Proc. Nati. Acad. Sci. USA Vol. 75, No. 7, pp. 3437-3439' July 1978
Medical Sciences
Growth hormone modulation of murine erythroleukemia cell growth in vitro (insulin/Friend leukemia/prolactin/somatomammotropin/somatotropin)
DAVID W. GOLDE*, NOELLE BERSCH*, AND CHOH HAO Lit *
Division of Hematology-Oncology, Department of Medicine, University of California Los Angeles School of Medicine, Los Angeles, California 90024
t Hormone Research Laboratory, University of California, San Francisco, California 94143
Contributed by Choh Hao Li, April 24, 1978
ABSTRACT There are few studies showing a biological effect of growth hormone (somatotropin) on cel proliferation in vitro at physiological concentrations. We report here that Friend virus-infected erythroleukemia cells are responsive to growth hormone in vitro. Using a serum-free clonogenic assay, we found as little as 0.1 ng of human growth hormone per ml caused a prominent stimulation of cell proliferation. Peak activity of human growth hormone occurred at 200 ng/ml, resulting in a 2-fold increase in cloning. Human chorionic somatomammotropin and the Cys(Cam)fi hGH(1-134) fragment of human growth hormone were also active, but a biologically inert oxidized human growth hormone had no growth-promoting effect in vitro. Cell proliferat was stimulated by insulin with peak potentiation occurring at 1 ng/ml, and prolactin had a demonstrable stimulatory effect between 50 and 100 ng/ml. These observations indicate that growth hormone and related polypeptides have a direct effect on the in vitro proliferation of e~ryhroleukemia cells in the absence of serum. The results confirm a direct action of growth hormone on mammalian cells and suggest that pituitary hormones may affect leukemic cell growth.
Growth hormone (somatotropin) is known to have important interactions with hematopoietic cells and certain lymphoid populations (1-4). Although there is considerable information on growth hormone binding to cell receptors, there are few studies on the physiologic effects of growth hormone on mammalian cells in vitro (5-8). We have recently reported a direct effect of growth hormone on potentiation of normal erythropoiesis in vttro (9). This effect was species specific and demonstrable with nanogram concentrations of hormone. We report here studies indicating that growth hormone stimulates murine erythroleukemia cell proliferation in a serum-free culture system. MATERIALS AND METHODS The dimethyl sulfoxide-inducible Friend leukemia cell line GM-86, clone 745, was obtained from the Institute for Medical Research, Camden, NJ, and maintained in continuous suspension culture with media containing 20% fetal calf serum. The erythroleukemia cells were grown in T-25 flasks and divided and fed with fresh medium 1 or 3 days before they were cloned in methylcellulose. The methylcellulose culture system was used as reported (9, 10), but 0.5% bovine serum albumin (Sigma Chemical Co., St. Louis, MO) was substituted for the fetal calf serum in the culture dishes. No erythropoietin or other stimulus was used. The cells were counted and directly plated in methylcellulose without washing. The cultures were incubated at 370 in a humidified atmosphere of 5% CO2 in air. Ten thousand cells were plated in each dish, and all clusters of eight The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adwrtisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
or more cells were enumerated 72 hr later, by using an inverted microscope. Highly purified human growth hormone (hGH) (11), bovine growth hormone (bGH) (12), human chorionic somatomammotropin (hCS) (13), and the plasmin-cleaved hGH fragment (14) Cys(Cam)53-hGH(1-134), were prepared as described. An oxidized and biologically inert hGH was produced by performic acid oxidation (15, 16). Ovine prolactin was prepared as reported (17). Porcine insulin was a gift of 0. Behren of Eli Lilly and Company. These hormones were dissolved in 0.1 M NaOH and diluted in phosphate-buffered saline (pH 9.3) before addition to the methylcellulose cultures. Appropriate cultures were established without hormone to control for diluent material. In selected experiments a plasma clot system was used to clone the erythroleukemia cells (18). Growth and differentiation in liquid culture was assessed by viable cell counts and differential counts after staining with benzidine (19).
