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Protein kinase C involvement in apoptosis
Pergamon
Gen. Pharmac. Vol. 26, No. 5, pp. 881-887, 1995 Copyright© 1995ElsevierScienceLid Printed in Great Britain.All rights reserved 0306-3623/95 $29.00+ 0.00
0306-3623(94)00295-9
REVIEW
Protein Kinase C Involvement in Apoptosis MIGUEL LUCAS* and VICTOR S,~NCHEZ-MARGALET Departamento de Bioquimica Mbdica y Biologia Molecular, Hospital Universitario Virgen Macarena, Facultad de Medicina, Avda. S~chez Pizju~n 4, 41009 Sevilla, Spain [Tel./Fax: 345.455. 73.52] (Received 20 October 1994)
Abstract--l. Conflicting observations on the involvement of PKC in apoptosis point to a great variability depending on cell type, agent or condition causing apoptosis, phase of the cell cycle and intracellular signaling pathway. 2. Inhibition by PKC of store-operated calcium entry mechanisms, which are sensitive to the oncoprotein bcl-2, should block the activation of calcium-dependent enzymes triggering the apoptotic cell death. 3. Activation of phosphatases by ceramide and inhibition of PKC by sphingosine seem to mediate the sphingomyelin pathway to apoptosis. 4. A putative target protein appears to be p34cdc2which is regulated by a network of kinases and phosphatases. The uncoupling of timing for p34cat2 activation and the completion of DNA replication results in the so-called "mitotic catastrophe" that shares some features with apoptosis. Key Words: Programmed cell death, calcium, ceramide, bcl-2, p34cdc2
INTRODUCTION
PATHWAYS TO A P O P T O S I S
The death of cells in normal tissue turnover is called apoptosis or programmed cell death (Kerr et al., 1972). Unlike necrotic cell death, the process requires that target cell be active in its own death since it depends on the integrity of the cell and the biosynthesis of R N A and proteins (Schwartz et al., 1990). However, the last requirement is not a general characteristic as in the case of TNF
Agents or conditions inducing apoptosis show variable degree of dependency on different pathways with the main feature relying on: (a) the induction of c-myc, p53 or bcl-2 transcripts; (b) the cell cycle phase; (c) the biosynthesis of proteins; (d) the activation of kinases; and (e) the hydrolysis of sphingomyelin. These apparently polymorphic pathways could be related to its intracellular control which may vary significantly as a function of cell type, the state of the cell and the apoptosis-inducing agent (Golstein et al., 1991). Oncogenes and tumor suppressor genes appears to be clearly involved: in fact, p53-dependent and independent pathways have been described (Lowe et al., 1993; Clarke et al., 1993) as well as altered expression of oncogenes c-fos and c-myc (Buttyan et al., 1988), whereas a protein encoded by the oncogene bcl-2 has been shown to block programmed cell death (Hockenberry et al., 1990). Indeed, apoptotic cell death induced by c-myc is inhibited by bcl-2, indicating a novel mechanism for oncogene interaction of potential interest in carcinogenesis (Bisonnette et al., 1992; Fanidi et al., 1992). Recently, the v-raf kinase has been described to trigger a pathway, alternative to bcl-2, which sup-
*To whom all correspondence should be addressed. 881
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Miguel Lucas and Victor Sfinchez-Margalet
presses apoptosis and promotes cell cycle progression and cell survival (Cleveland et al., 1994). A final common step should recruit different pathways to apoptosis and lead to the enzymatic degradation of the nuclear membrane, the fragmentation of DNA and the formation of apoptotic bodies. The main candidate for the final step seems to be calcium since a number of calcium-mediated events are crucial in the apoptotic process (Arends et al., 1990; Fesus et al., 1991; Sarin et al., 1993). These events include activation of enzymes, such as endonuclease, protease, transglutaminase and phospholipase, and changes in ionic and water fluxes through plasma membrane leading to condensation of cytosol. Moreover, DNA fragmentation and apoptosis have been related to influx of calcium (McConkey et al., 1989a), ATP-dependent Ca 2+ uptake in nuclei (Nicotera et aL, 1989) and to increases in intranuclear free Ca :+ concentration (Bellomo et al., 1992); nonetheless, calcium-independent pathways have been proposed (Lennon et al., 1992; Iseki et al., 1993; Baffy et al., 1993). An alternative point of view is the consideration of calcium and other events, such as the activation of kinases, as both regulatory signals of the pathways to apoptosis and effectors of the final reactions causing apoptosis. The role of PKC in the induction of apoptosis has been complicated by conflicting reports. For instance, the observation that activation of PKC by exposure to PMA, either alone or in combination with calciumionophore, induces apoptosis in lymphoid cells (Mercep et al., 1989) and that PKC inhibitors prevent glucocorticoid-induced apoptosis of thymocytes (Ojeda et al., 1990) suggest that PKC activation promotes apoptosis. On the other hand, the ability of PMA to oppose steroid-induced apoptosis in thymic lymphocytes (McConkey et al., 1989b), to prevent the death of T-lymphocytes deprived of interleukin-2 (Nieto and Lrpez-Rivas, 1989; Rodriguez-Tarducy and Lrpez-Rivas, 1989), radiation-induced apoptosis in vitro (Tomei et al., 1988) as well as serum deprivation-induced apoptosis of mature lymphocytes (Lucas et al., 1991) support an antagonistic effect of PKC in the apoptotic process (see also Table 1 that summarizes more recent data on the involvement of PKC in apoptotic cell death). It is conceivable that conflicting observations regarding the apparent role of PKC in the regulation of apoptosis reflect cell type-specific responses to triggering agents (Jarvis et al., 1994a). The present article deals with the implication of PKC in programmed cell death and in cell survival pointing to possible mechanisms and PKC-targets during the apoptotic process. The role of other kinases, such as tyrosine-kinases (Otani et al., 1993) or the double-stranded RNA-activated
kinase recently described to induce apoptosis (Lee and Esteban, 1994) is out of the scope of this review. STORE-OPERATED CALCIUM ENTRY, BCL-2 AND APOPTOSIS bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death (Hockenberry et al., 1990) by mechanisms which cannot be related to metabolic process such as ATP depletion since bcl-2 blocks apoptosis, induced by either serum deprivation or staurosporine, even in cells lacking mitochondrial DNA. Moreover bel-2 protein is known to be associated with the nuclear envelope and the endoplasmic reticulum as well as the mitochondrial membrane (Jacobson et al., 1993). An interesting observation was the inhibition by bcl-2 oncoprotein of the apoptosis induced by withdrawal of interleukins that was clearly associated with the repartitioning of intracellular calcium (Baffy et al., 1993). These observations have been reinforced by the experiments with thapsigargin, an inhibitor of the
1
2
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4
Fig. 1. Internucleosomal breakdown of DNA. Peripheral blood lymphocytes (PBL) were incubated for 72hr as follows: medium lacking fetal bovine serum (lanes 1 and 2) and supplemented with 50riM PMA (lane I); complete medium, including 10% heat-inactivated fetal bovine serum and 1/~M staurosporine (lane 4). DNA was extracted and labelled with [~32p]-dCTP and the Klenow fragment of DNA polimerase. Lane 3 was loaded with a 100 bp molecular size marker.
