0013-7227/84/1144-1352$02.00/0 Endocrinology Copyright © 1984 by The Endocrine Society

Vol. 114, No. 4 Printed in U.S.A.

New Powerful Insulin-Like Protein from Human Promyelocytic Leukemia Cells TOSHIKAZU YAMANOUCHI, YASUO AKANUMA, TOSHIO TSUSHIMA, KAZUO SHIZUME, HIDEAKI MIZOGUCHI, AND FUMIMARO TAKAKU The Third Department of Internal Medicine, University of Tokyo, Hongo, Tokyo 113, Japan; and Department of Internal Medicine, Tokyo Women's Medical College, The Institute for Growth Science, Kawadacho-10, Ichigaya, Shinjuku, Tokyo 162, Japan

ABSTRACT. Potent insulin-like activity was found in the conditioned medium of human promyelocytic leukemia (HL-60) cells. The conditioned medium of HL-60 cells at high density stimulated [3H] glucose incorporation into lipids in rat adipocytes in a time- and dose-dependent manner. The dose-response curve for this factor was not parallel to that for insulin, and the maximal effect achieved was much greater than that reached by insulin or multiplication-stimulating activity. Moreover, the maximal effect reached by either insulin or the conditioned medium was additive. The insulin-like activity was not suppressed in the presence of antiinsulin antibody. Insulin-like

I

T HAS BEEN recognized that human serum contains several peptides which show insulin-like biological activity in vitro as well as in vivo. In 1963 Froesch et al. (1) first reported on "nonsuppressible insulin-like activity (NSILA)" in human serum. Thereafter, NSILA-s was distinguished from NSILA-p based on its solubility in acid-ethanol (2). More recently, NSILA-s was found to consist of two distinct insulin-like growth factors (IGFI and II) (3). Insulin-like peptides have also been isolated from the conditioned media of a number of cultured cells. Multiplication-stimulating activity (MSA), a family of polypeptides synthesized from a rat liver cell line (BRA3A) in culture, resembles IGFs or insulin (4). MSA-like peptides have been isolated from the conditioned medium of human fibrosarcoma cells as well (5). The functional significance of these insulin-like substances is not yet fully understood. However, these factors might be involved in regulating cell function. HL-60 is a cell line established by Collins et al. (6) from leucocytes of a patient with acute promyelocytic leukemia, and can be induced to undergo terminal maturation by a number of chemical agents (7-14). The Received January 31,1983. All correspondence and requests for reprints should be addressed to: Toshikazu Yamanouchi, Third Department of Internal Medicine, University of Tokyo, Hongo, Tokyo 113, Japan.

activity was not detectable by radioreceptor assay for insulin, suggesting that the factor does not act through the insulin receptor. The factor in the conditioned medium of HL-60 cells was heat stable and sensitive to trypsin. When the conditioned medium was subjected to gel filtration on a Sephadex G-100 column, the major part of insulin-like activity eluted in the position corresponding to an apparent molecular weight between RNAase and insulin markers. The remaining activity, approximately 10% of the total, appeared with a larger molecular weight species. On isoelectric focusing of the smaller molecular species, insulin-like activity was largely focused in the position corresponding to pi 7.8-8.2. (Endocrinology 114: 1352, 1984)

present authors and others have reported on the presence of specific binding sites for insulin on HL-60 cells (15, 16). Recently, we found that HL-60 cells in culture release a potent insulin-like substance in the medium, and in the present report we describe the characteristics of this novel factor.

Materials and Methods Hormones and chemicals Monocomponent porcine insulin was a product of Ely Lilly (Indianapolis, IN). MSA was purchased from Collaborative Research Inc. (lot. 83-226, Lexington, MA). Somatomedin C was purified from human plasma by the method of Svoboda et al. (17). [125IlIodoinsulin and [125I]iodosomatomedin C were prepared by the Chloramine-T method modified as reported (18) to a SA of 100 mCi/mg. [D-3-3HlGlucose (SA, 11.5 Ci/ mmol) was obtained from New England Nuclear (Boston, MA). Antiporcine insulin antibody was prepared from guinea pigs in our laboratory. Antisomatomedin C antibody was obtained from National Institutes of Health (Bethesda, MD). Cells and cell culture HL-60 and human myeloid leukemia cells (K-562) were provided by Dr. S. Sato, the National Cancer Research Institute (Tokyo, Japan). Mouse myeloma cells (X63-Ag8653) and well differentiated hepatoma cells (Fao) derived from the Reuber 1352

