Studies of the differentiation of promyelocytic cells by phorbol ester

Biochimica et Biophysica Acta, 781 (1984) 239-246

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Elsevier BBA 91325

S T U D I E S OF T H E D I F F E R E N T I A T I O N O F P R O M Y E L O C Y T I C CELLS BY P H O R B O L E S T E R I. I N D U C T I O N OF D I S C R E T E M E M B R A N E P R O T E I N S C H A R A C T E R I S T I C OF M O N O C Y T E S AND E X P R E S S I O N O F M O T I L I T Y F U N C T I O N S IN HL-60 C E L L S F O L L O W I N G D I F F E R E N T I A T I O N BY P H O R B O L E S T E R

N1LI FEUERSTEIN * and HERBERT L. COOPER The Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205 (u.s.A,)

(Received August 1st, 1983) (Revised manuscript received December 6th, 1983)

Key words: Cell differentiation," Phorbol ester; Membrane protein; Protein synthesis induction," Promyelocytic cell," (Human myeloid cell)

Cells of the promyelocytic leukemia line, HL-60, differentiate into macrophage-like cells in response to phorbol ester. We report that this effect is associated with induction of spontaneous motility and a modest expression of chemotactic responsiveness to a formylated peptide. Using two-dimensional gel electrophoresis we demonstrate that the differentiation of HL-60 cells is associated with a differential effect on membrane vs. cytosol proteins. Changes in the cytosol characteristically involved reduction of synthesis of specific proteins. Changes in the membrane were mainly associated with either enhancement or de novo synthesis of discrete proteins. The hallmark of HL-60 cell differentiation appeared to be distinctive induction of a membrane-bound group of six proteins ( p l - - 5 , 4 5 - 7 0 kDa) and the de novo induction of a membrane protein indicated as ml0 (pl---7, 28 kDa). These discrete membrane proteins were found to be very prominently synthesized by human cultured monocytes, but virtually absent in lymphocytes. It is proposed that our ability to detect synthesis of proteins characteristic of different states of cellular differentiation will permit detailed studies of the biochemical changes which constitute this process. Definition of the role of these proteins and the mechanism regulating their synthesis will help to elucidate the biochemical basis of monocyte differentiation. Introduction The promyelocytic leukemic cells, HL-60, undergo terminal monocytoid differentiation upon exposure to phorbol ester (phorbol 12-myristate13-acetate) [1-4]. This process involves arrest of cell growth, induction of adherence to surfaces and acquisition of morphological, cytochemical and functional characteristics of macrophages. * To whom correspondence should be addressed at: Bldg. 10, Rm. B1-B40, National Institutes of Health, Bethesda, MD 20205, U.S.A. 0167-4781/84/$0.300 © 1984 Elsevier Science Publishers B.V.

Previous studies have demonstrated acquisition of directed migration capability (chemotaxis) by these cells following differentiation into granulocytes by dimethylsulfoxide [5,6]. In the present study we show that differentiation of HL-60 cells into macrophage-like cells by phorbol ester induces expression of motility functions. Using two-dimensional gel electrophoresis we further demonstrate that these functional changes are concomitant with discrete changes in synthesis of specific proteins. The hallmark of HL-60 cell differentiation appeared to be the distinctive induction of a membrane-bound group of six proteins ( p l --- 5, 45-70

240 kDa) and the de novo induction of a membrane protein indicated as ml0 (pI = 7, 28 kDa). These discrete membrane proteins were found to be very prominently synthesized by human cultured monocytes, but not in lymphocytes. Materials and Methods

HL-60 cell culture. The human myeloid cell line, HL-60, was established from a patient with acute promyelocytic leukemia. Characteristics of these cells have previously been reported [7]. The cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, penicillin-streptomycin (50 U / m l and 50 /xg/ml, respectively) and 2 mM glutamine (culture medium). For induction of differentiation, cells in their logarithmic growth phase were resuspended in fresh culture medium (5 • 105 ml) with or without phorbol-12-myristate-13acetate (Sigma), 10 _8 M, and incubated in petri dishes for 48 h. Lymphocyte preparation and cultivation. Human peripheral lymphocytes were prepared from fresh, heparinized venous blood of unselected normal donors as described [8]. The lymphocyte population typically obtained contained 85% T lymphocytes, 15% B lymphocytes, and < 1% monocytes [9]. Cultivation was in RPMI 1640 medium supplemented with 50 U / m l penicillin, 50 /xg/ml streptomycin, 2 mM glutamine, 10 mM Hepes pH 7.4, and 10% autologous plasma, at a density of 106 cells/ml in 5% CO 2 atmosphere at 37°C. Monocyte preparation and cultivation. Heparinized venous blood was diluted with an equal volume of RPMI 1640. In 50 ml centrifuge tubes, 35 ml of this mixture was underlaid with 15 ml of Ficoll-Isopaque solution (LSM solution, Litton Bionetics) and centrifuged at 600 x g for 20 rain at 20°C. Mononuclear ceils remaining at the interface were recovered, resuspended in RPMI, and centrifuged at 250 × g for 10 rain to remove platelets. The cell pellet was resuspended in RPMI 1640 and 10% autologous plasma at 2.106 cells/ml and incubated in plastic tissue culture flasks at 37°C for 24 h. Nonadherent cells (lymphocytes) were decanted and the adherent layer was washed three times with RPMI. The adherent cells were generally > 90% monocytes on microscopic examination. Monocytes were then cultured in RPMI 1640

