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Inhibition by N-(Phosphonacetyl)-l -aspartate of Ehrlich ascites tumour growth and glucose transport
Cancer Letters, 23 (1984) 183-188 Elsevier Scientific Publishers Ireland Ltd.
183
INHIBITION BY N-(PHOSPHONACETYL)-L-ASPARTATE OF EHRLICH ASCITES TUMOUR GROWTH AND GLUCOSE TRANSPORT
S.W. LEUNG, K.P. FUNG, Y.M. CHOY and C.Y. LEE* Department Kongl
of Biochemistry,
The Chinese University
of Hong Kong, Shatin, N.T. (Hong
(Received 7 March 1984) (Accepted 20 April 1984)
-
SUMMARY
N-(Phosphonacetyl)~L-aspartate (PALA) suppressed the growth of Ehrlich ascites tumour cells in vivo in a dose-dependent manner. Simultaneously as the growth rate decreased, the cellular uptake of glucose and the density of a class of glucose-reversible binding sites for cytochalasin B on the cell surface were also found to be reduced. There is a highly significant correlation between the magnitude of changes in the number of cytochalasin B binding sites and the magnitude of changes in glucose uptake. The physiological significance of these observations are discussed.
INTRODUCTION
Ehrlich ascites tumour cells depend primarily on glycolysis for the provision of energy. To maintain growth potential, it is essential that they possess a highly efficient system for glucose transport and metabolism. We have recently shown [2] that the ability of Ehrlich cells to take up glucose increased progressively during the course of tumour development. Simultaneously as the rate of uptake rose, the surface density of the putative glucose carrier molecules also increased. This was viewed as a compensatory response to the continuous fall in glucose level in the serum and peritoneal fluid accompanying tumour growth. PALA has been shown to arrest the growth of a wide spectrum of tumours [1,9] and to reduce the rate of glucose utilisation in one line of human cancer (HT29) cells studied [ 111. PALA acts specifically to inhibit aspartyl transcarbamylase and consequently de novo pyrimidine biosynthesis [ 31. To identify the biochemical events underlying the process of glucose trans-
*To whom all correspondence
should be addressed.
0304-3835/84/$03.00 0 1984 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
184
port, we examined the effect of PALA on the rate of glucose uptake and on the number of glucose carriers in Ehrlich ascites tumour cells. The results are reported here. MATERIALS
AND METHODS
Ehrlich ascites tumour, Ny Klein cell type, was maintained by weekly intraperitoneal implantation in albino mice (ICR strain). PALA was obtained from the Drug Synthesis and Chemistry Branch of the National Cancer Institute and used in 0.85% NaCl solution. Cytochalasin B and 2-deoxy-Dglucose were purchased from Sigma. [3H]Cytochalasin B (7.2 Ci/mmol) was obtained from New England Nuclear and 2deoxy-D-[ 3H]glucose (16 Ci/ mmol) was obtained from Amex&am. Mice weighing 30-35 g were inoculated i.p. with 10’ ascites tumour cells harvested from ‘Iday-old tumours in 0.2 ml phosphate-buffered saline (PBS) (pH 7.4), on day 0. The effect of PALA was examined in groups of at least 10 mice each. In the test groups, PALA (5-50 mg/kg body wt) was administered intraperitoneally daily from days l-6 inclusive. In the control group, only 0.85% saline was injected. The mice were killed by cervical dislocation on day 7. Tumour cells were collected by exhaustive drainage. Pooled cells were washed 5 times with half-isotonic saline to remove blood cells and harvested by centrifugation. The final cell suspension was prepared in PBS. Cells were counted with a haemocytometer and resuspended in the same buffer to the required concentration. Glucose uptake was measured as follows: Ehrlich ascites cell suspension (2 X lO’/ml in PBS) was equilibrated to 37°C. At 0 s, 0.2 ml of suspension was mixed with 0.2 ml of prewarmed 2deoxy-D-[ 3H]glucose (1 pCi/pmol) to give a final concentration of 0.25-4.0 mM. Reaction was stopped after 6 s by transferring 0.2 ml of the mixture to 1 ml of ice-cold PBS supplemented with 40 mM 2deoxy-D-glucose. Cells were collected by centrifugation at 15,000 X g for 10 s in an Eppendorf 5414 microcentrifuge. The supernate was removed and the cells washed with 1 ml of the same buffer. Cell lysis was accomplished by the addition of 0.2 ml 0.1% Triton X-100. One millilitre of Triton X-toluene scintillant [ 81 was added and the radioactivity counted in a Beckman LS7000 liquid scintillation counter. Nonspecific diffusion was corrected by substracting the uptake in the presence of 10 M cytochalasin B. The kinetic parameters V,.. and K, , representing respectively the maximal uptake and apparent half-saturation constant for the specific transport process, were determined by double-reciprocal plots. Equilibrium binding of cytochalasin B was performed according to Cuppoletti et al. [ 41 with minor modifications. For measuring total binding, 10’ cells in 1 ml PBS were incubated with 0.02 PCi [3H]cytochalasin B and 2 X 10e9-5 X 10e6 M cytochalasin B for 20 min at room temperature. After incubation, the supematants were separated from the pellets by centrifugation and the radioactivities in each fraction determined as previously
