. of glass beads were placed in the ServaliOmni-Mixer with 10 ml. of a 10 per cent cell suspensionin isotonic sucrose. The cells were then disrupted for a periodof 2 minutes at a rotor speed of 5000 r.p.m., at which speedthe rotor knife blades did not break up the cells in the absenceof the beads. When the nuclei obtained by this procedurewere examined under the phase microscope, very little cyto-plasmic contamination was present.

Differential centrifugation of the homogenatewas carried out in the following manner. Theascites cells, homogenized in 5 volumes of ice-coldcalcium-free Krebs-Ringer solution, were centrifuged at 20,000 X g for 20 minutes at l°-3°C.

in the Servali refrigerated centrifuge, and the

1105

Research. on January 15, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 2: The Hexose Monophosphate Shunt in Glucose Catabolism in ...cancerres.aacrjournals.org/content/canres/18/9/1105.full.pdf · The Hexose Monophosphate Shunt in Glucose Catabolism in

1106 Cancer Research Vol. 18, October, 1958

supernatant was decanted (supernatant I). Further centrifugation of supernatant I was carriedout at 40,000 X g for 50 minutes in the SpincoPreparative Model L centrifuge, and the decantedsupernatant was designated as supernatant II.

The gas phase was air for the cell suspensionsand 100 per cent oxygen for the tissue slices.After equilibration for 10 minutes at 38°C., sub

strate was tipped in, and the flasks were shakenfor 1 hour unless designated otherwise. Dilutesulfuric acid was then tipped in. Liberated CO?was trapped in the center well and isolated atthe end of the reaction as BaCOs following theaddition of 0.6 millimoles of carrier Na2C03. TheBaCOj was counted in "infinitely thick" layers

with a Micromil thin-window counter. Calculations were made as described previously (27).

Preliminary experiments were carried out inwhich the incorporation of C14 of glucose, labeled

in positions 1 or 6, into the respiratory COZwas measured. Carbon-6 oxidation was relativelyunaffected by glucose concentration. Carbon-1oxidation was found to be stimulated by increasingthe glucose concentration from 0.001 to 0.03 M,provided the incubation period was at least 15 minutes. The stimulation was insensitive to malonate,further evidence that the increased C-l oxidation was due to a stimulation of the initial enzymesof the hexose monophosphate shunt pathway.However, no stimulation was observed for incubation periods of less than 15 minutes.

The lack of stimulation of C-l oxidation atthe short incubation periods suggested that perhaps at the lower glucose concentrations the rate

TABLE1DESCRIPTIONOFMOUSEASCITESTUMORSSTUDIED

Ascites tumors:Anaplastic carcinomas:

Hyperdiploid Ehrlich (EL)Hypotetraploid Ehrlich Clone 2 (E2)Hypotetraploid Krebs-2 Clone D (K2D)

Lymphomas:A #1 lymphoma6C3HED lymphosarcomaDBA/2 lymphomaP288 lymph node leukemia

Sarcomas:MC1M (fibrosarcoma)

Solid tumors:Hyperdiploid Ehrlich (EL) carcinoma

Lactic acid was determined enzymatically bythe method of Horn and Bruns (14); pentoseand sedoheptulose were assayed by the use ofthe orcinol reagent (6, 28); and glucose was determined by the anthrone method (24) .2

RESULTSEffect of glucose concentration on hexose mono-

phosphate shunt.—In view of the observation ofRacker (22) that the ratio of C-l/C-6 oxidationof glucose by Ehrlich ascites cells is dependenton the concentration of glucose, we first examinedthe controlling effect of glucose concentration onthe rates of COz formation via the alternate oxi-dative pathway by ascites tumor cells.

1Uniformly labeled glucose (glucose-U-C14) and glucose-2-C" were obtained from H. S. Isbell of the NationalBureau of Standards, and glucose-6-C" and glucose-1-C14were purchased from the Volk Radiochemical Company. Lacticacid dehydrogenase was obtained from Worthington Biochemical Company. Malonate and dinitrophenol were recrystallizedfrom commercial preparations. Phenazine methosulfate, pyri-dine nucleotides, and 2X-crystalline yeast alcohol dehydrogenase were obtained from the Sigma Chemical Company.

Host strain

Ha/ICR SwissHa/ICR SwissHa/ICR Swiss

A/HaC3H/StDBA/2DBA/2

C3H,/He

Ha/ICR Swiss

Routine serialpassage in:

MalesFemalesMales

FemalesFemalesFemalesFemales

Males

Males

of COi formation via the hexose monophosphateshunt was linear for only a short time. Therefore,the effect of glucose concentration on carbon-1oxidation was measured in the presence of 0.017 Mmalonate, which completely inhibits oxidation ofcarbon-6. Under these conditions, carbon-1 oxidation is assumed to represent COt formation viathis pathway. As seen in Chart la, the initialrate of oxidation of carbon-1 of glucose by thehyperdiploid Ehrlich ascites cells was independentof glucose concentration in the range 5 X IO"3 Mto 5 X 10~4 M. However, a decline in the rate

of oxidation of carbon-1 was observed at thelower glucose concentrations, which can be explained by the rapid initial disappearance of glucose, as plotted in Chart Ife. The glucose whichdisappeared could be accounted for as lactic acid,although some lactic acid was found to disappearafter the first 10 minutes of reaction. Thus, itwould appear that the operation of the alternatepathway is not limited by glucose concentrationsabove 5 X 10~4 M except under conditions where

Research. on January 15, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 3: The Hexose Monophosphate Shunt in Glucose Catabolism in ...cancerres.aacrjournals.org/content/canres/18/9/1105.full.pdf · The Hexose Monophosphate Shunt in Glucose Catabolism in

WENNER et al.—Glucose Catabolism in Ascites Tumor Cells 1107

the substrate is removed by the glycolytic enzymes.

