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Is PKC activation required for leukemia cell differentiation?
Leukemia Research Vol. 21, No. 5, pp. 411-414, 1997. 0 1997 Elsevier Science Ltd. All rights reserved Prmted in Great Britain 0145-2126197 $17.00 + 0.00
Pergamon PII: SO145-2126(96)00121-X
COMMENTARY IS PKC ACTIVATION REQUIRED FOR LEUKEMIA DIFFERENTIATION?
CELL
Gayle E. Woloschak Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, Illinois. U.S.A. The role of protein kinase C (PKC) in the pathways of differentiation of leukemia cells is one of the most studied and least understood areas in hematology. It has been considered almost a universally accepted paradigm that PKC is essential for the induced differentiation of leukemic cells. The study of Manzel and Macfarlane [l] in this issue of Leukemia Research describes experiments demonstrating that PKC is not required for the dihydroxyvitamin Ds- or Transforming Growth Factor (TGF-Pi)-induced growth arrest (and consequently differentiation) in 10 different human leukemia cell lines. In these experiments, different isoforms (a, p, and y) of PKC were examined at the level of mRNA accumulation prior to or following exposure to 1,25dihydroxyvitamin Ds. The results of these expression experiments in the 10 different leukemia cell lines were correlated with induction of growth arrest (as a marker of differentiation) in the cells in the presence or absence of PKC inhibitors Bisindolylmaleimide I or II prior to exposure of the cells to TGFPt and 1,25-dihydroxyvitamin Ds. From those experiments, it can be concluded that TGF-fi and 1,25dihydroxyvitamin Da induce growth arrest via a PKC-independent mechanism. This finding is surprising in light of the wealth of data in other cell systems [2-4] and in leukemia cells [5-91 documenting an important role for PKC in growth arrest by other differentiating agents. Zeng and el-Deiry [6] recently have demonstrated that PKC is required for TPA-induced expression of the cyclin-dependent kinase (cdk)-inhibitor p21 WAFl/CIPl, which has been implicated in growth arrest caused by senescence, tumor suppression and terminal differentiation. In the report by Manzel and Macfarlane [l], cell growth arrest is assessed, but the relative contribution of differentiation and apoptosis to those results is not apparent. In fact, work by de Vente et al. [9] with U937 cells demonstrated that changes in PKC content and distribution within cells induce apoptosis rather than differentiation, suggesting that the decision of a cell to undergo death or differentiation in response to PKC activators may, in part, be modulated by PKC signal transduction pathway.
There may be other signals as well or, alternatively, PKC may influence both apoptosis and differentiation. Several reports have shown the importance of PKC activation for induction of apoptosis [l&12]. The mechanism of TGF-/It 1,25-dihydroxyvitamin Ds in induction of leukemia cell growth arrest appears to be different to those for TPA and other similar phorbol ester differentiation-inducing agents. There is a large body of evidence supporting the role of PKC in TPAinduced leukemia cell differentiation. Protein kinase C is the intracellular receptor for TPA and its derivative phorbol esters. Leukemic cells defective in PKC isoforms are not capable of differentiating in response to TPA [13-161. Other studies have shown that exposure of cells to antisense oligonucleotides to different PKC isoforms inhibits cell differentiation [ 171. Similarly, cells transfected with vectors causing over-expression of PKC are stimulated to undergo differentiation, even in the absence of exposure to differentiating agents [17, 181. There are also a wide variety of other agents (DNA damaging agents) capable of inducing leukemia cell differentiation via the PKC pathway [19-211. This has led to the idea that all cell differentiation in leukemia cells is PKC-dependent. Savickiene et al. P61, using a variety of different protein kinase inhibitors, similarly conclude that protein kinase activities in HL-60 cells are differentiation-stage-specific and that the specificity depends upon the actual inducing agents. Furthermore, HL-525 cells, which do not differentiate in response to TPA due to decreased PKC-fl expression relative to controls, can be induced to undergo differentiation in response to all-transretinoic acid, a pathway reported to be mediated by metabolically active vitamin D [27]. Together, these findings suggest that mechanisms regarding TPAinduced differentiation pathways may not be directly applicable to leukemia cell differentiation induced by vitamin D metabolites. In the studies of Manzel and Macfarlane [l], several cytokines were tested for the ability to modulate growth arrest induced by 1,25-dihydroxyvitamin Da-dihydroxy411