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NEW INSULIN-LIKE PROTEIN H-35 hepatoma were provided by Dr. M. Kasuga, Tokyo University (Tokyo, Japan). Human malignant B lymphoma cells (Raji) derived from Burkitt, and human T-cell leukemia cells (MOLT 4) were provided by Dr. H. Mizoguchi, Tokyo Women's Medical College (Tokyo, Japan). Cells were cultured at 37 C in RPMI-1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum, (FCS) (Boehringer, Mannheim, W. Germany, lot 674603) in a humidified atmosphere of 5% CO2 in air. Unless otherwise noted, HL-60 cells were plated at a density of 105/ml in 75 cm2 culture flasks and allowed to grow to a density of 106/ml, over a period of 4-5 days. The cells were pelleted by centrifugation (3000 rpm for 30 min) and the medium was collected and then concentrated by ultrafiltration with Amicon UM-05 membrane (nominal exclusion 5000 daltons), filtered through a 0.2-/x pore size membrane, and stored at 4 C. In some experiments, insulin-like cells grown to 106/ml were collected by centrifugation, washed twice with serum-free RPMI-1640 medium, and resuspended in serum-free KrebsRinger Bicarbonate (KRB) buffer with glucose (100 mg/ml). The cells were cultured at 37 C in a CO2 incubator for 24 h. The medium was collected, concentrated, and stored as described. Bioassay for insulin-like activity Epididymal adipocytes were prepared from the fat pads of 150-g male Wistar rats, as described by Gliemann (19). Isolated fat cells were washed twice with KRB buffer, pH 7.2, containing 0.1% BSA and 10 mg/dl glucose, and suspended in the buffer. The lipid content per milliliter of the suspension was determined as described (19). One-milliliter aliquots of the suspension were added to 20-ml scintillation vials containing 0.5 fxCi [3H]glucose in 20 fi\ KRB buffer and insulin standard solution or conditioned medium. The mixture was incubated at 37 C for 2 h. The reaction was terminated by adding 15 ml toluene-base scintillator according to the method of Moody et al. (20), and the radioactivity incorporated into lipids was determined.

Effect of heating The conditioned medium was heated to 60 C or 100 C for 1, 5, and 30 min. The medium was then dialyzed at 4 C against 50 vol PBS and assayed for insulin-like activity. Trypsin treatment One milliliter of the conditioned medium of HL-60 cells was incubated with 100 Mg/ml trypsin for 2 h at 37 C. The reaction was terminated by adding 1 mg soybean trypsin inhibitor. Three controls were used: 1) the same amount of trypsin inhibitor was added before trypsin in the medium; 2) standard medium with trypsin incubated before the addition of the soybean trypsin inhibitor; and 3) standard medium with trypsin and soybean trypsin inhibitor followed by incubation, were used. All three control mixtures incubated as cited above (for 2 h at 37 C).

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Antiinsulin antibody The conditioned medium was treated with several dilutions of guinea pig antiinsulin serum for 3 h at 37 C and then assayed for insulin-like activity in rat adipocytes. Gel chromatography The conditioned medium was subjected to gel chromatography on a Sephadex G-100 column (1.3 X 60 cm), equilibrated with sodium phosphate buffer, pH 7.4, containing 0.1 M NaCl. The column had been calibrated using BSA (mol wt 67 K), ovalbumin (43 K), ribonuclease A (RNase) (13.7 K), and porcine insulin (6 K). One-milliliter fractions were collected and assayed for biological activity and absorbance at 280 nm. Isoelectric focusing Active fractions from the Sephadex G-100 column were pooled and subjected to isoelectric focusing between pH 3.5 and pH 10.0 using preparative flat bed gel with LKB 2117 Multiphor (LKB, Bromma, Sweden). The gel consisted of 4 g Ultradex (LKB) in 10% (vol/vol) glycerol-distilled water plus Ampholine. Focusing was carried out at 4 C for 2 h at 30 W constant power. At the end of electrophoresis, the gel bed was sectioned with the aid of a fractioning grid, and each gel was transferred to a small column. The pH of each section was measured with a LKB 2117-111 Multiphor Surface pH electrode. The column was eluted with 1 M acetic acid, and the eluate was extensively dialyzed against water, reconstituted in KRB buffer, and assayed for biological activity. Radioreceptor assay (RRA) and RIA RRA for insulin was performed using a human placental membrane or rat adipocytes as the matrix. RRA was performed according to the method of Gambhir et al. (21). RIA for insulin was performed after the method of Yalow et al. (22). RIA for somatomedin C/IGF-I was performed by the method of Furlanetto et al. (23).

