BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
44,
175-180 (1990)
Accelerated Heme Synthesis and Degradation in Transformed Fibroblasts RIVKA MAMET,*
LEONARD
LEIBOVICI,~
YAEL TEITZ,$
AND NILI
SCHOENFELD*
*The Laboratory of Biochemical Pharmacology, and tThe Department of Internal Medicine B, Beilinson Medical Center, Petah Tiqva, and *The Department of Human Microbiology and The Sackler Faculty of Medicine, University of Tel Aviv, Ramat Aviv, Israel
Received April 20, 1990, and in revised form May 16, 1990
Porphobilinogen deaminase (PBGD) (EC 4.3.1.Q one of the enzymes of the heme biosynthetic pathway, is increased in erythrocytes of patients in the preleukemic state (1) and in lymphocytes of patients with chronic lymphatic leukemia (2). Elevated activity of this enzyme was also observed in fibroblastic cell lines transformed by different strains of Moloney leukemia virus (3). However, the increase in PBGD activity was shown to be positively correlated with growth rate in both nonmalignant and in malignant cell lines (4). Thus, one cannot rule out the possibility that the increased activity of PBGD in malignant cells is due to a faster growth rate and not to the malignant transformation per se. To address this issue, we determined the activity of PBGD and other parameters of the heme biosynthetic pathway in two cell lines, nonmalignant and malignant, both replicating at the same rate. METHODS AND MATERIALS Methods
Cultures of rat embryo fibroblastic cell lines were grown in Eagle’s minimal essential medium supplemented with 10% newborn calf serum as described previously (5). Cell lines were infected with both Moloney leukemia virus (MLV) and murine sarcoma virus (MS) (6) after four to five passages. Successful infection of cells was proven by high activity of reverse transcriptase, measured according to Teitz et al. (7). Transformation was assayed by the “cloning efficiency” method in semisolid agar as described by Bakhanoshvili et al. (6). The cells showed morphological changes typical of malignant transformation under light microscopy. Aminolevulinic acid synthase (ALAS), (EC 2.3.1.37) activity was determined according to Brooker et al. (8). A modification of a method described by us previously (9) was used for determining activity of aminolevulinic acid dehydratase (ALAD) (EC 4.2.1.24). PBGD activity was determined by the method 175 0885-4505/!20
$3.00
Copyright Q 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.
176
MAMET
ET AL.
TABLE I Growth Rates of Nontransformed and Transformed Cell Lines Growth rate
mpd, Day 2/ Cells
mpd, Day 1
m-4 Day 3/ mpd, Day 2
m-4 Day 41 mpd, Day 3
Nontransformed Transformed
1.83 k 0.20 2.04 + 0.30
2.03 k 0.52 2.53 f 0.16
1.25 k 0.13 1.49 + 0.24
Note. Total protein in each dish was measured daily during the 4 consecutive days of the experiment. Total protein on Day 1 was 0.15 f 0.05 mg protein/dish in both nontransformed and transformed cells. Rate of growth was calculated as mg protein/dish (mpd) on the indicated day divided by mg protein/dish on the preceding day. The results shown are the means 2 SD of four separate determinations.
of Magnussen et al. (10) as previously described for lymphocytes by Epstein et al. (11). Ferrochelatase (FC) (EC 4.99.1.1) was estimated by a modification of the method of Deybach et al. (12). The methods of Granick (13) and Morrison (14) were employed for the determination of the concentration of intracellular porphyrins and heme, respectively. The incorporation of 14C-ALA (0.26 &i/24 ,UM) into heme was measured according to Healey et al. (15) in sonicates of cells (2 mg protein/ml) incubated with the radioactive precursor for 45 min. The degradation of 14C-heme formed during the incubation was determined after a further 60-min incubation in the presence of desferrioxamine (0.5 mM), an inhibitor of heme synthesis. Protein was determined according to Lowry et al. (16). Materials Eagle’s minimal essential medium and newborn calf serum were purchased from Biological Industries (Kibbutz Beth-Haemek, Israel). The radiochemicals [2,3-‘4C]succinate (21.9 mCi/mmole), “FeSO, (309 mCi/mmole), and 5-amino[4“C]levulinic acid hydrochloride (56 mCi/mmole) were obtained from the Radiochemical Center (Amersham, UK). The chemicals used for the various enzymatic assays were obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents were of the highest purity available. Statistics Student’s t test and Pearson’s Product Moment for statistical evaluations.