Protein kinase C involvement in apoptosis
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Table I. Dnta supporting that PKC activation Mocks apopto~lis DNA fragmentation is associated with down regulation of PKC in promyclocytic leukemia cells (Jarvis et aL, 1994b, c) PKC inhibitors enhance apoptosis in mouse natural killer cells and cytotoxic T lymphocytes (Migliorati et al., 1994) The activation of PKC promotes cell survival of mature lymphocytes prone to apoptosis (Lucas et al., 1994) and protects from radiation-induced apoptony (Radford, 1994) Cyclosporine A proteCts B cells against calcium-dependent apoptosis, by blockade of the phosphoprotein phosphatase calcineurin, but not against the apoptosis triggered by the PKC inhibitor chelerytrine (Bonnefoyberard et al., 1994) Proper combination of calcium ionophore and protein kinase activator (PMA) inhibits corticosterone-induced apoptosis in lymphocytes (Iseki et al., 1993) Apoptosis, occurring at a high rate among B cells in germinal centers can be arrested by protein kinase C-activating phorbol esters (Knox et aL, 1993) Inhibition, as well as down regulation, of PKC cause apoptosis in freshly isolated rat bepatocytes (S/mchez et al., 1992) The inhibition of PKC by staurosporine triggers apoptosis of insulin-secreting RIN m5F cells without raising cytosolic free calcium (Shnchez-Margalet et al., 1993) Inhibitors of PKC block the prolongation of neutrophil survival induced by granulocyte colony-stimulatingfactor (G-CSF) and induce DNA fragmentation at concentrations that fail to alter the priming effect of G-CSF (Adachi et al., 1993) Translocation of PKC from the cytosol mediates phosphatidyl inositol-dependent pathway of rescue germinal center B cells from apoptosis (Knox and Gordon, 1994) Basic fibroblast growth factor (bFGF) and phorbol esters protect endothelial cells against radiation-induced apoptosis. The mechanism of action of bFGF involves activation of tyrosine-kinase which in turns causes the translocation of the alpha isotype of cytoplasmic PKC into the membrane (Haimovitzfriedman et al., 1994a) PKC inhibitors induce apoptosis of malignant glioma cells (Couldwell et aL, 1994) Selective PKC inhibitors block IL-2-mediated proliferation of routine T cells and cause apoptosis (G6mez et al., 1994) PKC activation blocks both radiation-induced sphingomyelin hydrolysis and apoptosis of aortic endothelial cells (Haimovitzfriedman et al., 1994b) Haggerty and Monroe (1994) describe a mutant of a B lymphocyte line sensitive to apoptosis caused by signaling components that lie downstream of PKC Data supporting that PKC activation promotes apoptosis Fragmentation of DNA induced by tyrosine kinase-inhibitors in mouse thymocytes is enhanced by phorbol esters capable of activating PKC (Azuma et al., 1993) Serine-threonine phosphatase inhibitors synergistically augment TNF-induced apoptosis in several TNF-sensitive tumor cell lines including histiocytic lymphoma, mammary carcinoma, and prostatic tumor cells (Wright et al., 1993) Phorbol ester induces apoptosis in promyelocytic leukemia cells (Macfarlane and O'Dnnnell, 1993; Macfarlane and Manzel, 1994) Phorbol ester enhances mitoxantrone-induced internucleosomal DNA fragmentation in human myeloid leukemia cells, whereas PKC inhibitors have no effects (Bhalla et al., 1994) Retinoic acid and activators of PKC enhance apoptosis of neuronal cells induced by serum deprivation down regulation of PKC reduces the ability of retinoic acid to induce apoptosis (Mailhos et al., 1994) Radiation-induced apoptosis of mouse thymocytes is prevented by a PKC inhibitor and. is potentiated by the PKC activator phorbol ester (Shaposhnikova et al., 1994) Phorbol esters leads to growth arrest an apoptosis of a continuously proliferating B lymphocyte line (Haggerty and Monroe, 1994)
calcium pumping ATPase of the endoplasmic reticulum that causes persistent depletion of intracellular calcium stores and produces apoptosis of hepatoma cell lines (Kaneko and Tsukamoto, 1994). This apparent paradox (the association of calcium depletion and apoptosis) can be explained taking into account
the so-called "capacitative" (store-operated) model o f c a l c i u m e n t r y t h a t is r e g u l a t e d by t h e d e g r e e of depletion of the endoplasmic reticulum calcium p o o l . I n t e r e s t i n g l y , this s t o r e - o p e r a t e d c a l c i u m e n t r y mechanism
is i n h i b i t e d by s t i m u l a t i o n o f p r o t e i n
k i n a s e C ( M o n t e r o e l a l . , 1993). T h e i n h i b i t i o n o f
Fig. 2. Thapsigargin (Tg), by inhibiting Ca2+-pumping ATPase in endoplasmic reticulum (er), depletes this intracellular calcium pool which in turn triggers the entry of calcium. The oncoprotein bcl-2 blocks the effect o f thapsigargin in the Ca2+-pumping ATPase. The activation of P K C inhibits the entry of calcium by the store operated "capacitative" mechanism (Montero et al., 1993).
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Miguel Lucas and Victor Shnchez-Margalet Sphingomyelin
Ceramide
Sphingosine
Phosphatase
Kinase
APOPTOSIS
Fig. 3. Scheme of the putative mechanism of the sphingomyelinase pathway to apoptosis. The hydrolysis of sphingomyelin produces ceramide and sphingosine which, by activating phosphatase and inhibiting kinase respectively, regulate the activity of target proteins (T.P.) hypothetical effectors of apoptosis. calcium entry should block the activation of calciumdependent enzymes triggering the apoptotic cell death. In fact Lam et al. (1994) have recently explained the role of bcl-2 in the repression of apoptosis by regulating endoplasmic reticulumassociated calcium fluxes. The induction of apoptosis by thapsigargin is blocked by bcl-2 and may be explained assuming that the oncoprotein, by inhibiting calcium leaking from the endoplasmic reticulum hinder the thapsigargin-induced store-operated calcium entry and therefore prevents the permanent increase of cytosolic free calcium that causes apoptosis. This could also be a general mechanism of the abrogation of apoptosis by phorbol esters since it inhibits the store-operated calcium entry into the cytosol (Montero et al., 1993). Nonetheless, a direct effect of PKC on bcl-2 has been described indicating that hematopoietic growth factors may inhibit apoptosis by a molecular mechanism involving activation and phosphorylation of bcl-2 (May et al., 1993).
acetate and synthetic diglycerides, suggesting opposite roles for digyiceride- and' ceramide-mediated signals in the regulation of apoptosis. Therefore, ceramide is a possible mediator of apoptosis in response to a number of agents, including interferon and hypoxia, that causes sphingomyelin hydrolysis. Besides, a ceramide-activated protein phosphatase could mediate the effects of ceramide (Dobrowsky and Hannum, 1992). On the other hand, sphingosine, a breakdown product of sphingolipids, is well known for its pharmacological inhibition of PKC. The coincidence of both complementary events may argue in favor of a PKC mediated pathway by inhibiting the phosphorlyation and activating the phosphatase of target proteins. Very recently, it has been described that ionizing radiation acts on cellular membranes of bovine and aortic endothelial cells to generate ceramide and initiate apoptosis, suggesting an alternative to the hypothesis that direct DNA damage mediates radiation-induced cell killing. The authors (Haimovitzfriedman et al., 1994a, b) indicate that PKC activation blocked both radiation-induced sphingomyelin hydrolysis and apoptosis. Cell type seems important since radiation-induced apoptosis of mouse thymocytes is prevented by PKC inhibitors
I Synthosis I P
J
Y
I
Mitosis I
P
f
T
Y
T
?l ? Y
T
PKC AND THE SPHINGOMYELIN PATHWAY
TO APOPTOSIS Sphingolipid breakdown products have anti-proliferative and tumor-suppressor properties (Hannun and Linardic, 1993) and the hydrolysis of sphingomyelin and ceramide generation have been implicated in a signal transduction pathway that mediates the effects of tumor necrosis factor alpha, TNF~. Moreover, it has been described that ceramide (Obeid e t al., 1993) as well as the activation of the sphingomyelin hydrolysis (Jarvis et al., 1994a) induce programmed cell death, a process that is inhibitible by the protein kinase C activators phorbol-myristate
Fig. 4. The entry into the mitosis phase requires the activation of p34~2 kinase following dephosphorylation and association with cyclins. The uncoupling of these events, by p34¢d¢2activation before DNA replication is completed, causes "mitosis catastrophe", a process quite similar to apoptosis. The triggering of apoptosis by the uncoupling of completion of DNA synthesis with events leading to nuclear membrane disintegration and apoptotic bodies formation is an interesting hypothesis with a number of interrogations (see also Oberharmer et al., 1994).
Protein kinase C involvement in apoptosis whereas it is potentiated by activation of PKC (Shaposnikova et al., 1994).