supplemented with glutamine, penicillin-streptomycin and 10% autologous serum. After 7 days in culture the cells were washed and labeled in monolayer as described. The labeled adherent cells were then detached by incubation with 0.2 M EDTA for 15 min, washed and prepared for twodimensional gel analysis as described. Radioactive labeling of cellular proteins. Cells were washed with phosphate-buffered isotonic saline (0.02 M, pH 7.4) and incubated with leucine-free RPMI 1640 supplemented with 10% dialyzed fetal calf serum and 150 t~Ci/ml [3H]leucine (Amersham, 130 Ci/mmol). Control cells and lymphocytes were labeled in suspension ( ( 3 - 5 ) " 10 6 cells/ml), while phorbol ester-treated HL-60 cells and monocytes were labeled in monolayer. Following 4 h incubation in labeling medium, cells were washed with cold phosphate buffer and exposed to further procedures as described. Cell fractionation. Following radioactive labeling, the cells were resuspended in Tris lysis buffer (20 mM Tris/3 mM CAC12/0.25 M sucrose/0.2 mM phenylmethylsulfonyl fluoride, pH 7.4) and sonicated. After nuclear residues and unbroken cells were removed by centrifugation at 2200 rpm, the crude membranes were pelleted by ultracentrifugation at 30000 rpm for 60 min at 4°C. The proteins in the soluble fraction were precipitated by addition of 5 vol. ice-cold acetone/NH4OH (30 : 1.7, v/v) followed after 10 min by centrifugation at 10000 rpm for 10 min at 4°C. Residual acetone was removed by evaporation. Adequacy of this fractionation technique was monitored by the distribution of identifiable endogenous marker proteins, as detected by two-dimensional gel electrophoresis and fluorography of [3H]leucine-labeled proteins. HLA heavy-chain proteins [10] were visualized only in the crude membrane fraction, while Hy +, a unique posttranslationally modified protein [11] which predominates in the cytosol [12], was virtually absent from the membrane fraction but evident in the cytosol. Sample preparation. The proteins of whole-cell pellets or the extracted proteins from the cytosol and crude membrane preparations were dissolved in isoelectrofocusing lysing solution (9.5 M urea/2% Nonidet P-40/5% 2-mercaptoethanol/2% LKB ampholytes, pH 3.5-10). Nuclear residues

241 were removed by centrifugation and the supernatants were stored at - 8 0 ° C until resolution by two-dimensional polyacrylamide gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis. Samples containing 2 . 1 0 6 cpm of 3H were analyzed by two-dimensional polyacrylamide gel electrophoresis as described [13]. First-dimension isoelectric focusing gels contained 2% LKB ampholytes (pH 3.5-10). Second-dimension gels were 12% acrylamide. G e l s were fixed, stained, dried and prepared for fluorography as described [131. Molecular weight standards ( M r = 1 4 - 9 2 kDa) were from Bio-Rad. Fluorography was carried out with Kodak XAR-5-film at - 7 0 ° C for 7 days. Chemotaxis (migration) assay. Following washing with phosphate-buffered isotonic saline (pH 7.4), cells were suspended in G e y ' s balanced salt solution ( 2 - 5 - 1 0 6 / m l ) aliquoted on the upper level of modified (blind) Boyden chambers and incubated for 4 h at 37°C. Thereafter the filters (nucleopore, 5 /~m) were removed, fixed, stained (Diff Quik), and applied on slides. The cells on the upper side of the filters were wiped off and the cells which migrated through the pores to the bottom side of the filters were counted. For assay of chemotaxis, fMLP was added to the lower chamber. Spontaneous migration was assayed

without additions to the lower chamber.

