Introduction

Protein kinase C (PKC) comprises a family of structurally related serine/threonine protein kinases that play crucial roles in transducing signals that regulate diverse cellular functions, including proliferation, differentiation, and apoptosis.^1,2,3,4 The PKC family is divided into three subgroups based on differences in their structures and co-factor requirements. The classical isoforms (, _I, _II, and ) are dependent upon Ca²⁺ and diacylglycerol (DAG) for their activation. The novel isoforms (, , , , and ) require DAG, but do not depend upon Ca²⁺. The atypical isoforms ( and /) do not require either DAG or Ca²⁺ for their biological activity, and in fact can be activated by the stress second-messenger molecule, ceramide.^5,6,7 The distribution of PKC isoforms appears to be tissue specific, suggesting that each isoform has a specific function depending upon the nature of stimuli and the specific cell type involved.⁴ In general, the classical PKC members such as PKC support signal-transduction pathways that promote cell survival and proliferation,^4,8,9 while atypical PKC members such as PKC likely have a role in stress-signaling pathways.^7,10

Recent data have shown that protection of cells during stress by PKC involves, at least in part, the regulation of Bcl2.^9,11,12 However, PKC has a number of downstream targets in addition to Bcl2, that can support cell survival. For instance, PKC can support cell proliferation through the mitogen-activated protein kinase (MAPK) cascade via Raf-1.^13,14,15,16 In addition, PKC regulation of the cell cycle is well documented.^4,17,18 Thus, PKC may affect the cell cycle or cell proliferation in such a way that supports cell survival.

As mentioned above, PKC is activated by DAG.^1,2 DAG and the sphingolipid ceramide have been suggested as comprising a 'cellular rheostat', since these molecules are oppositely regulated and have opposing effects as second signal molecules.^19,20,21,22 While DAG supports pro-growth signaling, ceramide induces programmed cell death, cell cycle arrest, or cell senescence in most cell types. Interestingly, ceramide promotes dephosphorylation and inactivation of both PKC and Bcl2 by protein phosphatase 2A (PP2A^23,24). This finding suggests a complex level of regulation of the entire Bcl2 pathway during apoptosis.^23,24,25,26 Whether a similar but opposite regulatory mechanism for the Bcl2 pathway during survival conditions exists is currently unknown. In this study, we show evidence for such a novel mechanism, since the overexpression of PKC in the acute lymphoblastic leukemia (ALL)-derived cell line REH results in the suppression of PP2A activity while promoting chemoresistance against the chemotherapeutic drug etoposide.

Materials and methods

Cell lines and cell viability

REH and OCI cells were obtained from the ATCC (Rockville, MD, USA) and maintained in RPMI 1640 + 10% bovine calf serum at 37°C in 5% CO₂. The REH/PKC transfectant cell lines (clone 3 and clone 5) used display near-identical phenotypic properties that have been previously described.⁹ Where appropriate, REH and REH/PKC cells were pretreated for 1 h with 10 nM okadaic acid (Calbiochem, San Diego, CA, USA) and then treated with varying doses of etoposide (Sigma, St Louis, MO, USA) for 24 h and viability was assessed by trypan blue staining as previously described.⁹

Cell proliferation and cell cycle analysis

REH and REH/PKC cells were seeded at 1 10⁵ cells/ml in six-well plates and aliquots were counted every 24 h by Coulter counter for five consecutive days. For cell cycle analysis, 5 10⁶ cells were washed in PBS and fixed in ice-cold 70% (v/v) ethanol and stained with propidium iodide (PI) solution (50 mg/ml PI, 180 U/ml RNAse, 0.1% Triton-100). The DNA content was determined using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA).

Analysis of apoptosis by annexin V staining

Cells were treated with various doses of etoposide (Sigma) or HA14-1 (Maybridge, Cornwall, UK) for 24 h. Cells were washed in PBS, resuspended in binding buffer containing annexin V (Roche Diagnostics, Indianapolis, IN, USA) and analyzed by flow cytometry using a Becton Dickinson FACScan after the addition of propidium iodide (Sigma).