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vitamin Da. Of the cytokines tested (TNFa, TGF/Jr, y-IFN, IL-l, and G-CSF), only TGF-/?r enhanced growth arrest in four out of 10 cell lines. The mechanisms of action of TGF-pi in this context are not clear, but are likely to be important, since TGF/Jr is released by many tumors and is found in inflammatory reactions. Reports in the literature on other cell types show that TGFflr is capable of inactivating PKCG by phosphorylation [28301, and that neutralization of TGFP with antibodies leads to enhanced PKC activation [31,32], suggesting that many TGF-fir effects are actually caused by a negative modulation of the PKC-dependent pathway [33-3.51. Whether TGF-P effects a change in PKC activity in leukemia cells is not clear at this point. Experiments by Manzel and Macfarlane [l] monitored mRNA expression, which was induced in some leukemic cells following vitamin D treatment, but did not measure PKC activation or translocation. It is interesting to speculate that the PKC-independent pathway for differentiation in leukemic cells is over-ridden by the PKC-dependent pathway and that the only way in which this pathway can be induced is if PKC is either depleted or rendered inactive in the cell. Further experiments aimed at determining the mechanism of differentiation pathway choice in leukemia cells will be important. It will be interesting also to explore whether other PKC-independent processes, such as cell surface antigen expression [36], also are stimulated by the vitamin D3 pathway of differentiation. For several years, different isoforms of PKC have been known, but it is only recently that the differential functions of these isoforms have begun to be elucidated. It is noteworthy that Manzel and Macfarlane [l] examined expression of several different isoforms of the enzyme, PKC-c(, p and 6. The PKC-y, which is found predominantly in brain and spinal cord [37], was not studied in these experiments and is not likely to be relevant to leukemia cell differentiation. Studies of PKC-E, zeta, and the newly discovered theta, would probably have led to more conclusive analysis of the association between vitamin D-induced differentiation and PKC expression. Studies by Kiley and Parker [38] have shown that individual PKCs localize to different subcellular compartments, suggesting a functional role for PKC sublocalization in the cell. Not all PKC isoforms are expressed in all leukemia cells. For example, U937 cells analyzed in the paper by Manzel and Macfarlane [l] are reported to express undetectable levels of PKC-a [38] and similarly show little mRNA accumulation [l]. TPA-induced leukemia cell differentiation is known to be isoform-specific [3841]. In addition, other studies by Macfarlane and Manzel [42] have demonstrated cell cycle regulation of PKC isoform expression. This leads to complications in understanding
the precise role of each PKC isoform in the differentiative process. Based on the studies of Manzel and Macfarlane [l, 421 and others in the literature, it is clear that differentiation of leukemic cells can take place by either PKC-dependent or PKC-independent pathways. This complicates our understanding of mechanisms underlying the differentiative process. Essential questions remain to be addressed: Are the two pathways ever activated at the same time? Which intracellular (intercellular?) signals transmit the pathway of choice for the cell? Do levels of activated PKC isoforms affect only the PKC-dependent pathway? Is it necessary to activate both pathways when using differentiating agents for therapy? Acknowledgements-The author wishes to thank MS Felicia King for her excellent assistance in preparing this manuscript and Drs Frank Collart and David Grdina for critical review prior to submission. This work was supported by the U.S. Department of Energy, Office of Health and Environmental Research, under Contract No. W-31-109-ENG-38.