RESULTS Growth hormone was found to stimulate erythroleukemic cell cloning at the high cloning efficiencies seen with 30% fetal calf serum; however, the serum-free system eliminated other serum humoral influences and allowed for a consistent demonstration of growth hormone effects at low concentration. The cloning efficiency of the erythroleukemia cells varied considerably, depending on when they were cloned in relationship to the previous feeding. The mean (+SEM) 72-hr cloning efficiency in the serum-free system was 256 + 60 colonies per 104 cells in 13 separate experiments. For convenience of comparison, the data are expressed as percentage of control value. Fig. 1 shows the effect of various hGH preparations and hCS on erythroleukemic cell colony formation. A prominent stimulatory effect of hGH was observable at concentrations of 10 ng/ml. Fig. 2 shows that the effect of as little as 0.1 ng of hGH per ml was discernible in this system (P < 0.05). Peak stimulation, 224% of control, occurred at 200 ng/ml, and 500 ng/ml caused greatly reduced potentiation of cloning. The dilution controls varied by no more than 1%. hCS and the hGH fragment were also active stimulators of erythroleukemic cell cloning but maximal stimulating concentrations (200 ng/ml) had approximately half the activity of hGH (Fig. 1). Oxidized hGH was inactive in this system and did not interfere with the response to hGH in mixing studies. bGH also stimulated erythroleukemia cell cloning but to a lesser degee than hGH: at peak stimulatory concentrations of bGH the maximal stimulation was 155% of control (data not shown). Insulin was found to potentiate cloning of the erythroleukemia cells in serum-free medium. A clear effect was discernible at 0.5 ng/ml, and peak stimulation was seen at 1 ng/ml (Fig. Abbreviations: bGH, bovine growth hormone; hCS, human chorionic
somatomammotropin; hGH, human growth hormone. 3437
Medical Sciences: Golde et al.
3438
Proc. Natl. Acad. Sci. USA 75 (1978) Table 1. Combination of various dosages of hGH with insulin (1 Ng) on erythroleukemia cloning
300 C
i0
hGH, ng/ml 0.1 0.5 1.0 5.0 10.0 100.0 * Mean + SEM.
0
is,0 200_ 0
I
x
- -----.
Q
._
% of control*
158 180 211 ± 173 i 167 150 ±
9 12 22 28 12 15
%
additivity 108 106 110 81 67 45
0
10
25
50
100 Hormone added, ng/ml
200
I, soo
FIG. 1. Effect of hGH preparations and hCS oin colony formation by erythroleukemia cells in methylcellulose. Thie mean (±SEM) cloning efficiency in these studies was 256 + 60 co]lonies per 104 cells in 13 separate experiments. The data are expressed percentage of control in three to seven experimt nts performed in duplicate. 0, hGH; *, hCS; 0, hGH fragment Cyrs(Cam)53-hGH(1134); &, performic acid-oxidized hGH.
2). Higher concentrations of insulin were less effective in promoting colony formation. Table 1 shows that the effects of insulin and hGH were additive at low concentrations but inhibition was observed with hGH concentration!s above 5 ng/ml Prolactin stimulated erythroleukemia cell proliferation at concentrations above 25 ng/ml and peak act ivity was seen at 100 ng/ml (Fig. 2). The effect of hGH on erythroleukemia cel 1 growth was observable in plasma clot culture and in liquid Esuspension. hGH at 1 ng/ml in serum-free liquid culture result ed in an increase in the viable cell count to 148% of control at '7 days. Although hGH stimulated cell proliferation in liquid clulture, it did not influence cell differentiation as measured biy the number of benzidine-positive cells seen in culture with a nd without 1.5% dimethyl sulfoxide.