APOPTOSIS VERSUS MITOSIS The uncoupling of timing for p34~c2 activation and the completion of DNA replication results in the so-called "mitotic catastrophe" that appears results from mitosis during DNA replication (Nurse, 1990; Heald et aL, 1993). P34~c2 is a highly regulated serine-threonine kinase that controls cell entry into mitosis. The regulation of p34cd~2is known to involve a network of kinases and phosphatases, that may respond to the state of DNA replication, as well as the formation of complexes with cyclins (Nurse, 1990). Entry into M-phase is determined by activation of p34¢dc2which requires p34°de dephosphorylation of phosphotyrosine and phosphothreonine and association with cyclins. The active form of the kinase leads to the phosphorylation of key substrates: H1hitone, p60src, iamins, centrosomal proteins and other proteins which need to be displaced from chromatin to allow chromosome condensation. The complex of p34~C2/cyclins initiates the dissolution of nuclear membrane and promote chromatin condensation, events that take place in both mitosis and apoptosis (Meikrantz et al., 1994). It has been recently proposed that premature p34 ~dc2 activation may be a general mechanism by which cells induced to undergo apoptosis initiate the disruption of the nucleus. This has been deduced from experiments with fragmentin and with staurosporine that induces dephosphorylation of p34~dc2 and apoptosis in lymphoma and mammary carcinoma cell lines (Shi et al., 1994). Staurosporine is a potent PKC inhibitor with a broad apoptotic activity in cell lines from different origins (Bertrand et al., 1994). This hypothesis may be questioned since Oberharmer et al. (1994) have recently shown that chromatin condensation during apoptosis seems to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34~¢2 kinase; in contrast to mitosis, dephosphorylation and activation of p34~dc2 does not occur in apoptotic cells.
REFERENCES Adachi S., Kubota M., Matsubara K., Wakazono Y., Hirota H., Kuwakado K., Akiyama Y. and Mikawa H. (1993) Role of protein kinase C in neutrophil survival enhanced by granulocyte colony-stimulating factor. Exp. Hemat. 21, 1709-1713. Arends M. J., Morris R. G., WyllieA. H. (1990) Apoptosis. The role of endonuclease. Am. J. Path. 136, 593-608. Azuma Y., Onishi Y., Sato-Y. and Kizaki H. (1993) Induction of mouse thymocyte apoptosis by inhibitors of tyrosine kinases is associated with dephosphorylation of nuclear proteins. Cell Immun. 152, 271-278.
885
Baffy G., Miyashita T., Williamson J. R. and Reed J. C. (1993) Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production. J. Biol. Chem. 268, 6511-6519. Bellomo G., Perotti M., Taddei F., Mirabelli F., Finardi G., Nicotera P. and Orrenius S. (1992) Tumor necrosis factor induces apoptosis in mammary adenocarcinoma cells by an increase in intranuclear free Ca2+ concentration and DNA fragmentation. Cancer Res. 52, 1342-1346. Bertrand R., Solary E., Oconnor P., Kohn K. W. and Pommier Y. (1994) Induction of a common pathway of apoptosis by staurosporine. Exp. Cell Res. 211, 314-321. Bhalla K., Ibrado A. M., Tourkina E., Tank C., Grant S., Bullock G., Huang Y,, Ponnathpur V. and Mahoney M. E. (1993) High-dose mitoxantrone induces programmed cell death or apoptosis in human myeloid leukemia cells. Blood 82, 3133-3140. Bissonnette R. P., Echeverri F., Mahboubi A. and Green R. (1992) Apoptotic cell death induced by C-myc is inhibited by bcl-2. Nature 359, 552-554. Bonnefoyberard N., Genestier L., Flacher M. and Revillard J. P. (1994) The phosphoprotein phosphatase calcineurin controls calcium-dependentapoptosis in B cell lines. Eur. J. Immun. 24, 325-329. Buttyan R., Zakeri Z., Lockshin R. and Wogelmuth D. (1988) Cascade induction of c-los, c-myc and heat shock 70 K transcripts during the regression of the ventral prostate glan. Molec. Endocr. 2, 650-657. Clarke A. R., Purdie C. A., Harrison D. J., Morris R. G., Bird C. C., Hooper M. L, and Wyllie A. H. (1993) Thymocyte apoptosis is induced by p53-dependent and independent pathways. Nature 362, 849-852. Cleveland F. L., Gonzfilez-GarciaM., Nufiez G., lhle J. H. and Rapp U. R. (1994) V-raf suppresses apoptosis and promotes growth of interleukin-3-dependent myeloid cells. Oncogen 9, 2217-2226. Collins M. K. L. and Lrpez-Rivas A. (1993) The control of apoptosis in mammalian cells. Trends Biochem Sci. 18, 307-309. Couldwell W. T., Hinton D. R., He S. K., Chen T. C., Sebat I., Weiss M. H. and Law R. E. (1994) Protein kinase C inhibitors induce apoptosis in human malignant glioma cell lines FEBS Lett. 345, 43-46. Dobrowsky R. T. and Hannun Y. A. (1992) Ceramide stimulates a cytosolic protein phosphatase. J. Biol. Chem. 267, 5048-5051. Fanidi A., Harrington E. A. and Evan G. I. (1992) Cooperative interaction between C-myc and bcl-2 protooncogenes. Nature 359, 554-556. Fesus L., Davies P. J. A, and Piacentini M. (1991) Apoptosis: molecular mechanismsin programmed cell death. Eur. J. Cell Biol. 56, 170-177. Golstein P., Ojcius D. M. and Young J. D. E. (1991) Cell death mechanism and the immune system. Immun. Rev. 121, 29-65. G6mez J., Delahera A., Silva A., Pitton C., Garcia A. and Rebollo A. (1994) Implication of protein kinase C in IL-2-Mediated proliferation and apoptosis in a murine T cell clone. Exp. Cell Res. 213, 178-182. Green D. R. and Scott D. W. (1994) Activation-induced apoptosis in lymphocytes. Curr. Opin. lmmun. 6, 476-487. Haggerty H. G. and Monroe J. G. (1994) A mutant of the WEHI-231 B lymphocyte line that is resistant to phorbol esters is still sensitiveto antigen receptor-mediated growth inhibition. Cell Immun. 154, 166-180. Haimovitzfriedman A., Balaban N., Mcloughlin M., Ehleiter D., MichaeliJ., Vlodavsky I. and Fuks Z. (1994a) Protein kinase C mediates basic fibroblast growth factor protection of endothelial cells against radiation-induced apoptosis. Cancer Res. 54, 2591-2597.
886
Mignel Lucas and Victor S~inchez-Margalet
Haimovitzfriedman A., Kan C. C., Ehleiter D., Persaud R. S., Mcloughlin M., Fuks Z. and Kolesnick R. N. (1994b) Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis. J. Exp. Med. 180, 525-535. Hannun Y. A. and Linardic C. M. (1993) Sphingolipid breakdown products: and anti-proliferative and tumorsuppressor lipids. Biochim. Biophys. Acta 1154, 223-236. Heald R., MacLoughlin M. and McKeon F. (1993) Human Weel maintains mitotic timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase. Cell 74, 463-474. Hockenberry D., Nufiez G., Milliman C., Schreiber R. D. and Korsmeyer S. J. (1990) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348, 334-336. Iseki R., Kudo Y. and lwata M. (1993) Early mobilization of Ca 2+ is not required for glucocorticoid°induced apoptosis in thymocytes, J. lmmun. 151, 5198-5207. Jacobson M. D., Burne J. F., King M. P., Miyashita T., Reed J. C. and Raft M. C. (1993) Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA. Nature 361,365-369. Jarvis W. D., Kolesnick R. N., Fornari F. A., Traylor R. S., Gerwitz D. A. and Grant S. (1994a) Induction of apoptotic D N A damage and cell death by activation of the sphingomyelin pathway. Proc. Natn Acad. Sci. U.S.A. 91, 73-77. Jarvis W. D., Turner A. J., Povirk L. F., Traylor R. S. and Grant S. (1994b) Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C. Cancer Res. 5,t, 1707-1774. Jarvis W. D., Povirk L. F., Turner A. J., Traylor R. S., Gewirtz D. A., Pettit G. R. and Grant S. T. I. (1994c) Effects of bryotstatin 1 and other pharmacological activators of protein kinase C on 1-[beta-D-arabinofuranosyl]cytosine-induced apoptosis in HL-60 human promyelocytic leukemia cells. Biochem. Pharmac. 47, 839-852. Kaneko Y. and Tsukamoto A. (1994) Thapsigargin-induced persistent intracellular calcium pool depletion and apoptosis in human hepatoma cells. Cancer Lett. 79, 147-155. Kerr J. F. R., Wyllie A. H. and Currie A. R. (1972) Apoptosis: a basic biological phenomenon with wideranging implications in tissue kinetics. Br. J. Cancer 26, 239-257. Knox K. A. and Gordon J. (1994) Protein tyrosine kinases couple the surface immunoglobulin of germinal center B cells to phosphatidylinositol-dependent and -independent pathways of rescue from apoptosis. Cell Immun. 155, 62-76. Knox K. A., Hhonson G. D. and Gordon J. (1993) Distribution of cAMP in secondary follicles and its expression in B cell apoptosis and CD40-mediated survival. Int. lmmun. 5, 1085-1091. Lam M., Dubyak G., Chen L., Nunez G., Miesfeld R. L. and Distelhorst C. W. (1994) Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulumassociated Ca 2+ fluxes. Proc. Natn Acad. Sci. U.S.A. 91, 6569-6573. Lee S. B. and Esteban M. (1994) The interferon-induced double-stranded RNA-activated protein kinase induces apoptosis. Virology 199, 491-496. Lennon S. V., Kilfeather S. A., Hallet M. B., Campbell A. K. and Gotter T. G. (1992) Elevations in cytosolic free Ca 2+ are not required to trigger apoptosis in human leukaemia cells. Clin. Exp. lmmun. 87, 565-571, Lowe S. W., Schmitt E. M., Smith S. W., Osborne B. A. and Jacks T. (1993) p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362, 847-849. Lucas M., Solano F. and Sanz A. (1991) Induction of programmed cell death (apoptosis) in mature lymphocytes. FEBS Lett. 1, 19-20.