Cytofluorometric analysis of mitochondrial membrane potential (_m)

To determine _m, cells treated with 1 M etoposide for 24 h were incubated with cationic lipophilic dye chlorophenyl-X-rosamine (CMXRos; Molecular Probes, Eugene, OR, USA) and CMXRos fluorescence was determined by FACScan as previously described.²⁷

Analysis of caspase activation

Cells were treated with 1 M etoposide for 24 h. Cell-permeable fluorigenic substrate Phi-Phi-Lux-G1D2 was administered to monitor caspase activity according to the manufacturer's recommendation (OncoImmunin, Kensington, MD, USA) and caspase activation was determined by FACScan as previously described.²⁷

Western blot analysis

Cells were sonicated in 200 l lysis buffer (62.5 mM Tris (pH 8.0), 2% SDS, 10% glycerol, 100 M AEBSF, 80 nM aprotinin, 5 M bestatin, 1.5 M E-64, 2 M leupeptin, 1 M Pepstatin, 500 M sodium orthovanadate, 500 M glycerol phosphate, 500 M sodium pyrophosphate, and 50 M DTT) and protein (5 10⁵ cell equivalents) was subjected to electrophoresis using 10–14% acrylamide/0.1% SDS gels. Proteins were transferred to a nitrocellulose membrane and Western blotting analysis was performed with antibodies against phospho-ERK (Cell Signaling, Beverly, MA, USA), ERK (Cell Signaling, Beverly, MA, USA), Bcl2 (Dako, Carpinteria, CA, USA), PP2A/A (Santa Cruz), PP2A/C (Santa Cruz), actin (Sigma) or PP2A/B56 . The PP2A/B56 antibody was produced at Alpha Diagnostics International (San Antonio, TX, USA) using a peptide (DGFTRKSVRKAQRQKR) which represents amino acids 22–37 of the PP2A/B56 protein sequence. Western blots were developed using an ECL kit (Amersham, Piscataway, NJ, USA) as previously described.⁹

In vitro ERK assay

ERK activity was determined using an in vitro MAPK kinase assay kit from Upstate Biotechnology (Lake Placid, NY, USA). Where indicated, cells were treated with 10 nM bryostatin-1 (Biomol, Plymouth Meeting, PA, USA) for 30 min. For each sample, ERK 1/2 was immunoprecipitated from 2 10⁷ cells using a specific anti-ERK 1/2 antibody conjugated to agarose. The ERK-containing agarose pellet was resuspended in Assay Buffer (20 mM MOPS (pH 7.2), 25 mM -glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 15 mM MgCl₂, and 100 M ATP). An inhibitor cocktail (PKC inhibitor peptide, PKA inhibitor peptide, and Compound R24571) was added to block the possible contaminating non-ERK kinases. Dephosphorylated myelin basic protein (MBP; 25 g) was added as substrate. The final volume of the reaction was 50 l. The kinase reaction was incubated at 30°C for 20 min with shaking, and spun to pellet the enzyme–agarose complex. Reaction mixture (2.5 l) containing 1 g MBP was electrophoresed using a 14% acrylamide/0.1% SDS gel and transferred to a nitrocellulose filter. Phosphorylation of MBP was observed by using an anti-phosphoMBP antibody and the Amersham ECL kit.

Metabolic labeling, immunoprecipitation, and immunoblotting analysis

Cells were labeled with [³²P] orthophosphoric acid, treated with 10 nM okadaic acid for 1 h, and Bcl2 was analyzed by immunoprecipitation as previously described,⁹ except that the anti-Bcl2 sera used was from Santa Cruz (Santa Cruz, CA, USA). Samples were electrophoresed in a 12% acrylamide/0.1% SDS gel, transferred to nitrocellulose, and exposed to Kodak X-Omat film at -80°C. The same blot was used for Western blotting with anti-Bcl2 antisera (Dako) and developed using an ECL kit (Amersham) as previously described.⁹

Protein phosphatase assay

Protein phosphatase activity of mitochondrial membrane fractions was determined as previously described.²³ Generation of free PO₄ from the phosphopeptide RRA(pT)VA was measured using the molybdate:malachite green:phosphate complex assay as described by the manufacturer (Promega, Madison, WI, USA). Mitochondrial membranes were prepared as previously described.⁹ The phosphatase assay was performed in a PP2A-specific reaction buffer (final 50 mM imidazole (pH 7.2)/0.2 mM EGTA/0.02% 2-mercaptoethanol/0.1 mg/ml BSA) using 100 M phosphopeptide substrate and 2 g of protein isolated from the mitochondrial membrane fraction. After incubation at 30°C for 30 min, molybdate dye was added and free phosphate was measured by absorbance at 590 nM. A standard curve with free phosphate was used to determine the amount of free phosphate generated. Phosphatase activity was defined as pmole free PO₄ generated/g protein/min.