An experiment with less tissue was carriedout to determine the effect of a wide range ofglucose concentrations on the rate of glycolysisand CÛ2 production via the shunt. Since thisexperiment, described in Table 2, was carriedout for very short incubation periods in whichoxidation of carbon-6 was negligible, it was foundunnecessary to add malonate to determine COsformation via the shunt. The rate of oxidationof carbon-1 of glucose, which is assumed to bederived entirely from the shunt, was independentof substrate concentrations as low as 2.5 X 10~5M.

Thus, the limitation in hexose monophosphate

oOX 15-

uo

cooc

10-

u

o_Jo

5-

¿CO

0.005M/ /

/0.0025M

O.OOIM

0.0005M

IO 20

TIME (MINUTES)30

CHABTlo.—Effect of time and substrate concentration onhexose monophosphate shunt decarboxylation by hyperdiploidEhrlich ascites cells. The molarities represent initial concentrations of glucose-1-C14.

shunt decarboxylation cannot be attributed toa low substrate affinity. The rate of glycolysiswas also maximal at low substrate concentrations,although at somewhat higher concentrations than2.5 X 10~6 M. Despite the higher concentrations

necessary for optimal rate, the glycolytic enzymeshave a marked competitive advantage, as seenby a comparison of the optimal velocities in this

experiment; i.e., for glycolysis, 0.17 jumóleglucosewas utilized per 10 minutes of incubation, and,for carbon-1 oxidation, only 0.005 /amoléwas utilized per 10 minutes.

As will be described in another section, pyruvatestimulates oxidation of carbon-1 of glucose byacting as an electron acceptor. This introducesthe complication that the glucose concentration

IO 20TIME (MINUTES)

CHAKT16.—Ascitescells (200 mg. fresh weight) were incubated in calcium-free Krebs-Ringer phosphate buffer for thedesignated time at 87.8°C. with air as the gas phase. Malonatewas present in a final concentration of 0.017 M.Hexose mono-phosphate shunt decarboxylation is considered to be equivalent to the amount of carbon-1 oxidation in this system.

might influence the shunt by its effect on thepyruvate level. To avoid this complication, theeffect of glucose was measured under conditionsin which the concentration of electron acceptorwas not such a crucial factor. That is, in thepresence of méthylèneblue, which is a more effective electron acceptor than pyruvate, the rateof carbon-1 oxidation was still found to be independent of substrate at concentrations as lowas 2 X IO-6 M.

Effect of artificial electron acceptors on oxidationof glucose-C1* by ascites cells.—Since glucose con

centration did not limit the initial rate of COs

Research. on January 15, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 4: The Hexose Monophosphate Shunt in Glucose Catabolism in ...cancerres.aacrjournals.org/content/canres/18/9/1105.full.pdf · The Hexose Monophosphate Shunt in Glucose Catabolism in

1108 Cancer Research Vol. 18, October, 1958

production via the shunt, the possibility that theelectron transport system was a rate-limiting stepwas examined by the use of artificial electronacceptors. Méthylèneblue, phenazine methosul-fate, and 2-methyl-l,4-naphthoquinone were foundto stimulate respiration from two- to five-fold,but the most marked effect of these substanceswas the preferential stimulation of oxidation ofcarbon-1 of glucose. As seen in Experiment I,Table 3, there was a minor enhancement of carbon-6 oxidation by the addition of méthylèneblue,but oxidation of carbon-1 was stimulated sevenfold. Furthermore, the stimulation of carbon-1oxidation was malonate-insensitive, indicatingthat the stimulation might be attributed to the

TABLE 2

EFFECTOFGLUCOSECONCENTRATIONONAEROBICGLY-COLYSISANDCO2FORMATIONVIATHEHEXOSEMONO-

PHOSPHATESHUNTPATHWAYFlasks containing 10 mg. of EL ascites cells (fresh tissue

weight) were equilibrated for 10 minutes at 38°C. in calcium-free Krebs-Ringer phosphate, after which procedure glucose-1-C" was added from the side arm. Flasks of each glucose concentration were then incubated for 5, 10, and 15 minutes. Thevalues recorded in this table represent the rate for the 10-minute period, at which time the velocity was linear.

Glucoseconcentration Lactic acid Glucose-C14 to C14Os

(Final molarity) (/imoles) (/¿atoms)1.0X10-« 0.34 0.0052.5X10-» 0.32 0.0055.0X10-4 0.30 0.0042.5X10-' 0.30 0.0041.0X10-4 0.26 0.0045.0X10-» 0.20 0.0052.5X10-5 0.16 0.005

initial reactions of the hexose monophosphateshunt. The most effective electron acceptor wasphenazine methosulfate, which gave a 15- to 30-fold stimulation of oxidation of carbon-1 by the hy-perdiploid and hypotetraploid Ehrlich and Krebs-2D ascites cells.

The manifold stimulation of carbon-1 oxidationby dyes suggests that one of the rate-limitingsteps for the reactions of the shunt may be theoxidation of TPNH. Therefore, it was of interest to examine the effect of 2-methyl-l,4-naphthoquinone, which has been shown to oxidizeTPNH more rapidly than DPNH (7). As seenin Experiment 5, this compound markedly increased carbon-1 oxidation but had no effect onthe oxidation of carbon-6, suggesting that it stimulated the hexose monophosphate shunt solely.

These electron acceptors also were found tostimulate the oxidation of carbon-2, which appeared to be related to the extent of enhancementof carbon-1 oxidation. Since carbon-2 can giverise to carbon-1 if recycling of the pentose cycleoccurs, it is presumed that the electron acceptorsstimulate the pentose cycle. Evidence compatiblewith the stimulation of the recycling process wasobtained by the assay of pentose and sedoheptu-lose in the experiments with méthylèneblue. Therewas no increase of either sugar (measured by theorcinol reaction) in the presence of méthylèneblue. The lack of accumulation of these intermediates suggests that reactions subsequent to theinitial dehydrogenations were not rate-limiting.