References Manzel, L. and Macfarlane, D. E. Protein kinase C is not necessary for transforming growth factor beta induced growth-arrest in leukemia cell lines. Leukemia Research, 1997, 21, 403. 2. Hong, Y., Martin, J. F., Vainchenker, W. and Erusalimsky, J. D., Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells. Blood, 1996, 87, 123. 3. Trubiani, O., Rana, R. A., Stuppia, L. and Di-Primio, R., Nuclear translocation of beta II PKC isoenzyme in phorbol ester-stimulated KM-3 pre-B human leukemic cells. Experimental Cell Research, 1995, 221, 172. 4. Schiemann, W. P. and Nathanson, N. M., Involvement of protein kinase C during activation of the mitogen-activated protein kinase cascade by leukemia inhibitory factor. Evidence for participation of multiple signaling pathways. Journal of Biological Chemistry, 1994, 269, 6376. 5. Lok, C. N., Chan, K. F. and Loh, T. T., Effects of protein kinase modulators on transferrin receptor expression in human leukaemic HL-60 cells. FEBS Letters, 1995, 365, 137. 6. Zeng, Y. X. and el-Deiry, W. S., Regulation of p21 WAFli CIPl expression by p53-independent pathways. Oncogene, 1996, 12, 1557. 7. Hooper, W. C., Abraham, R. T., Ashendel, C. L. and Woloschak, G. E., Differential responsiveness to phorbol esters correlates with differential expression of protein kinase C in KG-1 and KG-la human myeloid leukemia cells. Biochemica et Biophysics Actu, 1989, 1013, 47. 8. Homma, Y., Gemmell, M. A. and Huberman, E., Protein kinase C activates with different characteristics, including substrate specificity from two human HL-60 leukemia cell variants. Cancer Research, 1988, 48, 2744. 9. de Vente, J., Kiley, S., Garris, T., Bryant, W., Hooker, J., Posekany, K., Parker, P., Cook, P., Fletcher, D. and Ways, D. K., Phorbol ester treatment of U937 cells with altered protein kinase C content and distribution induces cell death
Is PKC activation required for leukemia cell differentiation? differentiation. and rather than Cell Growth Differentiation, 1995, 6, 371. 10. Zhu, W. H. and Loh, T. T., Differential effects of phorbol ester on apoptosis in HL-60 promyelocytic leukemia cells. Biochemistry and Pharmacology, 1996, 51, 1229. 11. Grant, S., Turner, A. J., Bartimole, T. M., Nelms, P. A., Joe, V. C. and Jarvis, W. D., Modulation of l-[beta-oarabinofuranosyl] cytosine-induced apoptosis in human myeloid leukemia cells by staurosporine and other pharmacological inhibitors of protein kinase C. Oncology Research, 1994, 6, 87. 12. Rinaudo, M. S., Su, K., Falk, L. A., Halder, S. and Mufson, R. A., Human interleukin-3 receptor modulates bcl-2 mRNA and protein levels through protein kinase C in TF-1 cells. Blood, 1995, 86, 80. 13. Tonetti, D. A., Henning-Chub, C., Yamanishi, D. T. and Huberman, E., Protein kinase C-p is required for macrophage differentiation of human HL-60 leukemia cells. Journal of Biological Chemistry, 1994, 269, 23230. 14. Tonetti, D. A., Horio, M., Collart, F. R. and Huberman, E., Protein kinase C b gene expression is associated with susceptibility of human promyelocytic leukemia cells to phorbol ester-induced differentiation. Cell Growth and Differentiation, 1992, 3, 739. 1.5. Rossi, F., McNagny, M., Smith, G., Frampton, J. and Graf, T., Lineage commitment of transformed haematopoietic progenitors is determined by the level of PKC activity. EMBO Journal, 1996, 15, 1894. 16. Wooten, M. W., Seibenhener, M. L. and Soh, Y., Expression of protein kinase C isoforms in HL60 and phorbol ester resistant HL525 cells. Cytobios, 1993,76,19. 17. Weinstein, I. B., Borner, C. M., Krauss, R. S., O’Driscoll, K., Choi, P. M., Moritomi, M., Hoshina, S., Hsieh, L.-L., Tchou-Wong, K.-M., Guadagno, S. N., Ueffing, M. and Guillem, J. Pleiotropic effects of protein kinase C and the concept of carcinogenesis as a progressive disorder in signal transduction. In Origins of Human Cancer: A Comprehensive Review, ed. J. Bruge, T. Curran, E. Harlow and F. McCormick. Cold Spring Harbor Laboratory Press, New York, 1991, pp. 113. 18. Ways, D. K., Posekany, K., devente, J., Garris, T., Chen, J., Hooker, J., Qin, W., Cook, P., Fletcher, D. and Parker, P., Overexpression of protein kinase C-zeta stimulates leukemic cell differentiation. Cell Growth and Differentiation, 1994, 5, 1195. 