DISCUSSION These observations indicate that malignant Friend virus-infected erythroleukernia cells retain the respons iveness to growth hormone characteristic of the normal comrnitted erythroid progenitor (9). Stimulation of cell proliferEation by growth hormone is observable as a prominent incre: ase in cloning in
-
0
260
° 220
100. 0.5 1.0 5 10 Hormone, ng/ml
hormone fragment (14) in this system roughly approximated
their growth-promoting activity in that stimulation of erythroleukemia cell colony formation was substantially less than seen with hGH. The biologically inactive oxidized hGH had no effect in vitro and did not interfere with the action of hGH, suggesting that the oxidized hormone did not block activation of a receptor mechanism. Unlike normal erythroid precursors (9), the erythroleukemia cells were stimulated by high concentrations of prolactin. Prolactin was effective in potentiating cell growth only at concentrations approximately 2 orders of magnitude greater than effective levels of hGH. This observation may relate to the high degree of molecular homology between prolactin and hGH. Insulin potentiated cell growth at physiologic concentrations below those known to be effective in other cell systems (20-23). The effects of these hormones were observable in other clonogenic systems (plasma clot culture) as well as in liquid suspension culture, indicating that the findings were not peculiar to methylcellulose culture. The relationship of pituitary hormones to normal hematopoiesis has been emphasized by other investigators (24). Growth hormone has also been shown to exhibit effects on hematopoietic malignancies, and hypophysectomy was reported to cause regression of certain leukemias in rats (25, 26). Results reported herein demonstrate that nanogram concentrations of hGH and related polypeptides directly stimulate in vitro erythroleukemia cell proliferation. The findings confirm a direct action of growth hormone on cells and suggest that pituitary hormones may affect leukemic cell growth. The system described offers a means to study the effects of growth hormones on mammalian cells in a serum-free system. This work was supported in part by U.S. Public Health Service Grants CA 15688 and CA 15619 (to D.W.G.) and AM-6097 and AM18677 (to C.H.L.).
180/ C
2 140
0
vitro. The absence of serum in this system obviated considerations of an indirect action of growth hormone such as mediation of serum somatomedin-like polypeptide binding to cells (20). The clear dose-response to growth hormone and the nanogram range of effective concentrations correlate well with the previously reported range for receptor binding studies using lymphoid cell lines (6). The effects of hCS (13) and the growth
5so
100
500 0o0o
FIG. 2. Effect of hGH (@), insulin (A), and1 prolactin (0) on erythroleukemia cell colony formation in vitro. Ain effect of hGH is noted at a concentration of 0.1 ng/ml. Results are expressed as mean (±SEM) percentage of control for five to seven [experiments performed in duplicate.
1. Fruhman, G. J., Gerstner, R. & Gordon A. S. (1954) Proc. Soc. Exp. Biol. Med. 85,93-96. 2. Peschle, C., Marone, G., Genovese, A., Sacchetti, L. & Condorelli, M. (1975) in Erythropoless. Proceedings of the 4th International Conference on Erythropoiesis, eds. Nakao, K., Fisher, J. W. & Takaku, F. (University of Tokyo Press, Tokyo), pp. 99-117. 3. Pandian, M. R. & Talwar, G. P. (1971) J. Exp. Med. 134,
1095-1113.
4. Arrenbrecht, S. (1974) Nature 252,255-257. 5. Lesniak, M. A., Roth, J., Gorden, P. & Gavin, J. R., III (1973) Nature New Biol. 421, 20-22.
Proc. Nati. Acad. Sci. USA 75 (1978)
Medical Sciences: Golde et al. 6. Lesniak, M. A. & Gorden, P. (1976) in Hormone-ReceptOr Inxi1teraction. Molecular Aspects. Modern ogy, ed. Levey, G. S. (Dekker, New York), Vol. 9, pp. 201219. 7. Ramachandran, J., Lee, V. & Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48,274-279. 8. Desai, L. S., Lazarus, H., Li, C. H. & Foley, G. E. (1973) Exp. Cell Res. 81, 330-332. 9. Golde, D. W., Bersch, N. & Li, C. H. (1977) Science 196, 1112-1113. 10. Golde, D. W., Bersch, N. & Cline, M. J. (1976) J. Clin. Invest. 57,
Phama~4logy-i
11. 12. 13. 14.
