COMMUNICATION
Protein Kinase Ciota Activity Is Necessary for Bcr-Abl-mediated Resistance to Drug-induced Apoptosis*

Lee JamiesonDagger , Lee Carpenter§, Trevor J. Biden§, and Alan P. FieldsDagger parallel **

From the Dagger  Sealy Center for Oncology and Hematology and the Departments of  Pharmacology and parallel  Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1048 and the § Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales 2010, Australia

    ABSTRACT
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Abstract
Introduction
References

K562 chronic myelogenous leukemia cells are highly resistant to chemotherapeutic drugs, such as taxol, that induce cell death by apoptosis. This resistance is mediated by the chimeric tyrosine kinase oncogene Bcr-Abl. However, little is known about the mechanism by which Bcr-Abl protects K562 cells from apoptosis. We recently demonstrated that expression of PKCiota is necessary for the resistance of K562 cells to taxol-induced apoptosis (Murray, N. R., and Fields, A. P. (1997) J. Biol. Chem. 272, 27521-27524). We now demonstrate that treatment of K562 cells with taxol leads to sustained activation of PKCiota . In contrast, Bcr-Abl-negative HL60 myeloid leukemia cells, which are sensitive to taxol-induced apoptosis, do not exhibit sustained PKCiota activation in response to taxol. Treatment of K562 cells with tyrphostin AG957, a selective Bcr-Abl inhibitor, blocks taxol-induced PKCiota activation and sensitizes these cells to taxol-induced apoptosis, indicating that PKCiota is a relevant downstream target of Bcr-Abl-mediated resistance. Furthermore, expression of constitutively active PKCiota by adenovirus-mediated gene transfer rescues AG957-treated K562 cells from taxol-induced apoptosis. Taken together, these results demonstrate that both Bcr-Abl and PKCiota activity are necessary for apoptotic resistance in K562 cells. Furthermore, they identify PKCiota as a critical downstream target of Bcr-Abl that is sufficient to mediate the anti-apoptotic effects of Bcr-Abl.

    INTRODUCTION
Top
Abstract
Introduction
References

Leukemia is a malignant disease of the bone marrow that is the leading cause of cancer death in children and adults under the age of 35 (1). Leukemias arise in hematopoietic progenitor cells and are characterized by impaired or blocked differentiation, uncontrolled proliferation, and resistance to apoptosis (2). Many leukemias can be effectively treated using one or combinations of chemotherapeutic agents, such as taxol, that induce apoptosis (reviewed in Ref. 3). A notable exception is chronic myelogenous leukemia (CML),1 which is highly resistant to most commonly utilized chemotherapeutic drugs, including taxol (4, 5). CML cells, such as K562 cells, are characterized by the presence of the Philadelphia chromosome, which results from a reciprocal translocation involving chromosome 9 and chromosome 22 (6, 7). In K562 cells, the Abl tyrosine kinase gene on chromosome 9 is translocated into the breakpoint cluster region (bcr) region on chromosome 22 producing a chimeric gene whose product, Bcr-Abl, expresses disregulated Abl tyrosine kinase activity (8). The Bcr-Abl gene is the transforming activity responsible for CML (9), and the resistance of K562 cells to drug-induced apoptosis is conferred by Bcr-Abl (5, 10, 11). Thus, inhibition of Bcr-Abl tyrosine kinase activity using selective inhibitors (12-15) or of Bcr-Abl expression using antisense oligonucleotides (16, 17) leads to increased sensitivity of K562 cells to apoptosis induced by many drugs including taxol.

Despite the importance of Bcr-Abl in resistance to drug-induced apoptosis, relatively little is known about the molecular mechanisms by which Bcr-Abl exerts its anti-apoptotic effects (18). Recently, we demonstrated that expression of the atypical PKC isozyme, PKCiota , is important for resistance to taxol-induced apoptosis (19). We have now further defined the role of PKCiota in this process. We find that treatment of K562 cells with taxol leads to potent and sustained activation of PKCiota . In contrast, treatment of HL60 cells, which are sensitive to taxol-induced apoptosis, does not lead to appreciable activation of PKCiota . Furthermore, inhibition of Bcr-Abl using the highly selective inhibitor tyrphostin AG957 blocks PKCiota activation and sensitizes K562 cells to taxol-induced apoptosis. Tyrphostin AG957-treated cells can be rescued from taxol-induced apoptosis by expression of constitutively active PKCiota . These data, along with our previous results (19), demonstrate that PKCiota activation is both necessary and sufficient to mediated the anti-apoptotic effects of Bcr-Abl.

