Different effects of the two protein kinase C activators bryostatin-1 and TPA on growth and differentiation of human monocytic leukemia cell lines

Leukemia Research Vol, 17, No. 10, pp. 897-901, 1993. Printed in Great Britain.

0145-2126/93 $6.00 + 0.00 © 1993 Pergamon Press Ltd

BRIEF COMMUNICATION D I F F E R E N T EFFECTS OF THE TWO P R O T E I N KINASE C A C T I V A T O R S BRYOSTATIN-1 A N D TPA ON G R O W T H A N D D I F F E R E N T I A T I O N OF H U M A N MONOCYTIC L E U K E M I A CELL LINES KLAUS G. STEUBE, DORTHE GRUNICKE, HILMAR OUENTMEIER and HANS G. DREXLER DSM--German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures, Braunschweig, Germany (Received 5 April 1993. Revision accepted 27 June 1993) Abstract--The effects of bryostatin-1 (Bryo) and 12-O-tetradecanoyl-phorbol 13-acetate (TPA), both activators of protein kinase C (PKC), on proliferation and differentiation of two monocytic leukemia cell lines, JOSK-I and JOSK-M, were investigated. Treatment with TPA or Bryo inhibited cellular proliferation in a dose-dependent manner. Both drugs induced distinct phenotypic changes associated with monocytic differentiation. Although c-myc mRNA is often found to be down-regulated during biomodulator-triggered in vitro myeiomonocytic differentiation, however, here the modulation of c-myc expression was less pronounced. All parameters studied were more prominently altered in TPA- than in Bryo-treated cells, and, were more distinct in JOSK-I than in JOSK-M. Since Bryo was able to antagonize the TPA-mediated effects on proliferation and morphological alterations, an (at least partially) different mode of action of these PKC activators on monocytic cell lines may be suggested. Key words: Bryostatin-1, monocytic differentiation, leukemia cell lines, protein kinase C.

with those of the phorbol ester TPA. Our results indicate that Bryo is capable of inducing differentiation in monocytic cells, albeit with a lower efficiency than TPA. While exhibiting low cytotoxicity, Bryo was found to be a strong anti-proliferative agent against JOSK-I cells.

INTRODUCTION MALIGNANThematopoietic cells are arrested in their normal maturation pathway and do not proceed to their final stage of development. Stimulation of differentiation of neoplastic cells, especially those of myelomonocytic lineages are thought to be important for the treatment of leukemia [1-3]. A possible candidate for this approach is the macrocyclic lactone bryostatin-1 (Bryo). This compound containing antineoplastic activity as well as differentiation inducing properties [4-7] was isolated recently from the marine mollusc Bugula neritina [4]. Bryo was found to inhibit growth of freshly explanted myeloid leukemia cells while supporting proliferation of multipotent stem cells [8--10]. Since most of the investigations on cell lines were undertaken with relatively immature myeloid leukemia cells, we chose two monocytic cell lines, JOSKM and JOSK-I, to study the effect of Bryo on more mature cell lines. The anti-proliferative and differentiation-inducing effects of Bryo were compared

M A T E R I A L S AND M E T H O D S Cell culture and chemicals JOSK-M and JOSK-I [11] are deposited at the German Collection of Microorganisms and Cell Cultures (DSM, Braunschweig, Germany). Originally, JOSK-M was derived from a 37-year-old man with chronic myeloid leukemia in myelomonocytic blast crisis and JOSK-I from a 72-year-old woman with acute myelomonocytic leukemia. Ceils were cultured at 37°C in a humidified atmosphere containing 5% CO2 using RPMI 1640 medium plus 10% heat inactivated fetal calf serum (Gibco). [Methyl-3H]thymidine (specific activity 74GBq/mmol) and [o,'-32p] deoxycytosinetriphosphate (specific activity 110 TBq/ mmol) were obtained from Amersham, TPA from Sigma. Bryo was kindly provided by Dr G. R. Pettit. The reagents were dissolved in dimethylsulfoxide and diluted prior to the experiments in RPMI 1640 medium.

Correspondence to: Dr K. Steube, DSM, Mascheroder Weg 1B, D-38124 Braunschweig, Germany (Tel: +49-5312616.159; Fax: +49-531-2616.150).