Correlation
test were employed
RESULTS
The growth rates of the nontransformed and the transformed cell lines did not differ significantly, as shown in Table 1. The various determinations were carried out on the third day of culture when the plates were fully covered. The activities of four enzymes of the heme bio-
HEME SYNTHESIS
IN TRANSFORMED
177
FIBROBLASTS
TABLE 2 Activities of Enzymes of the Heme Biosynthetic Pathway in Nontransformed and Transformed Cells Rat fibroblasts Enzyme ALAS, nmole B-ALA/mg protein/hr ALAD, nmole PBG/mg protein/hr PBGD, nmole porph/mg protein/hr FC, nmole mesoheme/mg protein/hr
Nontransformed 0.450 9.450 0.028
2 0.36 2 1.7 r 0.008
1.650 r 0.7
Transformed 1.460 46.900 0.055 2.910
rf- 0.37* -c 12* f 0.017* 2 0.9*
Note. The data shown are the means k SD obtained in six separate determinations. * P < 0.001.
synthetic pathway in nontransformed and transformed cells were measured and the results are shown in Table 2. As demonstrated in Table 2, ALAS, ALAD, PBGD, and FC are significantly increased in the transformed cells. It is interesting to note that a negative correlation exists between the activity of FC and the content of intracellular heme (n = 30, r = 0.65, P < 0.001). The concentration of intracellular heme was found to be 30 pmole/mg protein in nontransformed cells and 10 pmole/mg protein in transformed cells (Table 3). This observation might indicate an impairment in heme synthesis, acceleration in its degradation, or both. The incorporation of 5-amino-[‘4C]levulinate into porphyrins and heme and the degradation of the labeled product (14C-heme) formed were studied. As shown in Table 3, the rate of heme synthesis is threefold higher in transformed cells than in nontransformed cells. The degradation of the 14C-heme formed is also accelerated in the transformed cells (Table 3). In the nontransformed system only 3% of the newly synthetized heme degraded during a 1-hr incubation with desferrioxamine, compared to 45% in the transformed system (P < 0.001). The intracellular concentration of porphyrins was only slightly (20%) elevated in the transformed cells, despite a twofold increase in its synthesis. The increased activity of FC is probably responsible for this observation. DISCUSSION
To distinguish between the effects of accelerated growth and malignancy, this study was carried out in two cell lines, nontransformed and malignantly transformed, both replicating at the same rate. The activity of the four enzymes studied, ALAS, ALAD, PBGD, and FC, was found to be elevated in the transformed cell lines. Perturbances in the heme biosynthetic pathway were described in other malignant systems as well. However, no distinct trend has emerged. ALAS activity was reported to be decreased in erythrocytes of patients with refractory sideroblastic anemia associated with the preleukemic syndrome (1) and in hepatoma cells (17-19) and normal in lymphocytes of patients with CLL (20). In hepatoma cells a reduced FC activity
178
MAMET
ET AL.
