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Atypical Protein Kinase C ι Protects Human Leukemia Cells against Drug-induced Apoptosis

1997; Elsevier BV; Volume: 272; Issue: 44 Linguagem: Inglês

10.1074/jbc.272.44.27521

1083-351X

Nicole R. Murray, Alan P. Fields,

Venomous Animal Envenomation and Studies

Protein kinase C (PKC) isozymes play distinct roles in cellular function. In human K562 leukemia cells, PKC α is important for cellular differentiation and PKC βIIis required for proliferation. In this report, we assess the role of the atypical PKC isoform PKC ι in K562 leukemia cell physiology. K562 cells were stably transfected with expression plasmids containing the cDNA for human PKC ι in sense or antisense orientation to increase or decrease cellular PKC ι levels, respectively. Overexpression or inhibition of expression of PKC ι had no significant effect on the proliferative capacity of K562 cells nor their sensitivity to phorbol myristate acetate-induced cytostasis and megakaryocytic differentiation, suggesting that PKC ι does not play a critical role in these processes. Rather, PKC ι serves to protect K562 cells against drug-induced apoptosis. K562 cells, which are resistant to most apoptotic agents, undergo apoptosis when treated with the protein phosphatase inhibitor okadaic acid (OA). Overexpression of PKC ι leads to increased resistance to OA-induced apoptosis whereas inhibition of PKC ι expression sensitizes cells to OA-induced apoptosis. Overexpression of the related atypical PKC ζ has no protective effect, demonstrating that the effect is isotype-specific. PKC ι also protects K562 cells against taxol-induced apoptosis, indicating that it plays a general protective role against apoptotic stimuli. These data support a role for PKC ι in leukemia cell survival. Protein kinase C (PKC) isozymes play distinct roles in cellular function. In human K562 leukemia cells, PKC α is important for cellular differentiation and PKC βIIis required for proliferation. In this report, we assess the role of the atypical PKC isoform PKC ι in K562 leukemia cell physiology. K562 cells were stably transfected with expression plasmids containing the cDNA for human PKC ι in sense or antisense orientation to increase or decrease cellular PKC ι levels, respectively. Overexpression or inhibition of expression of PKC ι had no significant effect on the proliferative capacity of K562 cells nor their sensitivity to phorbol myristate acetate-induced cytostasis and megakaryocytic differentiation, suggesting that PKC ι does not play a critical role in these processes. Rather, PKC ι serves to protect K562 cells against drug-induced apoptosis. K562 cells, which are resistant to most apoptotic agents, undergo apoptosis when treated with the protein phosphatase inhibitor okadaic acid (OA). Overexpression of PKC ι leads to increased resistance to OA-induced apoptosis whereas inhibition of PKC ι expression sensitizes cells to OA-induced apoptosis. Overexpression of the related atypical PKC ζ has no protective effect, demonstrating that the effect is isotype-specific. PKC ι also protects K562 cells against taxol-induced apoptosis, indicating that it plays a general protective role against apoptotic stimuli. These data support a role for PKC ι in leukemia cell survival. Protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; nPKC, novel PKC; aPKC, atypical PKC; PMA, phorbol myristate acetate; OA, okadaic acid; gpIIIa/IIb, glycophorin IIIa/IIb. 1The abbreviations used are: PKC, protein kinase C; nPKC, novel PKC; aPKC, atypical PKC; PMA, phorbol myristate acetate; OA, okadaic acid; gpIIIa/IIb, glycophorin IIIa/IIb.is a family of at least 12 structurally related phospholipid-dependent serine/threonine protein kinases that are directly involved in the transmission of a wide variety of extracellular signals (1Clemens M.J. Trayner I. Menaya J. J. Cell Sci. 1992; 103: 881-887Crossref PubMed Google Scholar). The PKC enzyme family can be divided into three subgroups: the calcium-dependent or cPKCs (alpha (α), beta I (βI), beta II (βII), and gamma (γ)); the novel or nPKCs (delta (δ), epsilon (ε), eta (η), theta (θ), and mu (μ)); and the atypical or aPKCs (zeta (ζ), iota (ι), and lambda (λ)) (reviewed in Ref. 2Nishizuka Y. Nature. 