185
described [ 21. Cytochalasin B bound was calculated as percentage of total. To assess the glucose-reversible binding of cytochalasin B, total binding in the absence and presence of 500 mM D-glucose were measured and the difference obtained. Maximal glucose-sensitive binding and the apparent dissociation constant were determined by Scatchard analysis. RESULTS
AND DISCUSSION
Figure 1 shows that daily administration of PALA is capable of suppressing the growth of Ehrlich ascites tumour cells. The effect is dose dependent and quite dramatic. Six consecutive daily doses at 20 mg/kg body weight reduced the size of the tumour by as much as 75%. This would seem to place Ehrlich ascites cells in a category of murine tumours, including Lewis lung carcinoma and B6 melanoma [6], which are highly responsive to PALA treatment. Other murine cell lines such as L1210 leukemia and P388 leukemia are either completely refractory or only moderately sensitive to PALA [5]. It should also be noted here that Ehrlich ascites tumours, regardless of size, contained over 95% viable cells as determined by trypan blue exclusion. The uptake of hexoses by Ehrlich ascites tumour cells has previously been shown [ 21 to be mediated by a facilitated transport process. Such a process follows simple Michaelis-Menten kinetics and may adequately be described by the kinetic parameters V,,, and K, [4,9]. PALA also affected the rate
I
L
1
0
10
20 PALA
Dosage
1
30
I
I
40
50
CmglKgtday)
Fig. 1. Effect of PALA on the proliferation of Ehrlich ascites tumour cells. Tumourbearing mice were inoculated i.p. for 6 consecutive days with PALA in 0.85% NaCl at doses indicated. On day 7, cells were harvested and counted. Tumour size is expressed as percent of control (no PALA). Values are mean ?: S.E.M. for 3 separate experiments. Statistical analysis for significance of difference was performed using the Student t-test. *P-value for the significance of difference <0.005, **P-value for the significance of difference
186
of glucose uptake by Ehrlich ascites cells. Figure 2 shows that PALA administered at 20 mg/kg per day for 6 days reduced the value of V, ax for the transport of 2deoxy-D-glucose by about 50%. This reduction is highly significant (P < 0.005). By contrast, the K, value remained unchanged. These results were tentatively interpreted to mean that PALA reduced the rate of glucose uptake by lowering the number and/or operational efficiency of the glucose transporter molecules. The affinity between glucose and its transport component was, however, unaffected. To test this hypothesis, we examined the effect of PALA on the equilibrium binding of cytochalasin B by a class of glucose-sensitive sites on Ehrlich ascites tumour cells. These sites have been postulated to be identical to the glucose transporters in Ehrlich ascites cells [ 21 and a variety of other eucaryotic cell types [7,12,13]. Figure 3 shows that as the dose of PALA administered increased from 0 to 20 mg/kg per day, the number of binding sites decreased from 260 to 115 pmol/lO’ cells. The apparent dissociation constant, however, remained unchanged. When the corresponding data points in Figs. 2 and 3 were analysed, it was found that a highly significant correlation exists between the extent of reduction of glucose uptake induced by PALA and the extent of decrease of cytochalasin B binding sites (coefficient of correlation T = 0.99). It may therefore be concluded that changes in glucose uptake by Ehrlich ascites tumour cells are principally the result of changes in the number of transport carriers. While alteration in the turnover rate of the glucose carriers cannot be absolutely ruled out in the present
0
5
PALA
10
15
Dosage
hglKplday)
20
Fig. 2. Effect of PALA on the uptake of 2-deoxy-D-glucose by Ehrlich ascites tumour cells. Experimental details are as described in the text. Values are presented as mean f S.E.M. for 3 separate experiments. o-----o, maximal uptake rate (V,,, ); A----A, apparent half-saturation constant (K, ).