TABLE 3 Porphyrins and Heme in Nontransformed
and Transformed Cells Cells
Determination Intracellular concentration pmole/mg protein Porphyrins Heme Synthesis equivalent of 6ALA in pmole/mg protein/hr Porphyrins Heme Degradation % heme degraded/hr
Nontransformed
Transformed
27.8 2 2.2 (15) 30.4 + 4.4 (15)
34.4 t 3.5 (15)* 10.5 -c 2.6 (15)*
1.4 (3) 2.2 (3)
2.8 (3)* 5.1 (3)*
3 (3)
45 (3)*
Note. Intracellular porphyrins and heme were measured in sonicates of nontransformed and transformed cells. The sonicates were then incubated in the presence of “‘C-ALA for 45 min at the end of which r4C-porphyrins and 14C-heme were extracted and determined (see Methods and Materials). For measurement of 14C-heme degradation, desferrioxamine 0.5 mM was added and the sonicates were incubated for a further 60 min after which “C-heme was measured again. The numbers of separate determinations are given in parentheses. Porphyrins and heme formed were calculated as equivalents of 5aminolevulinate/mg protein/hr. * P < 0.001.
also was observed (21). However, the activity of PBGD was found to be elevated in our system as well as in all the other malignant systems in which the enzyme was determined (l-4), indicating probably that increased PBGD activity is a phenomenon characteristic of malignant cells. As shown in this study, the concentration of intracellular heme is markedly reduced in the transformed cells: 10 vs 30 pmole/mg protein in the nontransformed cells, although heme synthesis is threefold higher in the transformed cells. One can, therefore, assume that the low concentration of intracellular heme is a result of the accelerated rate of degradation of heme, which is probably caused by the increased activity of heme oxygenase (HO) (EC 1.14.99.3) in the transformed cells. These findings are in accordance with the literature. Reduced content of heme was found also in other malignant systems (18,19). Increased specific activity of HO in spleen microsomes of patients with CLL and with Hodgkin’s disease (22) and in liver cells (18,19) has previously been reported. A vital question in experimental systems comparing transformed to nontransformed cells is whether the differences between them are due to different growth rates or to the malignant transformation (23). The cell lines studied here have similar growth rates, and we can assume that the differences in the metabolic pathway of heme are specific to transformed cells. To the best of our knowledge, reduced intracellular heme appears to be a common feature to all malignant cells studied to date, and it is probably caused
HEME SYNTHESIS
IN TRANSFORMED
179
FIBROBLASTS
by the increased activity of heme oxygenase. This observation may point to a new approach to antitumor chemotherapy, tailored to take advantage of this difference between normal and transformed cells. SUMMARY
Various parameters of the heme biosynthetic pathway were studied in two cell lines, one nontransformed and the other malignantly transformed (MLV/MS), both replicating at the same rate. Using the above system enabled us to distinguish between phenomena characteristic of the malignant transformation per se and those due to accelerated growth rate. Heme synthesis and degradation as well as the activities of ALAS, ALAD, PBGD, and FC were found to be increased in the transformed cells. However, the concentration of intracellular heme was markedly reduced from 30.4 & 4.4 pmole/mg protein in nontransformed cells to 10.5 & 2.6 pmole/mg protein in transformed cells. These observations show that malignant transformation leads to changes in heme metabolism unrelated to growth rate in this cell line. ACKNOWLEDGMENTS We thank Mr. and Mrs. D. Salafor their generous financial support. We also thank Rachel Mevasser for her excellent technical assistance.
REFERENCES
1. Pasanen, A. V. O., Vuopio, P., Borgstrom, G. H., and Tenhunen, R.,
Scund.
J. Haematol.
27,
3.5 (1981). 2. Lahav, M., Epstein, O., Schoenfeld. N., Shaklai, M., and Atsmon, A., J. Amer. Med. Assoc. 257, 39 (1987). 3. Teitz, Y.. Schoenfeld, N., Epstein, O., and Atsmon, A., Med. Sci. Res. 16, 627 (1988). 4. Schoenfeld, N., Mamet, R., Leibovici, L., Epstein, O., T&z, Y., and A&non, A., B&hem, Med.
5. 6. 7. 8. 9. IO. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Metab.
Biol.
40, 213 (1988).