Lucas M., Sanchez-Margalet V., Sanz A. and Solano F. (1994) Protein kinase C activation promotes cell survival in mature lymphocytes prone to apoptosis. Biochem. Pharmac. 47, 667-672. Macfarlane D. E. and O'Donnell P. S. (1993) Phorbol ester induces apoptosis in HL-60 promyelocytic leukemia cells but not in HL-60 PET mutant. Leukemia 7, 1846-1851. Macfarlane D. E. and Manzel L. (1994) Activation of beta-isozyme of protein kinase C (PKC beta) is necessary and sufficient for phorbol ester-induced differentiation of HL-60 promydocytes. Studies with PKC beta-defective PET mutant. J. Biol. Chem. 269, 4327-4231. Mailhos C., Howard M. K. and Latchman D. S. (1994) A common pathway mediates retinoic acid and PMAdependent programmed cell death (apoptosis of neuronal cells). Brain Res. 644, %12. May W. S., Tyler P. G., Armstrong D. K. and Davidson N. E. (1993) Role for serine phosphorylation of Bcl-2 in an antiapoptotic signaling pathways triggered by IL-3, EPO and bryostain. Blood 82 (Suppl. 1), 1738. McConkey D. J., Nicotera P., Hartzell P., Bolloma G., Wyllie A. H. and Orrenius S. (1989a) Glucocorticoids activate a suicide process in thymocytes through an elevation of cytosolic Ca :+ concentration. A rchs Biochem. Biophys. 269, 365-370. McConkey D. J., Harztell P., Jondal M. and Orrenius S. (1989b) Inhibition of DNA fragmentation in thymocytes and isolated thymocyte nuclei by agents that stimulate protein kinase C. J. Biol. Chem. 264, 13399-13402. McConkey D. J., Orrenius S. and Jondal M. (1990) Cellular signaling in programmed cell death (apoptosis). lmmun. Today 11, 120-121. Meikrantz W., Gisselbrecht S., Tam S. W. and Schlegel R. (1994) Activation of cyclin A-dependent protein kinases during apoptosis Proc. Natn Acad. Sci. U.S.A. 91, 3754-3758. Mercep M., Noguchi P. D. and Ashwell J. D. (1989) The cell cycle book and lysis of an activated T-cell hybridoma are distinct processes with different Ca 2+ requirements and sensitivity to cyclosporine A. J. lmmun. 142, 4085-4092. Migliorati G., Nicoletti I., D'Adamio F., Spreca A., Pagliacci C. and Riccardi C. (1994) Dexamethasone induces apoptosis in mouse natural killer cells and cytotoxic T lymphocytes. Immunology $1, 21-26. Montero M., Garcia-Sancho J. and Alvarez J. (1993) Transient inhibition by chemotactic peptide of a storeoperated Ca 2+ entry pathway in human neutrophils. J. Biol. Chem. 268, 13055-13061. Nicotera P., McConkey D. J., Jones D. P. and Orrenius S. (1989) ATP stimulates Ca 2+ uptake and increases the free Ca -'+ concentration in isolated liver nuclei. Proc. Natn Acad. Sci. U.S.A. 86, 453-457. Nieto M. A. and L6pez-Rivas A. (1989) IL°2 protects T lymphocytes from glucocorticoid-induced DNA fragmentation and cell death, J. lmmun. 142, 4166-4170. Nurse P. (1990) Universal control mechanism regulating onset of M-phase. Nature 344, 503-509. Obeid U M., Linardic C. M., Karolak L. A. and Hannun Y. A. (1993) Programmed cell death induced by ceramide. Science 259, 1769-1771. Oberhammer F. A., Hocbegger K. and Froschl G. (1994) Chromatin condensation during apoptosis is accompanied by degradation of lamin A + B without activation of cdc2 kinase. 3". Cell BioL 126, 827-837. Ojeda F., Guarda M., Maldonado C. and Foleh H. (1990) Protein kinase C involvement in thymoeyte apoptosis induced by hydrocortisone. Cell Immun. 125, 535-539. Otani H., Erdos M. and Leonard W. J. (1993) Tyrosine kinase(s) regulate apoptosis and bcl-2 expression in a growth factor-dependent cell line. J. BioL Chem. 268, 22733-22736.
Protein kinase C involvement in apoptosis Radford I. R. (1994) Phorbol esters can protect mouse pre-T cell lines from radiation-induced rapid interphase apoptosis. Int. J. Radiat. Biol. 65, 345-355. Raft M. C. (1992) Social controls on cell survival and cell death. Nature 356, 397~,00. Rodriguez-Tarducy G. and L6pez-Rivas A. (1989) Phorbol esters inhibits apoptosis in IL-2-dependent T lymphocytes. Biochem. Biophys. Res. Commun. 164, 1069-1075. S~.nchez V., Lucas M., Sanz A. and Goberna R. (1992) Decrease protein kinase C activity is associated with programmed cell death (apoptosis) in freshly isolated rat hepatocytes. Biosci, Report 12, 199-206. Sanchez-Margalet V., Lucas M., Solano F. and Goberna R. (1993) Sensitivity of insulin-secreting RIN m5F cells to undergoing apoptosis by the protein kinase C inhibitor staurosporine. Exp. Cell Res. 209, 160-163. Sarin A., Adams D. H. and Henkart P. A. (1993) Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells. J. Exp. Med. 178, 1693-1700. Schwartz L. M., Kosz L. and Kay B. K. (1990) Gene activation is required for developmentally programmed cell death. Proc. Natn Acad. Sci. U.S.A. 87, 6594-6598.
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Shi L., Nishioka W. K., Th'ng J., Bradbury E. M., Litchfield D. W. and Greenberg A. H. (1994) Premature p34~c2 activation required for apoptosis. Science 263, 1143-1145. Shaposnikova V. V., Dobrovinskaya O. R., Eidus L. K. and Korystov Y. N. (t 994) Dependence of thymocyte apoptosis on protein kinase C and phospholipase A 2. FEBS Lett. 340, 319-319. Tomei D. L., Kanter P. and Wenner C. E. (1988) Inhibition of radiation-produced apoptosis in vitro by tumor promoters. Biochem. Biophys. Res. Commun. 155, 324-331. Wright S. C., Zheng H., Zhong J., Torti F. M. and Larrick J. W. (1993) Role of protein phosphorylation in TNFinduced apoptosis: phosphatase inhibitors synergize with TNF to activate DNA fragmentation in normal as well as TNF-resistant U937 variants. J. Cell Biochem. 53, 222-233. Wright S. C., Zhing J. and Larrick J. W. (1994) Inhibition of apoptosis as a mechanism of tumor promotion. FASEB Jl 8, 654-660. Wyllie A. H. (1988) Apoptosis. ISI Atlas of Science: Immunology 1, 192-196. Wyllie A. H., Kerr J. F. R. and Currie A. R. (1980) Cell death: the significance of apoptosis. Int. Rev. Cvtol. 68, 251-356.
