Effects of Inhibitors and Activators of Protein Kinase C on Late Erythroid Progenitor (CF'U-e) Colony Formation In Vitro

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1 Original Paper International Journal of Cell Cloning (1989) Effects of Inhibitors and Activators of Protein Kinase C on Late Erythroid Progenitor (CF'U-e) Colony Formation In Vitro Diane M. Jenis, Candace S. Johnson, Philip Fumnski Laboratory of Cell Biology, AMC Cancer Research Center, Denver, Colorado, USA Key Words. Protein kinase C Erythropoiesis * CFU-e PKC inhibitors and activators Abstract. Protein kinase C (PKC) plays a central role in external signal transduction for many cell types. To examine the involvement of PKC in the control of erythropoiesis, we tested the effects of PKC inhibitors on in vitro colony formation by late erythroid progenitors (CFUe) from normal and Friend virus-infected mice. Inhibitors of PKC and other kinases (H-7 and H-8) inhibited CFU-e at concentrations which inhibit PKC. HA1004, an inhibitor of the cyclic nucleotide-dependent kinases and a weak inhibitor of PKC, had little effect on CFU-e. In the absence of erythropoietin, a combination of phorbol ester and Ca++ ionophore significantly increased normal CFU-e. These results suggest PKC plays a role in the transduction of regulatory signals for the growth of CFU-e. Introduction The regulation of erythropoiesis is a function of erythropoietin (Epo) plus additional humoral factors and cellular interactions [l]. However, the mechanisms by which these external signals cause the cascade of events leading to production of mature, terminally differentiated erythrocytes remains unknown. One possible mechanism is through the activation of the calcium and phospholipiddependent protein kinase, protein kinase C (PKC), an enzyme involved in extracellular signal transduction in a variety of systems [2, 31. PKC has been shown to play a central role in the proliferation and differentiation of cells of the immune and hematopoietic lineages. Differentiation of HL-60 cells is induced by phorbol diesters, which activate PKC, as well as by inhibitors of PKC [4,5]. Phorbol esters stimulate colony formation by megakaryocyte [6,7] and granulocyte progenitors [ PKC is implicated in the in vim differentiation of Friend virus-infected Correspondence: Philip Furmanski, Ph.D., Laboratory of Cell Biology, AMC Cancer Research Center, 1600 Pierce Street, Denver, CO 80214, USA. Received January ; provisionally accepted February 13, 1989; accepted for publication March 2, /89/$2.00/0 0AlphaMed Press

2 PKC in Erythropoiesis 191 murine erythroleukemia cell lines induced by a number of mediators [l2-15]. Variable effects of PKC stimulators on colony formation by normal early erythroid progenitors (BFU-e) have been reported [16-181, although generally no effects have been seen on late erythroid progenitors (CFU-e). Recently, however, May e? al. [19] showed that bryostatins, potent activators of PKC, markedly stimulated colony formation by both normal human BFU-e and CFU-e. To further evaluate the role of PKC in late-stage erythropoietic regulation, we tested the effects of a series of inhibitors of PKC on in vitro colony formation by CFU-e. Target cells tested included CFU-e from normal spleen, fetal liver (a source of highly enriched, Epo-responsive erythroid progenitors) and cells (infected with Friend virus [FV] in vivo) whose responsiveness to the regulators of erythroid differentiation is altered. The inhibitors used were a series of recently described isoquinoline derivatives [20,21] with differential effects on various protein kinases: 1-(5-isoquinolinesulfonyl)-2-rnethylpiperazine dihydrochloride (H-7) and N-(2- methylaminoethyl)-5-isoquinolinesulfonamide dihydrochloride (H-8), preferential inhibitors of PKC, as well as other kinases and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide dihydrochloride (HAlW), an inhibitor of cyclic nucleotidedependent protein kinases. In addition, we tested the effects on CFU-e colony formation of staurosporine, an antibiotic recently isolated from cultures of Streptomyces sp. [22] and an extremely potent inhibitor of PKC with a significantly lesser effect on c-amp- and c-gmp-dependent kinases. We found that the PKC inhibitors suppressed colony formation by all target erythroid progenitors tested, including the Epo-independent CFU-e characteristic of infection with the FVP strain of Friend virus, whereas HA1004 did not. In addition, PKC activators caused a significant increase in CFU-e colony formation in the absence of exogenous Epo. The results suggest the involvement of PKC in late-stage erythropoietic proliferation and differentiation. Materials and Methods Animals These experiments utilized splenocytes or bone marrow cells obtained from BALBlc or NIHlPLCR mice which were inbred in our laboratories by brother-sister matings. Colonies were regularly mbnitored for the absence of adventitious viruses. NIHlPLCR mice were used for leukemic studies and were assessed by spleen palpation for leukemic status [23], which correlates well wi@ spleen weight, histopathology and virus titers. Fetal liver cells were obtained from BALB/c embryos at days of gestation and were prepared according to the technique of Dunn and Nupier [24]. Viruses The N-tropic, anemia-inducing (FVA) and polycythemia-inducing (FVP) strains of Friend virus were obtained from Dr. C. Friend and Dr. E. Mirand, respectively. Virus stocks were maintained by serial passage of cell-free virus stocks prepared from spleens of leukemic mice, as previously described [23]. Mice were inoculated i.p. with 0.5 ml of phosphate-buffered saline containing approximately 100 IDso of virus.