Results Insulin-like activity of the conditioned medium Insulin-like activity of the conditioned medium of HL60 cells grown to a density of 106/ml was compared to that of insulin (Table 1). As reported previously by Moody et al. (20), insulin stimulated [3H] glucose incorporation into lipids of rat adipocytes in a dose-related manner, and the maximal effect was obtained at a concentration of 10 ng/ml. That the conditioned medium contains a potent insulin-like activity can be seen (Table 1). At a concentration as low as 1 ng protein/ml, the partially purified preparation showed a significant activity. The maximal response was obtained at 20 ng/ml, and was approximately 2-fold greater than that obtained by 10 ng/ml insulin. The effect of 20 ng/ml of the crude preparation was additive to that produced by either 0.2

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NEW INSULIN-LIKE PROTEIN

TABLE 1. Bioactivityofthe substance derived from HL-60 conditioned medium Cone. (ng/ml) 0.2 10

Insulin Control medium (with 10% FCS) FCS MSA Protein preparation"

Protein preparation 20 + insulin

500 0.2 1 2 10 20 0.2 10

Added [3H]glucose uptake (cpm) 1086 ± 2302 ± 0 2166 ± 1724 ± 564 ± 1062 ± 2767 ± 5387 ± 5531 ± 6546 ± 8620 ±

102 301 405 440 86 212 230 382 396 426 760

Results are expressed as the mean ± SEM of the data derived from three separate experiments performed in triplicate. ° Protein preparation was partially purified by dialysis and Sephadex G-100 chromatography from HL-60 conditioned medium. TABLE 2. Comparison of the conditioned medium derived from several cell lines in the bioactivity Added [3H] glucose uptake (cpm) Control medium (without FCS) Control medium (with 10% FCS) HL-60 K-562 Raji MOLT 4 X63-Ag 8.6.5.3 Fao

0 248 ± 5242 ± 4681 ± 438 ± 276 ± 408 ± 333 ±

26 403 802 122 106 162 97

Cells are cultured for 24 h at a saturating density. Each conditioned medium contained 10% FCS, and 20 /*1 of the sample was used for bioassay. Results are expressed as the mean ± SEM of the data derived from three separate experiments performed in triplicate.

Endo • 1984 Volll4«No4

Stability of the HL-60 derived insulin-like activity The insulin-like activity of HL-60 conditioned medium was heat stable. Heating at 60 C for 30 min resulted in only a 10% decrease of the activity (Table 3). The insulin-like activity was sensitive to trypsin treatment, suggesting that the factor may be a protein. Effect of antiinsulin antibody The conditioned medium of HL-60 cells was mixed with several dilutions of antiinsulin antiserum, incubated for 3 h at 37 C, and assayed for insulin-like activity (Fig. 1). As expected, [3H]glucose incorporation by insulin was inhibited dose dependently by antiinsulin antibody, whereas the insulin-like activity was not affected in the presence of antiinsulin antibody. Time dependency of insulin-like activity Figure 2 illustrates a time course in [3H] glucose incorporation in rat adipocytes stimulated by either insulin or conditioned medium of HL-60 cells. In both cases, the [3H]glucose uptake reached the maximal level within 2 h. Gel chromatography The conditioned medium was chromatographed on a Sephadex G-100 column. As shown in Fig. 3, insulin-like activity appeared in two positions. A major part of the activity eluted in the position corresponding to an apparent molecular weight between the RNase (13.7 K) and insulin (6 K) markers. Insulin-like activity in this fraction was not detected by RRA for insulin, using either human placental membranes or rat adipocytes. Isoelectric focusing

or 10.0 ng/ml insulin. Control medium (RPMI-1640 supplemented with 10% FCS) showed some activity. FCS alone was also able to stimulate [3H]glucose uptake. However the maximal response by FCS never exceeded that reached by insulin (10 ng/ml) or MSA (500 ng/ml). In order to exclude the possibility that insulin-like activity of the conditioned medium of HL-60 is derived from FCS, HL-60 cells were cultured in serum-free RPMI1640 for 48 h. The serum-free, conditioned medium also showed a potent activity. We have examined insulin-like activity in the conditioned medium of other cell lines (Table 2). The conditioned medium of X63-Ag8653 and Fao did not contain any insulin-like activity. Interestingly, in other human leukemic cell lines, the medium of Raji and MOLT 4 did not reveal the insulin-like activity, whereas the medium of K-562 stimulated [3H]glucose incorporation to the same extent as that of HL-60.