19. Emoto, Y., Kisaki, H., Manome, Y., Kharbanda, S. and Kufe, D., Activation of protein kinase C delta in human myeloid leukemic cells treated with I-beta-oarabinofuranosylcytosine. Blood, 1996, 87, 1990. 20. Yoshida, M., Feng, W., Saijo, N. and Ikekawa, T., Antitumor activity of daphnane-type diterpene gnidimacrin isolated from Stellera chamaejasme L. International Journal of Cancer, 1996, 66, 268. 21. Leszczynski, D., Leszczynski, K. and Servomaa, K., Long wave ultraviolet radiation causes increase of membranebound fraction of protein kinase C in rat myeloid leukemia cells. Photodermatology, Photoimmunology and Photomedicine, 1995, 11, 124. 22. Weiss, R. H., Yabes, A. P. and Sinaee, R., TGF-beta and phorbol esters inhibit mitogenesis utilizing parallel protein kinase C-dependent pathways. Department of Internal Medicine, Northern California System of Clinics, U.S.A., 1995, 48, 738. 23. Lafon, C., Mazars, P., Guerrin, M., Barboule, N., Charcosset, J. Y. and Valette, A., Early gene responses associated with transforming -- growth factor-beta 1 growth
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inhibition and autoinduction in MCF-7 breast adenocarcinoma cells. Biochimica et Biophysics Acta, 1995, 1266, 288. Elias, J. A., Zheng, T., Whiting, N. L., Marcovici, A. and Trow, T. K., Cytokine-cytokine synergy and protein kinase C in the regulation of lung fibroblast leukemia inhibitory factor. American Journal of Physiology, 1994, 266, L426. Schwende, H., Fitzke, E., Ambs, P. and Dieter, P., Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin Da. Journal of Leukocyte Biology, 1996, 59, 555. Savickiene, J., Gineitis, A., Shanbhag, V. P. and Stigbrand, T., Protein kinase inhibitors exert stage specific and inducer dependent effects on HL-60 cell differentiation. Anticancer Research, 1995, 15, 687. Yang, K. D., Mizobuchi, T., Kharbanda, S. M., Datta, R., Huberman, E., Kufe, D. W. and Stone, R. M., All-transretinoic acid reverses phorbol ester resistance in a human myeloid leukemia cell line. Blood, 1994, 83, 490. Denning, M. F., Dlugosz, A. A., Threadgill, D. W., Magnuson, T. and Yuspa, S. H., Activation of the epidermal growth factor receptor signal transduction pathway stimulates tyrosine phosphorylation of protein kinase C delta. Journal of Biological Chemistry, 1996, 271, 5325. Geiges, D., Marks, F. and Gschwendt, M., Loss of protein kinase C delta from human HaCaT keratinocytes upon ras transfection is mediated by TGF alpha. Experimental Cell Research, 1995, 219, 299. Nishikawa, K., Yamamoto, S., Nagumo, H., Otsuka, C. and Kato, R., Suppressive effect of transforming growth factorbeta on the phosphorylation of endogenous substrates by conventional and novel protein kinase C in primary cultured mouse epidermal cells. Biochemistry and Biophysics Research Communications, 1993, 193, 384. Cowin, A. J., Heaton, E. L., Cheshire, S. H. and Bidey, U. K., The proliferative responses of porcine thyroid follicular cells to epidermal growth factor and thyrotrophin reflect the autocrine production of transforming growth factorbeta 1. Journal of Endocrinology, 1996, 148, 87. Studer, R. K., Craven, P. A. and DeRubertis, F. R., Lowdensity lipoprotein stimulation of mesangial cell fibronectin synthesis: role of protein kinase C and transforming growth factor-beta. Journal of Laboratory and Clinical Medicine, 1995, 125, 86. Sheu, S. J., Sakamoto, T., Osusky, R., Wang, H. M., Ogden, T. E., Ryan, S. J., Hinton, D. R. and Gopalakrishna, R., Transforming growth factor-beta regulates human retinal pigment epithelial cell phagocytosis by influencing a protein kinase C-dependent pathway. Graefes Archives of Clinical and Experimental Oththalmology, 1994, 232, 695. Bos, M. P., van der Meer, J. M. and Herrmann-Erlee, M. P., Regulation of protein kinase C activity by phorbol ester, thrombin, parathyroid hormone and transforming growth factor-beta 2 in different types of osteoblastic cells. Bone Mineralogy, 1994, 26, 141. Niles, R. M., Use of vitamins A and D in chemoprevention and therapy of cancer: control of nuclear receptor expression and function. Vitamins, cancer and receptors. Advances in Experimental Medicine and Biology, 1995, 375, 1. Hunakova, L., Sedlak, J., Klobusicka, M., Sulikova, M. and Chorvath, B., Phorbol ester (TPA)-induced differential modulation of cell surface antigens in human pluripotential leukemia (K-562) cell line: effects of protein kinase