Sherman, L., Eylan, E., and Teitz, Y., Arch. Viral. 66, 143 (1980). Bakhanosbvili, M., Wrescher, D. H., and Salzberg, S., Cancer Res. 43, 1289 (1983). Teitz, Y.. Lennette, E. H., Oshiro, I. S., and Cremer, N., J. Nat/. Cancer Inst. 46, 11 (1971). Brooker, J. D., Srivastava. G., May, B. K., and Elliott, W. H., Enzyme 28, 109 (1982). Schoenfeld, N.. Greenblat, Y., Epstein, 0.. and Atsmon, A., Biochim. Biophys. Acta 721, 408 (1982). Magnussen, C. R., Levine, J. B., Doherty, J. M., Chessman, J. O., and Tschudy, D. P., Blood 44, 857 (1974). Epstein, 0.. Lahav, M., Schoenfeld, N., Nemesh, L., Shaklai, M., and Atsmon, A., Cancer 52, 828 (1983). Deybach, J. C., de Verneuil, H., and Nordmann, Y., Hum. Genet. 58, 425 (1981). Granick. S., J. Biol. Chem. 241, 1359 (1966). Morrison, G. R., Anal. Gem. 37, 1124 (1967). Healey. J. F., Bonkowsky, H. L.. Sinclair, P. R., and Sinclair, J. F., B&hem. J. 198, 595 (1981). Lowry, 0. H.. Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265 (1951). Bonkowsky, H. L., Tschudy, D. P., Collins, A., and Doherty, J. M., J. NatI. Cancer Inst. 50, 1215 (1973). Stout, D. L., and Becker, F. F., Cancer Res. 46, 2756 (1986). Stout, D. L.. and Becker, F. F., Cancer Res. 47, %3 (1987).
180
MAMET
ET AL.
20. Schoenfeld, N., Epstein, O., Lahav, M., Mamet, R., Shaklai, M., and Atsmon, A., Cancer Lets 43, 43 (1988). 21. Daily, H. A., and Smith, A., Biochem. J. 223, 441 (1984). 22. Schacter, B. A., Yoda, B., and Israels, L. G., J. Lab. Clin. Med. 93, 838 (1979). 23. Weber, G., Cancer Res. 43, 3466 (1983).
MEDICINE
AND
METABOLIC
BIOLOGY
44,
175-180 (1990)
Accelerated Heme Synthesis and Degradation in Transformed Fibroblasts RIVKA MAMET,*
LEONARD
LEIBOVICI,~
YAEL TEITZ,$
AND NILI
SCHOENFELD*
*The Laboratory of Biochemical Pharmacology, and tThe Department of Internal Medicine B, Beilinson Medical Center, Petah Tiqva, and *The Department of Human Microbiology and The Sackler Faculty of Medicine, University of Tel Aviv, Ramat Aviv, Israel
Received April 20, 1990, and in revised form May 16, 1990
Porphobilinogen deaminase (PBGD) (EC 4.3.1.Q one of the enzymes of the heme biosynthetic pathway, is increased in erythrocytes of patients in the preleukemic state (1) and in lymphocytes of patients with chronic lymphatic leukemia (2). Elevated activity of this enzyme was also observed in fibroblastic cell lines transformed by different strains of Moloney leukemia virus (3). However, the increase in PBGD activity was shown to be positively correlated with growth rate in both nonmalignant and in malignant cell lines (4). Thus, one cannot rule out the possibility that the increased activity of PBGD in malignant cells is due to a faster growth rate and not to the malignant transformation per se. To address this issue, we determined the activity of PBGD and other parameters of the heme biosynthetic pathway in two cell lines, nonmalignant and malignant, both replicating at the same rate. METHODS AND MATERIALS Methods
Cultures of rat embryo fibroblastic cell lines were grown in Eagle’s minimal essential medium supplemented with 10% newborn calf serum as described previously (5). Cell lines were infected with both Moloney leukemia virus (MLV) and murine sarcoma virus (MS) (6) after four to five passages. Successful infection of cells was proven by high activity of reverse transcriptase, measured according to Teitz et al. (7). Transformation was assayed by the “cloning efficiency” method in semisolid agar as described by Bakhanoshvili et al. (6). The cells showed morphological changes typical of malignant transformation under light microscopy. Aminolevulinic acid synthase (ALAS), (EC 2.3.1.37) activity was determined according to Brooker et al. (8). A modification of a method described by us previously (9) was used for determining activity of aminolevulinic acid dehydratase (ALAD) (EC 4.2.1.24). PBGD activity was determined by the method 175 0885-4505/!20
$3.00
Copyright Q 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.
