Proc. Nat. Acad. Sci. USA Vol. 72, No. 4, pp. 1630-1634, April 1975

Purine Metabolism in Murine Virus-Induced Erythroleukemic Cells During Differentiation In Vitro (Friend leukemia cells/amidophosphoiribosyltransferase/5-phosphoribosylamine synthetase/cytidine deaminase) ,N

In's ..

GABRIELLE H. REEM* AND CHARLOTTE FRIENDt * Department of Pharmacology, New York University School of Medicine, New York, N.Y. 10016; and t the Center for Experimental Cell Biology, Mollie B. Roth Laboratory, Mount Sinai School of Medicine of the City University of New York, New York, N.Y. 10029

Communicated by Jerard Hurwitz, February 6, 1975 ABSTRACT Purine metabolism was studied in murine virus-induced erythroleukemic cells stimulated to differentiate in vitro in the presence of dimethylsulfoxide. The activities of the enzymes that catalyze the synthesis of the first intermediate of the de novo purine pathway, phosphoribosyl-1-amine, were decreased while the enzymes that catalyze the conversion of purine bases to purine ribonucleotides remained unchanged at the time the cells acquired the specialized function of hemoglobin synthesis. In addition, cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) activity increased with erythropoietic maturation, as it does during murine erythropoiesis in vivo. Stimulation of cellular proliferation of stationary erythroleukemic cells resulted in a marked increase in the activities of purine biosynthetic enzymes. These data provide a convincing example of repression and derepression of the PRA synthesizing enzymes in mammalian cells in vitro, and further evidence that the regulatory mechanisms operative in the normal development of erythrocytes can be activated by exposure of erythroleukemic cells to dimethylsulfoxide.

The regulatory adjustments that take place in the course of maturation of hematopoietic cells are reflected in alterations in the activity of a number of enzymes. For instance, differentiation of normal human and murine erythrocytes is associated with the loss of the ability to synthesize purines de now from small molecules, while the capacity to salvage purines by forming nucleotides is preserved (1-3). Specifically, differentiation is associated with the loss of a general cellular function and the acquisition of a specialized function, the synthesis of hemoglobin. These fundamental alterations in metabolism during the development of normal erythrocytes follow a complex program of selective gene activation and gene repression. In malignant cells this orderly program is disrupted and the sequential selection of gene repression and activation of normal development is perturbed. Under certain conditions, however, the malignant phenotype of hematopoietic cells can be reversed toward normalcy (4, 5). A means of investigating the biochemical events leading to the maturation of malignant cells, and of examining the possibility that the mechanism controlling these changes is closely related to those regulating gene expression in normal cell development, has been provided by cloned lines of murine virus-induced erythroleukemic cells (5) which can be stimu-

lated to differentiate along the erythroid pathway by dimethylsulfoxide (Me2SO) (6). Previous studies carried out in mice infected with Friend leukemia virus (FLV) indicated that marked changes in the catalytic activity of enzymes essential in purine and pyrimidine metabolism occurred during the development of leukemia (7-9). It, therefore, was of interest to investigate the activity of these enzymes during the differentiation of the erythroleukemic cells in titro. Two groups of enzymes of potential importance in the regulation of purine metabolism were studied: enzymes catalyzing the synthesis of purines de novo, and enzymes of the purine salvage pathway. Phosphoribosylpyrophosphate amidotransferase [ = amidophosphoribosyltransferase; 5-phosphoribosylamine: pyrophosphate phosphoribosyltransferase (glutamate-amidating), EC 2.4.2.14] and ribose-5-phosphate aminotransferase [= 5-phosphoribosylamine synthetase; ribose 5-phosphate: ammonia ligase (ADP forming), EC 6.3.4.7] catalyze the synthesis of phosphoribosyl 1-amine (PRibN), the first intermediate in purine biosynthesis, and are important in the regulation of this pathway (10-13). Hypoxanthine guanine phosphoribosyltransferase (HGPRT; IMP: pyrophosphate phosphoribosyl-transferase, EC 2.4.2.8) and adenine phosphoribosyltransferase (APRT; AMP: pyrophosphate phosphoribosyl-transferase, EC 2.4.2.7) catalyze the synthesis of purine ribonucleotides from purine bases by the salvage pathway. Changes in cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) activity, an enzyme important in the catabolism and reutilization of pyrimidines, were also monitored in this study. Elevation of cytidine deaminase activity has been reported to be associated not only with this virus-induced erythroleukemia, but specifically with accelerated erythroid differentiation in mice during stress erythropoiesis (14). The present communication describes the activities of these enzymes in control and differentiating erythroleukemic cells in tissue culture. Since the activities of these enzymes undergo characteristic changes during normal erythroid differentiation, they are markers of selective gene expression during development. MATERIALS AND METHODS