The active fractions (smaller molecular species) that eluted on the Sephadex G-100 column were pooled, conTABLE 3. Bioactivityofthe substance derived from HL-60 conditioned medium after various treatments Treatment None Heat 60 C 30 min 100 C 1 min 100 C 5 min Dialysis, 8000 mol wt exclusion Trypsin, 100 /ig/ml, 2 h, 37 C°

Added [3H]glucose uptake (cpm) 5621 ± 418 4498 ± 4829 ± 3380 ± 3341 ± 1209 ±

296 341 332 129 236

Results are expressed as the mean ± SEM of the data derived from three separate experiments performed in triplicate. 0 Three control studies revealed that trypsin and trypsin inhibitor did not affect the bioassay.

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NEW INSULIN-LIKE PROTEIN

1 250

FIG. 1. Effect of guinea pig insulin antiserum on bioactivity of the insulin-like substance derived from HL-60 conditioned medium. All data are expressed as a percentage of the increment of radioactivity above basal radioactivity per 5 mg lipid. Basal radioactivity is regarded as 100%. The values are the mean ± SE (n = 5). The presence of antiinsulin antibody at indicated dilutions significantly (P < 0.01; by Student's t test) suppressed the [3H] glucose incorporation by insulin, but failed to inhibit the activity of medium.

1

1355

1 i

i T

200

150

100

(-)

1:8000

1:4000

h

1:2000

1.1000

1:500

Anti insulin antibody (at each dilution) added

60

120

180

240

Time(min)

FIG. 2. Time course of the bioactivity on the [3H]glucose uptake by insulin (O) or by HL-60 conditioned medium (•). All data are expressed as a percentage of increased radioactivity per 5 mg lipid. Each point and bar represent the mean ± SE of triplicate determinations. The values obtained by HL-60 medium at indicated times were significantly (P < 0.01) higher compared to those by insulin.

centrated to 1 ju.g protein/ml, and subjected to isoelectric focusing (Fig. 4). Insulin-like activity was focused in the position between pH 7.6 and 8.2.

Discussion We have shown that the conditioned medium of HL60 cells contains a factor or factors with potent insulin like activity. Previously, Temin et al. (24) isolated a

family of peptides with insulin-like activity from calf serum. Consistent with their report, FCS was able to stimulate incorporation of [3H]glucose into rat adipocytes. Insulin-like activity in the conditioned medium of HL-60 cells is, however, not due to the presence of FCS in the medium. Nonconditioned medium supplemented with 10% FCS showed only slight activity. Furthermore, the maximal effect obtained by the addition of FCS was much less compared to that produced by the conditioned medium. More directly, the serum-free conditioned medium showed a significant insulin-like activity. The data demonstrate that the insulin-like factor(s) is derived from HL-60 cells. However, it is clear that the insulinlike activity is not insulin. The activity is not suppressed by antiinsulin antibody, and cannot be detected by RRA or RIA for insulin. The maximal effect (Emax) obtained by insulin never reached that of the conditioned medium. Since either agonist can be additive in its biological activity, it is implied that the insulin-like factors stimulate [3H] glucose uptake by a mechanism different from that of insulin. The dose-response curve for the conditioned medium was also divergent from that of MSA, which is very similar to human IGF-II in its structure and function. RIA for somatomedin C/IGF-I failed to detect any substances reactive to antisomatomedin C antibody. Both IGF-I and II have been reported to have insulin-like activity in a variety of bioassays (25, 26), but these peptides are only 1:100-1:50 as potent as insulin on a molar basis. The maximal effect obtained by either IGF-I or II is comparable to that by insulin. These observations strongly suggest that the insulin-like factors produced by HL-60 cells are not identical to human IGFI or II.