To determine whether the stimulatory effects

TABLE 3EFFECTOFARTIFICIALELECTRONACCEPTORSONGLUCOSE-C"OXIDATION

BYINTACTASCITESCELLSValues are based on tissue used (200 mg. fresh weight). The final concentrations were as follows: glucose (0.01 M), méthylèneblue (7 X IO-1M), phenazine methosulfate (3 X 10~4M), 2-methyl-l,4-naphthoquinone(l X 10~4M), dinitrophenol (1 X 10-'M).

EXPERIMENTno.:

ASCITES:

TIKE IMI-,. :

ADDITION:

Dye

Glucose carbonTotal

C-l

C-2

C-6

1

ELIff

MéthylèneBlue

1.73.9

0.92.40.100.610.190.350.050.10

MéthylèneBlue

8.8

4.3

1.2

0.24

*EL

30

None

3.6

1.2

0.19

0.09

SEL10Dinitro-

Phena-phenol zinefimoles

of oiygen

0.56.6 3.04E210Phena

zineconsumed

1.03.4

/latoms of glucose carbon to COi

3.0

0.35

0.22

0.294.280.061.680.020.790.0040.09

1.233.830.101.580.030.99

0.030.02

Phena-

zme

2.9

3.40

1.06

0.78

0.18

SK2D

10

None

0.75

0.37

0.08

0.05

0.02

2-Me-l,4-Naph-thoquinone

1.5

1.40

0.70

0.24

0.03

Research. on January 15, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 5: The Hexose Monophosphate Shunt in Glucose Catabolism in ...cancerres.aacrjournals.org/content/canres/18/9/1105.full.pdf · The Hexose Monophosphate Shunt in Glucose Catabolism in

WENNER et al.—Glucose Catabolism in Ascites Tumor Cells 1109

of the dyes could be attributed to an uncouplingof phosphorylation rather than their serving aselectron acceptors, carcinoma cells were incubatedwith 1 X 10~6 M dinitrophenol in the presenceof 0.01 M glucose-C14 labeled in positions 1 or

<5.As seen in Experiment 2, Table 3, no preferential stimulation of carbon-1 was observed, butoxidation of both carbons-1 and -6 was stimulatedto the same extent, suggesting that a symmetricalcleavage of glucose and oxidation via the citricacid cycle occurred. Thus it would appear thatthe stimulatory effects observed with electronacceptors could not be attributed to an increasedavailability of phosphate or phosphate acceptors.

Localization of a TPN-dependent hexose mono-phosphate shunt decarboxylative activity in the soluble

6-phosphogluconic dehydrogenase in the supernatant fraction of tissues from solid neoplasms (9).

As seen in Table 5, no oxidation of carbon-1by the hyperdiploid Ehrlich ascites particulate-free supernatant could be demonstrated unlesspyridine nucleotides were present. When glucose-6-phosphate served as substrate, there was appreciable oxygen consumption when TPN was added.However, no oxygen consumption was observedif DPN was added unless ATP was also present.Since TPN has been shown to be synthesized fromDPN and ATP in mammalian systems (20), itis conceivable that formation of TPN by thissystem permits appreciable oxygen consumption.Preliminary experiments indicate that a DPN ki-nase is present in the soluble fraction.

TABLE 4

INTRACELLULARDISTRIBUTIONOFHEXOSE MONOPHOSPHATESHUNT DECARBOXYLATIVE ACTIVITYOFHYPERDIPLOIDEHRLICH ASCITESCELLS

The following substances were in the designated final concentrations: MgSOj, 3 X 10~3M;cy-tochrome c, 4 X 10-*M;potassium chloride, 0.14 M;phosphate buffer, pH 7.4, 6 X IO"3M;TPN,3 X 10-«M;yeast hexokinase, 330 K.M. units at 25°C.; ATP, 2 X lp~3M;glucose, 0.01 M;phena-zine methosulfate, 2 X 10""*M; and 0.6 ml. of the tissue suspension in calcium-free Krebs-Ringerbuffer representing 120 mg. of tissue (fresh weight). The volume was brought to 1.6 ml., and thecells were incubated for 20 minutes with air as the gas phase at 38°C. The values are based ontime of incubation per tissue used. Hexokinase increased C-l oxidation of supernatant by 20%.

FRACTIONWhole cellsHomogenateSupernatant ISupernatant IIPart ¡culate

Oz UPTAKE

3.94.44.04.20.4

MATOMP OF GLUCOSE CAKBON TO COl*

Total C-I C-i C-6

3.35 1.86 0.12 0.054.85 1.80 0.43 0.092.79 1.81 0.07 0.043.10 1.96 0.17 0.05

0.10* Headings for separate columns indicate position of radioactive label in substrate used.

fraction.—Disruption of the cell membrane per

mitted further study as to the localization andthe establishment of a TPN-dependence of thestimulation of carbon-1 oxidation by phena/ine.Preliminary experiments with 0.25 M isotonic sucrose as the homogenization medium indicatedthat carbon-1 was oxidized primarily by the soluble fraction of the ascitic homogenate. Furtherdistribution studies were carried out with isotonicsalt solution as suspending medium. As seen inTable 4, appreciable oxidation of glucose by thehyperdiploid Ehrlich ascites homogenate was observed with supplements of phenazine methosulfate, pyridine nucleotides, and ATP.8 The rateof oxidation of glucose carbon-1 by the homogenate preparation was similar to that observedin the intact cells. Furthermore, all the activityof the homogenate could be accounted for bythe soluble fraction. It is presumed that the locus of hexose monophosphate shunt decarbox-ylation is the nonparticulate cytoplasmic fraction,which is in agreement with the compartmenta-tion of glucose-6-phosphate dehydrogenase and

Further data which are compatible with theTPN specificity for C-l oxidation are also shownwhen glucose-1-C14 serves as substrate. Since ATP

is required for the hexokinase reaction, additionof pyridine nucleotides alone does not permitoptimal oxidation of carbon-1. Therefore, a studyof the concentrations of DPN or TPN requiredto give optimal oxidative activity was made inthe presence of ATP. TPN was more effectivein stimulating hexose monophosphate shunt de-carboxylation than DPN, since a greater conver-