Monitoring radioactivity in specific proteins. Proteins were located in the gels by fluorography, excised, and incubated in 1 ml of 30% H202 at 75°C for 20 h (or until completely dissolved). Thereafter, Aquasol scintillation fluid (New England Nuclear, MA) was added and the samples were counted in liquid scintillation spectrometer. Pieces of gels from areas where radioactivity was not detected by fluorography were used as blanks. Results

Expression of spontaneous motifity in HL-60 ceils after differentiation by phorbol esters. HL-60 cells were suspended in fresh culture media with or without phorbol ester (10 -8 M) for 48 h. As previously described [1-3], we found that this treatment caused inhibition of cell growth and adherence of the cells to the plastic surface. The few cells which remained in suspension were generally dead. Following 48 h of phorbol ester treatment, cells were washed and motile function was examined by determining the ability of the cells to migrate through the pores of membrane filters in modified Boyden chambers. After 4 h incubation at 37°C, the filters were removed and stained. The cells on the upper side of the filters were then wiped off so

Fig. 1. Expression of spontaneous motility in HL-60 cells after differentiation induced by phorbol ester. Motility of control cells and cells treated with phorbol ester for 48 h was examined in modified (blind) Boyden chambers using 5~ nucleopore membrane filters. Following 4 h incubation at 37°C, the membrane filters were fixed and stained, and the cells on the upper side of the filters were wiped off so that the only cells visualized are those that migrated through the pores to the bottom side of the filters. Photographs show representative filters of control cells (left panel) and phorbol ester-differentiated cells (right panel). Small circles in left panel are membrane pores. Dark-staining material in right panel represents cells, singly and in clusters, migrating through pores.

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Fig. 2. Induction of discrete membrane proteins following differentiation of HL-60 cells by phorbol ester. Control or phorbol ester-differentiated cells were labeled with [3H]leucine for 4 h and then lysed, sonicated, and sedimented at 30000 rpm (60 min at 4°C). The particulate fractions (membrane preparations) were dissolved in lysis solution and analyzed by two-dimensional gel electrophoresis. Left panel: control cells. Right panel: differentiated cells. Arrows and numbers indicate proteins that undergo changes upon phorbol ester treatment. Circles indicate the expected location of these proteins in control cells.

that the only cells visualized were those that m i g r a t e d t h r o u g h the pores of the filters. Photog r a p h s of r e p r e s e n t a t i v e m e m b r a n e filters of these e x p e r i m e n t s are shown in Fig, 1. As d e m o n s t r a t e d in the left panel, we c o u l d never detect any migration of u n d i f f e r e n t i a t e d H L - 6 0 cells (very few cells were d e t e c t e d at the b o t t o m chamber). In contrast, the d i f f e r e n t i a t e d cells expressed a high rate of m o t i l i t y as i n d i c a t e d b y the a b u n d a n c e of cells that m i g r a t e d t h r o u g h the filter (right panel).

Examination of chemotactic responsiveness to the formylated peptide (fMLP) following differentiation by phorbol ester. F u r t h e r e x p e r i m e n t s were u n d e r t a k e n in o r d e r to e x a m i n e the p o s s i b i l i t y that in a d d i t i o n to s p o n t a n e o u s motility, H L - 6 0 also acquire c h e m o t a c t i c responsiveness after differe n t i a t i o n b y p h o r b o l ester. In these e x p e r i m e n t s the d i f f e r e n t i a t e d cells (after 48 h t r e a t m e n t with p h o r b o l ester) were w a s h e d a n d further i n c u b a t e d with fresh culture m e d i u m without p h o r b o l ester for 24 h. These p r o c e d u r e s were designed to e l i m i n a t e the presence of p h o r b o l ester a n d p e r m i t the cells to achieve a resting c o n d i t i o n before e x p o s u r e to the c h e m o a t t r a c t a n t . The results of these e x p e r i m e n t s showed that in the absence of

p h o r b o l ester the d i f f e r e n t i a t e d cells c o n t i n u e d to express a high level of s p o n t a n e o u s m i g r a t i o n as i n d i c a t e d b y the n u m b e r of cells that migrated t h r o u g h the filters (1573 + 19.83 per mm2). The c h e m o a t t r a c t a n t f M L P (10 - s M) caused a 30% increase in cell m i g r a t i o n ( P < 0.001), suggesting that, in a d i t i o n to i n d u c t i o n of s p o n t a n e o u s motility, a m o d e r a t e c h e m o t a c t i c response m a y be assoc i a t e d with the d i f f e r e n t i a t i o n of HL-60 cells into macrophages. TABLE I MEASUREMENT OF RADIOACTIVITY IN THE DIFFERENTIATION MARKER PROTEINS ml-m5 AND ml0 BEFORE AND AFTER DIFFERENTIATION Proteins were located by fluorography and excised from the gels. The gel pieces were dissolved and counted as described. Results represent cpm in specific proteins.