    EXPERIMENTAL PROCEDURES

Cell Culture and Drug Treatments-- Human K562 chronic myelogenous leukemia cells and HL60 promyelocytic leukemia cells were maintained in suspension culture as described previously (19). Cells at a density of 2 × 105 cells/ml were treated with paclitaxel (taxol) at the concentrations and for the time periods indicated in the figure legends. In some cases, the Bcr-Abl-selective inhibitor tyrphostin AG957 or the PI 3-kinase inhibitors wortmannin or LY 294002 were added to K562 cell cultures at the concentrations indicated in the figure legends prior to treatment with taxol. Tyrphostin AG957 is a highly selective Bcr-Abl inhibitor that blocks Bcr-Abl-mediated cell growth and resistance to apoptosis in K562 cells (12, 20). Apoptosis was assessed by the presence of apoptotic bodies or chromosomal condensation and by trypan blue exclusion as described previously (19). These morphological manifestations of apoptosis correlate directly with the apoptotic DNA fragmentation induced by taxol and provide a convenient method to quantitate the apoptotic response (19). For assessment of nuclear DNA morphology, 1 × 105 cells were fixed and permeablized with 1% paraformaldehyde phosphate-buffered saline for 15 min at 37 °C, stained with 4',6-diamidino-2-phenylindole for 5 min, and observed under phase-fluorescence optics. A minimum of 500 cells for each treatment were scored for the presence of apoptotic bodies.

Immunoblot Analysis for PKCiota -- K562 and HL60 cell extracts were lysed directly into SDS sample buffer and subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis for PKCiota as described previously (19). We have previously demonstrated that K562 cells express PKCiota but no detectable PKCzeta (19). Protein quantitation was carried out using BCA protein assay reagent (Pierce). Antigen/antibody complexes were detected using ECL (Amersham Pharmacia Biotech).

Immunoprecipitation and PKCiota Kinase Assay-- For immunoprecipitation kinase assay of PKCiota , K562 and HL60 cells were lysed in RIPA lysis buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) at 4 °C in the presence of 1 mM phenylmethylsulfonyl fluoride and 20 µg/ml leupeptin. Lysates were precleared by incubation with 50 µl of protein A-Sepharose beads (Sigma) for 1 h at 4 °C. PKCiota antibody was first bound to 50 µl of protein A-Sepharose at 4 °C for 1 h prior to addition to clarified lysates and further incubation at 4 °C for 1 h. Immunoprecipitates were washed once with RIPA buffer and once with kinase assay buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 0.5 mM EGTA, 0.1 mM CaCl2) to remove unbound proteins. Washed beads were resuspended in kinase buffer containing 40 µg/ml phosphatidylserine, 10 µg of histone substrate, 10 µM ATP, and 10 µCi of [gamma -32P]ATP. The reactions were incubated at room temperature for 30 min and terminated by addition of SDS buffer and boiling prior to SDS-polyacrylamide gel electrophoresis and autoradiography.

Generation and Use of Recombinant Adenoviruses-- Constitutively active PKCiota was generated by PCR amplification of a fragment containing an A120E substitution within the pseudosubstrate domain of human PKCiota . Such a mutation has previously been shown to induce constitutive activity in PKCiota (21). The EcoRV/NdeI-digested fragment was subcloned into similarly digested full-length PKCiota cDNA and sequenced to confirm the presence of this single mutation. PKCiota A120E (designated caPKCiota ) was then subcloned into pXCX.CMV, a transfer vector based on pXCX2 (22) containing the expression cassette from pRcCMV (Stratagene). Recombinant adenoviruses containing either the green fluorescent protein (GFP) (a kind gift from Phil Poronik, University of Sydney, NSW, Australia) or constitutively active PKCiota were produced by homologous recombination with pJM17 (a circular form of the adenovirus genome) in HEK 293 cells as described (23). Once recombinant viruses were plaque purified and characterized by Western blot analysis, concentrated viral lysates were generated by infection of HEK 293 cells as described (23). Viral lysates (titers of ~1012 plaque-forming unit/ml) were used to infect K562 cells. Briefly, viral lysate was transfected into K562 cells using the LipofectAMINE reagent (Life Technologies, Inc.). After 24 h, cells were treated with taxol and/or AG957 for 24 h, at which time cells were assayed for apoptosis as described above. Infection efficiency was routinely >95% as judged by fluorescence microscopy for GFP expression. caPKCiota expression was confirmed by immunoprecipitation kinase assay of total cell lysates from cells 24 h after infection as described above.