176
MAMET
ET AL.
TABLE I Growth Rates of Nontransformed and Transformed Cell Lines Growth rate
mpd, Day 2/ Cells
mpd, Day 1
m-4 Day 3/ mpd, Day 2
m-4 Day 41 mpd, Day 3
Nontransformed Transformed
1.83 k 0.20 2.04 + 0.30
2.03 k 0.52 2.53 f 0.16
1.25 k 0.13 1.49 + 0.24
Note. Total protein in each dish was measured daily during the 4 consecutive days of the experiment. Total protein on Day 1 was 0.15 f 0.05 mg protein/dish in both nontransformed and transformed cells. Rate of growth was calculated as mg protein/dish (mpd) on the indicated day divided by mg protein/dish on the preceding day. The results shown are the means 2 SD of four separate determinations.
of Magnussen et al. (10) as previously described for lymphocytes by Epstein et al. (11). Ferrochelatase (FC) (EC 4.99.1.1) was estimated by a modification of the method of Deybach et al. (12). The methods of Granick (13) and Morrison (14) were employed for the determination of the concentration of intracellular porphyrins and heme, respectively. The incorporation of 14C-ALA (0.26 &i/24 ,UM) into heme was measured according to Healey et al. (15) in sonicates of cells (2 mg protein/ml) incubated with the radioactive precursor for 45 min. The degradation of 14C-heme formed during the incubation was determined after a further 60-min incubation in the presence of desferrioxamine (0.5 mM), an inhibitor of heme synthesis. Protein was determined according to Lowry et al. (16). Materials Eagle’s minimal essential medium and newborn calf serum were purchased from Biological Industries (Kibbutz Beth-Haemek, Israel). The radiochemicals [2,3-‘4C]succinate (21.9 mCi/mmole), “FeSO, (309 mCi/mmole), and 5-amino[4“C]levulinic acid hydrochloride (56 mCi/mmole) were obtained from the Radiochemical Center (Amersham, UK). The chemicals used for the various enzymatic assays were obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents were of the highest purity available. Statistics Student’s t test and Pearson’s Product Moment for statistical evaluations.
Correlation
test were employed
RESULTS
The growth rates of the nontransformed and the transformed cell lines did not differ significantly, as shown in Table 1. The various determinations were carried out on the third day of culture when the plates were fully covered. The activities of four enzymes of the heme bio-
HEME SYNTHESIS
IN TRANSFORMED
177
FIBROBLASTS
TABLE 2 Activities of Enzymes of the Heme Biosynthetic Pathway in Nontransformed and Transformed Cells Rat fibroblasts Enzyme ALAS, nmole B-ALA/mg protein/hr ALAD, nmole PBG/mg protein/hr PBGD, nmole porph/mg protein/hr FC, nmole mesoheme/mg protein/hr
Nontransformed 0.450 9.450 0.028
2 0.36 2 1.7 r 0.008
1.650 r 0.7
Transformed 1.460 46.900 0.055 2.910
rf- 0.37* -c 12* f 0.017* 2 0.9*
Note. The data shown are the means k SD obtained in six separate determinations. * P < 0.001.