187
0
5
PALA
10 Dosage
15
20
(mglKglday)
Fig. 3. Effect of PALA on the-binding of dytochalasin B by Ehrlich ascites tumour cells. Glucose-sensitive binding of cytochalasin B was determined as described in the text. Values are presented as mean ?: S.E.M. triplicate determinations in 3 separate experiments. o---o, maximal glucose-reversible binding (B,); A-A, apparent dissociation constant (Kd ).
study, its contribution to the overall transport process is at best minimal. A similar conclusion has been reached in our previous work [ 21 with the effect of methotrexate on glucose transport in Ehrlich cells and it was suggested that the depletion of nucleotides by methotrexate and the consequent inhibition of DNA, RNA and protein synthesis might have constituted the primary chain of events leading to reduced carrier production. The present work with PALA which acts by uniquely inhibiting de novo pyrimidine nucleotide biosynthesis, in contrast to the more divergent action of folate acid derivatives, would lend further strength to this view. Tsuboi and Kwong [ll] showed that treatment of the human colonic adenocarcinoma cell line HT-29 with PALA was accompanied by a decreased rate of glucose utilisation. Since normal HSOOOl cells were unaffected, they attributed the reduction to the presence of dying cells in the HT-29 culture. They further suggested that cell death was the result of the failure of the neoplastic cells to switch to a quiescent G, state upon pyrimidine deprivation. Our present data on cytochalasin B binding and cellular viability do not support this view. Instead, decreased synthesis of glucose carriers resulting from PALA-induced pyrimidine nucleotide starvation is likely the primary cause for the lowered rate of glucose consumption. In view of the recent observation [l] that the extent of cell death under the influence of PALA varies widely from one cell type to another even though the specific activity of aspartyl transcarbamylase in the cell lines are essentially the same, it would be of interest to examine the possibility that cell death might have
188
arisen from glucose starvation as well as to explore the extent to which the salvage pathway for pyrimidine nucleotide synthesis operates in HSOOl, HT-29 and Ehrlich ascites tumour cells.
ACKNOWLEDGEMENT
This work was supported (H.K.).