Cells. Clone 745A of an established line of Friend erythroleukemia cells was maintained in suspension culture. The properties of these cell lines and their responses to Me2SO have been recently reviewed (15). The cells, seeded at 1 X 105/ml, were grown in dehydrated Gibco Eagle's basal medium in Earle's balanced salt solution supplemented with 15%o fetal

Abbreviations: MeSO, dimethylsulfoxide; PRibPP, phosphoribosylpyrophosphate; Rib-5-P, ribose 5-phosphate; PRibN, 5-phosphoribosyl-1-amine; HGPRT, hypoxanthine guanine phosphoribosyltransferase; APRT, adenine phosphoribosyltransferase; PEI, polyethyleneimine.

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calf serum, 250 units/ml of penicillin, and 0.2 mg/ml of streptomycin, except for the experiments recorded in Table 5 for which RPMI 1640 medium was used. Certified reagent grade Me2SO (Fisher) was added to the medium on a volume per volume (v/v) basis to a final concentration of 2% directly before use. Cultures were incubated at 370 in 5% CO2 in air. Materials. [8-14C]Adenine (30 Ci/mol) and [8-14C]hypoxanthine (62 Ci/mol) were purchased from Amersham/Searle, and [2-14C]cytidine (28 Ci/mol) from Schwarz/Mann. Scintillation fluid consisted of 5 g of 2,5-diphenyloxazole (PPO), 1000 ml of toluene and 500 ml of Triton X-100. Radioactivity was measured in a Beckman liquid scintillation counter. For thinlayer chromatography, plastic-backed, precoated thin-layer plates manufactured by Macherey Nagel Co., distributed by Brinkmann, were used. Sodium phosphoribosylpyrophosphate (PRibPP), ribose-5-phosphate (Rib-5-P), and glycine were bought from Sigma Chemical Co., glutamine from Calbiochem, and ammonium chloride from Fisher Scientific Corp. All reagents and chemicals were of the purest grade available. All buffers contained 1 mM EDTA and 0.1 mM 2-mercaptoethanol. Enzyme Assays. Cell-free extracts were prepared at 4°. Cells were harvested by centrifugation at 650 X g and washed three times in 0.9% NaCl buffered with phosphate (pH 7.0). The pellet was resuspended in deionized water (approximately 108 cells per ml) and cells were disrupted by freeze-thawing five times. After centrifugation at 100,000 X g for 60 min, the cellfree supernatant fractions were dialyzed against 10 mM Tris HCI, pH 7.3. Activities of the enzymes that synthesize PRibN, PRibPP amidotransferase and Rib-S-P aminotransferase, were measured as previously described (11-13). APRT activity was assayed by measuring AMP synthesis from [8-'4C]adenine. The reaction mixture contained 0.2 mM PRibPP, 5 mM MgCl2, 0.03 uCi of [8-'4C]adenine, 50 mM Tris- HCl pH 8.0, and 10-50 ug of enzyme protein in a total volume of 100 yd. After incubation for 10 min at 370, the reaction was stopped by freezing and the product was determined by thin-layer chromatography on polyethylenimine (PEI) plastic-backed thin-layer plates developed with 0.2 M sodium chloride. The radioactive spots were cut out and their radioactivity was measured as described above. HGPRT activity was assayed by determining IMP production from [8-'4C]hypoxanthine. The reaction mixture contained 1 mM PRibPP, 2 mM MgCl2, 50 mM Tris'HCl, pH 8.0 and 0.12 MCi of [8-'4C]hypoxanthine in a final volume of 100 ul; incubations were carried out for 10 min at 370. IMP was identified by thin-layer chromatography as described for AMP. Intact cells were incubated in serumless medium with [8-14C]adenine or [8-'4C]hypoxanthine for the determination of purine ribonucleotide synthesis. After 1 hr at 370, cells were harvested by centrifugation, the medium was decanted, and purine ribonucleotides were extracted from the pellet with 100 jul of 2 N ice-cold perchloric acid. After neutralization of the extract with 2 N KOH, the reaction products were identified by thin-layet chromatography on PEI-cellulose. Duplicate plates were developed in two solvent systems: 0.2 M lithium chloride and 1.8 M lithium chloride. Cytidine deaminase activity was assayed in cell-free extracts by measuring uridine and uracil production from [2-