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inhibitors with broad- and PKC selective inhibitory activity. Neoplasma, 1995, 42, 249. 37. Baumgartner, R. A., Ozawa, K., Cunha-Melo, J. R., Yamada, K., Gusovsky, F. and Beaven, M. A., Studies with transfected and permeabilized RBL-2H3 cells reveal unique inhibitory properties of protein kinase C gamma. Molecular Biology of the Cell, 1994, 5, 475. 38. Kiley, S. C. and Parker, P. J., Differential localization of protein kinase C isozymes in U937 cells: evidence for distinct isozyme functions during monocyte differentiation. Journal of Cell Science, 1995, 108, 1003. 39. Zheng, B., Chambers, T. C., Raynor, R. L., Markham, P. N., Gebel, H. M., Vogler, W. R. and Kuo, J. F., Human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (protein phosphates inhibitor)
lacks protein kinase C-epsilon, exhibits multidrug resistance phenotype, and expresses drug pump P-glycoprotein. Journal of Biological Chemistry, 1994, 269, 12332. 40. Ryves, W. J., Dimitrijevic, S., Gordge, P. C. and Evans, F. J., HL-60 cell differentiation induced by phorbol- and 12deoxyphorbol esters. Carcinogenesis, 1994, 15, 2501. 41. Murray, N. R., Burns, D. J. and Fields, A. P., Presence of a beta II protein kinase C-selective nuclear membrane activation factor in human leukemia cells. Journal of Biological Chemistry, 1994, 269, 21385. 42. Macfarlane, D. E. and Manzel, L., Activation of betaisozyme of protein kinase C (PKC beta) is necessary and sufficient for phorbol ester-induced differentiation of HL60 promyelocytes. Studies with PKC beta-defective PET mutant. Journal of Biological Chemistry, 1994, 269, 4327.
Pergamon PII: SO145-2126(96)00121-X
COMMENTARY IS PKC ACTIVATION REQUIRED FOR LEUKEMIA DIFFERENTIATION?
CELL
Gayle E. Woloschak Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, Illinois. U.S.A. The role of protein kinase C (PKC) in the pathways of differentiation of leukemia cells is one of the most studied and least understood areas in hematology. It has been considered almost a universally accepted paradigm that PKC is essential for the induced differentiation of leukemic cells. The study of Manzel and Macfarlane [l] in this issue of Leukemia Research describes experiments demonstrating that PKC is not required for the dihydroxyvitamin Ds- or Transforming Growth Factor (TGF-Pi)-induced growth arrest (and consequently differentiation) in 10 different human leukemia cell lines. In these experiments, different isoforms (a, p, and y) of PKC were examined at the level of mRNA accumulation prior to or following exposure to 1,25dihydroxyvitamin Ds. The results of these expression experiments in the 10 different leukemia cell lines were correlated with induction of growth arrest (as a marker of differentiation) in the cells in the presence or absence of PKC inhibitors Bisindolylmaleimide I or II prior to exposure of the cells to TGFPt and 1,25-dihydroxyvitamin Ds. From those experiments, it can be concluded that TGF-fi and 1,25dihydroxyvitamin Da induce growth arrest via a PKC-independent mechanism. This finding is surprising in light of the wealth of data in other cell systems [2-4] and in leukemia cells [5-91 documenting an important role for PKC in growth arrest by other differentiating agents. Zeng and el-Deiry [6] recently have demonstrated that PKC is required for TPA-induced expression of the cyclin-dependent kinase (cdk)-inhibitor p21 WAFl/CIPl, which has been implicated in growth arrest caused by senescence, tumor suppression and terminal differentiation. In the report by Manzel and Macfarlane [l], cell growth arrest is assessed, but the relative contribution of differentiation and apoptosis to those results is not apparent. In fact, work by de Vente et al. [9] with U937 cells demonstrated that changes in PKC content and distribution within cells induce apoptosis rather than differentiation, suggesting that the decision of a cell to undergo death or differentiation in response to PKC activators may, in part, be modulated by PKC signal transduction pathway.