3It is somewhat surprising that carbon-6 oxidation wasas rapid in the soluble portion as in the whole cells. However,this finding is not necessarily contradictory to the establishedfact that mitochondria are involved in C-6 oxidation. Thereis a significant time lag for incorporation of C14from carbon-6of glucose into the respiratory CO2 by the intact ascites cell;this can probably be attributed to the dilution of the radioactive intermediates by the numerous intermediary metabolitesinvolved in citric acid cycle oxidation. In view of the shortincubation period in this experiment, only a relatively smallamount of C-6 oxidation is observed. This oxidation can probably be attributed to hexose monophosphate shunt decarbox-ylation by a randomization of the isotope, e.g., resynthesisof hexose from symmetrical 3-carbon units.

Research. on January 15, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 6: The Hexose Monophosphate Shunt in Glucose Catabolism in ...cancerres.aacrjournals.org/content/canres/18/9/1105.full.pdf · The Hexose Monophosphate Shunt in Glucose Catabolism in

GLUCOSE-e-PO,Oxygencon

sumed(/¿moles)000.88.28.6GLUCOSB-I-C"Oiygenconsumed(pinoles)00411.93.33.3Glucosecarbon-1to

COj(/¿atoms)00.041.380.871.511.53

TABLE 5

TPN-DEPENDENCE FOR GLUCOSE OXIDATION BY EL ASCITES SUPERNATANT II

The reaction mixture was essentially the same as described in Table 4, except that hexo-kinase was not present and nucleotides were added as specified below. The values are basedon the time of incubation (1 hour). Substrate concentrations: glucose-6-PO«and glucose-1-C",0.01 M each.

SUBSTBATÕ:

ADDITIONSATP DPN TPN(u) («) (M)

0.0010.0014

0.001 0.00140.001 0.00014

0.00140.001 0.00140.001 0.00014

TABLE 6ANAEROBICFORMATIONOF CO2 IN ASCITESCELLS*

GLUCOSECARBONTOCOatGAS (iiatoms/hour/flask)

ASCITES PHAS* Total C-I Oí C-«

6C3HED lymphoma Nt 0.20 0.19 0.01 0.01Air 0.58 0.30 0.07 0.07

K2D carcinoma NI 0.22 0.17 0.003 0.02Air 1.53 0.45 0.17 0.15

Hyperdiploid (EL) carcinoma Nj 0.19 0.10 0.003 0.004Air 1.55 0.25 0.11 0.10

MC1M fibrosarcoma Nj 0.37 0.20 0.05 0.06Air 4.04 0.79 0.57 0.44

DBA/2 lymphoma Nt 0.25 0.15 0.007 0.02Air 0.62 0.29 0.06 0.13

P288 lymph node leukemia Nt 0.38 0.24 0.02 0.02Air 1.42 0.48 0.22 0.19

* Each flask contained 100 mg. (wet wt.) ascites cells in 3 ml. calcium-free Krebs-Ringer phosphate buffer. After 10 minutes' equilibration, glucose was added from the side arm to a final concentration of 0.01 M.

For anaerobic experiments, Linde High Purity Nitrogen (specified 99.99 per cent), passedthrough three successive solutions of alkaline anthroquinone-hydrosulfite (8), was bubbled throughthe medium before the experiment and flushed through the flasks for 10 minutes after they wereon the manometers. The subsequent 10-minute equilibration period provided added assurance thattraces of oxygen would be consumed by respiration before the addition of labeled glucose. The reaction was stopped after 60 minutes of incubation at 38°,during which interval no measurablerespiration occurred. CO2trapped in the center well was counted as BaCO. at infinite thickness.

t Headings for separate columns indicate position of radioactive label in substrate used.

TABLE 7

EFFECT OFPYRUVATEON GLUCOSEOXIDATION BYASCITESCELLSAND BYMOUSELIVERSLICESUNDER AEROBICAND ANAEROBICCONDITIONS

Experimental conditions are the same as described in Table 6 except that in the experiment with mouse liver 200 mg. oftissue slices (fresh weight) were used.

GLUCOSECABBONTOCOj* OXYGENGAB PYBOVATB C-l C-ÃŽ C-6 Total UPTAKE

TISSUE PHASE (0.01 M) (fiatoms per hour per flask) (timóles)K -1 ' carcinoma Ns

Air - 0.32 0.12 0.10 1.07 3.43.7

DBA/2 lymphoma

Air 0.29 0.06 0.13 0.62 3.03.0

Liver N,

Air 0.17 0.05 0.25 8.7+ 0.19 0.03 0.48 9.8

* Headings for separate columns indicate position of radioactive label in substrate used.

0.120.390.320.410.150.370.290.370.010.020.170.190.0020.040.120.100.010.040.060.050.010.020.100.080.020.040.130.110.0080.0050.050.030.150.491.070.980.250.590.620.650.020.020.250.48

Research. on January 15, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 7: The Hexose Monophosphate Shunt in Glucose Catabolism in ...cancerres.aacrjournals.org/content/canres/18/9/1105.full.pdf · The Hexose Monophosphate Shunt in Glucose Catabolism in

WENNERe¿al.—GlucloseCatabolism in Ascites Tumor Cells lili

sion of glucose carbon-1 to COj was observedby the addition of TPN at a concentration of0.0001 M. Thus, it would appear that oxidationof carbon-1 is TPN-dependent in this system.

In a similar manner, the distribution and TPNdependence of the carbon-1 oxidative system wasstudied with a solid Ehrlich tumor derived fromthe hyperdiploid Ehrlich ascites. Carbon-1 oxidation by a Potter-Elvehjem homogenate of this tumor could also be attributed to a TPN-dependentoxidative system localized in the nonparticulatecytoplasmic fraction.