synthetic pathway in nontransformed and transformed cells were measured and the results are shown in Table 2. As demonstrated in Table 2, ALAS, ALAD, PBGD, and FC are significantly increased in the transformed cells. It is interesting to note that a negative correlation exists between the activity of FC and the content of intracellular heme (n = 30, r = 0.65, P < 0.001). The concentration of intracellular heme was found to be 30 pmole/mg protein in nontransformed cells and 10 pmole/mg protein in transformed cells (Table 3). This observation might indicate an impairment in heme synthesis, acceleration in its degradation, or both. The incorporation of 5-amino-[‘4C]levulinate into porphyrins and heme and the degradation of the labeled product (14C-heme) formed were studied. As shown in Table 3, the rate of heme synthesis is threefold higher in transformed cells than in nontransformed cells. The degradation of the 14C-heme formed is also accelerated in the transformed cells (Table 3). In the nontransformed system only 3% of the newly synthetized heme degraded during a 1-hr incubation with desferrioxamine, compared to 45% in the transformed system (P < 0.001). The intracellular concentration of porphyrins was only slightly (20%) elevated in the transformed cells, despite a twofold increase in its synthesis. The increased activity of FC is probably responsible for this observation. DISCUSSION
To distinguish between the effects of accelerated growth and malignancy, this study was carried out in two cell lines, nontransformed and malignantly transformed, both replicating at the same rate. The activity of the four enzymes studied, ALAS, ALAD, PBGD, and FC, was found to be elevated in the transformed cell lines. Perturbances in the heme biosynthetic pathway were described in other malignant systems as well. However, no distinct trend has emerged. ALAS activity was reported to be decreased in erythrocytes of patients with refractory sideroblastic anemia associated with the preleukemic syndrome (1) and in hepatoma cells (17-19) and normal in lymphocytes of patients with CLL (20). In hepatoma cells a reduced FC activity
178
MAMET
ET AL.
TABLE 3 Porphyrins and Heme in Nontransformed
and Transformed Cells Cells
Determination Intracellular concentration pmole/mg protein Porphyrins Heme Synthesis equivalent of 6ALA in pmole/mg protein/hr Porphyrins Heme Degradation % heme degraded/hr
Nontransformed
Transformed
27.8 2 2.2 (15) 30.4 + 4.4 (15)
34.4 t 3.5 (15)* 10.5 -c 2.6 (15)*
1.4 (3) 2.2 (3)
2.8 (3)* 5.1 (3)*
3 (3)
45 (3)*
Note. Intracellular porphyrins and heme were measured in sonicates of nontransformed and transformed cells. The sonicates were then incubated in the presence of “‘C-ALA for 45 min at the end of which r4C-porphyrins and 14C-heme were extracted and determined (see Methods and Materials). For measurement of 14C-heme degradation, desferrioxamine 0.5 mM was added and the sonicates were incubated for a further 60 min after which “C-heme was measured again. The numbers of separate determinations are given in parentheses. Porphyrins and heme formed were calculated as equivalents of 5aminolevulinate/mg protein/hr. * P < 0.001.
also was observed (21). However, the activity of PBGD was found to be elevated in our system as well as in all the other malignant systems in which the enzyme was determined (l-4), indicating probably that increased PBGD activity is a phenomenon characteristic of malignant cells. As shown in this study, the concentration of intracellular heme is markedly reduced in the transformed cells: 10 vs 30 pmole/mg protein in the nontransformed cells, although heme synthesis is threefold higher in the transformed cells. One can, therefore, assume that the low concentration of intracellular heme is a result of the accelerated rate of degradation of heme, which is probably caused by the increased activity of heme oxygenase (HO) (EC 1.14.99.3) in the transformed cells. These findings are in accordance with the literature. Reduced content of heme was found also in other malignant systems (18,19). Increased specific activity of HO in spleen microsomes of patients with CLL and with Hodgkin’s disease (22) and in liver cells (18,19) has previously been reported. A vital question in experimental systems comparing transformed to nontransformed cells is whether the differences between them are due to different growth rates or to the malignant transformation (23). The cell lines studied here have similar growth rates, and we can assume that the differences in the metabolic pathway of heme are specific to transformed cells. To the best of our knowledge, reduced intracellular heme appears to be a common feature to all malignant cells studied to date, and it is probably caused
HEME SYNTHESIS
IN TRANSFORMED
179
FIBROBLASTS
by the increased activity of heme oxygenase. This observation may point to a new approach to antitumor chemotherapy, tailored to take advantage of this difference between normal and transformed cells. SUMMARY
Various parameters of the heme biosynthetic pathway were studied in two cell lines, one nontransformed and the other malignantly transformed (MLV/MS), both replicating at the same rate. Using the above system enabled us to distinguish between phenomena characteristic of the malignant transformation per se and those due to accelerated growth rate. Heme synthesis and degradation as well as the activities of ALAS, ALAD, PBGD, and FC were found to be increased in the transformed cells. However, the concentration of intracellular heme was markedly reduced from 30.4 & 4.4 pmole/mg protein in nontransformed cells to 10.5 & 2.6 pmole/mg protein in transformed cells. These observations show that malignant transformation leads to changes in heme metabolism unrelated to growth rate in this cell line. ACKNOWLEDGMENTS We thank Mr. and Mrs. D. Salafor their generous financial support. We also thank Rachel Mevasser for her excellent technical assistance.