Enzyme Control in Differentiating Leukemic Cells

1631

CL

'_20 0

E Z

lo 72

1014 6

10

andgltamne(boto), ro PR~bP

OibPanaoia(dle

72 96 120 144 168 HOURS

FIG. 1. Effect of Me2SO on synthesis of PRibN from PRibPP and glutamine (bottom), from PRibPP and ammonia (middle) and from Rib-5-P and ammonia (top), in cultured erythroleukemic cells. Cultures were maintained for 7 days in Eagle's medium with (solid bars) or without (empty bars) 2% Me2SO. Cells were harvested and enzyme extracts were prepared as described in Materials and Methods. The standard incubation mixture for the determination of glutamine PRibPP amidotransferase activity contained 0.5 mM PRibPP, 2 mM glutamine, 2 mM MgCl2, 0. 5 mM ATP, 2 mM glycine, and 100 mM Tris * HC1, pH 8.5, in 206 jul. For PRibN synthesis from PRibPP and ammonia, glutamine was omitted and replaced by 5 mM ammonia. Rib-S-P aminotransferase activity was measured by substituting 1 mM Rib-S-P and 5 mM ammonia as substrates. Incubations were carried out with 50-lOOug of enzyme protein at 370 for 20 min. Under these conditions, assays were linear with varying protein concentrations.

14C]cytidine. The reaction mixture contained in a final volume of 40 pil, 0.05 MCi of cytidine, 50 mM Tris-HCl, pH 8.0, and 10-50 ug of enzyme protein. Reactions were carried out for 10 min at 37° and stopped by the addition of 20 ul of 3.0 N HCl containing 5 mM uridine, 5 mM uracil, and 5 mM cytidine. The products of the reaction were separated by thinlayer chromatography on cellulose developed in butanol: water: formic acid 77: 13: 10 (14). The addition of Me2SO to cell-free extracts did not interfere with the enzyme assays, nor did brief exposure (90 min) of cell cultures to Me2SO result, in changes in enzyme activities. Protein concentrations were determined according to the method of Lowry (16). RESULTS PRibN Synthesizing Enzymes. The specific activity of PRibPP amidotransferase and Rib-S-P aminotransferase was monitored daily in control cultures and in cultures grown in media containing 2% Me2SO. No medium changes were made during the 7-day period. A significant difference in glutamine PRibPP amidotransferase activity between Me2SO-treated cultures and controls was detected after 72 hr, when the cells were still actively proliferating and had not reached maximum density (Fig. 1, bottom). No significant changes in enzyme activities were detectable before 72 hr. Enzyme activity declined in both treated and untreated cells after the fourth day, the decline being more rapid in Me2SO-treated cultures. Glutamine PRibPP amidotransferase activity decreased in

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TABL9 1. Enzyme activities in logarithmically growing cultures PRibN* formed after addition of: Duration of experi- PRibPP + Gln PRibPP + NH8 Rib-S-P ment (hr) Control Me2SO Control Me2SO Control 27.8 6.8 19.8 1.5 6.3 72 17.9 11.1 26.9 1.2 5.5 96