Statistics

Statistical analysis was performed using standard t-test analysis with Sigma Stat computer software (SSPS, Chicago, IL, USA).

Results

PKC overexpression in REH cells does not affect cell cycle or cell proliferation

A potential mechanism for PKC 's role in chemoresistance in ALL-derived REH cells likely involves Bcl2.^9,28 PKC , however, has diverse functions and has many molecular targets.^1,2,3,4 It is possible that other mechanisms may be involved in the promotion of chemoresistance in REH cells by PKC . PKC is an important regulator of cell cycle and cell proliferation pathways,^{4,15,16,17,18} and thus raises the possibility that PKC -mediated chemoresistance in REH cells may occur via these processes. To investigate this possibility, cell cycle profiles of REH cells overexpressing PKC and parental REH cells were compared (Figure 1a). Cells in the logarithmic phase of growth were stained with propidium iodide and the cell cycle was analyzed using FACScan. As demonstrated in Figure 1a, REH/PKC transfectant cells from clones 3 and 5 and REH parental cells display nearly identical cell cycle profiles, with 49% of cells in G0/G1 phase, 33% of cells in S phase, and 16% of cells in G2/M phase. Though cell cycle effects are unlikely, a possibility remained that PKC overexpression in REH cells could alter cell proliferation, since PKC is an activator of the MAPK pathway.^13,14,15,16 Cell growth profiles reveal that there is little difference between cell proliferation rates when comparing REH parental cells and both REH clones overexpressing PKC (Figure 1b). These results demonstrate that it is unlikely that PKC promotes survival by alteration of either the cell cycle or cell proliferation in REH cells.

Figure 1.

Overexpression of PKC does not alter cell cycle or cell proliferation in REH cells. (a) The cell cycle was analyzed by quantitation of G1, S, and G2/M phase cells using propidium iodide staining of fixed cells by flow cytometry as described in Materials and methods. Error bars represent the means.d. from two separate experiments. (b) Cell proliferation rates were determined by counting cells every 24 h using a Coulter counter for five consecutive days. Error bars represent the means.d. from three separate experiments.

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REH cells exhibit little if any basal MAPK activity, and overexpression of PKC does not activate MAPK in these cells

Since PKC did not affect cell proliferation in REH cells, activity of the MAPK kinases ERK1/2 was examined in REH and two REH/PKC transfectants. As a control, human acute myeloid leukemia OCI cells were used for comparison since these cells display robust basal activation of ERK1/2.²⁷ Phosphorylated ERK1/2 is detected in OCI cells, but not in REH parental cells or REH/PKC transfectant cells (Figures 2a and b). Since bryostatin-1 (bryo) has been shown to promote ERK1/2 activation,²⁹ total and phosphorylated ERK levels were next examined in REH and REH/PKC transfectant cells treated with 10 nM bryo for 30 min. As shown in Figure 2b, ERK 1/2 can be activated in both REH parental cells and in REH/PKC transfectant cells. To confirm that ERK1/2 was not active in untreated REH or REH/PKC transfectant cells, ERK1/2 was isolated from these cells for use in an in vitro kinase assay using myelin basic protein (MBP) as a substrate (Figure 2c). ERK1/2 from untreated REH cells or cells from a representative REH/PKC transfectant clone failed to phosphorylate MBP. Consistent with the ability of bryo to promote ERK 1/2 phosphorylation in REH parental and REH/PKC transfectant cells, the drug stimulated ERK1/2 activity in both cell lines (Figure 2c). These results suggests that simple overexpression of PKC in REH cells is not sufficient to activate ERK1/2. The finding that REH/PKC transfectants exhibit little or no ERK1/2 activity provides the first evidence that PKC can act as a direct Bcl2 kinase, since ERK1/2 has also been identified as a Bcl2 kinase.³⁰

Figure 2.

REH and REH PKC transfectant cells do not display basal ERK activity. (a) Total and phospho-ERK1/2 levels in control OCI cells, REH parent cells, and REH/PKC clone 5 cells were determined by Western blot analysis, as described in Materials and methods. (b) Cells were treated with 10 nM bryo for 30 min where appropriate. Total and phospho-ERK1/2 levels in REH parental cells and cells from REH/PKC clone-3 and -5 cells were determined by Western blot analysis as described in Materials and methods. (c) MAPK kinase activity was determined using an in vitro assay with immunoprecipitated ERK1/2 from REH parent cells, parental cells treated with bryostatin-1, REH PKC clone-5 cells, and clone-5 cells treated with bryostatin-1. The MAPK kinase assay used myelin basic protein (MBP) as a substrate and was performed as described in Materials and methods.