3 Jenis/Johnson/Furmanski 192 Protein Kinase Inhibitors and Activators The protein kinase inhibitors H-7, H-8 and HA1004 were purchased from Seikagaku America, Inc., St. Petersburg, FL. Staurosporine was purchased from Karniya Biomedical Company, Thousand Oaks, CA. The phorbol esters, phorbol 12-myristate U-acetate (PMA) and 4-a-phorbol 12-myristate &acetate (4aPMA), were obtained from L.C. Services Corp., Woburn, MA. The Ca++ ionophore A23187 was obtained from Sigma Chemical Co., St. Louis, MO. All reagents were of the highest grade commercially available and used without further purification. Erythmid Progenitor Assays CFU-e were cultured using the plasma clot method of McLeod et al. [25]. Clots were fixed, stained with dimethoxybenzidine and hematoxylin. Clusters of 8 or more hemoglobinized cells were counted as colonies. Six clots were counted for each data point. Each experiment was replicated at least 3 times. Protein kinase activators and inhibitors were freshly prepared and added in the indicated concentrations directly to the culture mixtures. Experiments were performed in subdued light to prevent inactivation of the inhibitors. Vehicle controls were included in all experiments. Results Inhibition of Normal CFU-e Colony Formation by Inhibitors of PKC The protein kinase inhibitors H-7 and H-8 decreased colony formation by normal mouse spleen cells in plasma clot culture in a dose-dependent manner (Fig. 1). Significant inhibition was observed with 5-10 pm inhibitor and in replicate experiments caused a 50% reduction in colony formation at concentrations of The enzyme-inhibitor dissociation constants (Ki s) for c-ampdependent, c-gmpdejmdent protein kinases, calmodulindependent phosphorylation and PKC are 3.0 pm, 5.8 pm, 97.0 pm and 6.0 pm for H-7, respectively, and 1.2 +, 0.48 pm, 68.0 and 15 ph4 for H-8, respectively [20]. HA1004, the cyclic nucleotidedependent protein kinase inhibitor, had little to no effect on colony formation at concentrations as high as 40 pm, well above its Ki s for c- AMP- and c-gmp-dependent kinases (2.3 pm and 1.3 pm, respectively). Staurosporine, a more potent inhibitor of protein kinases, sharply inhibited normal spleen CFU-e colony formation at concentrations as low as nm (Fig. 2), levels approximating its Ki for PKC (0.7 nm) and much lower than its Ki for the cyclic nucleotide-dependent kinases ( nm). Interestingly, inhibition by staurosporine was biphasic; dosedependent inhibition of colony formation at concentrations between 1 and 50 nm reproducibly occurred at a much lower rate than that observed between 0 and 1 nm. Similar results were obtained with CFU-e from normal bone marrow and with cells isolated from both BALB/c and NIH/PLCR strains of mice. Inhibition of Colony Formation by Fetal Liver CFU-e Erythropoietic progenitors constitute only a very small fraction of spleen and bone marrow cell populations. Effects of the protein kinase inhibitors tested could

4 PKC in Erythropoiesis 193 Fig. 1. Suppression of normal spleen CFU-e colony formation in vitro by protein kinase inhibitors H-7 (0-0), H-8 (m-m) and HA1004 (A-A) at pu final concentrations. Values expressed as a percent of the number of colonies in controls without inhibitors (142 f 41 colonies), mean of 6 cultures f SE. Data points at concentrations equal to or greater than 20 pa4 (H-7 and H-8) were significantly different from control without inhibitor (raw data), p < (Student s t test). thus result from indirect effects mediated through other cells in the cultures. To examine this possibility, the effects of the inhibitors on CFU-e were determined using fetal liver cells, a source highly enriched in Epo-responsive erythroid progenitors [26]. Fetal liver cells were very sensitive to the effects of H-7 and H-8 (Fig. 3). CFU-e colony formation by fetal liver cells was also reproducibly decreased by HA1004, but only at concentrations >20 pm, well above its Ki for cyclic nucleotide-dependent kinases, and thus probably reflecting inhibition of PKC, nonspecific toxicity or effects on another biochemical pathway. Inhibition of Leukemic Erythropoiesis by Inhibitors of PKC As described above for normal erythroid progenitors, CFU-e from animals