CL

C' 30-

+ NH3 12.6 18.1

TABLE 2. Effect of fresh media on growing and stationary cultures

PRibN* formed after addition of:

peri- Fresh PRibPP + Gin PRibPP + NH, Rib-S-P + NH,

ment media (hr) added Control Me2SO Control MegSO Control MeSO 16.8 26.6 10.0 21.7 0.85 5.9 72 13.9 27.0 5.9 18.8 0.78 4.6 96 13.0 38.8 5.3 18.5 3.9 0.74 + 13.0 25.3 5.3 1.2 16.1 3.3 120 13.0 4.8 30.8 13.8 0.3 3.5 144 12.9 24.5 4.7 0.2 28.0 6.7 +

Enzyme activities were determined prior to the addition of the appropriate fresh media (72 and 120 hr) and 24 hr later (96 and 144 hr, respectively) and compared with enzyme activities in unsupplemented cultures. * nmol/min per mg of protein.

NH, Rib-5-P

NH3

r1

20E

1, 15_

control cultures from 4.4 to 2.5 nmol/min per mg of protein. When ammonia was substituted for glutamine for the determination of PRibPP amidotransferase activity, a comparable difference in specific activity between control and treated cultures was observed after 72 hr; this difference was maintained between days 4 and 7 (Fig. 1, middle). The difference in Rib-5P amidotransferase activity between control and treated cells was not detected until the 96th hr after exposure to Me2SO (Fig. 1, top). In order to determine whether these changes in enzyme activities were related to the rate of proliferation, the appropriate fresh media were added daily to maintain the cells in logarithmic growth. Activities of the PRibN synthesizing enzymes were determined in these actively growing cultures at 72 and 96 hr after the initial seeding (Table 1). In spite of the fact that control and treated cultures multiplied at approximately the same rate, the activities of the PRibN synthesizing enzymes retained significantly lower in the treated cultures. The greatest decrease was observed in glutamine PRibPP amidotransferase activity of Me2SO-treated cells. Under these experimental conditions, the depression of Rib-S-P aminotransferase activity of treated cells was already detectable after 72 hr, as was the decrease in PRibPP amidotransferase activity.

+

rm

25-

Me2SO

Comparison of the activities of the PRibN synthesizing enzymes of erythroleukemic cells during logarithmic growth. Cells were seeded at a density of 1 X 106 cells per ml in Eagle's medium with and without Me2SO and diluted with appropriate media daily. Cells were harvested 72 and 96 hr after the initial seeding, and enzyme activities were determined as described (see Fig. 1 and refs. 12 and 13). * nmol/min per mg of protein.

Duration of ex-

PRi bPP

E

10-

PRibPP

Z

5

+

Gin

Hi

t O i

1.

FIG. 2. Effect of the addition of fresh media on enzyme activities 144 hr after seeding. Control cultures (hatched bars) and Me2SO-treated cultures (solid bars) to which no fresh media were added were compared with control cultures (bars with open circles) and Me2SO-treated cultures (bars with solid circles) supplemented with the appropriate fresh media 24 hr later.

The effect of the addition of fresh medium to growing and stationary cultures was then examined. Parallel cultures were seeded and supplemented with fresh medium either after 72 hr, when the cells were still multiplying, or after 120 hr, when cell growth had virtually ceased. Enzyme activities were measured 24 hr after the addition of fresh medium and compared with those of cultures not supplemented for the duration of the experiment. Twenty-four hours after the addition of fresh medium at 72 hr there was no significant change in the specific activities of the PRibN synthesizing enzymes, although the differences between the enzyme activities in control and Me2SO-treated cultures were maintained (Table 2, 96 hr). The addition of fresh medium after 120 hr, when cultures were stationary, resulted in a doubling in the number of cells in control cultures within 24 hr and in an approximately 2-fold increase in PRibN synthesis from PRibPP and glutamine, and from PRibPP and ammonia, while Rib-S-P aminotransferase activity was unchanged (Table 2, 144 hr). This effect is more clearly represented in Fig. 2. In Me2SO-treated cultures, the addition of fresh medium 120 hr after seeding failed to stimulate cell division appreciably and the activity of PRibN synthesizing enzymes remained low. This failure to respond could be due to the toxic effects of prolonged exposure to Me2SO. Purine Nucleotide Synthesis by the Salvage Pathway. The specific activity of HGPRT and APRT in control and Me2SOtreated cultures after 96 hr of exposure to Me2SO was not found to be significantly different (Table 3). Since purine ribonucleotide synthesis by the salvage pathway is the source of purines in mature erythrocytes, the rate of ribonucleotide