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PKC overexpression blocks caspase activation and mitochondrial membrane depolorization in cells treated with etoposide

Etoposide-induced apoptosis was examined in REH parental cells and REH/PKC clone-3 and -5 cells. Cells were treated for 24 h with 1 M etoposide and apoptosis was then measured using Annexin V binding assay (Figure 3a). As shown in Figure 3a, etoposide-treated REH cells displayed a significant fraction of cells undergoing programmed cell death (>40%), while both PKC transfectant cell lines exhibited significantly less apoptosis in response to the drug (<10%). These results are consistent with previous data in which REH cells exhibit an IC₅₀ of 1 M for 24 h treatment with etoposide, while cells overexpressing PKC show a >10-fold increase.⁹ The molecular changes accompanying apoptosis in REH and REH/PKC transfectant cells were further characterized by measuring loss of mitochondrial membrane potential. Untreated cells and cells treated with 1 M etoposide for 24 h were stained with CMXRos, a dye that measures active respiration in the mitochondrial membranes. CMXRos-positive (ie living) and -negative (ie apoptotic) cells were identified by FACSCAN. As demonstrated in Figure 3b, REH cells treated with etoposide showed significant loss of mitochondrial membrane potential (35%), while REH/PKC clone 5 cells treated with etoposide resembled untreated cells (5%).

Figure 3.

Overexpression of PKC in REH cells abrogates the lethality of etoposide, reduces damage to mitochondrial membrane potential and prevents caspase activation. (a) Apoptosis was observed using an annexin V binding assay on REH parental and REH/PKC transfectant clone-3 and -5 cells, as described in Materials and methods. Where appropriate, cells were treated with 1 M etoposide (VP16) for 24 h (P0.001). (b) Mitochondrial membrane potential (_m) was assessed by measuring CMXRos fluorescence by flow cytometry on REH and REH/PKC clone-5 cells that were untreated or treated with 1 M etoposide (VP16) for 24 h as described in Materials and methods. (c) Activation of caspases in REH and REH/PKC clone-5 cells that were untreated or treated with 1 M etoposide (VP16) for 24 h was detected using cell-permeable fluorigenic substrate Phi-Phi-Lux-G1D2 in a flow cytometery assay, as described in Materials and methods.

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A hallmark feature of programmed cell death pathways is the activation of caspases.³¹ The ability of PKC overexpression to block caspase activation in cells treated with 1 M etoposide for 24 h was examined using the Casptag assay (Figure 3c). Consistent with the suppression of apoptosis, REH/PKC clone 5 cells display no activation of caspase, while parental REH cells demonstrate significant activation of caspase in response to etoposide. These data demonstrate that PKC can suppress the apoptotic machinery in REH cells.

Figure 3.

Continued.

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Overexpression of PKC does not effectively protect REH cells from HA14-1-induced apoptosis

Overexpression of PKC promotes Bcl2 phosphorylation.⁹ However, the results presented above demonstrate that PKC does not activate many of the non-Bcl2 survival pathways that PKC regulates, thus suggesting that PKC -mediated chemoresistance requires Bcl2. This possibility was tested by utilizing HA14-1, a cytotoxic, small molecular ligand that disrupts Bcl2's antiapoptotic function by binding to the hydrophobic region of the BH3 domain of Bcl2.^32,33,34,35 REH and REH/PKC transfectant clone 5 cells were treated with various concentrations of HA14-1 (5–50 M) for 24 h, and apoptosis was measured by annexin V binding assay (Figure 4a). As shown in Figure 4a, HA14-1 induced apoptosis in a dose-dependent manner in both cell lines. REH PKC transfectants did not display any significant resistance to HA14-1 compared to the parental cell line. The respective IC₅₀ values for parental REH cells and REH/PKC clone 5 cells treated with HA14-1 for 24 h are 16 and 20 M, respectively. In addition, protection of REH cells from etoposide imparted by PKC was completely abrogated when cells were also treated with HA14-1 (Figure 4b). While PKC overexpression protects REH cells from etoposide, ectopic PKC expression cannot efficiently protect cells from HA14-1. These results suggest that PKC promotes chemoresistance in REH cells by a mechanism that requires Bcl2.

Figure 4.