1988; 334: 661-665Crossref PubMed Scopus (3532) Google Scholar). These groupings are based on the presence or absence of functional domains that confer isotype-specific co-factor and activator requirements. Biochemical and immunologic studies indicate that multiple PKC isotypes are expressed in virtually all cell and tissue types (3Wetsel W.C. Khan W.A. Merchenthaler I. Rivera H. Halpern A.E. Phung H.M. Negro-Vilar A. Hannun Y.A. J. Cell Biol. 1992; 117: 121-133Crossref PubMed Scopus (393) Google Scholar, 4Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1217) Google Scholar), suggesting a universal role in cellular function. The expression of individual PKC isotypes is developmentally regulated (5Saxon M.L. Zhao X. Black J.D. J. Cell Biol. 1994; 126: 747-763Crossref PubMed Scopus (120) Google Scholar, 6Yoshida Y. Huang F.L. Nakabayshi H. Huang K.-P. J. Biol. Chem. 1988; 263: 9868-9873Abstract Full Text PDF PubMed Google Scholar) and is responsive to the differentiation state of many cell and tissue types (7Makowske M. Ballester R. Cayre Y. Rosen O.M. J. Biol. Chem. 1988; 263: 3402-3410Abstract Full Text PDF PubMed Google Scholar, 8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). For these reasons, it is thought that PKC isotypes fulfill distinct, nonredundant functions within the cell. In human leukemia cells, we have provided direct evidence for PKC isotype specific function in the control of cellular proliferation and differentiation (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). The two conventional PKC isotypes expressed in these cells, PKC α and PKC βII play distinct roles in these cellular processes (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). PKC α is involved in cellular differentiation and overexpression of PKC α leads to gene dose-dependent cytostasis and increased sensitivity to differentiating agents such as phorbol myristate acetate (PMA) (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar, 9Melloni E. Pontremoli S. Michetti M. Sacco O. Cakiroglu A.G. Jackson J.F. Rifkind R.A. Marks P.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5282-5286Crossref PubMed Scopus (83) Google Scholar, 10Murray N.R. Thompson L.T. Fields A.P. Parker P.J. Dekker L.V. Protein Kinase C. R. G. Landes Press, Austin, TX1997: 97-120Google Scholar). In contrast, the levels of PKC βII correlate with the proliferative state of these cells, being lost when cells differentiate in response to PMA (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). Furthermore, cellular proliferation is blocked when PKC βII expression is specifically inhibited by antisense oligonucleotides directed against PKC βII, demonstrating that it is required for leukemia cell proliferation (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). In the present report, we identify and determine the function of the third major PKC isotype expressed in K562 cells, the atypical PKC ι. Using isotype-specific antibodies to atypical PKC ζ and PKC ι we demonstrate that K562 cells express PKC ι, but no detectable PKC ζ. We further demonstrate that PKC ι regulates cellular susceptibility to drug-induced apoptosis. Our results indicate that PKC ι plays an important role in leukemia cell survival and suggest that PKC ι may be an attractive target for development of new chemotherapeutic agents in the treatment of leukemia. Human erythroleukemia (K562) cells were maintained in suspension culture and induced to undergo cytostasis and megakaryocytic differentiation in response to PMA as described previously (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). Apoptosis was induced in cells plated at 4 × 105 cells/ml in growth medium containing okadaic acid (OA; Alexis Biochemicals) or taxol at the concentrations and for the times indicated in the figure legends. Parental K562 cells and transfectants overexpressing PKC isotypes (see below) were monitored for proliferation rate by daily cell counting using a hemacytometer. Megakaryocytic differentiation was monitored by assessing expression of the megakaryocytic cell surface marker glycophorin IIIa/IIb (gpIIIa/IIb) by fluorescence activated cell sorting of cells stained with fluorescein-labeled monoclonal antibody to gpIIIa/IIb (Dako) as described previously (11Hocevar B.A. Morrow D.M. Tykocinski M.L. Fields A.P. J. Cell Sci. 1992; 101: 671-679Crossref PubMed Google Scholar). Cell viability was determined