by a grant from the World Health Foundation
REFERENCES 1 Baillon, J., Guichard, M., Malaise, E.P. and Herve, G. (1983) Kinetic parameters of aspartate transcarbamylase in human normal and tumoural cell lines. Cancer Res., 43, 2277-2282. 2 Chan, T.W., Fung, K.P., Choy, Y.M. and Lee, C.Y. (1983) Glucose transport in developing Ehrlich ascites tumour cells. Parallel changes in rate of glucose uptake and cytochalasin B binding activity during tumour development and methotrexate treatment, Arch. B&hem. Biophys., 225,458-466. 3 Collins, K.D. and Stark, G.R. (1971) Aspartate transcarbamylase. Interaction with the transition state analogue N-(phosphoacetyl-L-aspartate. J. Biol. Chem., 246, 65996605. 4 Cuppoletti, J., Mayhew, E. and Jung, C.Y. (1981) Cytochalasin B binding to Ehrlich ascites tumour cells and its relationship to glucose carrier. Biochim. Biophys. Acta, 642,392-404. 5 Johnson, R.K., Inouye, T., Goldin, A. and Stark, G.R. (1976) Antitumour activity of N-(phosphonacetyl)-L-aspartic acid, a transition-state inhibitor of aspartate transcarbamylase. Cancer Res., 36, 2720-2725. 6 Johnson, R.K., Swyryd, E.A. and Stark, G.R. (1978) Effects of N-(phosphonacetyl)L-aspartate on murine tumours and normal tissues in vivo and in vitro and the relationship of sensitivity to rate of proliferation and level of aspartate transcarbamylase. Cancer Res., 38, 371-378. 7 Karnieli, E., Zarnowsky, M.J., Hissin, P.J., Simpson, LA., Salans, L.B. and Cushman, S.W. (1981) Insulin-stimulated tram&cation of glucose transport systems in the isolated rat adipose cell. J. Biol. Chem., 256, 4772-4777. 8 Patterson, MS. and Greene, R.C. (1965) Measurement of low beta-emitters in aqueous solution by liquid scintillation counting of emulsions. Anal. Chem., 37, 854857. 9 Plageman, P.G.W. and Richey, D.P. (1974) Transport of nucleosides nucleic acid bases, choline and glucose by animal cells in culture. Biochim. Biophys. Acta, 344, 263-305. 10 Rozencweig, M., Abele, R., Piccart, M., Van Hoff, D.D. and Muggia, F.M. (1980) N-(Phosphonacetyl)-L-aspartate (PALA): Current status. In: Recent Results in Cancer Research, Vol. 74, Cancer Chemo- and Immunopharmacology, pp. 72-77. Editors: G. Mathe and F.M. Muggia. Springer-Verlag, Berlin, Heidelberg, New York. 11 Tsuboi, K.K. and Kwong, L.K. (1978) Antiproliferative agents and differential survival between normal and cancer cells. Cancer Res., 38, 3745-3750. 12 Sogin, D.C. and Hinkle, P.C. (1980) Binding of cytochalasin B to human erythrocyte glucose transporter. Biochemistry, 19, 5417-5420. 13 Suzuki, K. and Kono, T. (1980) Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc. Natl. Acad. Sci. U.S.A., 77, 2542-2545.
183
INHIBITION BY N-(PHOSPHONACETYL)-L-ASPARTATE OF EHRLICH ASCITES TUMOUR GROWTH AND GLUCOSE TRANSPORT
S.W. LEUNG, K.P. FUNG, Y.M. CHOY and C.Y. LEE* Department Kongl
of Biochemistry,
The Chinese University
of Hong Kong, Shatin, N.T. (Hong
(Received 7 March 1984) (Accepted 20 April 1984)
-
SUMMARY
N-(Phosphonacetyl)~L-aspartate (PALA) suppressed the growth of Ehrlich ascites tumour cells in vivo in a dose-dependent manner. Simultaneously as the growth rate decreased, the cellular uptake of glucose and the density of a class of glucose-reversible binding sites for cytochalasin B on the cell surface were also found to be reduced. There is a highly significant correlation between the magnitude of changes in the number of cytochalasin B binding sites and the magnitude of changes in glucose uptake. The physiological significance of these observations are discussed.
INTRODUCTION
Ehrlich ascites tumour cells depend primarily on glycolysis for the provision of energy. To maintain growth potential, it is essential that they possess a highly efficient system for glucose transport and metabolism. We have recently shown [2] that the ability of Ehrlich cells to take up glucose increased progressively during the course of tumour development. Simultaneously as the rate of uptake rose, the surface density of the putative glucose carrier molecules also increased. This was viewed as a compensatory response to the continuous fall in glucose level in the serum and peritoneal fluid accompanying tumour growth. PALA has been shown to arrest the growth of a wide spectrum of tumours [1,9] and to reduce the rate of glucose utilisation in one line of human cancer (HT29) cells studied [ 111. PALA acts specifically to inhibit aspartyl transcarbamylase and consequently de novo pyrimidine biosynthesis [ 31. To identify the biochemical events underlying the process of glucose trans-
*To whom all correspondence
should be addressed.