Gen. Pharmac. Vol. 26, No. 5, pp. 881-887, 1995 Copyright© 1995ElsevierScienceLid Printed in Great Britain.All rights reserved 0306-3623/95 $29.00+ 0.00
0306-3623(94)00295-9
REVIEW
Protein Kinase C Involvement in Apoptosis MIGUEL LUCAS* and VICTOR S,~NCHEZ-MARGALET Departamento de Bioquimica Mbdica y Biologia Molecular, Hospital Universitario Virgen Macarena, Facultad de Medicina, Avda. S~chez Pizju~n 4, 41009 Sevilla, Spain [Tel./Fax: 345.455. 73.52] (Received 20 October 1994)
Abstract--l. Conflicting observations on the involvement of PKC in apoptosis point to a great variability depending on cell type, agent or condition causing apoptosis, phase of the cell cycle and intracellular signaling pathway. 2. Inhibition by PKC of store-operated calcium entry mechanisms, which are sensitive to the oncoprotein bcl-2, should block the activation of calcium-dependent enzymes triggering the apoptotic cell death. 3. Activation of phosphatases by ceramide and inhibition of PKC by sphingosine seem to mediate the sphingomyelin pathway to apoptosis. 4. A putative target protein appears to be p34cdc2which is regulated by a network of kinases and phosphatases. The uncoupling of timing for p34cat2 activation and the completion of DNA replication results in the so-called "mitotic catastrophe" that shares some features with apoptosis. Key Words: Programmed cell death, calcium, ceramide, bcl-2, p34cdc2
INTRODUCTION
PATHWAYS TO A P O P T O S I S
The death of cells in normal tissue turnover is called apoptosis or programmed cell death (Kerr et al., 1972). Unlike necrotic cell death, the process requires that target cell be active in its own death since it depends on the integrity of the cell and the biosynthesis of R N A and proteins (Schwartz et al., 1990). However, the last requirement is not a general characteristic as in the case of TNF
Agents or conditions inducing apoptosis show variable degree of dependency on different pathways with the main feature relying on: (a) the induction of c-myc, p53 or bcl-2 transcripts; (b) the cell cycle phase; (c) the biosynthesis of proteins; (d) the activation of kinases; and (e) the hydrolysis of sphingomyelin. These apparently polymorphic pathways could be related to its intracellular control which may vary significantly as a function of cell type, the state of the cell and the apoptosis-inducing agent (Golstein et al., 1991). Oncogenes and tumor suppressor genes appears to be clearly involved: in fact, p53-dependent and independent pathways have been described (Lowe et al., 1993; Clarke et al., 1993) as well as altered expression of oncogenes c-fos and c-myc (Buttyan et al., 1988), whereas a protein encoded by the oncogene bcl-2 has been shown to block programmed cell death (Hockenberry et al., 1990). Indeed, apoptotic cell death induced by c-myc is inhibited by bcl-2, indicating a novel mechanism for oncogene interaction of potential interest in carcinogenesis (Bisonnette et al., 1992; Fanidi et al., 1992). Recently, the v-raf kinase has been described to trigger a pathway, alternative to bcl-2, which sup-
*To whom all correspondence should be addressed. 881
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Miguel Lucas and Victor Sfinchez-Margalet
presses apoptosis and promotes cell cycle progression and cell survival (Cleveland et al., 1994). A final common step should recruit different pathways to apoptosis and lead to the enzymatic degradation of the nuclear membrane, the fragmentation of DNA and the formation of apoptotic bodies. The main candidate for the final step seems to be calcium since a number of calcium-mediated events are crucial in the apoptotic process (Arends et al., 1990; Fesus et al., 1991; Sarin et al., 1993). These events include activation of enzymes, such as endonuclease, protease, transglutaminase and phospholipase, and changes in ionic and water fluxes through plasma membrane leading to condensation of cytosol. Moreover, DNA fragmentation and apoptosis have been related to influx of calcium (McConkey et al., 1989a), ATP-dependent Ca 2+ uptake in nuclei (Nicotera et aL, 1989) and to increases in intranuclear free Ca :+ concentration (Bellomo et al., 1992); nonetheless, calcium-independent pathways have been proposed (Lennon et al., 1992; Iseki et al., 1993; Baffy et al., 1993). An alternative point of view is the consideration of calcium and other events, such as the activation of kinases, as both regulatory signals of the pathways to apoptosis and effectors of the final reactions causing apoptosis. The role of PKC in the induction of apoptosis has been complicated by conflicting reports. For instance, the observation that activation of PKC by exposure to PMA, either alone or in combination with calciumionophore, induces apoptosis in lymphoid cells (Mercep et al., 1989) and that PKC inhibitors prevent glucocorticoid-induced apoptosis of thymocytes (Ojeda et al., 1990) suggest that PKC activation promotes apoptosis. On the other hand, the ability of PMA to oppose steroid-induced apoptosis in thymic lymphocytes (McConkey et al., 1989b), to prevent the death of T-lymphocytes deprived of interleukin-2 (Nieto and Lrpez-Rivas, 1989; Rodriguez-Tarducy and Lrpez-Rivas, 1989), radiation-induced apoptosis in vitro (Tomei et al., 1988) as well as serum deprivation-induced apoptosis of mature lymphocytes (Lucas et al., 1991) support an antagonistic effect of PKC in the apoptotic process (see also Table 1 that summarizes more recent data on the involvement of PKC in apoptotic cell death). It is conceivable that conflicting observations regarding the apparent role of PKC in the regulation of apoptosis reflect cell type-specific responses to triggering agents (Jarvis et al., 1994a). The present article deals with the implication of PKC in programmed cell death and in cell survival pointing to possible mechanisms and PKC-targets during the apoptotic process. The role of other kinases, such as tyrosine-kinases (Otani et al., 1993) or the double-stranded RNA-activated
kinase recently described to induce apoptosis (Lee and Esteban, 1994) is out of the scope of this review. STORE-OPERATED CALCIUM ENTRY, BCL-2 AND APOPTOSIS bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death (Hockenberry et al., 1990) by mechanisms which cannot be related to metabolic process such as ATP depletion since bcl-2 blocks apoptosis, induced by either serum deprivation or staurosporine, even in cells lacking mitochondrial DNA. Moreover bel-2 protein is known to be associated with the nuclear envelope and the endoplasmic reticulum as well as the mitochondrial membrane (Jacobson et al., 1993). An interesting observation was the inhibition by bcl-2 oncoprotein of the apoptosis induced by withdrawal of interleukins that was clearly associated with the repartitioning of intracellular calcium (Baffy et al., 1993). These observations have been reinforced by the experiments with thapsigargin, an inhibitor of the
1
2
3
4
Fig. 1. Internucleosomal breakdown of DNA. Peripheral blood lymphocytes (PBL) were incubated for 72hr as follows: medium lacking fetal bovine serum (lanes 1 and 2) and supplemented with 50riM PMA (lane I); complete medium, including 10% heat-inactivated fetal bovine serum and 1/~M staurosporine (lane 4). DNA was extracted and labelled with [~32p]-dCTP and the Klenow fragment of DNA polimerase. Lane 3 was loaded with a 100 bp molecular size marker.