by trypan blue exclusion and was >95% in all untreated cultures. The full-length human PKC ι cDNA (kindly provided by Dr. T. Biden, Garvan Institute of Biomedical Research, Sydney, Australia) was excised from pAXNeoRX using (5′)SalI and (3′) BamHI and cloned into the (5′)XhoI and (3′) BamHI sites within the multiple cloning site of pREP4 and pREP10 to achieve sense and antisense orientations, respectively. The full-length human PKC ζ cDNA (kindly provided by Sphinx Pharmaceutical Co.) was excised from pBluescript using (5′) SpeI and (3′) KpnI and cloned into the (5′) NheI and (3′) KpnI sites of pREP10 in sense orientation. K562 cells were transfected with plasmid containing PKC ι or PKC ζ cDNA or with control plasmid and transfectants were selected and maintained as described previously (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). Transfectants were screened for PKC isotype expression by immunoblotting as described below. K562 cell transfectants were screened for PKC isotype expression by immunoblot analysis using isotype-specific PKC antibodies. Antibodies specific to PKC α and PKC βII were produced against peptides corresponding to the carboxyl terminus of each isotype and were characterized previously (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). Antibody to PKC ζ/ι was produced against peptide corresponding to the carboxyl terminus (amino acids 576–592) of rat PKC ζ (12Baldassare J.J. Henderson P.A. Burns D. Loomis C. Fisher G.J. J. Biol. Chem. 1992; 267: 15585-15590Abstract Full Text PDF PubMed Google Scholar). Isotype-specific antibodies to PKC ζ and PKC ι (Santa Cruz Biotechnology, Inc.) were generated against peptides corresponding to amino acids 2–21 within the divergent amino terminus of human PKC ζ and PKC ι, respectively. Following drug treatments, cells were harvested and DNA isolated as described previously (13Ray S. Ponnathpur V. Huang Y. Tang C. Mahoney M.E. Ibrado A.M. Bullock G. Bhalla K. Cancer Chemother. Pharmacol. 1994; 34: 365-371Crossref PubMed Scopus (57) Google Scholar). Briefly, cells were lysed at 1 × 106 cells/100 μl in lysis buffer containing 0.5% SDS and 0.2 mg/ml proteinase K and incubated at 50 °C for 3 h. RNase A was added to a concentration of 0.2 mg/ml and incubated at 37 °C overnight. Cell lysates were extracted with phenol/chloroform (1:1, v/v) and precipitated with ethanol. The dried pellet was resuspended in 10 mm Tris, pH 8.0, 1 mm EDTA and DNA concentration determined spectrophotometrically. 2 μg of DNA/lane was subjected to electrophoresis in 1.5% agarose and visualized by staining with ethidium bromide. Cellular DNA morphology was assessed by staining with 4′,6-diamidino-2-phenylindole (DAPI) and observation under phase-fluorescence optics as described previously (14Thompson L.T. Fields A.P. J. Biol. Chem. 1996; 271: 15045-15053Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Images were captured using a Hamamatsu video camera and processed using Photoshop 5.0. In previous studies, we determined that the two calcium-dependent PKC isotypes expressed in human K562 leukemia cells, PKC α and PKC βII, play distinct functional roles in K562 cell physiology (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). PKC α is involved in induction of cytostasis and differentiation, while PKC βII is required for cellular proliferation (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). In addition to these two classical PKC isotypes, we detected expression of an atypical PKC, which we identified as PKC ζ based on immunoreactivity with an antipeptide antibody against the carboxyl terminus of rat PKC ζ (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). Subsequent to our report, a second atypical PKC isotype was identified and cloned, termed PKC λ in mouse and PKC ι in human, that has high sequence homology to PKC ζ (15Akimoto K. Mizuno K. Osada S. Hirai S. Tanuma S. Suzuki K. Ohno S. J. Biol. Chem. 1994; 269: 12677-12683Abstract Full Text PDF PubMed Google Scholar,16Selbie L.A. Schmitz-Peiffer C. Sheng Y. Biden T.J. J. Biol. Chem. 1993; 268: 24296-24302Abstract Full Text PDF PubMed Google Scholar). Comparison of the carboxyl-terminal regions of human PKC ζ and PKC ι reveals extensive homology in the peptide region used to generate the PKC ζ antibody, raising the possibility that this antibody may