15.
57-62. Li, C. H., Liu, W.-K. & Dixon, J. S. (1962) Arch. Biochem. Biophys., Suppl. 1, 327-32. Li, C.H. (1954) J. Biol. Chem. 211,555-558. Li, C. H. (1970) Ann. Sclavo 12,651-662. Li, C. H. & Graf, L. (1974) Proc. Natl. Acad. Sci. USA 71, 1197-1201. Bewley, T. A., Brovetto-Cruz, J. & Li, C. H. (1969) Biochemistry 8,4701-4708.
3439
Fonss-Bech, P. & Schmidt, K. D. (1969) Int. J. Protein Research - &-15-92. 17. Li, C. H., Dixon, J. S., Lo, T. B., Schmidt, K. D. & Pankov, Y. A. (1970) Arch. Biochem. Bwophys. 141, 705-737. 18. Stephenson, J. R., Axelrad, A. A., McLeod, D. L. & Shreeve, M. M. (1971) Proc. NatI. Acad. Sci. USA 68, 1542-1546. 19. Friend, C., Scher, W., Holland, J. G., Sato, T. (1971) Proc. NatI. Acad. Sd. USA 68,378-382. 20. Moses, A. C., Nissley, S. P., Cohen, K. L. & Rechler, M. M. (1976) 16.
Nature 263, 137-140. 21. Gospodarowicz, D. & Moran, J. S. (1974) Proc. Natl. Acad. Sci. USA 71, 4584-4588. 22. Gospodarowicz, D. & Moran, J. S. (1976) Annu. Rev. Biochem. 44,531-558. 23. Hayashi, I. & Sato, G. H. (1976) Nature 259, 132-134. 24. Reddi, A. H. & Huggins, C. B. (1976) Nature 263,514-515. 25. Huggins, C. & Oka, H. (1972) Cancer Res. 32,239-242. 26. Bentley, H. P., Hughes, E. R. & Peterson, R. D. A. (1974) Nature 252,747-748.
Medical Sciences
Growth hormone modulation of murine erythroleukemia cell growth in vitro (insulin/Friend leukemia/prolactin/somatomammotropin/somatotropin)
DAVID W. GOLDE*, NOELLE BERSCH*, AND CHOH HAO Lit *
Division of Hematology-Oncology, Department of Medicine, University of California Los Angeles School of Medicine, Los Angeles, California 90024
t Hormone Research Laboratory, University of California, San Francisco, California 94143
Contributed by Choh Hao Li, April 24, 1978
ABSTRACT There are few studies showing a biological effect of growth hormone (somatotropin) on cel proliferation in vitro at physiological concentrations. We report here that Friend virus-infected erythroleukemia cells are responsive to growth hormone in vitro. Using a serum-free clonogenic assay, we found as little as 0.1 ng of human growth hormone per ml caused a prominent stimulation of cell proliferation. Peak activity of human growth hormone occurred at 200 ng/ml, resulting in a 2-fold increase in cloning. Human chorionic somatomammotropin and the Cys(Cam)fi hGH(1-134) fragment of human growth hormone were also active, but a biologically inert oxidized human growth hormone had no growth-promoting effect in vitro. Cell proliferat was stimulated by insulin with peak potentiation occurring at 1 ng/ml, and prolactin had a demonstrable stimulatory effect between 50 and 100 ng/ml. These observations indicate that growth hormone and related polypeptides have a direct effect on the in vitro proliferation of e~ryhroleukemia cells in the absence of serum. The results confirm a direct action of growth hormone on mammalian cells and suggest that pituitary hormones may affect leukemic cell growth.