0304-3835/84/$03.00 0 1984 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
184
port, we examined the effect of PALA on the rate of glucose uptake and on the number of glucose carriers in Ehrlich ascites tumour cells. The results are reported here. MATERIALS
AND METHODS
Ehrlich ascites tumour, Ny Klein cell type, was maintained by weekly intraperitoneal implantation in albino mice (ICR strain). PALA was obtained from the Drug Synthesis and Chemistry Branch of the National Cancer Institute and used in 0.85% NaCl solution. Cytochalasin B and 2-deoxy-Dglucose were purchased from Sigma. [3H]Cytochalasin B (7.2 Ci/mmol) was obtained from New England Nuclear and 2deoxy-D-[ 3H]glucose (16 Ci/ mmol) was obtained from Amex&am. Mice weighing 30-35 g were inoculated i.p. with 10’ ascites tumour cells harvested from ‘Iday-old tumours in 0.2 ml phosphate-buffered saline (PBS) (pH 7.4), on day 0. The effect of PALA was examined in groups of at least 10 mice each. In the test groups, PALA (5-50 mg/kg body wt) was administered intraperitoneally daily from days l-6 inclusive. In the control group, only 0.85% saline was injected. The mice were killed by cervical dislocation on day 7. Tumour cells were collected by exhaustive drainage. Pooled cells were washed 5 times with half-isotonic saline to remove blood cells and harvested by centrifugation. The final cell suspension was prepared in PBS. Cells were counted with a haemocytometer and resuspended in the same buffer to the required concentration. Glucose uptake was measured as follows: Ehrlich ascites cell suspension (2 X lO’/ml in PBS) was equilibrated to 37°C. At 0 s, 0.2 ml of suspension was mixed with 0.2 ml of prewarmed 2deoxy-D-[ 3H]glucose (1 pCi/pmol) to give a final concentration of 0.25-4.0 mM. Reaction was stopped after 6 s by transferring 0.2 ml of the mixture to 1 ml of ice-cold PBS supplemented with 40 mM 2deoxy-D-glucose. Cells were collected by centrifugation at 15,000 X g for 10 s in an Eppendorf 5414 microcentrifuge. The supernate was removed and the cells washed with 1 ml of the same buffer. Cell lysis was accomplished by the addition of 0.2 ml 0.1% Triton X-100. One millilitre of Triton X-toluene scintillant [ 81 was added and the radioactivity counted in a Beckman LS7000 liquid scintillation counter. Nonspecific diffusion was corrected by substracting the uptake in the presence of 10 M cytochalasin B. The kinetic parameters V,.. and K, , representing respectively the maximal uptake and apparent half-saturation constant for the specific transport process, were determined by double-reciprocal plots. Equilibrium binding of cytochalasin B was performed according to Cuppoletti et al. [ 41 with minor modifications. For measuring total binding, 10’ cells in 1 ml PBS were incubated with 0.02 PCi [3H]cytochalasin B and 2 X 10e9-5 X 10e6 M cytochalasin B for 20 min at room temperature. After incubation, the supematants were separated from the pellets by centrifugation and the radioactivities in each fraction determined as previously
185
described [ 21. Cytochalasin B bound was calculated as percentage of total. To assess the glucose-reversible binding of cytochalasin B, total binding in the absence and presence of 500 mM D-glucose were measured and the difference obtained. Maximal glucose-sensitive binding and the apparent dissociation constant were determined by Scatchard analysis. RESULTS
AND DISCUSSION
Figure 1 shows that daily administration of PALA is capable of suppressing the growth of Ehrlich ascites tumour cells. The effect is dose dependent and quite dramatic. Six consecutive daily doses at 20 mg/kg body weight reduced the size of the tumour by as much as 75%. This would seem to place Ehrlich ascites cells in a category of murine tumours, including Lewis lung carcinoma and B6 melanoma [6], which are highly responsive to PALA treatment. Other murine cell lines such as L1210 leukemia and P388 leukemia are either completely refractory or only moderately sensitive to PALA [5]. It should also be noted here that Ehrlich ascites tumours, regardless of size, contained over 95% viable cells as determined by trypan blue exclusion. The uptake of hexoses by Ehrlich ascites tumour cells has previously been shown [ 21 to be mediated by a facilitated transport process. Such a process follows simple Michaelis-Menten kinetics and may adequately be described by the kinetic parameters V,,, and K, [4,9]. PALA also affected the rate
I
L
1
0
10
20 PALA
Dosage
1
30
I
I
40
50
CmglKgtday)