cross-react with PKC ι. These findings led us to test the specificity of this antibody and to reexamine the expression of atypical PKC isotypes in K562 cells using isotype-specific antibodies to PKC ζ and PKC ι, generated against the divergent amino-terminal regions of these isotypes (Fig. 1). As observed previously (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar), the COOH-terminal anti-rat PKC ζ antibody recognizes a single band of molecular mass 74 kDa in K562 cell lysates (Fig. 1 A, lane 1). In addition, this antibody recognizes human PKC ζ when overexpressed in K562 cells (Fig. 1 A, lane 3) and purified recombinant baculovirus-expressed human PKC ζ (Fig. 1 A, lane 4). However this antibody also recognized a 74-kDa band of increased intensity in lysates from K562 cells transfected with the human PKC ι cDNA (Fig. 1 A, lane 2), indicating that it also recognizes PKC ι. In contrast, an isotype-specific PKC ζ antibody recognizes PKC ζ overexpressed in K562 cells and recombinant purified PKC ζ (Fig. 1 B, lanes 3 and 4) but fails to recognize a band in either parental K562 cells or cell transfectants overexpressing PKC ι (Fig.1 B, lanes 1 and 2). Conversely, a PKC ι-specific antibody recognizes a 74-kDa band in K562 cells (Fig.1 C, lane 1) whose intensity is increased in PKC ι-overexpressing cells (Fig. 1 C, lane 2). This PKC ι antibody does not recognize PKC ζ when expressed in K562 cells or recombinant baculovirus-expressed human PKC ζ (Fig. 1 C, lanes 3 and 4), demonstrating its specificity for PKC ι. From these results we conclude that the carboxyl-terminal rat PKC ζ antibody used in our previous studies (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar) recognizes both human PKC ζ and PKC ι, whereas the amino-terminal PKC ζ and PKC ι antibodies recognize their respective antigens specifically. Furthermore, our results demonstrate that K562 cells express PKC ι, but no detectable PKC ζ. Having identified the atypical PKC isotype expressed in K562 cells as PKC ι, we wished to evaluate its functional role. For this purpose, we stably transfected K562 cells with expression plasmids containing the full-length cDNA for human PKC ι in either sense or antisense orientation or with cDNA for human PKC ζ. Immunoblot analysis with the PKC ι-specific antibody demonstrated that these cell lines express either enhanced (Fig.2 A, lane 2) or reduced (Fig.2 A, lane 3) levels of PKC ι, respectively, when compared with control vector-transfected cells (Fig. 2 A, lane 1) or those expressing PKC ζ (Fig. 2 A, lane 4). Importantly, the changes in the expression level of PKC ι or PKC ζ seen in these transfectants had no effect on the level of expression of PKC α and PKC βII (Fig. 2, B and C). Therefore, the changes in PKC ι and PKC ζ expression do not lead to compensatory changes in the levels of other PKC isotypes. Having established cell lines selectively overexpressing or inhibited from expressing PKC ι, we next investigated the role of PKC ι in K562 cell physiology. First, we assessed whether, like PKC βII (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar), PKC ι was involved in K562 cell proliferation (Fig.3 A). Neither overexpression or antisense inhibition of expression of PKC ι had a significant effect on the proliferation rate of K562 cells, suggesting that PKC ι functions in signaling pathways that are not rate-limiting for normal K562 cell proliferation. It is possible that the very low level of PKC ι expressed in antisense PKC ι cells is sufficient to support cellular proliferation; however, these results are clearly distinct from our previous findings with PKC βII, in which antisense inhibition of PKC βII expression led to dramatic inhibition of K562 cell proliferation (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). We next determined whether PKC ι is important for K562 cell differentiation. Treatment of K562 cells with PMA induces cytostasis and megakaryocytic differentiation (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). We demonstrated previously that PMA-induced cytostasis is mediated, at least in part, by PKC α (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar). PMA induces increased expression of PKC α, and overexpression of PKC α confers a gene dose-dependent reduction in proliferative capacity and increased susceptibility to