    RESULTS

K562 Chronic Myelogenous Leukemia Cells Are Resistant to Taxol-induced Apoptosis-- Recently, we demonstrated that PKCiota expression is important for resistance of K562 cells to apoptotic agents including taxol (19). Specifically, overexpression of PKCiota leads to enhanced resistance to taxol-induced apoptosis, whereas antisense inhibition of PKCiota expression leads to sensitization to taxol-induced apoptosis (19). These results suggested that changes in PKCiota expression and/or activity may play a critical role in the survival of K562 cells to apoptotic stress. To determine whether the level of PKCiota expression is a key determinant of resistance to drug-induced apoptosis, we studied two human leukemic cell lines that differ in their sensitivity to taxol-induced apoptosis. HL60 cells are sensitive to taxol, undergoing dose-dependent apoptosis such that in the presence of 100 nM taxol, >90% of HL60 cells are apoptotic after 24 h (Fig. 1A). In contrast, K562 cells are highly resistant to taxol-induced apoptosis showing little or no apoptosis after 24 h in up to 325 nM taxol (Fig. 1B).


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Fig. 1.   K562 chronic myelogenous leukemia cells are resistant to taxol-induced apoptosis. K562 (A) and HL60 (B) leukemia cells were incubated with the indicated concentration of taxol for 24 h and assessed for apoptosis as described under "Experimental Procedures." Results are plotted as percentages of normal or apoptotic cells.

PKCiota Is Activated in Response to Taxol in K562 but Not HL60 Cells-- To assess whether this difference in resistance to taxol-induced apoptosis correlates with PKCiota expression, both cell lines were treated with 100 nM taxol for up to 50 h, and total cell lysates were subjected to immunoblot analysis for PKCiota as described previously (19) (Fig. 2). Interestingly, these cell lines express similar basal levels of PKCiota (Fig. 2, A and B). Furthermore, PKCiota levels do not vary in response to taxol-induced apoptotic stress, suggesting that differences in resistance to apoptosis are not due to differences in PKCiota expression. Therefore, we measured PKCiota activity in K562 and HL60 cells during taxol-induced apoptotic stress (Fig. 2C). Cells were treated with 100 nM taxol and PKCiota immunoprecipitated from cell lysates at the indicated times and assayed for PKCiota activity as described under "Experimental Procedures." Immunoblot analysis demonstrated that >90% of total cellular PKCiota is captured by this antibody (data not shown). Basal PKCiota activity is similar in K562 and HL60 cells prior to exposure to taxol, consistent with the similar levels of PKCiota expression in these cells. When HL60 cells are treated with 100 nM taxol, very little change in PKCiota activity is observed over the first 25 h, by which time >90% of HL60 cells are apoptotic. After 25 h, PKCiota activity drops to undetectable levels by 36 h. This loss of PKCiota activity is not due to proteolytic degradation because the levels of PKCiota do not change over this time course (Fig. 2A).


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Fig. 2.   Immunoblot and activity analysis of PKCiota in K562 and HL60 cells during taxol-induced apoptotic stress. K562 (A) and HL60 (B) cells were incubated with 100 nM taxol, and at the indicated times cell lysates were prepared and subjected to immunoblot analysis for PKCiota as described under "Experimental Procedures." C, K562 (K) and HL60 (H) cells were treated with taxol as described above, and at the indicated times cell lysates were prepared and subjected to immunoprecipitation and PKCiota kinase analysis using histone H1 as substrate as described under "Experimental Procedures."

In contrast, PKCiota activity in K562 cells is dramatically induced in response to taxol (Fig. 2C). Increased PKCiota activity is seen at 6 h, peaks at 12 h, and is sustained over the subsequent 36 h. Quantitation of the data indicates a 6.5-fold increase in PKCiota activity by 12 h. These data demonstrate that endogenous PKCiota is activated in response to apoptotic stress in K562 cells but not HL60 cells, suggesting that sustained PKCiota activation may be important in the resistance of K562 cells to taxol-induced apoptosis.