Further evidence in support of the TPN-dependent oxidation of carbon-1 of glucose by thesupernatant fraction was obtained spectrophoto-metrically. No measurable glucose-6-phosphatedehydrogenase activity was observed if DPNserved as coenzyme under the conditions of assaydescribed by De Moss (5). However, when TPNserved as coenzyme, the rate of TPNH productionby the supernatant fraction was QTrNH= 24at 30°C. Thus, C02 formation by way of the

hexose monophosphate shunt is apparently notlimited by this dehydrogenase. Since hexokinaseincreases the phenazine-stimulated C-l oxidation,it may limit the enhanced decarboxylation.

Effect of oxygen on glucose-C1*oxidation.—Sincethe in vitro limitation imposed on the oxidationof TPNH may be overcome in vivo by a numberof reductive syntheses which require TPNH asthe electron donor, it is important to know theextent to which oxygen is used as an electronacceptor for the hexose monophosphate shunt.In Table 6 are described the results of studiescarried out with a number of ascites tumors onthe rates of oxidation of the different carbonatoms of radioactive glucose to carbon dioxideunder anaerobic and aerobic conditions. Thesestudies have revealed that, in all the ascites cellsexamined, there is an appreciable anaerobic formation of carbon dioxide from glucose.4 Therewas a significant oxidation of carbon-1 of glucose,which accounted for the major portion of theglucose carbon to CO2. The oxidation of carbon-1proceeded anaerobically at about 10—40per centof the aerobic rate and accounted for approximately 55-90 per cent of the total glucose carbonoxidized to COS. The anaerobic formation of carbon dioxide can probably be attributed to thepresence of endogenous substances which can actas electron acceptors for TPNH oxidation.

As seen in Table 7, the addition of 10~2Msodium

4Although the rate of anaerobic formation of carbon dioxideis small in relation to the oxygen consumption (S-10 per cent),it may in part account for respiratory quotients greater than1 observed by previous investigators (2, 19).

pyruvate in the medium resulted in a threefoldstimulation of the anaerobic oxidation of glucosecarbon-1 by the Krebs-2 carcinoma and DBA/2lymphoma.6 It is presumed that pyruvate mightserve as an electron acceptor for the TPNH generated by the initial enzymes of the hexose mono-phosphate shunt. It is striking that pyruvatestimulates carbon-1 oxidation more than does oxygen, and in the presence of pyruvate oxygendoes not increase carbon-1 oxidation. The failureof pyruvate and oxygen to stimulate additivelysuggests that intermediary metabolites such aspyruvate may serve as the principal electron acceptors for the hexose monophosphate shunt inascites cells and that the role of oxygen in thispathway may be to favor the accumulation ofsuch intermediates.

The stimulation of carbon-1 oxidation by theaddition of pyruvate under anaerobic or aerobicconditions was also observed with three othermouse ascites tumors, namely, the MClM fibrosarcoma, 6C3HED lymphoma, and the hyperdiploid Ehrlich (EL) carcinoma. Thus, it appearsthat the stimulatory effect of pyruvate as wellas the anaerobic formation of COa is a generalproperty of ascites tumors.

It was also of interest to study anaerobic C-loxidation of glucose by mouse liver slices, a non-neoplastic tissue which has hexose monophosphateshunt activity. As seen in Table 7, no significantanaerobic CÜ2production from carbon-1 of glucose was observed in the presence or absenceof pyruvate. Thus, the anaerobic oxidation whichis observed with neoplastic tissues is not associatedwith all tissues having hexose monophosphateshunt activity.

Coupling of hexose monophosphate shunt dehy-drogenases with lactic acid dehydrogenase.—Themechanisms by which pyruvate might serve asan electron acceptor are manifold. Of the possibleconsiderations, TPNH might be reoxidized via lactic acid dehydrogenase; or by the TPN-dependentmalic enzyme, which could account for the anaerobic COj fixation into pyruvate observed by Craneand Ball (4) for ox retina; or by enzymes producingpropanediol phosphate from pyruvate (10); orby transhydrogenase as an alternate potentialmediator of DPNH oxidation. Malic enzyme didnot seem to be involved, since the addition of

'An unexpected stimulation of C-6 oxidation was alsoobserved by the addition of pyruvate, which should be acompetitive substrate for that coming from glucose-6-C14.It is conceivable that this enhancement of the relativelyminor C-6 oxidation might also be attributed to hexose mono-phosphate shunt decarboxylation if randomization of isotopeoccurred, e.g., the resynthesis of hexose from glycolytic intermediates.

Research. on January 15, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 8: The Hexose Monophosphate Shunt in Glucose Catabolism in ...cancerres.aacrjournals.org/content/canres/18/9/1105.full.pdf · The Hexose Monophosphate Shunt in Glucose Catabolism in

1112 Cancer Research Vol. 18, October, 1958

bicarbonate was not required for maximal rateof carbon-1 oxidation in the presence of pyruvate.The most likely explanation of the stimulatoryeffect of pyruvate on carbon-1 oxidation is thatthe TPN-dependent dehydrogenases of the shuntare linked with the conversion of pyruvate tolactate. Evidence for a TPN-linked lactic aciddehydrogenase can be demonstrated under theexperimental conditions in which a stimulationof glucose carbon-1 oxidation by pyruvate wasobserved. As shown in Table 8, the soluble fractionof ascites cells catalyzes a rapid oxidation of

TABLE 8

OXIDATIONOFTPNHBYPYRUVATECATALYZEDBYELASCITESSUPERNATANT*

MHOLSS AK I Ml 3 Horns'

INCUBATION

Net Amólespyridine Net Amóles

nucleotide lactateoxidized produced

0.90 0.801.3 1.3

INITIAL HATFopOXIDATION OK

PYRIDINE

NUCLEOTIDE

Qoiid. PNt

36940

PrBIDINENUCLKOTIDE

TPNHDPNHTPNH+DPN 2

*The reaction vessels contained ascitic supernatant II(equivalent to 30 mg. of fresh weight of ascites cells) in 3.4 ml.of calcium-free Ringer phosphate buffer containing 0.01 M pyruvate. The oxidation of the reduced pyridine nucleotide wasmeasured by determining the change in optical density at 340m/¿at 30°C. in the Gary spectrophotometer. The reaction wasstarted by the addition of 0.9 /¿molesof TPNH or 1.8 /¿molesof DPNH to the sample compartment, and stopped by theaddition of trichloroacetic acid for assay of lactic acid. For initial rate studies, less tissue was used to observe rates whichwere linear with respect to enzyme concentration. The rate ofTPNH and DPNH oxidation in the absence of pyruvate wasnegligible.

tQ oxid. PN refers to /¿Iof pyridine nucleotide oxi-dized/mg dry wt/hour.