PMA-induced cytostasis (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar, 10Murray N.R. Thompson L.T. Fields A.P. Parker P.J. Dekker L.V. Protein Kinase C. R. G. Landes Press, Austin, TX1997: 97-120Google Scholar). The expression of PKC ι is also induced by PMA treatment (8Murray N.R. Baumgardner G.P. Burns D.J. Fields A.P. J. Biol. Chem. 1993; 268: 15847-15853Abstract Full Text PDF PubMed Google Scholar), suggesting that PKC ι may also participate in PMA-induced cytostasis and differentiation. Therefore, we evaluated the effect of overexpression and antisense inhibition of expression of PKC ι on PMA-induced cytostasis and megakaryocytic differentiation (Fig.3 B). Cells expressing increased or decreased levels of PKC ι showed the same susceptibility to PMA-induced cytostasis as cells carrying control vector. Furthermore, expression of the megakaryocytic marker gpIIIa/IIb was unaffected by changes in PKC ι expression (Fig.3 C). These results indicate that PKC ι does not play a determinant role in PMA-induced cytostasis or megakaryocytic differentiation. We next determined whether PKC ι plays a role in K562 cell survival and drug-induced apoptosis. K562 cells are resistant to many apoptotic agents (17Ritke M.K. Rusnak J.M. Lazo J.S. Allan W.P. Dive C. Heer S. Yalowich J.C. Mol. Pharmacol. 1994; 46: 605-611PubMed Google Scholar, 18Ray S. Bullock G. Nunez G. Tang C. Ibrado A.M. Huang Y. Bhalla K. Cell Growth Diff. 1996; 7: 1617-1623PubMed Google Scholar) but can be induced to undergo apoptosis in response to the protein phosphatase inhibitor OA (19Zheng B. Chambers T.C. Raynor R.L. Markham P.N. Gebel H.M. Vogler W.R. Kuo J.F. J. Biol. Chem. 1994; 269: 12332-12338Abstract Full Text PDF PubMed Google Scholar). Therefore, we determined whether K562 cells overexpressing or inhibited from expressing PKC ι differ in their sensitivity to OA-induced apoptosis. First, parental K562 cells were treated with increasing concentrations of OA to establish conditions that induce apoptosis as measured by a standard interchromosomal DNA fragmentation assay (Fig.4 A). OA induces dose-dependent apoptosis when cells are treated for 24 h, with a maximal effective dose of about 45 nm. These results are consistent with the reported IC50 of 10 nm for OA-induced cytotoxicity in K562 cells (19Zheng B. Chambers T.C. Raynor R.L. Markham P.N. Gebel H.M. Vogler W.R. Kuo J.F. J. Biol. Chem. 1994; 269: 12332-12338Abstract Full Text PDF PubMed Google Scholar). Based on these data, we chose to use 30 nm OA, a dose that gave demonstrable, but submaximal, DNA fragmentation, to assess the effect of selective overexpression and antisense inhibition of expression of PKC ι on OA-induced apoptosis (Fig. 4 B). Overexpression of PKC ι leads to enhanced resistance to OA-induced DNA fragmentation when compared with control cells (Fig. 4 B, compare lanes 4 and 2). In contrast, antisense inhibition of PKC ι leads to increased sensitivity to OA-induced apoptosis (Fig. 4 B, compare lanes 8 and2). The protective effect of PKC ι is isotype-selective, since overexpression of the related atypical PKC isotype, PKC ζ to similar levels gave no demonstrable protection from OA-induced apoptosis (Fig. 4 B, compare lanes 6 and2). Interestingly, overexpression of PKC ι also protects K562 cells from apoptosis induced by taxol (Fig. 4 C, lane 4), whereas inhibition of expression of PKC ι enhances taxol-induced apoptosis (Fig. 4 C, lane 6). These results demonstrate that the protective effects of PKC ι are not specific for OA-induced apoptosis. As an independent measure of apoptosis, we next examined nuclear DNA morphology of control and OA-treated cells. Cells undergoing apoptosis exhibit apoptotic nuclear DNA morphology characterized by highly condensed chromosomal masses, nuclear swelling, and formation of apoptotic bodies (20Arends M.J. Morris R.G. Wyllie A.H. Am. J. Pathol. 1990; 136: 593-608PubMed Google Scholar, 21Wyllie A.H. Nature. 