Immunophenotypic analysis The following mAb were applied for cell surface marker 897

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analysis: OKM-1 (CDllb), MCS-2 (CD13), FMC-17 (CD14), VIM-D5 (CD15) and MY-9 (CD33). Binding of the mAb was assessed by flow cytometry (FACScan, Becton Dickinson) using fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin heavy chain specific antisera. Non-reactive antibodies were applied as controls. Addition of propidium iodide (10 Ixg/ml) to the cells prior to the analysis allowed gating out of the dead cells.

RNA isolation and Northern blot analysis Cellular RNA was isolated by the guanidinium isothiocyanate/cesium chloride method, separated in a 1% agarose/2.2 M formaldehyde gel, transferred to the nylon membrane (Schleicher and Schiill) and cross-linked with UV. Filters were hybridized overnight at 62°C with a 32p_ labeled probe (USB Random Primed Labeling Kit) specific for c-myc (1.5 kb HindlII-EcoR1 fragment from pBR328).

A. Effect of TPA 100

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B. Effect of Bryostatin-1 RESULTS

Growth inhibition of the cell lines Figure 1 shows the anti-proliferative effect of the two PKC activators in the concentration range between 0.1 and 1000nM. While 10nM TPA inhibited growth of JOSK-I and JOSK-M down to 2 and 37%, respectively, (part A), 50 nM Bryo must be applied to reduce growth of JOSK-I to 47% relative to the untreated control (part B). Higher Bryo concentrations did not further decrease growth of JOSK-I cells. Interestingly, the [3H]-thymidine incorporation of JOSK-M cells remained almost unaffected by Bryo, even with concentrations of up to 1 IxM. Staining of the cells with trypan blue revealed no significant differences in the viability between cells treated with 10 nM TPA or 100 nM Bryo and untreated cells. In order to monitor cellular proliferation with a second assay, all experiments were repeated and hence confirmed using the MTT assay. Antagonistic effect of Bryo on TPA-treated cultures We next examined whether Bryo could antagonize the TPA-induced growth inhibition in JOSK-I and JOSK-M when applied simultaneously with TPA to the cultures (Table 1). While Bryo did not affect growth of JOSK-M cells, it was indeed able to reverse almost completely the TPA-mediated growth inhibition in JOSK-M. In JOSK-I cells where 5 nM TPA stopped proliferation completely, the simultaneous addition of 50 or 100 nM Bryo plus 5 nM TPA resulted in a [3H]-thymidine incorporation value of 40% relative to untreated controls. Higher Bryo concentrations added together with TPA did not further reverse the TPA-mediated growth inhibition of JOSK-I. Similar results were obtained when Bryo was added 2 h prior to TPA to the cultures, but not when added 12 h after TPA addition.

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FIG. 1. [3H]-thymidineincorporation of JOSK-I and JOSKM cells. Cells (1 × 105/ml) were seeded in 100 Id aliquots into 96-well flat bottom culture plates. Different concentrations of TPA (A) and Bryo (B) were added from a tenfold stock solution. Radioactive incorporation of [aH]thymidine was determined over a period of 3 h initially and after the cells had been exposed for 48 h with or without the drugs. The incorporation difference (48 minus 0 h) for untreated cultures were set at 100% and the data for the drug-treated cultures were related to it. All experiments were repeated at least 3 times and the individual data represent the means of experiments carried out in triplicate.

Morphological changes Both cell lines usually grew as single cells in suspension and did not attach to the bottom of the culture flasks. Upon TPA treatment the cells became adherent, developed pseudopodia and formed cell aggregates. The sizes of the vacuoles and the cytoplasma increased, while the size of the nuclei decreased. The morphological changes observed after TPA exposure were more prominent in JOSKI than in JOSK-M cells (not shown). When Bryo was added 1 h prior to TPA, these phorbol ester-induced

Effects of protein kinase C activators on leukemia cell lines TABLE 1. GROWTH RESTORING EFFECT OF TREATED CELLS