Fig. 1. Effect of PALA on the proliferation of Ehrlich ascites tumour cells. Tumourbearing mice were inoculated i.p. for 6 consecutive days with PALA in 0.85% NaCl at doses indicated. On day 7, cells were harvested and counted. Tumour size is expressed as percent of control (no PALA). Values are mean ?: S.E.M. for 3 separate experiments. Statistical analysis for significance of difference was performed using the Student t-test. *P-value for the significance of difference <0.005, **P-value for the significance of difference
186
of glucose uptake by Ehrlich ascites cells. Figure 2 shows that PALA administered at 20 mg/kg per day for 6 days reduced the value of V, ax for the transport of 2deoxy-D-glucose by about 50%. This reduction is highly significant (P < 0.005). By contrast, the K, value remained unchanged. These results were tentatively interpreted to mean that PALA reduced the rate of glucose uptake by lowering the number and/or operational efficiency of the glucose transporter molecules. The affinity between glucose and its transport component was, however, unaffected. To test this hypothesis, we examined the effect of PALA on the equilibrium binding of cytochalasin B by a class of glucose-sensitive sites on Ehrlich ascites tumour cells. These sites have been postulated to be identical to the glucose transporters in Ehrlich ascites cells [ 21 and a variety of other eucaryotic cell types [7,12,13]. Figure 3 shows that as the dose of PALA administered increased from 0 to 20 mg/kg per day, the number of binding sites decreased from 260 to 115 pmol/lO’ cells. The apparent dissociation constant, however, remained unchanged. When the corresponding data points in Figs. 2 and 3 were analysed, it was found that a highly significant correlation exists between the extent of reduction of glucose uptake induced by PALA and the extent of decrease of cytochalasin B binding sites (coefficient of correlation T = 0.99). It may therefore be concluded that changes in glucose uptake by Ehrlich ascites tumour cells are principally the result of changes in the number of transport carriers. While alteration in the turnover rate of the glucose carriers cannot be absolutely ruled out in the present
0
5
PALA
10
15
Dosage
hglKplday)
20
Fig. 2. Effect of PALA on the uptake of 2-deoxy-D-glucose by Ehrlich ascites tumour cells. Experimental details are as described in the text. Values are presented as mean f S.E.M. for 3 separate experiments. o-----o, maximal uptake rate (V,,, ); A----A, apparent half-saturation constant (K, ).
187
0
5
PALA
10 Dosage
15
20
(mglKglday)
Fig. 3. Effect of PALA on the-binding of dytochalasin B by Ehrlich ascites tumour cells. Glucose-sensitive binding of cytochalasin B was determined as described in the text. Values are presented as mean ?: S.E.M. triplicate determinations in 3 separate experiments. o---o, maximal glucose-reversible binding (B,); A-A, apparent dissociation constant (Kd ).
study, its contribution to the overall transport process is at best minimal. A similar conclusion has been reached in our previous work [ 21 with the effect of methotrexate on glucose transport in Ehrlich cells and it was suggested that the depletion of nucleotides by methotrexate and the consequent inhibition of DNA, RNA and protein synthesis might have constituted the primary chain of events leading to reduced carrier production. The present work with PALA which acts by uniquely inhibiting de novo pyrimidine nucleotide biosynthesis, in contrast to the more divergent action of folate acid derivatives, would lend further strength to this view. Tsuboi and Kwong [ll] showed that treatment of the human colonic adenocarcinoma cell line HT-29 with PALA was accompanied by a decreased rate of glucose utilisation. Since normal HSOOOl cells were unaffected, they attributed the reduction to the presence of dying cells in the HT-29 culture. They further suggested that cell death was the result of the failure of the neoplastic cells to switch to a quiescent G, state upon pyrimidine deprivation. Our present data on cytochalasin B binding and cellular viability do not support this view. Instead, decreased synthesis of glucose carriers resulting from PALA-induced pyrimidine nucleotide starvation is likely the primary cause for the lowered rate of glucose consumption. In view of the recent observation [l] that the extent of cell death under the influence of PALA varies widely from one cell type to another even though the specific activity of aspartyl transcarbamylase in the cell lines are essentially the same, it would be of interest to examine the possibility that cell death might have
188
arisen from glucose starvation as well as to explore the extent to which the salvage pathway for pyrimidine nucleotide synthesis operates in HSOOl, HT-29 and Ehrlich ascites tumour cells.