1980; 284: 555-556Crossref PubMed Scopus (4146) Google Scholar). Therefore, control cells and those expressing increased or decreased levels of PKC ι were treated with 30 nm OA and nuclear morphology was visualized using the fluorescent DNA dye DAPI (Fig. 4 D). The nuclear morphology of cells expressing enhanced or reduced levels of PKC ι was identical to control cells in the absence of OA, indicating that changes in PKC ι expression have no direct effect on DNA morphology (data not shown). However, control and PKC ι-overexpressing cells treated with 30 nm OA (Fig. 4 D, panels 2 and 3, respectively) displayed mitotic chromosomal condensation, consistent with the known mitotic arrest induced by OA in K562 cells (22Zheng B. Woo C.F. Kuo J.F. J. Biol. Chem. 1991; 266: 10031-10034Abstract Full Text PDF PubMed Google Scholar). In contrast, cells expressing reduced levels of PKC ι exhibit a more pronounced apoptotic effect on DNA morphology, including the presence of apoptotic bodies (Fig. 4 D, panel 4, arrowhead). These results are consistent with the DNA fragmentation data (Fig. 3), and support the conclusion that the level of expression of PKC ι is a key determinant of cellular susceptibility to drug-induced apoptosis. PKC ι is a member of the atypical PKC subfamily. These enzymes lack a calcium-binding domain and contain only one cysteine-rich zinc finger (15Akimoto K. Mizuno K. Osada S. Hirai S. Tanuma S. Suzuki K. Ohno S. J. Biol. Chem. 1994; 269: 12677-12683Abstract Full Text PDF PubMed Google Scholar) and, as such, are not calcium-dependent and are not activated by phorbol esters or diacylglycerol (15Akimoto K. Mizuno K. Osada S. Hirai S. Tanuma S. Suzuki K. Ohno S. J. Biol. Chem. 1994; 269: 12677-12683Abstract Full Text PDF PubMed Google Scholar). Relatively little is known about the in vivo activators and cofactor requirements of the atypical PKCs. Recent studies indicate that the atypical PKCs can be activated by phosphatidylinositol phosphates, specifically those phosphorylated in the 3 position by phosphatidylinositol 3-kinase (23Nakanishi H. Breuer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar), and by ceramide generated by sphingomyelinase (24Mueller G. Ayoub M. Storz P. Rennecke J. Fabbro D. Pfizenmaier K. EMBO J. 1995; 14: 1961-1969Crossref PubMed Scopus (469) Google Scholar). These second messengers have been linked to a number of cellular functions including mitogenesis and cell survival. Early reports suggested that PKC λ/ι is critical forXenopus maturation and cellular proliferation in mouse fibroblasts (25Dominguez I. Diaz-Meco M.T. Muncio M.M. Berra E. Garcia de Herreros A. Cornet M.E Sanz L. Moscat J. Mol. Cell. 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Chem. 1996; 271: 31262-31268Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 30Diaz-Meco M.T. Municio M.M. Frutos S. Sanchez P. Lozano J. Sanz L. Moscat J. Cell. 1996; 86: 777-786Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Our present data demonstrate that PKC ι plays a critical role in human leukemia cell survival, particularly in the presence of apoptotic stimuli, and indicate that the atypical PKC isotypes may be involved in cell survival in response to many types of cellular stress. Further studies will be required to elucidate the specific pathways in which PKC ι participates to mediate its anti-apoptotic effects. Future studies will focus on the role of cellular phospholipids, including phosphatidylinositol phosphates and ceramides, and of the recently identified atypical PKC binding proteins (30Diaz-Meco M.T. Municio M.M. Frutos S. Sanchez P. Lozano J. Sanz L. Moscat J. Cell. 1996; 86: 777-786Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 31Diaz-Meco M.T. Munico M.M. Sanchez P. Lozano J. Moscat J. Mol. Cell. Biol. 1996; 16: 105-114Crossref PubMed Google Scholar, 32Puls A. Schmidt S. Grawe F. Stabel S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6191-6196Crossref PubMed Scopus (193) Google Scholar), in regulating PKC ι function and human leukemia survival. The recent finding that expression of the atypical PKC-binding protein, prostate apoptosis regulator 4 (PAR-4), is induced during apoptosis (33Sells S.F. Woods D.P. Joshi-Bavre S.S. Muthukumar S. Jacob R.J. Crist S.A. Humphreys S Rangnekar V.M. Cell Growth Diff. 1994; 5: 457-466PubMed Google Scholar) provides an intriguing potential mechanism by which atypical PKC activity, and thereby cell survival, may be regulated. We thank Dr. T. Biden (Garvan Institute of Medical Research, Sydney, Australia) for generously providing the human PKC ι cDNA, Sphinx Pharmaceutical Inc. for kindly providing the human PKC ζ cDNA and anti-rat PKC ζ antibody, and Y. Ye for technical advice and helpful discussions.