Cell line

0

JOSK-I + 5 nM TPA JOSK-M + 10 nM TPA

Bryo IN T P A -

Bryo concentration [nM] 10 50 100

1

7

42

40

37

91

92

96

The cells were cultured for 48 h in the presence of 5 nM TPA (JOSK-I) or 10 nM TPA (JOSK-M) and the different concentrations of Bryo as indicated. Simultaneous cultures without TPA and Bryo were grown as controls. The experiments were evaluated by [3H]-thymidine incorporation. The incorporation difference (48 minus 0 h) for untreated cultures (no TPA and no Bryo) were set at 100% and the data for the drug-treated cultures were related to it. The experiments were repeated twice and were carried out in triplicate. TABLE2. SURFACEMARKEREXPRESSIONOFJOSK-M AND JOSK-I BEFOREAND AFTERTREATMENTWITHTPA AND Bryo Cell line

CDllb

JOSK-M + TPA + Bryo JOSK-I + TPA + Bryo

30 83 63 36 89 86

Surface marker C D 1 3 C D 1 4 C D 1 5 CD33 99 98 97 99 95 99

<1 3 <1 <1 <1 <1

98 97 96 nd. nd. nd.

98 99 97 nd. nd. nd.

For each experiment 8 x 10 6 cells were incubated for 48 h in culture medium without or with 5 x 10 -9 M TPA (JOSK-M), 2 × 10-9M TPA (JOSK-I) or 10-TM Bryo (both cell lines). The data (average of two experiments) represent percentages of positive viable cells analyzed by flow cytometry as outlined in 'Materials and Methods'. nd. = not determined.

effects on the morphology could be inhibited. After cultivation of JOSK-I cells solely in the presence of Bryo, an increase in the number of enlarged, granulalike particles containing cells and development of pseudopodia were observed, but no cell clusters were detected. Also the cells were only loosely attached to the plastic surface. JOSK-M cells, however, incubated with 10 or 100nM Bryo remained morphologically unchanged (not shown) which was in accordance with the weak anti-proliferative effect described above.

Cell surface marker expression Changes in the expression of cell surface markers analysed by flow cytometry are given in Table 2. Uninduced cells were weakly positive for CD11b,

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strongly positive for CD13, CD15 and CD33 and negative for CD14. Upon TPA or Bryo treatment C D l l b expression was markedly increased. Again the effect of Bryo was stronger in JOSK-I than in JOSK-M. Besides a very weak increase of CD14 (from 0.14 to 3.1%) in TPA-treated JOSK-M cells all other surface markers remained essentially unchanged. C-myc proto-oncogene expression Changes in c-myc expression were analyzed by Northern blotting of total cellular RNA (Fig. 2). Staining of the gels with ethidium bromide ensured that equal amounts of RNA were applied on the gels and subsequently transferred onto nylon membranes. In both cell lines the mRNA for c-myc was expressed. Exposure of the cells for various times to TPA or Bryo did not alter the c-myc level dramatically. A weak increase of the proto-oncogene mRNA could be observed in TPA-treated JOSK-M cells. In a control experiment, TPA, but not Bryo, was able to decrease c-myc mRNA in the cell line U-937 (not shown). DISCUSSION Tumor-promoting phorbol esters are potent inducers of maturation of a wide variety of leukemia cells. The initial step in the induction of differentiation is the binding to and subsequent activation of protein kinase C [12]. Like TPA, Bryo binds to and activates PKC [13, 14], but unlike TPA it clearly lacks tumorpromoting activity [15]. Our results indicate that TPA and Bryo could induce differentiation of JOSKI as evidenced by changes in morphology, surface marker expression and growth modulation. These changes are well-known parameters of differentiation in myeloid leukemia cells [16]. Although a decrease in c-myc expression is thought to be necessary for induction of differentiation, the level of c-myc mRNA in TPA- or Bryo-treated JOSK-I cells remained almost constant. We suggest that down-regulation of c-myc does not play a major role for differentiation of JOSK-I cells as is suggested for more immature cell lines [5,6, 17]. Comparing the changes induced by the same molar concentrations of TPA and Bryo, TPA was always more effective in both cell lines than Bryo. The extent of Bryo-induced growth inhibition in other myelomonocytic [5, 7] or lymphocytic [9, 18] cell lines was also found to be variable. JOSK-M, however, turned out to be less amenable for induction of differentiation. Similar results have been reported for several HL-60 sublines [6] or the erythroleukemia cell line K-562 [19]. In T-cells, Bryo could