ACKNOWLEDGEMENT
This work was supported (H.K.).
by a grant from the World Health Foundation
REFERENCES 1 Baillon, J., Guichard, M., Malaise, E.P. and Herve, G. (1983) Kinetic parameters of aspartate transcarbamylase in human normal and tumoural cell lines. Cancer Res., 43, 2277-2282. 2 Chan, T.W., Fung, K.P., Choy, Y.M. and Lee, C.Y. (1983) Glucose transport in developing Ehrlich ascites tumour cells. Parallel changes in rate of glucose uptake and cytochalasin B binding activity during tumour development and methotrexate treatment, Arch. B&hem. Biophys., 225,458-466. 3 Collins, K.D. and Stark, G.R. (1971) Aspartate transcarbamylase. Interaction with the transition state analogue N-(phosphoacetyl-L-aspartate. J. Biol. Chem., 246, 65996605. 4 Cuppoletti, J., Mayhew, E. and Jung, C.Y. (1981) Cytochalasin B binding to Ehrlich ascites tumour cells and its relationship to glucose carrier. Biochim. Biophys. Acta, 642,392-404. 5 Johnson, R.K., Inouye, T., Goldin, A. and Stark, G.R. (1976) Antitumour activity of N-(phosphonacetyl)-L-aspartic acid, a transition-state inhibitor of aspartate transcarbamylase. Cancer Res., 36, 2720-2725. 6 Johnson, R.K., Swyryd, E.A. and Stark, G.R. (1978) Effects of N-(phosphonacetyl)L-aspartate on murine tumours and normal tissues in vivo and in vitro and the relationship of sensitivity to rate of proliferation and level of aspartate transcarbamylase. Cancer Res., 38, 371-378. 7 Karnieli, E., Zarnowsky, M.J., Hissin, P.J., Simpson, LA., Salans, L.B. and Cushman, S.W. (1981) Insulin-stimulated tram&cation of glucose transport systems in the isolated rat adipose cell. J. Biol. Chem., 256, 4772-4777. 8 Patterson, MS. and Greene, R.C. (1965) Measurement of low beta-emitters in aqueous solution by liquid scintillation counting of emulsions. Anal. Chem., 37, 854857. 9 Plageman, P.G.W. and Richey, D.P. (1974) Transport of nucleosides nucleic acid bases, choline and glucose by animal cells in culture. Biochim. Biophys. Acta, 344, 263-305. 10 Rozencweig, M., Abele, R., Piccart, M., Van Hoff, D.D. and Muggia, F.M. (1980) N-(Phosphonacetyl)-L-aspartate (PALA): Current status. In: Recent Results in Cancer Research, Vol. 74, Cancer Chemo- and Immunopharmacology, pp. 72-77. Editors: G. Mathe and F.M. Muggia. Springer-Verlag, Berlin, Heidelberg, New York. 11 Tsuboi, K.K. and Kwong, L.K. (1978) Antiproliferative agents and differential survival between normal and cancer cells. Cancer Res., 38, 3745-3750. 12 Sogin, D.C. and Hinkle, P.C. (1980) Binding of cytochalasin B to human erythrocyte glucose transporter. Biochemistry, 19, 5417-5420. 13 Suzuki, K. and Kono, T. (1980) Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc. Natl. Acad. Sci. U.S.A., 77, 2542-2545.