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Endo • 1984 Volll4«No4

60 Vo

BSA(67,000)

RNase( 13,700) j

50 Ovalbumin(43,000)

in

40

-0.1

30

0.05

20

10

F r a c t i o n number

FIG. 3. Sephadex G-100 column chromatograms of HL-60 conditioned medium. One and a half milliliter of serum-free conditioned medium of HL-60 cells were applied to a Sephadex G-100 column (1.3 x 60 cm) as described in the text. One milliliter each of the eluates was collected and assayed for absorbancy at 280 nm (O O). Twenty microliters of each eluate were assayed for bioactivity ( • • ) . The values are the mean of triplicate determinations. The variation of the values was within 5% of the mean.

300

200

Q.

100

0

1

2

3

4

5

6

7

Distance from top of gel (cm) FIG. 4. Isoelectric focusing of HL-60 conditioned medium. Fractions showing maximal bioactivity in Sephadex G-100 chromatography were pooled and subjected to isoelectric focusing. The gel was fractionated into 30 zones, eluated by 0.5 ml distilled water, and 20 /xl of each eluate were assayed for bioactivity. The pH ( ) of each eluate was determined. The values are the mean of triplicate determinations. The variation of the values was within 10% of the mean.

HL-60 derived insulin-like activity was sensitive to trypsin, suggesting that the factor is a polypeptide. On Sephadex G-100 chromatography, insulin-like activity eluted in two peaks. The major activity appeared in the position corresponding to an apparent molecular weight

between RNase and insulin markers, and minor activity between ovalbumin and BSA. The relationship between the two species has not yet been determined. Isoelectrofocusing experiments also revealed a heterogeneity of the insulin-like factor. The major activity was focused between pH 7.2 and 8.2, but a small peak of activity was detected at the position corresponding to pi 4.8-5.0. Thus, the conditioned medium of HL-60 cells may have multiple insulin-like substances. We noticed that the conditioned medium of another cell line of human leukemic cells, K-562, contain a potent insulin-like activity comparable to that produced by HL-60 cells. It remains to be determined whether the insulin-like factors found in the conditioned medium of the two cell lines are identical. Recently, a number of insulin-like or somatomedinlike substances have been identified in the conditioned media of cultured cells. MSA has been purified from the conditioned medium of a rat hepatocyte cell line (BRL3A) (4). De Larco and Todaro (5) reported isolation and characterization of MSA-like peptides produced by a human sarcoma cell line 8387. The functional significance of these factors is not yet fully understood. They might be involved in regulating cell function. It has been reported that some human cell lines require factors produced by themselves for growth or maturation (27, 28). Recently, Brennan et al. (27) reported on the presence of autostimulative growth factors in the conditioned medium of HL-60 cells. Growth-stimulating activity of the growth factors was heat stable and inactivated by treatment with trypsin. Mol wt was estimated at 13,000, as based on behavior in gel chromatography. These prop-

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NEW INSULIN-LIKE PROTEIN

erties are similar to those of the insulin-like factors that we have obtained in the present study. However, further study is required to determine the relationship between the two activities.

References 1. Froesch ER, Biirgi H, Ramseier EB, Bally P, Labhart A 1963 Antibody-suppressible and nonsuppressible insulin-like activities in human serum and their physiologic significance. An insulin assay with adipose tissue of increased precision and specificity. J Clin Invest 42:1816 2. Jakob A, Hauri C, Froesch ER 1968 Nonsuppressible insulin-like activity of human serum. III. Differentiation of two distinct molecules with nonsuppressible ILA. J Clin Invest 47:2678 3. Rinderknecht E, Humbel RE 1976 Polypeptides with non-suppressible insulin-like and cell-growth promoting activities in human serum: Isolation, chemical characterization, and some biological properties of forms I and II. Proc Natl Acad Sci USA 73:2365 4. Moses AC, Nissley SP, Short PA, Rechler MM, Podskalny JM 1980 Purification and characterization of multiplication stimulating activity (MSA): insulin-like growth factors purified from rat liver cell conditioned media. Eur J Biochem 103:387 5. De Larco JE, Todaro GJ 1978 A human fibrosarcoma cell line producing multiplication stimulating activity (MSA)-related peptides. Nature 272:356 6. Collins SJ, Gallo RC, Gallagher RE 1977 Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature 270:347 7. Collins SJ, Ruscetti FW, Gallagher RE, Gallo RC 1978 Terminal differentiation of human promyelocytic leukemic cells induced by dimethylsulfoxide and other polar solvents. Proc Natl Acad Sci USA 75:2458 8. Huberman E, Callahan MF 1979 Induction of terminal differentiation in human promyelocytic leukemia cells by tumor-promoting agents. Proc Natl Acad Sci USA 76:1293 9. Rovera G, Santoli D, Damsky C 1979 Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester. Proc Natl Acad Sci USA 76:2779 10. Lotem J, Sachs L 1979 Regulation of normal differentiation in mouse and human myeloid leukemic cells by phorbol esters and the mechanism of tumor promotion. Proc Natl Acad Sci USA 76:5158 11. Honma Y, Takenaga K, Kasukabe T, Hozumi M 1980 Induction of differentiation of cultured human promyelocytic leukemia cells by retinoids. Biochem Biophys Res Commun 95:507 12. Breitman TR, Selonick SE, Collins SJ 1980 Induction of differentiation of the human promyelocytic leukemia cell-line (HL-60) by