TABLE 3. Me2SO does not affect the specific activity of the enzymes of the purine salvage pathway in cell-free extracts prepared from erythroleukemic cells in culture HGPRT activity* Control Me2SO 25.5 24.6

APRT activity* Control Me2SO 64.8 64.6

The specific activities of HGPRT and APRT of erythroleukemic cells in culture when compared. Enzyme activities were determined after 96 hr as described in Materials and Methods. * nmol/min per mg of protein.

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TABLE 4. Me2SO does not affect purine ribonucleotide synthesis by the salvage pathway in intact erythroleukemic cells Ribonucleotide synthesis (cpm) Additions

Control

Me2SO

[8-14C]Adenitie

30,500 15,400

31,100 15,900

[8-14C]Hypoxanthine

TABLE 5. Cytidine deaminase activity in cell-free extracts of erythroleukemic cells in culture

Exp. no. 1

2 Purine ribonucleotide synthesis was determined after 96 hr as described in Materials and Methods.

also measured in intact cells. No significant synthesis difference between control and treated cultures exposed to Me2SO for 96 hr was detected (Table 4). was

Cytidine Deaminase Activity. The activity of cytidine deaminase was monitored in control and treated cultures 48 and 96 hr after the initial seeding (Table 5, Exp. 1). At 48 hr, cytidine deaminase was low and there was no significant difference between the control and treated cultures. After 96 hr, when the majority of the cell population was synthesizing hemoglobin, cytidine deaminase activity had increased almost 3-fold in Me2SO-treated cells, but remained unchanged in control cells. This rise in cytidine deaminase activity during erythroid differentiation was in contrast to the observed fall in the activity of the PRibN synthesizing enzymes (Fig. 1). The addition of fresh medium after 72 hr did not alter the pattern of cytidine deaminase activity in either control or treated cells (Table 5, Exp. 2).

DISCUSSION Our results demonstrate that cultured erythroleukemic cells maintain the biochemical characteristics of the cells from which they originated. The level of activity of the purine biosynthetic enzymes of the cells of the established lines of erythroleukemic cells was found to be comparable to that of the leukemic spleens (7, 8). Our earlier studies had shown that, unlike mature erythrocytes, the immature cells in spleens of virus-infected leukemic mice have the enzyme systems required for de novo synthesis of purines and that the activity of the purine biosynthetic enzymes increased in a characteristic pattern during the course of the disease. Similarly, the activity of cytidine deaminase, an enzyme important in pyrimidine metabolism, was comparable in erythroleukemic cells in culture and in the cells of leukemic spleens (9, 14). When the erythroleukemic cells were induced to differentiate by the addition of Me2SO, they expressed some of the properties of normally differentiating erythroid cells in that they synthesized hemoglobin characteristic of adult mice (5). The present studies show that other genetic traits of the normally differentiating erythroid cells were also expressed by the erythroleukemic cells during exposure to Me2SO. In these maturing erythroleukemic cells, the purine biosynthetic enzymes were repressed as they are in developing normal erythrocytes, while the activity of the enzymes of the salvage pathway was preserved, as demonstrated by the incorporation of purine bases into purine ribonucleotides by cell-free extracts and in intact cells. In mature mouse erythrocytes, the enzyme activities of the salvage pathway serve to recover purine bases generated by catabolic reactions. It is interesting to note that the activity of cytidine de-