K. G. STEUBEel al.

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JOSK-M

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2

6

12

2

6

12

24

JOSK-I

24

0

2

6

12

24

FIG. 2. c-myc expression in JOSK-I and JOSK-M. Cells were cultured at 2 x 106/ml in either (A): TPA (10 nM for JOSK-M and 5 nM for JOSK-I) or (B): Bryo (100 nM both cell lines) for the indicated hours. Cells were sedimented, washed in PBS and immediately frozen. Total cellular RNA was isolated for Northern analysis (10 I~g per lane) with the 32p-labeled c-myc probe. The figure depicts a representative photography out of three Northern hybridizations from individual experiments.

efficiently antagonize phorbol ester-induced cell proliferation [20], whereas in the two B-cell lines EHEB and JVM-2 both biomodulators did not act in an antagonistic or synergistic fashion [18]. From these findings a different or partly different mode of action for Bryo and TPA on leukemia cell lines can be suggested. The differences might be due to variations in the expression and/or differential activation and translocation of PKC isotypes [12, 19, 21]. Different binding affinities of TPA and Bryo could also explain our finding that Bryo, although unable to inhibit growth of JOSK-M, could antagonize some of the TPA-mediated effects in this cell line. Furthermore, some of the Bryo-triggered events, which ordinarily are associated with cell differentiation, might have

been dissociated from the complete maturation process in JOSK-M. In conclusion, Bryo is clearly able to induce differentiation in monocytic leukemia cells. Its lack of tumor-promoting activity, while supporting growth of stem cells [8-10] suggests that Bryo might be a useful agent for further studies of in vivo differentiation of myelomonocytic leukemia.

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12. Nishizuka Y. (1989) The protein kinase C family. Heterogeneity and its implications. A. Rev. Biochem. 58, 31. 13. Berkow R. L. & Kraft A. S. (1985) Bryostatin, a nonphorbol macrocyclic lactone, activates intact human polymorphonuclear leukocytes and binds to the phorbol ester receptor. Biochem. biophys. Res. Commun. 131, 1109. 14. Smith J. B., Smith L. & Pettit G. R. (1985) Bryostatins: potent, new mitogens that mimic phorbol ester tumor promotors. Biochem. biophys. Res. Commun. 132, 939. 15. Wender P. A., Cribbs C. M., Koehler K. F., Sharkey N. A., Herald C. L., Kamano Y., Pettit G. R. & Blumberg P. M. (1988) Modeling of the bryostatins to the phorbol ester pharmacophore on protein kinase C. Proc. natn. Acad. Sci. U.S.A. 85, 7197. 16. Rovera G., Ferraro D., Pagliardi G. L., Vartikar J., Pessano S., Bottera L., Abraham S. & Lebman D. (1982) Induction of differentiation of human myeloid leukemias by phorboi esters: phenotypic changes and mode of action. Ann. N.Y. Acad. Sci. 397, 211. 17. Bernstein S. E., Kharbanda S. M., Sherman M. L., Stone R. M. & Kufe D. W. (1991) Inhibition of protein kinase C is associated with a decrease in c-myc expression in human myeloid leukemia cells. FEBS Lett. 294, 73. 18. Hu Z., Gignac S. M., Uphoff C. C., Quentmeier H., Steube K. G. & Drexler H. G. (1993) Induction of differentiation of B-cell leukemia cell lines JVM-2 and EHEB by bryostatin 1. Leukemia Lymphoma 10, 135. 19. Hocevar B. A., Morrow D. M., Tykocinski M. L. & Fields A. P. (1992) Protein kinase C isotypes in human erythroleukemia cell proliferation and differentiation. J. Cell Sci. 101, 671. 20. Hess A. D., Silanskis M. K., Esa A. H., Pettit G. R. & May W. S. (1988) Activation of human T lymphocytes by bryostatin. J. Immun. 141, 3263. 21. Kraft A. S., Reeves J. A. & Ashendel C. L. (1988) Differing modulation of protein kinase C by bryostatin 1 and phorbol esters in JB 6 mouse epidermal cells. J. biol. Chem. 263, 8437.