5 Jenis/Johnson/Furmanski 194 Fig. 2. Suppression of CFU-e colony formation by the protein kinase C inhibitor staurosporine at nm concentrations. Values expressed as a percent of the number of colonies in controls without inhibitors (232 f 33 colonies), mean of 6 cultures f SE. Data points at concentrations equal to or greater than 3.5 nm were significantly different from control without inhibitor (raw data), p < 0.01 (Student s t test). infected by Friend virus were also sensitive to inhibition by H-7, H-8 and staurosporine, but not HA1004 (except at concentrations CFU-e infected with the FVA and FVP strains were equally sensitive to the inhibitors, as were Epoindependent, FVP CFU-e in both the presence or absence of Epo. Representative data for FVP in the presence of Epo are shown in Figures 4 and 5. Effects of PKC Activation on CFU-e Colony Formation In addition to studies utilizing protein kinase inhibitors, we have investigated the ability of PKC activators to influence CFU-e colony formation. In accordance with previous reports [16-181, several tumor-promoting phorbol esters at vwing concentrations had no effect on CFU-e in standard culture conditions (saturating

6 PKC in Erythropoiesis 195 Fig. 3. Suppression of fetal liver CFU-e colony formation in vitro by protein kinase inhibitors H-7 (0-O), H-8 (H-H) and HA1004 (A- A) at fl final concentrations. Values expressed as a percent of the number of colonies in controls without inhibitors (323 f 59 colonies), mean of 6 cultures f SE. Data points at concentrations equal to or greater than 10 fl(h-7), 20fl(H-8), or 30a(HA1004) were significantly different from control without inhibitor (raw data), p < (Student s t test). quantities of Epo). In the absence of Epo, modest, but significant, dosedependent increases in colony formation were observed with the active phorbol ester PMA (10 ng/ml to 1 pg/ml), but not with the inactive ester 4aPMA (data not shown). The Ca++ ionophore A23187 caused a small, but significant, increase in CFU-e colony formation only in the presence of Epo (Table I), as previously reported [27]. Combinations of A23187 and PMA (at a concentration that itself had no effect on CFU-e) significantly stimulated colony formation, but only in the absence of Epo. No enhancement of colony formation was seen in fetal liver cell cultures. Dose-response studies (data not shown) revealed a narrow concentration range for the stimulatory effects of A23187 on colony formation, in accor-

7 Jenis/Johnson/Furmanski 196 Fig. 4. Suppression of FVP-induced leukemic CFU-e colony formation in vitro by protein kinase inhibitors H-7 (0-0), H-8 (1-1 ) and HA1004 (A- A) at pm final concentrations. Values expressed as a percent of the number of colonies in controls without inhibitors (382 f 73 colonies), mean of 6 cultures f SE. Data points at concentrations equal to or greater than 10 pm (H-7) or 20 pm (H-8) were significantly different from control without inhibitor (raw data), p < (Student s t test). dance with other studies which show that effects of Cat+ ionophores often occur in limited concentration ranges and that the agents are toxic to mouse cells at higher doses [27, 281. Discussion The results of this study provide evidence for the involvement of PKC in the proliferation and/or differentiation of late erythroid progenitor cells. While experiments based on inhibitors must be interpreted with caution due to inherent uncertainties regarding specificity and accessibility to the target, the findings that

8 PKC in Erythropoiesis 197 Fig. 5. Suppression of FVP-induced leukemic CFU-e colony formation by the protein kinase C inhibitor staurosporine at nm concentrations. Values expressed as a percent of the number of colonies in controls without inhibitors (296 f 84 colonies), mean f SE. Data points at concentrations greater than or equal to 1.0 nm were significantly different from control without inhibitor (raw data), p < 0.01 (Student s t test). a series of different inhibitors reduced CFU-e colony formation in approximate relation to their Ki s for PKC and that PKC activators increased colony formation are consistent with the hypothesis. A number of other investigators have made use of the series of isoquinoline sulfonamide inhibitors to evaluate the role of PKC and other kinases in various physiological processes. For example, Clark et al. [29] showed that antigen- and L-2-induced in vitro proliferation of T cells (events which are thought to be PKCmediated) was inhibited by H-7 at concentrations similar to those used here (1 to 20 CLM), while HA1004 had no consistent effect. Viability of the T cells was not affected by H-7 at these concentrations. Similarly, Rush et al. [30] showed that H-7 and H-8 (at ClM) inhibited B cell activation. Berkow et al. [31] found