TPNH by pyruvate. The oxidation of TPNHis accompanied by an almost stoichiometric production of lactic acid, which is indicative that thereaction is due to lactic acid dehydrogenase.6

The enzyme appears to be similar to the TPN-linked lactic acid dehydrogenase of the solublefraction of rat liver as described by Navazioet al. (21). The initial rate of TPNH oxidationby the tumor enzyme ranged from l/25th tol/40th the rate of DPNH oxidation, a somewhathigher relative activity than for the liver enzymestudied at the same pH. As has also been observedwith the liver preparation, TPNH oxidation wasmarkedly inhibited by the addition of DPN.

The possibility was considered that the oxidation of TPNH was catalyzed by a DPN-specificlactic dehydrogenase mediated by transhydrogen-

6This finding might also explain the observation of Kit(17) that, in the presence of fluoride and pyruvate, the oxidation of carbon-1 of glucose by the Gardner and Ehrlichtumors under aerobic conditions was stimulated.

äse.In order to test for the presence of transhydrogenase in the soluble fraction, an experiment,described in Table 9, was carried out in whicha DPN-specific enzyme, alcohol dehydrogenase,and its substrate, acetaldehyde, were added tothe supernatant in the presence of TPNH. Onewould expect a catalysis of the oxidation of TPNHif transhydrogenase were present. However, nooxidation of TPNH was observed under conditionsin which pyruvate was reduced by TPNH. Furthermore, transhydrogenase could not be detectedin the supernatant by an assay involving theoxidation of TPNH by DPN in the presenceof acetaldehyde and alcohol dehydrogenase. Therefore, it is presumed that the oxidation of TPNHby pyruvate is catalyzed by a TPN-linked lacticacid dehydrogenase.

Although the enzyme catalyzes a much sloweroxidation of TPNH than of DPNH, it is presentin the ascites supernatant with a capacity to

TABLE 9

ABSENCEOFTRANSHYDROGENASEINELASCITESSUPERNATANT*

ADDITIONS A OPTICALDENSITY

Substrate

Pyruvate

PyruvateDPN

Alcohol MINUTEXÕOMG.dehydrogenase FRESHWEIGHT-Hacetaldebyde DPNH TPNH

- 0.023 0.011

7.5 0.180+ 3.6 0.015+ 0.170+ 0.011

* The reaction vessels contained ascitic supernatant H(equivalent to 20 mg. fresh weight or less) in a total volumeof 1.1 ml. of calcium-free Krebs-Ringer phosphate buffer.Additions as described above were made to both reference andsample cells except that pyridine nucleotide was not added tothe reference cells. The reaction was started by the addition ofreduced pyridine nucleotide to the sample compartment, andthe decrease in optical density at 340 m/¿at 30°C. was recorded. For the extremely rapid oxidations of DPNH, the initial reaction rate was measured with less tissue, and the valuerepresented above was calculated for the equivalent of 20 mg.of tissue from the initial reaction velocity. Concentrationsused: pvruvate, 0.03 M; alcohol dehydrogenase and acetaldehyde, 0".003M; DPN, DPNH, and TPNH, 1.2 X 10~*M.

oxidize TPNH which is more than sufficient tosatisfy the stoichiometric requirements of carbon-1oxidation. Thus, since two molecules of TPNHmust be oxidized for each molecule of CO2 formedvia the shunt, a QOIM.TPN= 36 would permita rate of CO2 formation from carbon-1 of glucoseequivalent to a Q value of 18. However, the highestrate of CO2 production from carbon-1 of glucoseby the intact ascites cell in the presence of pyruvate had a Q value of 1, which is far belowits potential. The rate of formation of TPNHis equivalent to a Q of 24 as measured eitherspectrophotometrically with the ascites supernatant or by carbon-1 oxidation in the phenazine-

Research. on January 15, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 9: The Hexose Monophosphate Shunt in Glucose Catabolism in ...cancerres.aacrjournals.org/content/canres/18/9/1105.full.pdf · The Hexose Monophosphate Shunt in Glucose Catabolism in

WENNERet al.—GlucoseCatabolism in Ascites Tumor Cells 1113

stimulated intact cell. Therefore, the failure ofpyruvate to stimulate carbon-1 oxidation by theintact cell to the level of the soluble fractioncannot be attributed to dehydrogenases as rate-limiting steps. The most likely explanation forthe lack of realization of the full capacity ofcarbon-1 oxidation by the addition of pyruvateis that the intracellular level of DPN is sufficientto cause an inhibition in the oxidation of TPNHby pyruvate. As has been shown in Table 8,equimolar concentrations of DPN inhibit markedly TPNH oxidation. Furthermore, the additionof DPN has also been found to cause a markedinhibition of C-l oxidation by pyruvate catalyzedby the EL ascites supernatant. Thus, in additionto the availability of an electron acceptor suchas pyruvate which might limit the operation ofthe shunt, the relative concentrations of DPNand TPNH would also appear to be importantin the regulation of TPN-dependent dehydrog-enations.