Growth hormone (somatotropin) is known to have important interactions with hematopoietic cells and certain lymphoid populations (1-4). Although there is considerable information on growth hormone binding to cell receptors, there are few studies on the physiologic effects of growth hormone on mammalian cells in vitro (5-8). We have recently reported a direct effect of growth hormone on potentiation of normal erythropoiesis in vttro (9). This effect was species specific and demonstrable with nanogram concentrations of hormone. We report here studies indicating that growth hormone stimulates murine erythroleukemia cell proliferation in a serum-free culture system. MATERIALS AND METHODS The dimethyl sulfoxide-inducible Friend leukemia cell line GM-86, clone 745, was obtained from the Institute for Medical Research, Camden, NJ, and maintained in continuous suspension culture with media containing 20% fetal calf serum. The erythroleukemia cells were grown in T-25 flasks and divided and fed with fresh medium 1 or 3 days before they were cloned in methylcellulose. The methylcellulose culture system was used as reported (9, 10), but 0.5% bovine serum albumin (Sigma Chemical Co., St. Louis, MO) was substituted for the fetal calf serum in the culture dishes. No erythropoietin or other stimulus was used. The cells were counted and directly plated in methylcellulose without washing. The cultures were incubated at 370 in a humidified atmosphere of 5% CO2 in air. Ten thousand cells were plated in each dish, and all clusters of eight The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adwrtisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
or more cells were enumerated 72 hr later, by using an inverted microscope. Highly purified human growth hormone (hGH) (11), bovine growth hormone (bGH) (12), human chorionic somatomammotropin (hCS) (13), and the plasmin-cleaved hGH fragment (14) Cys(Cam)53-hGH(1-134), were prepared as described. An oxidized and biologically inert hGH was produced by performic acid oxidation (15, 16). Ovine prolactin was prepared as reported (17). Porcine insulin was a gift of 0. Behren of Eli Lilly and Company. These hormones were dissolved in 0.1 M NaOH and diluted in phosphate-buffered saline (pH 9.3) before addition to the methylcellulose cultures. Appropriate cultures were established without hormone to control for diluent material. In selected experiments a plasma clot system was used to clone the erythroleukemia cells (18). Growth and differentiation in liquid culture was assessed by viable cell counts and differential counts after staining with benzidine (19).
RESULTS Growth hormone was found to stimulate erythroleukemic cell cloning at the high cloning efficiencies seen with 30% fetal calf serum; however, the serum-free system eliminated other serum humoral influences and allowed for a consistent demonstration of growth hormone effects at low concentration. The cloning efficiency of the erythroleukemia cells varied considerably, depending on when they were cloned in relationship to the previous feeding. The mean (+SEM) 72-hr cloning efficiency in the serum-free system was 256 + 60 colonies per 104 cells in 13 separate experiments. For convenience of comparison, the data are expressed as percentage of control value. Fig. 1 shows the effect of various hGH preparations and hCS on erythroleukemic cell colony formation. A prominent stimulatory effect of hGH was observable at concentrations of 10 ng/ml. Fig. 2 shows that the effect of as little as 0.1 ng of hGH per ml was discernible in this system (P < 0.05). Peak stimulation, 224% of control, occurred at 200 ng/ml, and 500 ng/ml caused greatly reduced potentiation of cloning. The dilution controls varied by no more than 1%. hCS and the hGH fragment were also active stimulators of erythroleukemic cell cloning but maximal stimulating concentrations (200 ng/ml) had approximately half the activity of hGH (Fig. 1). Oxidized hGH was inactive in this system and did not interfere with the response to hGH in mixing studies. bGH also stimulated erythroleukemia cell cloning but to a lesser degee than hGH: at peak stimulatory concentrations of bGH the maximal stimulation was 155% of control (data not shown). Insulin was found to potentiate cloning of the erythroleukemia cells in serum-free medium. A clear effect was discernible at 0.5 ng/ml, and peak stimulation was seen at 1 ng/ml (Fig. Abbreviations: bGH, bovine growth hormone; hCS, human chorionic
somatomammotropin; hGH, human growth hormone. 3437
Medical Sciences: Golde et al.
3438
Proc. Natl. Acad. Sci. USA 75 (1978) Table 1. Combination of various dosages of hGH with insulin (1 Ng) on erythroleukemia cloning
300 C
i0
hGH, ng/ml 0.1 0.5 1.0 5.0 10.0 100.0 * Mean + SEM.