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of** Time Tim.o. Cytidine deaimmiase addition of Duration activity* fresh media of experiControl Me2SO ment (hr) (hr) 0.72 0.59 48 0 1.92 0.52 96 0 1.61 0.36 96 0 1.80 0.46 96 72

* nmol/min per mg of protein. In Exp. no. 1, no fresh media were added. In Exp. no. 2, the appropriate fresh media were added at 72 hr and enzyme activity was determined 24 hr later as described in Materials and Methods.

aminase increased during Me2SO-stimulated differentiation while the activity of the purine biosynthetic enzymes decreased. This elevation in cytidine deaminase activity also occurs during accelerated erythropoiesis in mice and is thought to accompany the development of erythroid cells in vivo (14). The present observation that the specific activity of the PRibN-synthesizing enzymes was repressed in differentiating cells in vitro stresses the importance of these enzymes in the regulation of de novo purine biosynthesis. Repression and derepression of purine biosynthetic enzymes have been observed in bacteria (17), but not in mammalian cells, although such changes had been searched for in cultured hepatoma cells (18). An increase in the specific activity of PRibPP amidotransferase in mammalian cells has so far only been observed in vivo in the course of virus-induced erythroleukemia in mice (7, 8). The current investigation extends this finding to erythroleukemic cells in vitro and provides evidence that this enzyme activity could be repressed by stimulating erythroid differentiation in the presence of Me2SO, and derepressed in stationary cells induced to proliferate by the addition of fresh medium. The synthesis of PRibN has been considered to be the ratelimiting step of the purine biosynthetic pathway (10, 19) and there is evidence that PRibN synthesis in human cells is catalyzed by more than one enzyme activity (11-13). PRibN can be synthesized enzymatically by PRibPP amidotransferase and by Rib-5-P aminotransferase. Recent studies have suggested that PRibN synthesis from PRibPP and ammonia in human lymphoblasts may be catalyzed by an ammonia-requiring enzyme activity, distinct from glutamine PRibPP amidotransferase, or by a subunit of glutamine PRibPP amidotransferase that has lost the capacity to release ammonia from glutamine (12). The changes observed in the activity of the PRibN-synthesizing enzymes following exposure to Me2SO are consistent with the hypothesis that more than one enzyme activity catalyzes PRibN synthesis from PRibPP, since the decline in glutamine PRibPP amidotransferase activity during differentiation was more precipitous and more marked than that of the enzyme activity that catalyzes PRibN synthesis from PRibPP and ammonia (Fig. 1). Our studies illustrate the significant contribution of the PRibN-synthesizing enzymes in the regulation of purine biosynthesis. The understanding of the regulation of this pathway in malignant diseases and in gout is of particular interest.

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Cell Biology: Reem and Friend

We are presently exploring the activity of the enzymes that catalyze the synthesis of the substrates required for PRibN synthesis, since it has been postulated that cellular PRibPP levels may be rate limiting for PRibN synthesis by glutamine PRibPP amidotransferase (20). Indeed, abnormally high rates of de novo purine biosynthesis in several gouty patients have been associated with mutant forms of PRibPP synthetase which generate abnormally high levels of PRibPP (21, 22). In previous studies the initiation of the synthesis of macromolecules associated with erythroid differentiation occurred within the first 2 days of Me2SO treatment of the erythroleukemic cells. It was only by the second day that elevations in 6-aminolevulinic acid synthetase, heme, globin mRNA, and globin were observed (23-26). Hemoglobin was not detected in identifiable quantities until the third day of treatment (6). The results of our present report are consistent with these earlier findings, since the marked differences in the purine biosyntietic enzymes and cytidine deaminase activities also occurred approximately 3 days after exposure to Me2SO. Demonstration of the alterations of purine and pyrimidine metabolism in differentiating erythroleukemic cells provides further evidence that growth in the presence of Me2SO results in the switching on of the regulatory mechanisms operative in normal erythrocytes. These findings suggest that factors which regulate gene expression during normal development can, when activated in cancer cells, alter the expression of the malignant phenotype. The erythroleukemic cells in culture thus provide a useful model for studying gene expression under controlled conditions. We wish to acknowledge the excellent technical assistance of Mrs. Carolann LandeenMrs. Karin Reimann, and Mr. J. Gilbert Holland. This work was supported in part by U.S. Public Health Service Grants AM 15262, CA 10,000, and CA 13,047. 1. Lowy, B. A., Williams, M. & London, I. M. (1962) J. Biol. Chem. 237, 1622-1625. 2. Reem, G. H. (1974). in Purine Metabolism in Man, eds. Sperling, O., de Vries, A. & Wyngaarden, J. V. (Plenum Press, New York), pp. 245-253.