Protein kinase C involvement in apoptosis
883
Table I. Dnta supporting that PKC activation Mocks apopto~lis DNA fragmentation is associated with down regulation of PKC in promyclocytic leukemia cells (Jarvis et aL, 1994b, c) PKC inhibitors enhance apoptosis in mouse natural killer cells and cytotoxic T lymphocytes (Migliorati et al., 1994) The activation of PKC promotes cell survival of mature lymphocytes prone to apoptosis (Lucas et al., 1994) and protects from radiation-induced apoptony (Radford, 1994) Cyclosporine A proteCts B cells against calcium-dependent apoptosis, by blockade of the phosphoprotein phosphatase calcineurin, but not against the apoptosis triggered by the PKC inhibitor chelerytrine (Bonnefoyberard et al., 1994) Proper combination of calcium ionophore and protein kinase activator (PMA) inhibits corticosterone-induced apoptosis in lymphocytes (Iseki et al., 1993) Apoptosis, occurring at a high rate among B cells in germinal centers can be arrested by protein kinase C-activating phorbol esters (Knox et aL, 1993) Inhibition, as well as down regulation, of PKC cause apoptosis in freshly isolated rat bepatocytes (S/mchez et al., 1992) The inhibition of PKC by staurosporine triggers apoptosis of insulin-secreting RIN m5F cells without raising cytosolic free calcium (Shnchez-Margalet et al., 1993) Inhibitors of PKC block the prolongation of neutrophil survival induced by granulocyte colony-stimulatingfactor (G-CSF) and induce DNA fragmentation at concentrations that fail to alter the priming effect of G-CSF (Adachi et al., 1993) Translocation of PKC from the cytosol mediates phosphatidyl inositol-dependent pathway of rescue germinal center B cells from apoptosis (Knox and Gordon, 1994) Basic fibroblast growth factor (bFGF) and phorbol esters protect endothelial cells against radiation-induced apoptosis. The mechanism of action of bFGF involves activation of tyrosine-kinase which in turns causes the translocation of the alpha isotype of cytoplasmic PKC into the membrane (Haimovitzfriedman et al., 1994a) PKC inhibitors induce apoptosis of malignant glioma cells (Couldwell et aL, 1994) Selective PKC inhibitors block IL-2-mediated proliferation of routine T cells and cause apoptosis (G6mez et al., 1994) PKC activation blocks both radiation-induced sphingomyelin hydrolysis and apoptosis of aortic endothelial cells (Haimovitzfriedman et al., 1994b) Haggerty and Monroe (1994) describe a mutant of a B lymphocyte line sensitive to apoptosis caused by signaling components that lie downstream of PKC Data supporting that PKC activation promotes apoptosis Fragmentation of DNA induced by tyrosine kinase-inhibitors in mouse thymocytes is enhanced by phorbol esters capable of activating PKC (Azuma et al., 1993) Serine-threonine phosphatase inhibitors synergistically augment TNF-induced apoptosis in several TNF-sensitive tumor cell lines including histiocytic lymphoma, mammary carcinoma, and prostatic tumor cells (Wright et al., 1993) Phorbol ester induces apoptosis in promyelocytic leukemia cells (Macfarlane and O'Dnnnell, 1993; Macfarlane and Manzel, 1994) Phorbol ester enhances mitoxantrone-induced internucleosomal DNA fragmentation in human myeloid leukemia cells, whereas PKC inhibitors have no effects (Bhalla et al., 1994) Retinoic acid and activators of PKC enhance apoptosis of neuronal cells induced by serum deprivation down regulation of PKC reduces the ability of retinoic acid to induce apoptosis (Mailhos et al., 1994) Radiation-induced apoptosis of mouse thymocytes is prevented by a PKC inhibitor and. is potentiated by the PKC activator phorbol ester (Shaposhnikova et al., 1994) Phorbol esters leads to growth arrest an apoptosis of a continuously proliferating B lymphocyte line (Haggerty and Monroe, 1994)
calcium pumping ATPase of the endoplasmic reticulum that causes persistent depletion of intracellular calcium stores and produces apoptosis of hepatoma cell lines (Kaneko and Tsukamoto, 1994). This apparent paradox (the association of calcium depletion and apoptosis) can be explained taking into account
the so-called "capacitative" (store-operated) model o f c a l c i u m e n t r y t h a t is r e g u l a t e d by t h e d e g r e e of depletion of the endoplasmic reticulum calcium p o o l . I n t e r e s t i n g l y , this s t o r e - o p e r a t e d c a l c i u m e n t r y mechanism
is i n h i b i t e d by s t i m u l a t i o n o f p r o t e i n
k i n a s e C ( M o n t e r o e l a l . , 1993). T h e i n h i b i t i o n o f
Fig. 2. Thapsigargin (Tg), by inhibiting Ca2+-pumping ATPase in endoplasmic reticulum (er), depletes this intracellular calcium pool which in turn triggers the entry of calcium. The oncoprotein bcl-2 blocks the effect o f thapsigargin in the Ca2+-pumping ATPase. The activation of P K C inhibits the entry of calcium by the store operated "capacitative" mechanism (Montero et al., 1993).
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Miguel Lucas and Victor Shnchez-Margalet Sphingomyelin
Ceramide
Sphingosine
Phosphatase
Kinase
APOPTOSIS
Fig. 3. Scheme of the putative mechanism of the sphingomyelinase pathway to apoptosis. The hydrolysis of sphingomyelin produces ceramide and sphingosine which, by activating phosphatase and inhibiting kinase respectively, regulate the activity of target proteins (T.P.) hypothetical effectors of apoptosis. calcium entry should block the activation of calciumdependent enzymes triggering the apoptotic cell death. In fact Lam et al. (1994) have recently explained the role of bcl-2 in the repression of apoptosis by regulating endoplasmic reticulumassociated calcium fluxes. The induction of apoptosis by thapsigargin is blocked by bcl-2 and may be explained assuming that the oncoprotein, by inhibiting calcium leaking from the endoplasmic reticulum hinder the thapsigargin-induced store-operated calcium entry and therefore prevents the permanent increase of cytosolic free calcium that causes apoptosis. This could also be a general mechanism of the abrogation of apoptosis by phorbol esters since it inhibits the store-operated calcium entry into the cytosol (Montero et al., 1993). Nonetheless, a direct effect of PKC on bcl-2 has been described indicating that hematopoietic growth factors may inhibit apoptosis by a molecular mechanism involving activation and phosphorylation of bcl-2 (May et al., 1993).
acetate and synthetic diglycerides, suggesting opposite roles for digyiceride- and' ceramide-mediated signals in the regulation of apoptosis. Therefore, ceramide is a possible mediator of apoptosis in response to a number of agents, including interferon and hypoxia, that causes sphingomyelin hydrolysis. Besides, a ceramide-activated protein phosphatase could mediate the effects of ceramide (Dobrowsky and Hannum, 1992). On the other hand, sphingosine, a breakdown product of sphingolipids, is well known for its pharmacological inhibition of PKC. The coincidence of both complementary events may argue in favor of a PKC mediated pathway by inhibiting the phosphorlyation and activating the phosphatase of target proteins. Very recently, it has been described that ionizing radiation acts on cellular membranes of bovine and aortic endothelial cells to generate ceramide and initiate apoptosis, suggesting an alternative to the hypothesis that direct DNA damage mediates radiation-induced cell killing. The authors (Haimovitzfriedman et al., 1994a, b) indicate that PKC activation blocked both radiation-induced sphingomyelin hydrolysis and apoptosis. Cell type seems important since radiation-induced apoptosis of mouse thymocytes is prevented by PKC inhibitors
I Synthosis I P
J
Y
I
Mitosis I
P
f
T
Y
T
?l ? Y
T
PKC AND THE SPHINGOMYELIN PATHWAY
TO APOPTOSIS Sphingolipid breakdown products have anti-proliferative and tumor-suppressor properties (Hannun and Linardic, 1993) and the hydrolysis of sphingomyelin and ceramide generation have been implicated in a signal transduction pathway that mediates the effects of tumor necrosis factor alpha, TNF~. Moreover, it has been described that ceramide (Obeid e t al., 1993) as well as the activation of the sphingomyelin hydrolysis (Jarvis et al., 1994a) induce programmed cell death, a process that is inhibitible by the protein kinase C activators phorbol-myristate
Fig. 4. The entry into the mitosis phase requires the activation of p34~2 kinase following dephosphorylation and association with cyclins. The uncoupling of these events, by p34¢d¢2activation before DNA replication is completed, causes "mitosis catastrophe", a process quite similar to apoptosis. The triggering of apoptosis by the uncoupling of completion of DNA synthesis with events leading to nuclear membrane disintegration and apoptotic bodies formation is an interesting hypothesis with a number of interrogations (see also Oberharmer et al., 1994).
Protein kinase C involvement in apoptosis whereas it is potentiated by activation of PKC (Shaposnikova et al., 1994).