DISCUSSIONThis study points out that the competitive

advantage of glycolysis over the shunt pathwayfor glucose utilization by neoplastic ascites cellscannot be attributed to a limitation in substratesupply. Judging from the marked stimulation ofcarbon-1 oxidation by artificial electron carriersobserved in the present experiments, a more likelyrate-limiting factor in the in vitro operation ofthe hexose monophosphate shunt is the availabilityof a hydrogen acceptor, in which case the capacityof the hexose monophosphate shunt to generatereduced TPN would exceed the rate of oxidationof TPNH. This is in agreement with the suggestion (15,18) that the hexose monophosphate shuntmay function to provide TPNH for directingspecific reductive syntheses.

Evidence compatible with this suggestion isobtained by the demonstration of the shunt underanaerobic conditions, when endogenous substratescould serve as oxidants for TPNH. The markedstimulation of anaerobic glucose decarboxylationby pyruvate provides an example for TPNH oxidation by fermentation intermediates.

Since oxygen does not increase glucose carbon-1oxidation in the presence of pyruvate, it is presumed that intermediary metabolites serve as theprincipal electron acceptors for the hexose mono-phosphate shunt and that oxygen favors theaccumulation of suitable electron acceptors forTPNH. That oxygen is not directly involvedin the oxidation of TPNH is also suggested byour failure to observe appreciable activity of TPN-cytochrome c reductase in homogenates and of

transhydrogenase in the soluble fraction of thetumor cells. Furthermore, Reynafarje and Potter(23) have reported that TPN-cytochrome c reductase as well as transhydrogenase is virtually absentin the Novikoff hepatoma.

The availability of electron acceptors, however,cannot be considered as the only rate-limitingstep in the operation of the hexose monophosphateshunt in ascites cells. If the mechanism by whichpyruvate stimulates is via the TPN-linked lacticacid dehydrogenase, consideration of the relativeconcentrations of TPNH and DPN must be made.Since the intracellular concentration of TPNHis low with respect to DPN, the presence ofDPN in the soluble fraction could readily exerta regulatory effect on TPN-dependent dehydrogenases.

Although an anaerobic oxidation of carbon-1of glucose could not be demonstrated with mouseliver slices in the presence or absence of pyruvate,these properties are not unique to the neoplastictissues. Kinoshita (16) has reported an anaerobicoxidation of carbon-1 of glucose which could bestimulated by pyruvate beyond the aerobic level.Furthermore, Dr. Leonard Cohen has independently observed this phenomenon in the retinaof the 5-day-old rabbit.7

A possible alternate function of the hexosemonophosphate shunt in ascites cells is the synthesis of ribose-5-phosphate for nucleic acids andcoenzymes. The hexose monophosphate shuntpathway would be a very direct pathway for pen-tose formation involving oxidative decarboxylation of glucose-6-phosphate. However, from ourresults, which have been described in a preliminary report (26), it seems that these tumor cellssynthesize ribose-5-phosphate predominantly bya C-3, C-2 condensation, presumably from trans-ketolase and transaldolase reactions.

SUMMARYExamination of the rate-controlling factors in

the hexose monophosphate shunt pathway in ascites tumor cells was made by studying the incorporation of C14of glucose—labeled uniformly orin carbons-1, -2, and -6—into the respiratoryCÛ2 under varied conditions. A study of theeffect of glucose concentration on the operationof the alternate pathway in a hyperdiploid Ehrlichascites tumor revealed that the initial rate ofCO2 production by the shunt was independentof substrate concentration in the range of 2.5 XIO"6 M tO 1 X IO"2 M.

Artificial electron acceptors such as méthylène7L. IL Cohen and W. K. Noell, Glucose Oxidation in the

Developing Retina (in preparation).

Research. on January 15, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 10: The Hexose Monophosphate Shunt in Glucose Catabolism in ...cancerres.aacrjournals.org/content/canres/18/9/1105.full.pdf · The Hexose Monophosphate Shunt in Glucose Catabolism in

1114 Cancer Research Vol. 18, October, 1958

blue, menadione, and phenazine methosulfatestimulated carbon-1 oxidation from six- to 30-foldwith only a slight stimulatory effect on the oxidation of carbon-6, suggesting that one of the rate-limiting factors in the operation of the alternatepathway is the availability of the electron transport system. This stimulation was also observedwith homogenates of ascites cells when a sourceof TPN was supplied. This TPN-dependent oxida-tive system for carbon-1 was localized in the soluble fraction, which also contained a lactic aciddehydrogenase that catalyzed the oxidation ofTPNH by pyruvate.

A significant oxidation of carbon-1 of glucoseby intact tumor cells was observed under anaerobic•conditions.The rate of oxidation of carbon-1 wasstimulated by pyruvate to that observed in thepresence of oxygen. Oxygen did not increase carbon-1 oxidation by the intact cells in the presenceof moderate pyruvate levels, indicating that intermediary metabolites such as pyruvate may serveas the principal electron acceptors for the hexosemonophosphate shunt. From the data, it is concluded that the prime function of the hexosemonophosphate shunt is to provide reduced tri-phosphopyridine nucleotide for specific reductivesyntheses.

ACKNOWLEDGMENTS

The authors are indebted to Dr. T. S. Hauschka forfurnishing the animals bearing the tumors used in this study,and to Dr. L. H. Cohen for helpful suggestions.

REFERENCES1. ABRABAM,S.; HILL, R.; and CHAIKOFF,I. L. Concerning

1'athways of Glucose Utilization in Mouse Liver andHepatoma. Cancer Research, 15:177-80, 1955.

2. BLOCH-FRANKENTHAL,L., and WEINHOUSE,S. Metabolismof Neoplastic Tissue. XII. Effects of Glucose Concentration on Respiration and Glycolysis of Ascites TumorCells, Studied with Carbon-14 Labeling. Cancer Research,17:1082-90,1957.