Bcr-Abl Is Involved in PKCiota Activation during Taxol-induced Apoptotic Stress-- The Bcr-Abl tyrosine kinase is the oncogene responsible for CML and is highly expressed in K562 cells but is absent from HL60 cells. Bcr-Abl is required for both transformation and resistance to drug-induced apoptosis of K562 cells (5, 10, 11). However, the proximal downstream targets of Bcr-Abl critical for the resistance phenotype are not known. Given the involvement of PKCiota activation in taxol resistance, we determined whether taxol-induced PKCiota activation in K562 cells was dependent upon Bcr-Abl kinase activity. Furthermore, because Bcr-Abl can directly bind, phosphorylate, and activate PI 3-kinase (24, 25) to generate PI 3,4,5-trisphosphate, which can in turn activate PKCiota (26), we also determined whether PI 3-kinase activity was required for taxol-induced PKCiota activation. K562 cells were treated with 100 nM taxol in the presence or absence of the Bcr-Abl inhibitor tyrphostin AG957 (12, 20), or the PI 3-kinase inhibitors wortmannin (27) or LY294002 (28) for 12 h. PKCiota activity was subsequently determined by immunoprecipitation kinase assay. Taxol treatment leads to potent activation of PKCiota above the basal activity seen in the absence of taxol (Fig. 3A). However, tyrphostin AG957 completely abolishes taxol-induced PKCiota activation, whereas neither wortmannin (100 nM) nor LY294002 (100 µM) affected PKCiota activation despite the fact that they were used at concentrations well above their IC50 values (5 nM for wortmannin (27) and 1.4 µM for LY294002 (28)). Tyrphostin AG957-mediated inhibition of taxol-induced PKCiota activation is dose-dependent and is nearly complete at 30 µM consistent with the effective doses of the compound that inhibit Bcr-Abl activity (12, 20). Furthermore, tyrphostin AG957 leads to dose-dependent sensitization of K562 cells to taxol-induced apoptosis consistent with its ability to inhibit PKCiota activation (Fig. 3C). Neither wortmannin nor LY294002 had any effect on taxol-induced apoptosis, consistent with the lack of inhibition of PKCiota observed with these PI 3-kinase inhibitors. Taken together, these data indicate that Bcr-Abl is an upstream regulator of taxol-induced PKCiota activation, that PKCiota activation is important for resistance of K562 cells to taxol, and that Bcr-Abl-mediated PKCiota activation does not require PI 3-kinase activity.


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Fig. 3.   Tyrphostin AG957 inhibits taxol-induced PKCiota activation and sensitizes K562 cells to taxol-induced apoptosis. K562 cells were treated with 100 nM taxol in the absence or presence of 100 µM LY294002, 100 nM wortmannin, and tyrphostin AG957 at the indicated concentrations. At 12 h, cell lysates were prepared and subjected to immunoprecipitation PKCiota kinase and immunoblot analysis (A and B) and for measurement of apoptosis (C) as described under "Experimental Procedures." White bars, nonapoptotic; cross-hatched bars, apoptotic.

Tyrphostin AG957-blocked K562 Cells Can Be Rescued from Taxol-induced Apoptosis by Constitutively Active PKCiota -- To determine whether PKCiota activity is sufficient to protect K562 cells from taxol-induced apoptosis, we used recombinant adenovirus gene transfer to express constitutively active PKCiota in K562 cells. Constitutively active PKCiota adenovirus (caPKCiota -Ad) was produced by mutation of alanine 120 to glutamic acid in the pseudosubstrate region of PKCiota as described under "Experimental Procedures." This mutation has been demonstrated to lead to constitutive activation of PKCiota (21). K562 cells were infected with either GFP or caPKCiota adenovirus for 24 h. GFP immunofluorescence demonstrated that >95% of cells are infected under these conditions. Immunoprecipitation kinase assays demonstrate that GFP-Ad-infected cells contain PKCiota activity comparable with uninfected cells (Fig. 4A). In contrast, cells infected with caPKCiota -Ad contain 3-5-fold higher levels of PKCiota activity. Adenovirus-infected cells were next treated with taxol and tyrphostin AG957 for 24 h, and the level of apoptosis was assessed (Fig. 4B). Neither taxol nor tyrphostin AG957 alone induced apoptosis in GFP- or caPKCiota -expressing K562 cells. However, in the presence of tyrphostin AG957, taxol induced apoptosis in GFP-expressing cells. In contrast, expression of caPKCiota blocked taxol-induced apoptosis in tyrphostin AG 957-treated cells. These results demonstrate that PKCiota is a downstream target of Bcr-Abl and that PKCiota activity is sufficient to mediate the anti-apoptotic effects of Bcr-Abl.


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Fig. 4.   Expression of constitutively active PKCiota rescues AG957-treated K562 cells from taxol-induced apoptosis. A, K562 cells were infected with constitutively active (CA-PKCiota -Ad) or GFP-expressing adenovirus for 48 h. Infected cells were lysed and cellular PKCiota immunoprecipitated and assessed for kinase activity as described under "Experimental Procedures." B, K562 cells were infected with constitutively active PKCiota (aPKCiota ) or GFP-expressing adenovirus as described for A. 24 h after infection, cells were treated with taxol and/or tyrphostin AG957 for a further 24 h as indicated. Cells were fixed and scored for apoptosis as described under "Experimental Procedures."