There may be other signals as well or, alternatively, PKC may influence both apoptosis and differentiation. Several reports have shown the importance of PKC activation for induction of apoptosis [l&12]. The mechanism of TGF-/It 1,25-dihydroxyvitamin Ds in induction of leukemia cell growth arrest appears to be different to those for TPA and other similar phorbol ester differentiation-inducing agents. There is a large body of evidence supporting the role of PKC in TPAinduced leukemia cell differentiation. Protein kinase C is the intracellular receptor for TPA and its derivative phorbol esters. Leukemic cells defective in PKC isoforms are not capable of differentiating in response to TPA [13-161. Other studies have shown that exposure of cells to antisense oligonucleotides to different PKC isoforms inhibits cell differentiation [ 171. Similarly, cells transfected with vectors causing over-expression of PKC are stimulated to undergo differentiation, even in the absence of exposure to differentiating agents [17, 181. There are also a wide variety of other agents (DNA damaging agents) capable of inducing leukemia cell differentiation via the PKC pathway [19-211. This has led to the idea that all cell differentiation in leukemia cells is PKC-dependent. Savickiene et al. P61, using a variety of different protein kinase inhibitors, similarly conclude that protein kinase activities in HL-60 cells are differentiation-stage-specific and that the specificity depends upon the actual inducing agents. Furthermore, HL-525 cells, which do not differentiate in response to TPA due to decreased PKC-fl expression relative to controls, can be induced to undergo differentiation in response to all-transretinoic acid, a pathway reported to be mediated by metabolically active vitamin D [27]. Together, these findings suggest that mechanisms regarding TPAinduced differentiation pathways may not be directly applicable to leukemia cell differentiation induced by vitamin D metabolites. In the studies of Manzel and Macfarlane [l], several cytokines were tested for the ability to modulate growth arrest induced by 1,25-dihydroxyvitamin Da-dihydroxy411
412
G. E. Woloschak
vitamin Da. Of the cytokines tested (TNFa, TGF/Jr, y-IFN, IL-l, and G-CSF), only TGF-/?r enhanced growth arrest in four out of 10 cell lines. The mechanisms of action of TGF-pi in this context are not clear, but are likely to be important, since TGF/Jr is released by many tumors and is found in inflammatory reactions. Reports in the literature on other cell types show that TGFflr is capable of inactivating PKCG by phosphorylation [28301, and that neutralization of TGFP with antibodies leads to enhanced PKC activation [31,32], suggesting that many TGF-fir effects are actually caused by a negative modulation of the PKC-dependent pathway [33-3.51. Whether TGF-P effects a change in PKC activity in leukemia cells is not clear at this point. Experiments by Manzel and Macfarlane [l] monitored mRNA expression, which was induced in some leukemic cells following vitamin D treatment, but did not measure PKC activation or translocation. It is interesting to speculate that the PKC-independent pathway for differentiation in leukemic cells is over-ridden by the PKC-dependent pathway and that the only way in which this pathway can be induced is if PKC is either depleted or rendered inactive in the cell. Further experiments aimed at determining the mechanism of differentiation pathway choice in leukemia cells will be important. It will be interesting also to explore whether other PKC-independent processes, such as cell surface antigen expression [36], also are stimulated by the vitamin D3 pathway of differentiation. For several years, different isoforms of PKC have been known, but it is only recently that the differential functions of these isoforms have begun to be elucidated. It is noteworthy that Manzel and Macfarlane [l] examined expression of several different isoforms of the enzyme, PKC-c(, p and 6. The PKC-y, which is found predominantly in brain and spinal cord [37], was not studied in these experiments and is not likely to be relevant to leukemia cell differentiation. Studies of PKC-E, zeta, and the newly discovered theta, would probably have led to more conclusive analysis of the association between vitamin D-induced differentiation and PKC expression. Studies by Kiley and Parker [38] have shown that individual PKCs localize to different subcellular compartments, suggesting a functional role for PKC sublocalization in the cell. Not all PKC isoforms are expressed in all leukemia cells. For example, U937 cells analyzed in the paper by Manzel and Macfarlane [l] are reported to express undetectable levels of PKC-a [38] and similarly show little mRNA accumulation [l]. TPA-induced leukemia cell differentiation is known to be isoform-specific [3841]. In addition, other studies by Macfarlane and Manzel [42] have demonstrated cell cycle regulation of PKC isoform expression. This leads to complications in understanding
the precise role of each PKC isoform in the differentiative process. Based on the studies of Manzel and Macfarlane [l, 421 and others in the literature, it is clear that differentiation of leukemic cells can take place by either PKC-dependent or PKC-independent pathways. This complicates our understanding of mechanisms underlying the differentiative process. Essential questions remain to be addressed: Are the two pathways ever activated at the same time? Which intracellular (intercellular?) signals transmit the pathway of choice for the cell? Do levels of activated PKC isoforms affect only the PKC-dependent pathway? Is it necessary to activate both pathways when using differentiating agents for therapy? Acknowledgements-The author wishes to thank MS Felicia King for her excellent assistance in preparing this manuscript and Drs Frank Collart and David Grdina for critical review prior to submission. This work was supported by the U.S. Department of Energy, Office of Health and Environmental Research, under Contract No. W-31-109-ENG-38.
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