REFERENCES
1. Pasanen, A. V. O., Vuopio, P., Borgstrom, G. H., and Tenhunen, R.,
Scund.
J. Haematol.
27,
3.5 (1981). 2. Lahav, M., Epstein, O., Schoenfeld. N., Shaklai, M., and Atsmon, A., J. Amer. Med. Assoc. 257, 39 (1987). 3. Teitz, Y.. Schoenfeld, N., Epstein, O., and Atsmon, A., Med. Sci. Res. 16, 627 (1988). 4. Schoenfeld, N., Mamet, R., Leibovici, L., Epstein, O., T&z, Y., and A&non, A., B&hem, Med.
5. 6. 7. 8. 9. IO. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Metab.
Biol.
40, 213 (1988).
Sherman, L., Eylan, E., and Teitz, Y., Arch. Viral. 66, 143 (1980). Bakhanosbvili, M., Wrescher, D. H., and Salzberg, S., Cancer Res. 43, 1289 (1983). Teitz, Y.. Lennette, E. H., Oshiro, I. S., and Cremer, N., J. Nat/. Cancer Inst. 46, 11 (1971). Brooker, J. D., Srivastava. G., May, B. K., and Elliott, W. H., Enzyme 28, 109 (1982). Schoenfeld, N.. Greenblat, Y., Epstein, 0.. and Atsmon, A., Biochim. Biophys. Acta 721, 408 (1982). Magnussen, C. R., Levine, J. B., Doherty, J. M., Chessman, J. O., and Tschudy, D. P., Blood 44, 857 (1974). Epstein, 0.. Lahav, M., Schoenfeld, N., Nemesh, L., Shaklai, M., and Atsmon, A., Cancer 52, 828 (1983). Deybach, J. C., de Verneuil, H., and Nordmann, Y., Hum. Genet. 58, 425 (1981). Granick. S., J. Biol. Chem. 241, 1359 (1966). Morrison, G. R., Anal. Gem. 37, 1124 (1967). Healey. J. F., Bonkowsky, H. L.. Sinclair, P. R., and Sinclair, J. F., B&hem. J. 198, 595 (1981). Lowry, 0. H.. Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265 (1951). Bonkowsky, H. L., Tschudy, D. P., Collins, A., and Doherty, J. M., J. NatI. Cancer Inst. 50, 1215 (1973). Stout, D. L., and Becker, F. F., Cancer Res. 46, 2756 (1986). Stout, D. L.. and Becker, F. F., Cancer Res. 47, %3 (1987).
180
MAMET
ET AL.
20. Schoenfeld, N., Epstein, O., Lahav, M., Mamet, R., Shaklai, M., and Atsmon, A., Cancer Lets 43, 43 (1988). 21. Daily, H. A., and Smith, A., Biochem. J. 223, 441 (1984). 22. Schacter, B. A., Yoda, B., and Israels, L. G., J. Lab. Clin. Med. 93, 838 (1979). 23. Weber, G., Cancer Res. 43, 3466 (1983).