0
is,0 200_ 0
I
x
- -----.
Q
._
% of control*
158 180 211 ± 173 i 167 150 ±
9 12 22 28 12 15
%
additivity 108 106 110 81 67 45
0
10
25
50
100 Hormone added, ng/ml
200
I, soo
FIG. 1. Effect of hGH preparations and hCS oin colony formation by erythroleukemia cells in methylcellulose. Thie mean (±SEM) cloning efficiency in these studies was 256 + 60 co]lonies per 104 cells in 13 separate experiments. The data are expressed percentage of control in three to seven experimt nts performed in duplicate. 0, hGH; *, hCS; 0, hGH fragment Cyrs(Cam)53-hGH(1134); &, performic acid-oxidized hGH.
2). Higher concentrations of insulin were less effective in promoting colony formation. Table 1 shows that the effects of insulin and hGH were additive at low concentrations but inhibition was observed with hGH concentration!s above 5 ng/ml Prolactin stimulated erythroleukemia cell proliferation at concentrations above 25 ng/ml and peak act ivity was seen at 100 ng/ml (Fig. 2). The effect of hGH on erythroleukemia cel 1 growth was observable in plasma clot culture and in liquid Esuspension. hGH at 1 ng/ml in serum-free liquid culture result ed in an increase in the viable cell count to 148% of control at '7 days. Although hGH stimulated cell proliferation in liquid clulture, it did not influence cell differentiation as measured biy the number of benzidine-positive cells seen in culture with a nd without 1.5% dimethyl sulfoxide.
DISCUSSION These observations indicate that malignant Friend virus-infected erythroleukernia cells retain the respons iveness to growth hormone characteristic of the normal comrnitted erythroid progenitor (9). Stimulation of cell proliferEation by growth hormone is observable as a prominent incre: ase in cloning in
-
0
260
° 220
100. 0.5 1.0 5 10 Hormone, ng/ml
hormone fragment (14) in this system roughly approximated
their growth-promoting activity in that stimulation of erythroleukemia cell colony formation was substantially less than seen with hGH. The biologically inactive oxidized hGH had no effect in vitro and did not interfere with the action of hGH, suggesting that the oxidized hormone did not block activation of a receptor mechanism. Unlike normal erythroid precursors (9), the erythroleukemia cells were stimulated by high concentrations of prolactin. Prolactin was effective in potentiating cell growth only at concentrations approximately 2 orders of magnitude greater than effective levels of hGH. This observation may relate to the high degree of molecular homology between prolactin and hGH. Insulin potentiated cell growth at physiologic concentrations below those known to be effective in other cell systems (20-23). The effects of these hormones were observable in other clonogenic systems (plasma clot culture) as well as in liquid suspension culture, indicating that the findings were not peculiar to methylcellulose culture. The relationship of pituitary hormones to normal hematopoiesis has been emphasized by other investigators (24). Growth hormone has also been shown to exhibit effects on hematopoietic malignancies, and hypophysectomy was reported to cause regression of certain leukemias in rats (25, 26). Results reported herein demonstrate that nanogram concentrations of hGH and related polypeptides directly stimulate in vitro erythroleukemia cell proliferation. The findings confirm a direct action of growth hormone on cells and suggest that pituitary hormones may affect leukemic cell growth. The system described offers a means to study the effects of growth hormones on mammalian cells in a serum-free system. This work was supported in part by U.S. Public Health Service Grants CA 15688 and CA 15619 (to D.W.G.) and AM-6097 and AM18677 (to C.H.L.).