9 JenisNohnsonlFurmanski 198 lhble I. Effect of PMA and CA" ionophore A23187 on CFU-e colony formation in vitro by adult mouse spleen cells CFU-ell X lo5 cells Solvent control f 10.ga 10.7 f 1.2 A23187 (5 ng/ml) f 4.3 (+23)b 6.0 f 1.0 (-44) PMA (5 ng/ml) f 2.7 (-4) 12.0 f 2.9 (+12) A23187 (5 ng/ml) + PMA (5 ng/ml) f 8.7 (+7) 59.0 f 6.9 (+451)' 'Average number of colonies f SE; numbers in parentheses are percent change from control. bsignificantly different from control, p < 0.05 'p < differential effects of H-7 on release of superoxide anion by neutrophils dependent on the stimulus used. Radtioch et al. [32] showed that H-7 blocked macrophage activation by interferon-/3 (but not interferon-y) and had no effect on cell viability. The correlation of such inhibitor effects with other evidence for PKC involvement in these systems and the general lack of effect of the inhibitors on cell viability suggest that the results with inhibitors are indicative of a role for PKC in the events examined. Suppression of CFU-e colony fornlation by PKC inhibitors in this study was observed with both normal adult spleen and bone marrow cells, as well as with cells from animals infected by either FVA or FVP. Late erythroid progenitor cells from the latter characteristically have lost reponsiveness to Epo [33]. These results suggest either that the involvement of PKC in erythropoiesis is unrelated to cellular regulation by Epo or that the lesion in FVP-infected cells occurs at a locus prior to intracellular transduction of the signal arising from interaction of Epo with its receptor. As discussed below, other studies also suggest the involvement of PKC in aspects of erythropoietic regulation that do not involve Epo. Suppression of CFU-e colony formation by PKC inhibitors could also be due to indirect effects of the agents, through nonerythroid cells present in the assay cultures. Conversely, colony formation by CFU-e in fetal liver preparation, estimated to consist of 7040% Epo-sensitive erythroid cells [26], was similarly responsive to all the inhibitors tested. Moreover, cell populations from FVA and FVP leukemic spleens, also enriched for CFU-e, provided similar results. Several previous studies [ have examined the effects on erythroid progenitors of tumor-promoting phorbol esters, which are now known to activate PKC. Generally, no effect was observed on in vitro colony formation by CFU-e, while

10 PKC in Erythropoiesis 199 BFU-e colony formation has been variably found to be stimulated [17] or inhibited [18] by PMA. However, May et al. recently showed that bryostatin, a powerful activator of PKC [34], markedly stimulated colony formation by both human CFU-e and BFU-e in plasma clot cultures [19]. The differences in these studies may relate to changes in methodology or cell sources, or may reflect differences between effects of phorbol esters and other PKC activators on the enzyme. In this regard, it has been shown that while phorbol esters and bryostatins both activate PKC, only the phorbol esters are tumor promoters; bryostatins lack tumorpromoting activity and are antineoplastic [34]. In addition, while HL-60 cells are induced to differentiate by phorbol esters, bryostatins are inactive in this system WI. Studies of FV-induced erythroleukemia cell lines, which grow and differentiate independently of Epo, but often respond to nonphysiological inducers of differentiation such as dimethyl sulfoxide (DMSO) and N,N -hexamethylene bisacetamide, also demonstrate an involvement of PKC in erythropoiesis. A number of investigators have shown that phorbol esters inhibit differentiation in this model of erythroid development [l2, 131. Balazuvich et al. recently showed that differentiation induced with DMSO was associated with translocation and activation of PKC, although they concluded that activation of the enzyme itself was insufficient to cause commitment to differentiation [B]. Interestingly, a role for PKC was recently suggested in the regulation of Epo production by a human renal cell carcinoma line in vitro [36]. CFU-e colony formation by adult spleen cells was increased by phorbol ester plus Ca++ ionophore in the absence of Epo. This Epo sparing effect is in accord with the possibility that signals from the primary regulator of erythropoiesis are transduced intracellularly through PKC. Synergistic effects of PMA and A23198 have been observed in several other PKC-mediated responses [2,3, 371, the result of phorbol ester-induced translocation of PKC to its membrane-active site and ionophore-increased intracellular Ca++ levels. We also found that fetal liver CFU-e colonies were not increased by PMA or PMA plus A These data are in accord with those of Fibuch et ul. [17] and may reflect the higher sensitivity of fetal liver CFU-e to Epo, the presence of high concentrations of serum in the plasma clot cultures and the resultant high background (without added exogenous Epo) of colonies. In accord with previous reports [27], the addition of Ca++ ionophore A23187 itself caused a modest stimulation in CFU-e colony formation [27]. CFU-e colony formation is inhibited by the Ca++ chelator ethyleneglycol-bis-(/3-aminoethyl ether)-n,n,n, -tetraacetic acid [27], while Epo increases Ca++ activity in human bone marrow cells in culture [38]. These findings support a role for Ca++ in latestage erythropoiesis, which could be related to the role of PKC in the process. The data presented here support the involvement of PKC in regulatory signal transduction for late events in erythropoiesis. They do not exclude the participation of other protein kinases, nor do they establish the locus of the regulatory interactions involved, which are the subjects of further study.