APOPTOSIS VERSUS MITOSIS The uncoupling of timing for p34~c2 activation and the completion of DNA replication results in the so-called "mitotic catastrophe" that appears results from mitosis during DNA replication (Nurse, 1990; Heald et aL, 1993). P34~c2 is a highly regulated serine-threonine kinase that controls cell entry into mitosis. The regulation of p34cd~2is known to involve a network of kinases and phosphatases, that may respond to the state of DNA replication, as well as the formation of complexes with cyclins (Nurse, 1990). Entry into M-phase is determined by activation of p34¢dc2which requires p34°de dephosphorylation of phosphotyrosine and phosphothreonine and association with cyclins. The active form of the kinase leads to the phosphorylation of key substrates: H1hitone, p60src, iamins, centrosomal proteins and other proteins which need to be displaced from chromatin to allow chromosome condensation. The complex of p34~C2/cyclins initiates the dissolution of nuclear membrane and promote chromatin condensation, events that take place in both mitosis and apoptosis (Meikrantz et al., 1994). It has been recently proposed that premature p34 ~dc2 activation may be a general mechanism by which cells induced to undergo apoptosis initiate the disruption of the nucleus. This has been deduced from experiments with fragmentin and with staurosporine that induces dephosphorylation of p34~dc2 and apoptosis in lymphoma and mammary carcinoma cell lines (Shi et al., 1994). Staurosporine is a potent PKC inhibitor with a broad apoptotic activity in cell lines from different origins (Bertrand et al., 1994). This hypothesis may be questioned since Oberharmer et al. (1994) have recently shown that chromatin condensation during apoptosis seems to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34~¢2 kinase; in contrast to mitosis, dephosphorylation and activation of p34~dc2 does not occur in apoptotic cells.
REFERENCES Adachi S., Kubota M., Matsubara K., Wakazono Y., Hirota H., Kuwakado K., Akiyama Y. and Mikawa H. (1993) Role of protein kinase C in neutrophil survival enhanced by granulocyte colony-stimulating factor. Exp. Hemat. 21, 1709-1713. Arends M. J., Morris R. G., WyllieA. H. (1990) Apoptosis. The role of endonuclease. Am. J. Path. 136, 593-608. Azuma Y., Onishi Y., Sato-Y. and Kizaki H. (1993) Induction of mouse thymocyte apoptosis by inhibitors of tyrosine kinases is associated with dephosphorylation of nuclear proteins. Cell Immun. 152, 271-278.
885
Baffy G., Miyashita T., Williamson J. R. and Reed J. C. (1993) Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production. J. Biol. Chem. 268, 6511-6519. Bellomo G., Perotti M., Taddei F., Mirabelli F., Finardi G., Nicotera P. and Orrenius S. (1992) Tumor necrosis factor induces apoptosis in mammary adenocarcinoma cells by an increase in intranuclear free Ca2+ concentration and DNA fragmentation. Cancer Res. 52, 1342-1346. Bertrand R., Solary E., Oconnor P., Kohn K. W. and Pommier Y. (1994) Induction of a common pathway of apoptosis by staurosporine. Exp. Cell Res. 211, 314-321. Bhalla K., Ibrado A. M., Tourkina E., Tank C., Grant S., Bullock G., Huang Y,, Ponnathpur V. and Mahoney M. E. (1993) High-dose mitoxantrone induces programmed cell death or apoptosis in human myeloid leukemia cells. Blood 82, 3133-3140. Bissonnette R. P., Echeverri F., Mahboubi A. and Green R. (1992) Apoptotic cell death induced by C-myc is inhibited by bcl-2. Nature 359, 552-554. Bonnefoyberard N., Genestier L., Flacher M. and Revillard J. P. (1994) The phosphoprotein phosphatase calcineurin controls calcium-dependentapoptosis in B cell lines. Eur. J. Immun. 24, 325-329. Buttyan R., Zakeri Z., Lockshin R. and Wogelmuth D. (1988) Cascade induction of c-los, c-myc and heat shock 70 K transcripts during the regression of the ventral prostate glan. Molec. Endocr. 2, 650-657. Clarke A. R., Purdie C. A., Harrison D. J., Morris R. G., Bird C. C., Hooper M. L, and Wyllie A. H. (1993) Thymocyte apoptosis is induced by p53-dependent and independent pathways. Nature 362, 849-852. Cleveland F. L., Gonzfilez-GarciaM., Nufiez G., lhle J. H. and Rapp U. R. (1994) V-raf suppresses apoptosis and promotes growth of interleukin-3-dependent myeloid cells. Oncogen 9, 2217-2226. Collins M. K. L. and Lrpez-Rivas A. (1993) The control of apoptosis in mammalian cells. Trends Biochem Sci. 18, 307-309. Couldwell W. T., Hinton D. R., He S. K., Chen T. C., Sebat I., Weiss M. H. and Law R. E. (1994) Protein kinase C inhibitors induce apoptosis in human malignant glioma cell lines FEBS Lett. 345, 43-46. Dobrowsky R. T. and Hannun Y. A. (1992) Ceramide stimulates a cytosolic protein phosphatase. J. Biol. Chem. 267, 5048-5051. Fanidi A., Harrington E. A. and Evan G. I. (1992) Cooperative interaction between C-myc and bcl-2 protooncogenes. Nature 359, 554-556. Fesus L., Davies P. J. A, and Piacentini M. (1991) Apoptosis: molecular mechanismsin programmed cell death. Eur. J. Cell Biol. 56, 170-177. Golstein P., Ojcius D. M. and Young J. D. E. (1991) Cell death mechanism and the immune system. Immun. Rev. 121, 29-65. G6mez J., Delahera A., Silva A., Pitton C., Garcia A. and Rebollo A. (1994) Implication of protein kinase C in IL-2-Mediated proliferation and apoptosis in a murine T cell clone. Exp. Cell Res. 213, 178-182. Green D. R. and Scott D. W. (1994) Activation-induced apoptosis in lymphocytes. Curr. Opin. lmmun. 6, 476-487. Haggerty H. G. and Monroe J. G. (1994) A mutant of the WEHI-231 B lymphocyte line that is resistant to phorbol esters is still sensitiveto antigen receptor-mediated growth inhibition. Cell Immun. 154, 166-180. Haimovitzfriedman A., Balaban N., Mcloughlin M., Ehleiter D., MichaeliJ., Vlodavsky I. and Fuks Z. (1994a) Protein kinase C mediates basic fibroblast growth factor protection of endothelial cells against radiation-induced apoptosis. Cancer Res. 54, 2591-2597.
886
Mignel Lucas and Victor S~inchez-Margalet
Haimovitzfriedman A., Kan C. C., Ehleiter D., Persaud R. S., Mcloughlin M., Fuks Z. and Kolesnick R. N. (1994b) Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis. J. Exp. Med. 180, 525-535. Hannun Y. A. and Linardic C. M. (1993) Sphingolipid breakdown products: and anti-proliferative and tumorsuppressor lipids. Biochim. Biophys. Acta 1154, 223-236. Heald R., MacLoughlin M. and McKeon F. (1993) Human Weel maintains mitotic timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase. Cell 74, 463-474. Hockenberry D., Nufiez G., Milliman C., Schreiber R. D. and Korsmeyer S. J. (1990) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348, 334-336. Iseki R., Kudo Y. and lwata M. (1993) Early mobilization of Ca 2+ is not required for glucocorticoid°induced apoptosis in thymocytes, J. lmmun. 151, 5198-5207. Jacobson M. D., Burne J. F., King M. P., Miyashita T., Reed J. C. and Raft M. C. (1993) Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA. Nature 361,365-369. Jarvis W. D., Kolesnick R. N., Fornari F. A., Traylor R. S., Gerwitz D. A. and Grant S. (1994a) Induction of apoptotic D N A damage and cell death by activation of the sphingomyelin pathway. Proc. Natn Acad. Sci. U.S.A. 91, 73-77. Jarvis W. D., Turner A. J., Povirk L. F., Traylor R. S. and Grant S. (1994b) Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C. Cancer Res. 5,t, 1707-1774. Jarvis W. D., Povirk L. F., Turner A. J., Traylor R. S., Gewirtz D. A., Pettit G. R. and Grant S. T. I. (1994c) Effects of bryotstatin 1 and other pharmacological activators of protein kinase C on 1-[beta-D-arabinofuranosyl]cytosine-induced apoptosis in HL-60 human promyelocytic leukemia cells. Biochem. Pharmac. 47, 839-852. Kaneko Y. and Tsukamoto A. (1994) Thapsigargin-induced persistent intracellular calcium pool depletion and apoptosis in human hepatoma cells. Cancer Lett. 79, 147-155. Kerr J. F. R., Wyllie A. H. and Currie A. R. (1972) Apoptosis: a basic biological phenomenon with wideranging implications in tissue kinetics. Br. J. Cancer 26, 239-257. Knox K. A. and Gordon J. (1994) Protein tyrosine kinases couple the surface immunoglobulin of germinal center B cells to phosphatidylinositol-dependent and -independent pathways of rescue from apoptosis. Cell Immun. 155, 62-76. Knox K. A., Hhonson G. D. and Gordon J. (1993) Distribution of cAMP in secondary follicles and its expression in B cell apoptosis and CD40-mediated survival. Int. lmmun. 5, 1085-1091. Lam M., Dubyak G., Chen L., Nunez G., Miesfeld R. L. and Distelhorst C. W. (1994) Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulumassociated Ca 2+ fluxes. Proc. Natn Acad. Sci. U.S.A. 91, 6569-6573. Lee S. B. and Esteban M. (1994) The interferon-induced double-stranded RNA-activated protein kinase induces apoptosis. Virology 199, 491-496. Lennon S. V., Kilfeather S. A., Hallet M. B., Campbell A. K. and Gotter T. G. (1992) Elevations in cytosolic free Ca 2+ are not required to trigger apoptosis in human leukaemia cells. Clin. Exp. lmmun. 87, 565-571, Lowe S. W., Schmitt E. M., Smith S. W., Osborne B. A. and Jacks T. (1993) p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362, 847-849. Lucas M., Solano F. and Sanz A. (1991) Induction of programmed cell death (apoptosis) in mature lymphocytes. FEBS Lett. 1, 19-20.