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3. Fontenelle, L. J. & Henderson, J. F. (1969) Biochim. Biophys. Acta 177, 175- 176. 4. Paran, M., Sachs, L., Barak, Y. & Resnitzky, P. (1970) Proc. Nat. Acad. Sci. USA 67,1542-1549. 5. Friend, C., Patuleia, M. C. & de Harven, E. (1966) Nat. Cancer Inst. Monogr. 22, 505-522. 6. Friend, C., Scher, W., Holland, J. G. & Sato, T. (1971) Proc. Nat. Acad. Sci. USA 68, 378-382. 7. Reem, G. H. & Friend, C. (1967) Science 157, 1203-1204. 8. Reem, G. H. & Friend, C. (1969) Biochim. Biophys. Acta 171, 58-66. 9. Rothman, I. K., Malathi, N. G. & Silber, R. (1971) Cancer Res. 31, 274-276. 10. Wyngaarden, J. B., Silberman, H. R. & Sadler, J. H. (1958) Ann. N.Y. Acad. Sci. 75, 45-60. 11. Reem, G. H. (1972) J. Clin. Invest. 51, 1058-1062. 12. Reem, G. H. (1974) J. Biol. Chem. 249, 1696-1703. 13. Reem, G. H. (1968) J. Biol. Chem. 243, 5695-5701. 14. Rothman, I. K., Zanjani, E. D., Gordon, A. S. & Silber, R. (1970) J. Clin. Invest. 49, 2051-2067. 15. Friend, C., Preisler, H. D. & Scher, W. (1974) in Current Topics in Developmental Biology, eds. Monroy, A. & Moscona, A. A. (Academic Press, New York), pp. 81-101. 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 17. Nierlich, D. P. & Magasanik, B. (1971) Biochim. Biophys. Acta 230, 349-361. 18. Martin, D. W. & Owen, N. T. (1972) J. Biol. Chem. 17, 54775485. 19. Wyngaarden, J. B. (1972) in Current Topics in Cellular Regulation, eds. Horecker, B. & Stadtman, E. R. (Academic Press, New York), Vol. V, pp. 135-176. 20. Sperling, O., Wyngaarden, J. B. & Starmer, C. S. (1973) J. Clin. Invest. 52, 2468-2485. 21. Sperling, O., Persky-Brosh, S., Boer, P. & deVries, A. (1973) Biochem. Med. 7, 389-394. 22. Becker, M. A., Meyer, WK. J., Wood, A. W., & Seegmiller, J. E. (1973) Science 179, 1123-1126. 23. Ikawa, Y., Ross, J., Hayashi, K. Ebert, P., Gielen, J. & Leder, P. (1973) in Proc. VIth Symp. Comp. Luk. Res. Program and Abstracts (Nagoya/Ise-Shima), p. 65. 24. Ross, J., Ikawa, Y. & Leder, P. (1972) Proc. Nat. Acad. Sci. USA 69, 3620-3623. 25. Preisler, H. D., Housman, D., Scher, W. & Friend, C. (1973) Proc. Nat. Acad. Sci. USA 70,2956-2959. 26. Friend, C., Scher, W. & Preisler, H. D. (1974) Ann. N.Y. Acad. Sci. 241, 582-588.