    DISCUSSION

PKC is a family of serine/threonine, lipid-dependent kinases implicated in the regulation of cellular proliferation, differentiation, and apoptosis (19, 29-32). We have characterized the function of the three PKC isozymes expressed in K562 cells, PKCalpha , beta II, and iota , through selective overexpression and inhibition of expression of each isozyme (19, 31-33). Overexpression of PKCalpha leads to gene dose-dependent cytostasis and increased sensitivity to PMA-induced megakaryocytic differentiation, whereas inhibition of PKCalpha expression blocks PMA-induced differentiation, demonstrating that PKCalpha plays a direct role in this process (31). In contrast, overexpression of PKCbeta II leads to a small increase in proliferation and resistance to PMA-induced differentiation (31). Inhibition of PKCbeta II expression blocks proliferation, demonstrating that PKCbeta II is required for this process (31). The proliferative effects of PKCbeta II are mediated, at least in part, through the cell cycle-regulated translocation and activation of the enzyme at the nucleus (32, 33). Nuclear PKCbeta II directly phosphorylates the nuclear envelope protein lamin B at sites involved in mitotic nuclear lamina disassembly and cell cycle progression through the G2/M phase (33-35).

Atypical PKCiota plays no obvious role in K562 cell proliferation or differentiation (19). Rather, overexpression of PKCiota leads to enhanced resistance to drug-induced apoptosis, and antisense inhibition of PKCiota expression leads to increased sensitivity to apoptosis, indicating an important role in resistance to drug-induced apoptosis (19). Atypical PKC isozymes can also protect NIH 3T3 cells against UV-induced apoptosis (21), indicating that atypical PKCs may play a general role in cell survival and protection from apoptotic stress. However, the mechanism by which PKCiota protects cells from apoptosis is not known. In NIH 3T3 cells, atypical PKC activity is inhibited by UV exposure prior to the onset of apoptosis, suggesting that atypical PKC activity is required for cell survival (21). In this and our previous studies, we provide direct evidence that PKCiota activity is both necessary and sufficient for resistance of K562 cells to drug-induced apoptosis.

Our results with PKCiota are reminiscent of those described previously for Bcr-Abl, whose expression and tyrosine kinase activity is required for resistance of K562 cells to drug-induced apoptosis (5, 10, 11-15). Indeed, although both HL60 and K562 cells express similar levels of PKCiota , the pattern of PKCiota activity in these cells during apoptotic stress is distinct. During apoptotic stress, sustained activation of PKCiota is observed in K562 cells but not HL60 cells, suggesting that K562 cells possess an upstream regulator of PKCiota activity that is either not present or is defective in HL60 cells. Furthermore, we find that inhibition of Bcr-Abl blocks taxol-induced PKCiota activation and induces apoptosis in K562 cells, demonstrating that Bcr-Abl is required for taxol-induced PKCiota activation. An interesting aspect of our results is the fact that PI 3-kinase activity is not required for Bcr-Abl-mediated PKCiota activation or protection from taxol-induced apoptosis. PI 3,4,5-trisphosphate can stimulate atypical PKC activity in vivo (24). Our results do not preclude involvement of the PI 3-kinase pathway in the activation of PKCiota in response to other cellular stimuli.

Bcr-Abl is a well characterized oncoprotein whose transforming phenotype is linked to its anti-apoptotic properties. Inhibition of Bcr-Abl activity with the tyrosine kinase inhibitors quercetin or genistein (15) or Bcr-Abl expression with antisense oligonucleotides against Bcr-Abl (16, 17) sensitizes K562 cells to apoptosis. Our results using the highly selective Bcr-Abl inhibitor tyrphostin AG957 are consistent with these studies and demonstrate that PKCiota activation is a requisite downstream target of Bcr-Abl necessary for its anti-apoptotic function. Future studies will focus on determining the molecular mechanism by which Bcr-Abl activates PKCiota and on identification of the downstream targets of PKCiota that mediate its anti-apoptotic effects.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant CA56869 (to A. P. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** Leukemia Society of America Scholar. To whom correspondence should be addressed: Sealy Center for Oncology & Hematology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1048.

The abbreviations used are: CML, chronic myelogenous leukemia; PKC, protein kinase C; PI, phosphatidylinositol; GFP, green fluorescent protein.
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