Lucas M., Sanchez-Margalet V., Sanz A. and Solano F. (1994) Protein kinase C activation promotes cell survival in mature lymphocytes prone to apoptosis. Biochem. Pharmac. 47, 667-672. Macfarlane D. E. and O'Donnell P. S. (1993) Phorbol ester induces apoptosis in HL-60 promyelocytic leukemia cells but not in HL-60 PET mutant. Leukemia 7, 1846-1851. Macfarlane D. E. and Manzel L. (1994) Activation of beta-isozyme of protein kinase C (PKC beta) is necessary and sufficient for phorbol ester-induced differentiation of HL-60 promydocytes. Studies with PKC beta-defective PET mutant. J. Biol. Chem. 269, 4327-4231. Mailhos C., Howard M. K. and Latchman D. S. (1994) A common pathway mediates retinoic acid and PMAdependent programmed cell death (apoptosis of neuronal cells). Brain Res. 644, %12. May W. S., Tyler P. G., Armstrong D. K. and Davidson N. E. (1993) Role for serine phosphorylation of Bcl-2 in an antiapoptotic signaling pathways triggered by IL-3, EPO and bryostain. Blood 82 (Suppl. 1), 1738. McConkey D. J., Nicotera P., Hartzell P., Bolloma G., Wyllie A. H. and Orrenius S. (1989a) Glucocorticoids activate a suicide process in thymocytes through an elevation of cytosolic Ca :+ concentration. A rchs Biochem. Biophys. 269, 365-370. McConkey D. J., Harztell P., Jondal M. and Orrenius S. (1989b) Inhibition of DNA fragmentation in thymocytes and isolated thymocyte nuclei by agents that stimulate protein kinase C. J. Biol. Chem. 264, 13399-13402. McConkey D. J., Orrenius S. and Jondal M. (1990) Cellular signaling in programmed cell death (apoptosis). lmmun. Today 11, 120-121. Meikrantz W., Gisselbrecht S., Tam S. W. and Schlegel R. (1994) Activation of cyclin A-dependent protein kinases during apoptosis Proc. Natn Acad. Sci. U.S.A. 91, 3754-3758. Mercep M., Noguchi P. D. and Ashwell J. D. (1989) The cell cycle book and lysis of an activated T-cell hybridoma are distinct processes with different Ca 2+ requirements and sensitivity to cyclosporine A. J. lmmun. 142, 4085-4092. Migliorati G., Nicoletti I., D'Adamio F., Spreca A., Pagliacci C. and Riccardi C. (1994) Dexamethasone induces apoptosis in mouse natural killer cells and cytotoxic T lymphocytes. Immunology $1, 21-26. Montero M., Garcia-Sancho J. and Alvarez J. (1993) Transient inhibition by chemotactic peptide of a storeoperated Ca 2+ entry pathway in human neutrophils. J. Biol. Chem. 268, 13055-13061. Nicotera P., McConkey D. J., Jones D. P. and Orrenius S. (1989) ATP stimulates Ca 2+ uptake and increases the free Ca -'+ concentration in isolated liver nuclei. Proc. Natn Acad. Sci. U.S.A. 86, 453-457. Nieto M. A. and L6pez-Rivas A. (1989) IL°2 protects T lymphocytes from glucocorticoid-induced DNA fragmentation and cell death, J. lmmun. 142, 4166-4170. Nurse P. (1990) Universal control mechanism regulating onset of M-phase. Nature 344, 503-509. Obeid U M., Linardic C. M., Karolak L. A. and Hannun Y. A. (1993) Programmed cell death induced by ceramide. Science 259, 1769-1771. Oberhammer F. A., Hocbegger K. and Froschl G. (1994) Chromatin condensation during apoptosis is accompanied by degradation of lamin A + B without activation of cdc2 kinase. 3". Cell BioL 126, 827-837. Ojeda F., Guarda M., Maldonado C. and Foleh H. (1990) Protein kinase C involvement in thymoeyte apoptosis induced by hydrocortisone. Cell Immun. 125, 535-539. Otani H., Erdos M. and Leonard W. J. (1993) Tyrosine kinase(s) regulate apoptosis and bcl-2 expression in a growth factor-dependent cell line. J. BioL Chem. 268, 22733-22736.
Protein kinase C involvement in apoptosis Radford I. R. (1994) Phorbol esters can protect mouse pre-T cell lines from radiation-induced rapid interphase apoptosis. Int. J. Radiat. Biol. 65, 345-355. Raft M. C. (1992) Social controls on cell survival and cell death. Nature 356, 397~,00. Rodriguez-Tarducy G. and L6pez-Rivas A. (1989) Phorbol esters inhibits apoptosis in IL-2-dependent T lymphocytes. Biochem. Biophys. Res. Commun. 164, 1069-1075. S~.nchez V., Lucas M., Sanz A. and Goberna R. (1992) Decrease protein kinase C activity is associated with programmed cell death (apoptosis) in freshly isolated rat hepatocytes. Biosci, Report 12, 199-206. Sanchez-Margalet V., Lucas M., Solano F. and Goberna R. (1993) Sensitivity of insulin-secreting RIN m5F cells to undergoing apoptosis by the protein kinase C inhibitor staurosporine. Exp. Cell Res. 209, 160-163. Sarin A., Adams D. H. and Henkart P. A. (1993) Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells. J. Exp. Med. 178, 1693-1700. Schwartz L. M., Kosz L. and Kay B. K. (1990) Gene activation is required for developmentally programmed cell death. Proc. Natn Acad. Sci. U.S.A. 87, 6594-6598.
887
Shi L., Nishioka W. K., Th'ng J., Bradbury E. M., Litchfield D. W. and Greenberg A. H. (1994) Premature p34~c2 activation required for apoptosis. Science 263, 1143-1145. Shaposnikova V. V., Dobrovinskaya O. R., Eidus L. K. and Korystov Y. N. (t 994) Dependence of thymocyte apoptosis on protein kinase C and phospholipase A 2. FEBS Lett. 340, 319-319. Tomei D. L., Kanter P. and Wenner C. E. (1988) Inhibition of radiation-produced apoptosis in vitro by tumor promoters. Biochem. Biophys. Res. Commun. 155, 324-331. Wright S. C., Zheng H., Zhong J., Torti F. M. and Larrick J. W. (1993) Role of protein phosphorylation in TNFinduced apoptosis: phosphatase inhibitors synergize with TNF to activate DNA fragmentation in normal as well as TNF-resistant U937 variants. J. Cell Biochem. 53, 222-233. Wright S. C., Zhing J. and Larrick J. W. (1994) Inhibition of apoptosis as a mechanism of tumor promotion. FASEB Jl 8, 654-660. Wyllie A. H. (1988) Apoptosis. ISI Atlas of Science: Immunology 1, 192-196. Wyllie A. H., Kerr J. F. R. and Currie A. R. (1980) Cell death: the significance of apoptosis. Int. Rev. Cvtol. 68, 251-356.