ml m2 m3 m4 ml0

Control

Phorbol ester

Phorbo ester/control

2129 1557 3186 1160 567

32650 14400 9490 13750 7253

15.3 9.2 2.9 11.9 12.8

243

Induction of discrete membrane proteins is associated with differentiation of HL-60 cells by phorbol ester. HL-60 cells were induced to differentiate by phorbol ester as described. After 48 h control or phorbol ester-treated cells were washed and radiolabeled as in Methods. The cells were then sonicated and fractionated as described. Membrane proteins of control and differentiated cells were resolved on two-dimensional gel electrophoresis (Fig. 2). The most prominent changes consistently associated with differentiation of HL-60 cells are indicated on Fig. 2 by arrows. We would like especially to note two distinctive features of HL-60 differentiation: the prominent induction of a group of membrane proteins, m l - m 6 , which are closely related in their isoelectric point (approx. 4.8-5), but range in molecular weights from 45 to 70 kDa, and furthermore the de novo appearance of a more basic, membrane protein, ml0, at 28 kDa and p I = 7.0. Direct measurement of radioactivity in several of these specific proteins showed that there was an average of 10.4 + 2.4-fold increase in the radioactivity in these specific proteins following differentiation (Table I).

Reduced synthesis of discrete cytosol proteins following differentiation of HL-60 cells. The effect of H+

differentiation by phorbol ester on cytosol proteins is presented in Fig. 3. Several changes which were found to be prominent and reproducible are indicated. Notably, whereas the changes observed in the membrane following phorbol ester treatment (Fig. 2) typically involved induction of proteins, many of the specific changes which occur in the cytosol apparently involve reduction of protein synthesis. Protein ml0, which was exhibited in the membrane (Fig. 2, right panel), was also found, although less abundantly, in the cytosol of phorbol ester-treated cells (Fig. 3, right panel). This protein is not at all detected in the cytosol of control cells suggesting that de novo synthesis of this protein is correlated with differentiation of HL-60 cells.

Identification of membrane proteins induced during differentiation as characteristic proteins of human peripheral monocytes but not of lymphocytes. In order to analyze which of the changes detected in Figs. 2 and 3 are most relevant to induction of monocyte-specific functions, we compared the pattern of protein expression before and after differentiation to the pattern of whole cell protein expression in either peripheral adherent cells (monocytes) or peripheral nonadherent cells (lymphocytes). Monocytes and lymphocytes were OH-

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Fig. 3. Effectof phorbol ester-induceddifferentiationon synthesisof cytosolproteins. Control or differentiatedcells were labeled with [3H]leucine for 4 h and then lysed, sonicated, and exposed to fractionation procedures as described. The cytosol proteins were precipitated by acetone:NH4OH (30:1.7), dissolved in lysis solution, and analyzed by two-dimensional gel electrophoresis. Left panel: control cells. Right panel: differentiatedcells.

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21Fig. 4. Membrane proteins induced during HL-60 differentiation are characteristic of human peripheral monocytes but not of tymphocytes. Two-dimensional gel analysis of [ 3H]leucine-labeled membrane proteins in HL-60 cells, before and after differentiation, are compared with whole cell [3H]leucine-labeled proteins of human peripheral monocytes and lymphocytes. Arrows indicate the proteins which are induced in HL-60 cells by differentiation and are also prominent in monocytes. Protein numbers are identical to those indicated in Fig. 2. Upper left: HL-60, control cells. Upper right: HL-60 differentiated cells. Lower left: lymphocytes. Lower right: monocytes.

245

separated from human fresh peripheral blood as described. Monocytes were further cultured 7 days in autologous serum. The cells were labeled with [3H]leucine for 4 h and lysed in lysis solution, and the whole cell proteins were resolved by two-dimensional gel electrophoresis. Fig. 4 strikingly demonstrates that the group of membrane-bound acidic proteins ml-m6 and protein ml0 which were induced during differentiation are very prominently synthesized proteins of human monocytes but are much reduced or absent in human lymphocytes. Other membrane proteins which were induced during differentiation, such as m8, m9, m l l and m12, were not found to be prominent proteins synthesized by monocytes. Discussion