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retinoic acid. Proc Natl Acad Sci USA 77:2936 13. Mendelsohn N, Michi J, Gilbert HS, Acs G, Christman JK 1980 L-ethionine as an inducer of differentiation in human promyelocytic leukemia cells (HL-60). Cancer Res 40:3206 14. Honma Y, Fujita Y, Okabe J, Kasukabe T, Hozumi M 1980 Induction of differentiation of human promyelocytic leukemia cell (HL-60) by arginase. Cancer Lett 10:287 15. Yamanouchi T, Tsushima T, Murakami H, Sato Y, Shizume K, Oshimi K, Mizoguchi H 1982 Differentiation of human promyelocytic leukemia cells is accompanied by an increase in insulin receptors. Biochem Biophys Res Commun 108:414 16. Abita J, Gauville C, Saal F 1982 Characterization of insulin receptors in human promyelocytic leukemia cell HL 60. Biochem Biophys Res Commun 106:574 17. Svoboda ME, Van Wyk JJ, Klapper DG, Fellows RE, Grissum FE, Schlueter RJ 1980 Purification of somatomedin-C from human plasma: Chemical and biological properties, partial sequence analysis, and relationship to other somatomedins. Biochemistry 19:790 18. Freychet P, Kahn R, Roth J, Neville JR DM 1972 Insulin Interactions with liver plasma membranes. J Biol Chem 247:3953 19. Gliemann J 1967 Assay of insulin-like activity by the isolated fat cell method. I. Factors influencing the response to crystalline insulin. Diabetologia 3:382 20. Moody AJ, Stan MA, Stan M 1974 A simple free fat cell bioassay for insulin. Horm Metab Res 6:12 21. Gambhir KK, Archer JA, Curter L 1977 Insulin radioreceptor assay for human erythrocytes. Clin Chem 23:1590 22. Yalow RS, Berson SA 1960 Immunoassay of endogenous plasma insulin in man. J Clin Invest 39:1157 23. Furlanetto RW, Underwood LE, Van Wyk JJ, D'Ercole AJ 1977 Estimation of somatomedin-C levels in normals and patients with pituitary disease by radioimmunoassay. J Clin Invest 60:648 24. Temin HM, Pierson Jr RW, Dulak NC 1972 The role of serum in the control of multiplication of avian and mammalian cells in culture. In: Cristofalo VI, Rothblat G (ed) Growth, Nutrition, and Metabolism of Cells in Culture. Academic Press, New York, p 50 25. Zapf J, Schoenle E, Froesch ER 1978 Insulin-like growth factors I and II: some biological actions and receptor binding characteristics of two purified constituents of nonsuppressible insulin-like activity of human serum. Eur J Biochem 87:285 26. Zapf J, Rinderknecht E, Humbel RE, Froesch ER 1979 Nonsuppressible insulin-like activity (NSILA) from human serum: recent accomplishments and their physiologic implications. Metabolism 27:1803 27. Brennan JK, Abboud CN, Di Persio JF, Barlow GH, Lichtman MA 1981 Autostimulation of growth by human myelogenous leukemia cells (HL-60). Blood 58:803 28. Fibach E, Peled T, Rachmilewitz EA 1982 Self-renewal and commitment to differentiation of human leukemic promyelocytic cells (HL-60). J Cell Physiol 113:152

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