180/ C
2 140
0
vitro. The absence of serum in this system obviated considerations of an indirect action of growth hormone such as mediation of serum somatomedin-like polypeptide binding to cells (20). The clear dose-response to growth hormone and the nanogram range of effective concentrations correlate well with the previously reported range for receptor binding studies using lymphoid cell lines (6). The effects of hCS (13) and the growth
5so
100
500 0o0o
FIG. 2. Effect of hGH (@), insulin (A), and1 prolactin (0) on erythroleukemia cell colony formation in vitro. Ain effect of hGH is noted at a concentration of 0.1 ng/ml. Results are expressed as mean (±SEM) percentage of control for five to seven [experiments performed in duplicate.
1. Fruhman, G. J., Gerstner, R. & Gordon A. S. (1954) Proc. Soc. Exp. Biol. Med. 85,93-96. 2. Peschle, C., Marone, G., Genovese, A., Sacchetti, L. & Condorelli, M. (1975) in Erythropoless. Proceedings of the 4th International Conference on Erythropoiesis, eds. Nakao, K., Fisher, J. W. & Takaku, F. (University of Tokyo Press, Tokyo), pp. 99-117. 3. Pandian, M. R. & Talwar, G. P. (1971) J. Exp. Med. 134,
1095-1113.
4. Arrenbrecht, S. (1974) Nature 252,255-257. 5. Lesniak, M. A., Roth, J., Gorden, P. & Gavin, J. R., III (1973) Nature New Biol. 421, 20-22.
Proc. Nati. Acad. Sci. USA 75 (1978)
Medical Sciences: Golde et al. 6. Lesniak, M. A. & Gorden, P. (1976) in Hormone-ReceptOr Inxi1teraction. Molecular Aspects. Modern ogy, ed. Levey, G. S. (Dekker, New York), Vol. 9, pp. 201219. 7. Ramachandran, J., Lee, V. & Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48,274-279. 8. Desai, L. S., Lazarus, H., Li, C. H. & Foley, G. E. (1973) Exp. Cell Res. 81, 330-332. 9. Golde, D. W., Bersch, N. & Li, C. H. (1977) Science 196, 1112-1113. 10. Golde, D. W., Bersch, N. & Cline, M. J. (1976) J. Clin. Invest. 57,
Phama~4logy-i
11. 12. 13. 14.
15.
57-62. Li, C. H., Liu, W.-K. & Dixon, J. S. (1962) Arch. Biochem. Biophys., Suppl. 1, 327-32. Li, C.H. (1954) J. Biol. Chem. 211,555-558. Li, C. H. (1970) Ann. Sclavo 12,651-662. Li, C. H. & Graf, L. (1974) Proc. Natl. Acad. Sci. USA 71, 1197-1201. Bewley, T. A., Brovetto-Cruz, J. & Li, C. H. (1969) Biochemistry 8,4701-4708.
3439
Fonss-Bech, P. & Schmidt, K. D. (1969) Int. J. Protein Research - &-15-92. 17. Li, C. H., Dixon, J. S., Lo, T. B., Schmidt, K. D. & Pankov, Y. A. (1970) Arch. Biochem. Bwophys. 141, 705-737. 18. Stephenson, J. R., Axelrad, A. A., McLeod, D. L. & Shreeve, M. M. (1971) Proc. NatI. Acad. Sci. USA 68, 1542-1546. 19. Friend, C., Scher, W., Holland, J. G., Sato, T. (1971) Proc. NatI. Acad. Sd. USA 68,378-382. 20. Moses, A. C., Nissley, S. P., Cohen, K. L. & Rechler, M. M. (1976) 16.
Nature 263, 137-140. 21. Gospodarowicz, D. & Moran, J. S. (1974) Proc. Natl. Acad. Sci. USA 71, 4584-4588. 22. Gospodarowicz, D. & Moran, J. S. (1976) Annu. Rev. Biochem. 44,531-558. 23. Hayashi, I. & Sato, G. H. (1976) Nature 259, 132-134. 24. Reddi, A. H. & Huggins, C. B. (1976) Nature 263,514-515. 25. Huggins, C. & Oka, H. (1972) Cancer Res. 32,239-242. 26. Bentley, H. P., Hughes, E. R. & Peterson, R. D. A. (1974) Nature 252,747-748.