The present study demonstrates that differentiation of HL-60 cells into monocytes by phorbol ester is associated with induction of motility functions. This was found to be accompanied by induction of specific membrane proteins characteristic of monocytes and reduced synthesis of specific proteins in the cytosol. Increase in chemotactic responsiveness to the chemotactic peptide (fMLP) has been reported in HL-60 cells following DMSO-induced differentiation into granylocytes [5,6]. A modest chemotactic responsiveness to fMLP is also exhibited by the HL-60 cells treated by phorbol ester. The possibility that the induction of biological functions associated with acquisition of macrophage functions might be related to induction of specific cytosol or membrane proteins was studied by analysis of protein synthesis using two-dimensional gel electrophoresis. We demonstrate that the differentiation of HL-60 by phorbol ester is associated with a differential effect on membrane versus cytosol proteins. While the changes in the cytosol apparently involve reduction of synthesis of specific proteins, the changes in the membrane were mainly associated with either enhacement or de novo synthesis of many specific proteins. Distinctive membrane associated changes were: (a) prominent induction of a membrane-bound group of proteins ( p I = 5, 45-70 kDa), indicated as ml-m6, and (b) de novo appearance of a membrane protein indicated as ml0 (pI ~ 7, 28 kDa).

A previous study [14] reported protein changes in the HL-60 following differentiation. However, since in the previous study molecular weight markers were not delineated and subcellular fractionation was not done, the results of this study could not be compared with the data presented here. Two major effects were associated with phorbol ester treatment of HL-60 cells: (1) arrest of cell growth and (2) acquisition of macrophage-like characteristics. It is possible that the reduced synthesis of certain cytosol proteins in response to phorbol ester treatment exemplifies changes related to cessation of cell growth. Further study will be required to clarify this point. However, certain of the membrane-associated changes in protein synthesis are evidently related to the differentiation of these cells in response to phorbol ester. Comparison of HL-60 cell proteins, before and after differentiation, with proteins of monocytes and lymphocytes (Fig. 4), clearly indicates that the group of membrane-bound proteins ml-m6, as well as protein ml0, are very prominently synthesized by cultured human monocytes. These proteins were much reduced or absent in human lymphocytes. Thus, macrophage-like differentiation of HL-60 cells is associated with the appearance of a distinctive set of biochemical characteristics, namely, the active synthesis of a group of specific membrane proteins. In the accompanying study [15], we show that inhibition of these differentiation functions in HL-60 cells by a methyltransferase inhibitor is correlated with selective inhibition of the synthesis of the differentiation marker proteins. Our ability to detect synthesis of proteins characteristic of different states of cellular differentiation will permit detailed studies of the biochemical changes which constitute this process. Futhermore, eventual definition of the role of these proteins, and of the mechanisms regulating their synthesis, will help to elucidate the biochemical basis of monocyte-specific functions and thus to understand defects associated with phagocytic leukocyte dysfunctions. References 1 Huberman, E. and Callahan, M.F. (1979) Proc. Natl. Acad. Sci. U.S.A. 76 1293-1297

246 2 Rovera, G., O'Brien, T.G. and Diamond, L. (1979) Science 204, 868-870 3 Rovera, G., Santoli, D. and Damsky, C. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2779-2783 4 Todd, R.F., Griffin, J.D., Ritz, J., Nadler, L.M., Abrams, T. and Schlossman, S.F. (1981) Leukemia Res. 5, 491-495 5 Fontana, J.A., Wright, D.G., Schiffmam E., Corcoran, B.A. and Deisseroth, A.B. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3664-3669 6 Niedel, J., Kahane, I., Lachman, L. and Cuatrecasas, P. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1000-1005 7 Collins, S.J., Galo, R.C. and Gallagher, R.E. (1977) Nature 270, 347-349 8 Cooper, H.L. (1974) Methods Enzymol. 32, 633-639

9 Cooper, H.L. Fagnani, R., London, J., Trepel, J. and Lester, E. (1982) J. Immunol. 128, 828-833 10 Monos, D.S. and Cooper, H.L. (1983) J. Immunol. 131, 341 346 11 Cooper, H.L., Park, M.H. and Folk, J.E. (1982) Cell 29, 791-797 12 Cooper, H.L., Park, M.H., Folk, J.E., Safer, B. and Braverman, R. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1854 1857 13 O'Farrel, P. (1975) J. Biol. Chem. 250, 4007-4021 14 Liebermann, D., Hoffman-Liebermann, B. and Sachs, L. (1981) int. J. Cancer 28, 285-290 15 Feuerstein, N. and Cooper, H.L. (1984) Biochim. Biophys. Acta 781,247-256