S. COLOWICK,S. P., and KAPLAN,N. O. General PreparativeProcedures. Methods in Enzymology I, p. 69, 1955.

4. CRANE,R. K., and BALL, E. G. Relationship of C14OZFixation to Carbohydrate Metabolism in Retina. J. Biol.Chem., 189:269-76, 1951.

5. DE Moss, R. D. Glucose-6-phosphate and 6-Phospho-gluconic Dehydrogenases from Leuconostoc mesmleroides.Methods in Enzymology, 1:328-32, 1955.

0. DRURT, H. F. Identification and Estimation of Pentosesin the Presence of Glucose. Arch. Biochem., 19:455-66,1948.

7. ENOLAHD,S., and STRECKER,II. J. Oxidation of ReducedPyridine Nucleotides in Brain. Fed. Proc., 15:248, 1956.

8. FIESER, L. Experiments in Organic Chemistry, ad ed.,p. 393. Boston: Heath, 1950.

9. CLOCK,G. E., and McLEAN, P. Levels of Enzymes ofthe Direct Oxidative Pathway of Carbohydrate Metabo

lism in Mammalian Tissues and Tumors. Biochem. J.,66:171-75,1954.

10. GHOTH,D. P.; LEPAGE, G. A.; HEIDELBERGER,C.; andSTOESZ,P. A. Metabolism of Pyruvate in Tumor Homogenates. Cancer Research, 12:529-34, 1952.

11. HAUSCHKA,T. S. Cell Population Studies on Mouse AscitesTumors. Trans. New York Acad. Se. S. II, Vol. 16:64-73,1953.

12. HAUSCHKA,T. S., and FURTH,J. The Pathophysiologyand Immunogenetics of Transplan table Leukemia. Proc.Henry Ford International Symposium, The Leukemias:Etiology and Pathophysiology, pp. 87-120. New York:Academic Press, 1957.

13. HORECKEH,B. L., and HIATT,H. II. Pathways of Carbohydrate Metabolism in Normal and Neoplastic Cells.New Eng. J. Med., 258:177-84, 1958.

14. HORN, H. and BRUNS,F. H. Quantitative Bestimmungvon l(+)-Milchsäure mit Milchsäuredehydrogenase. Bio-chim. & Biophys. Acta, 21:378-80, 1956.

15. KAPLAN,N. O.; SWARTZ,M. N.; FRECH, M. E.; andCIOTTI,M. M. Phosphorylative and NonphosphorylativePathways of Electron Transfer in Rat Liver Mitochondria.Proc. Nat. Acad. Se., 42:481-87, 1956.

16. KiNosHiTA,J. H. The Stimulation of the PhosphogluconateOxidation Pathway by Pyruvate in Bovine Corneal Epithelium. J. Biol. Chem., 228:247-53, 1957.

17. KIT, S. The Role of the Hexosemonophosphate Shuntin Tumors and Lymphatic Cells. Cancer Research, 16:70-76, 1956.

18. KREBS, H. A. Considerations Concerning the Pathwaysof Syntheses in Living Matter. Bull. John Hopkins Hosp.,96:19-23, 1954.

19. KUN, E., TALALAY,P., and WILLIAMS-ASHMAN,H. G.Studies on the Ehrlich Ascites Tumor. I. The Enzymicand Metabolic Activities of the Ascites Cells and theAscitic Plasma. Cancer Research, 11:855-63, 1951.

20. MEHLEB,A. H.; KORNBERG,A.; CRISOLA,S.; and OCHOA,S. The Enzymatic Mechanism of Oxidation-Reductionsbetween Malate or Isocitrate and Pyruvate. J. Biol. Chem.,174:961-77, 1948.

21. NAVAZIO,F.; ERNSTER,B.; and ERNSTER,L. Studieson TPN-linked Oxidations. II. The Quantitative Significance of Liver Lactic Dehydrogenase as a Catalyzerof TPNH Oxidation. Biochim. & Biophys. Acta, 26:416-21,1957.

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

23. RETNAFARJE,B., and POTTER,V. R. Comparison of Trans-hydrogenase and Pyridine Nucleotide-Cytochrome c Rc-ductase Activities in Rat Liver and Novikoff Hepatoma.Cancer Research, 17:1112-19, 1957.

24. SEIFTER,S.; DAYTON,S.; Novic, B.; and MUNTWYLEH,E.Estimation of Glycogen with Anthrone Reagent. Arch.Biochem., 25:191-200, 1950.

25. WEINHOUSE,S. Studies on the Fate of Isotopically LabeledMetabolites in the Oxidative Metabolism of Tumors. Cancer Research, 11:585-91, 1951.

26. WENNER,C. E.; HACKNET,J.; and HERBERT,J. Pathwaysof Glucose Metabolism in Ascites Cells. Proc. Am. Assoc.Cancer Research, 2:259, 1957.

27. WENNER,C. E., and WEINHOUSE,S. Metabolism of Neoplastic Tissue. IX. An Isotope Tracer Study of GlucoseCatabolism Pathways in Normal and Neoplastic Tissues.J. Biol. Chem., 222:399-414, 1956.

28. WOOD.W. A., and SCHWERDT,R. F. Carbohydrate Oxidation by Pseudomonas fluorescens. II. Mechanism ofHexose Phosphate Oxidation. J. Biol. Chem., 206:625-35,1954.

Research. on January 15, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from

Page 11: The Hexose Monophosphate Shunt in Glucose Catabolism in ...cancerres.aacrjournals.org/content/canres/18/9/1105.full.pdf · The Hexose Monophosphate Shunt in Glucose Catabolism in

1958;18:1105-1114. Cancer Res   Charles E. Wenner, John H. Hackney and Francis Moliterno  Ascites Tumor CellsThe Hexose Monophosphate Shunt in Glucose Catabolism in

  Updated version

  http://cancerres.aacrjournals.org/content/18/9/1105

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

  [email protected] 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/18/9/1105To request permission to re-use all or part of this article, use this link

Research. on January 15, 2020. © 1958 American Association for Cancercancerres.aacrjournals.org Downloaded from