11 JenislJohnsonlFurmanski 200 Acknowledgments We thank Stacey Ho$man for technical assistance and Cathrine Allen for preparation of the manuscript. This research was supported by grant from the National Cancer Institute and by a gift to the AMC Cancer Research Center from Richard L. Robinson References Nathan DG, Sytkowski A. Erythropoietin and the regulation of erythropoiesis. N Engl J Med 1983;308: Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 1984; 308 : Nishizuka Y. Studies and perspectives of protein kinase C. Science 1986;233: Huberman E, Callaham M. Induction of terminal differentiation in human promyelocytic leukemia cells by tumor promoting agents. Proc Natl Acad Sci USA 1979;76: Okazaki T, Kato Y, Mochizuki T, llshima M, Sawada H, Uchino H. Staurosporine, a navel protein kinase inhibitor, enhances HL-60-cell differentiation induced by various compounds. Exp Hematol 1988;16: Long MW, Smolen JE, Szczepauski P, Boxer LA. Role of phorbol diesters in in vitro murine megakaryocyte colony formation. J Clin Invest 1984;74: Long MW, Gragowski LL, Heffner CH, Boxer LA. Phorbol diesters stimulate the development of an early murine progenitor cell: the burst-forming unit-megakaryocyte. J Clin Invest 1985;76: Stuart RK, Hamilton JA. 'hmor-promoting phorbol esters stimulate hematopoietic colony formation in vitro. Science 1980;208: Stuart RK, Sensenbrenner LL, Shadduck RK, Waheed A, Caramatti C. Phorbol esterstimulated murine myelopoiesis: role of colony-stimulating factors. J Cell Physiol 1983 ;I Stuart RK, Hamilton JA, Sensenbrenner LL, Moore MAS. Regulation of myelopoiesis in vim: partial replacement of colony-stimulating factors by tumor-promoting phorbol esters. Blood 1981; Griffin JD, Larcom P, Kufe DW. TPA induces differentiation of purified human myeloblasts in the absence of proliferation. Exp Hematol l985;l3: Rovera G, O'Brien TG, Diamond L. lbmor promoters inhibit spontaneous differentiation of Friend erythroleukemia cells in culture. Proc Natl Acad Sci USA 1977; 74: Yamasaki H, Fibach E, Nude1 U, Weinstein IB, Rifkind RA, Marks PA. 'hmor promoters inhibit spontaneous and induced differentiation of murine erythroleukemia cells in culture. Proc Natl Acad Sci USA 1977;74: Melloni E, Pontremoli S, Michetti M, et al. Protein kinase C activity and hexamethylene bisacetamide-induced erythroleukemia cell differentiation. Proc Natl Acad Sci USA 1987;84: Balazavich KJ, Portnow D, Boxer LA, Prochownik EV. Changes in protein kinase C activity are associated with the differentiation of Friend erythroleukemia cells. Biochem Biophys Acta Bmckbank KBM. Influence of the tumor promoter TPA upon erythroid burst formation in vitro. Br J Haematol 1983;53: Fibach E, Marks PA, Rifkind RA. lbmor promoters enhance myeloid and erythroid colony formation by normal mouse hematopoietic cells. Proc Natl Acad Sci USA 1980;

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