REH and REH PKC transfectant cells are similarly sensitive to the Bcl2 inhibitor, HA14-1. (a) Cells were treated with varying doses of HA14-1 for 24 h and apoptosis measured by annexin V staining as described in Materials and methods. Error bars represent the means.d. from three separate experiments. (b) Cells were treated with 1 M etoposide, 20 M HA14-1, or a combination of both drugs for 24 h. Error bars represent the means.d. from three separate experiments.

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PKC overexpression suppresses mitochondrial PP2A activity

Negative regulation of PKC by PP2A is well documented;^35,36 however, the ability of any of the PKC isoforms to regulate PP2A has yet to be demonstrated. Since PKC and PP2A both target Bcl2 at mitochondrial membranes, mitochondrial PP2A activity was compared in REH and REH/PKC clone 5 cells using a molybdate dye assay with a synthetic phosphopeptide substrate. As shown in Figure 5a, the mitochondrial PP2A activity in REH/PKC transfectants is nearly half of that observed in parental REH cells. Overexpression of PKC did not suppress expression levels of the catalytic C subunit of PP2A or the scaffold A subunit (Figure 5b). However, as shown in Figure 4b, there is a significant reduction of protein levels of the PP2A/B56 subunit. The suppressed expression of this particular B subunit is significant, since it was recently found that the PP2A/B56 subunit is the regulatory subunit that comprises the PP2A isoform that acts as the Bcl2 phosphatase.²⁴ Thus, at least one mechanism as to how PKC may suppress mitochondrial PP2A activity involves the regulation of PP2A/B56 expression. Since phosphorylation of PP2A can regulate its activity,^37,38,39 it will be important to determine if PKC can phosphorylate any of the PP2A subunits. This possibility is currently under investigation.

Figure 5.

PP2A activity is suppressed in REH cells overexpressing PKC . (a) Protein (2 g) from isolated mitochondria was used in an in vitro phosphatase assay and free PO₄ measured by absorbance at 590 nM, as described in Materials and methods. A standard curve with free phosphate was used to determine the amount of free phosphate generated. Phosphatase activity was defined as pmole free PO₄ generated/g protein/min. Error bars represent the means.d. from three separate experiments. (b) Western blot analysis was performed as described in Materials and methods using PP2A/A antisera, PP2A/C antisera, B56 antisera, and actin antisera on total protein lysate (5 10⁵ cell equivalents) from REH cells (lane 1) and REH PKC transfectant cells (lane 2). P-value was determined to be 0.004 when comparing the two sets.

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Suppression of PP2A in REH cells promotes chemoresistance to etoposide

If PKC promotes chemoresistance in REH cells by a mechanism involving the suppression of PP2A, one would predict that blocking PP2A activity in these cells would promote chemoresistance. Okadaic acid (OA) is a PP2A inhibitor that is relatively specific for PP2A at low concentrations (ie 10 nM⁴⁰). Parental REH cells treated with 10 nM OA prior to exposure to various concentrations of etoposide for 24 h became >10-fold more resistant to the drug, as indicated by the IC₅₀ values shown in Figure 6a.

Figure 6.

OA promotes cell survival and Bcl2 phosphorylation in REH cells. (a) Cells were treated with 10 nM OA 3 h prior to addition of varying doses of etoposide for 24 h. Cell survival was assessed by trypan blue staining at 24 h and IC₅₀ determined as the concentration resulting in 50% cell death. Error bars represent the means.d. from three separate experiments. (b) Cells were treated with 10 nM OA for 3 h and Bcl2 phosphorylation status was determined as described in Materials and methods.

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Furthermore, 10 nM OA promotes Bcl2 phosphorylation in parental REH cells (Figure 6b). Thus, the effects of suppression of PP2A on chemoresistance and Bcl2 phosphorylation status in REH cells mimic those effects imparted by PKC overexpression.

Discussion

In this study, the mechanistic role of PKC overexpression in the attenuation of etoposide-induced apoptosis in ALL-derived REH cells was explored. Previous data suggested that PKC acted in this process by a mechanism involving the phosphorylation of Bcl2.⁹ However, other potential non-Bcl2 regulatory pathways were not examined in that study. While Bcl2 has a potent antiapoptotic function, other PKC-regulated pathways may also promote cell survival in REH cells. For instance, PKC could influence cytoprotection through the cell cycle or cell proliferation. Several reports have demonstrated that forced overexpression of different PKC isoforms alter the cell cycle and may influence the response of transfected cell lines to chemotherapy.^41,42,43,44 The results presented here show that PKC overexpression does not influence the cell cycle in REH cells. While PKC may not affect the cell cycle in REH cells, an effect on cell proliferation seemed possible since the MAPK cascade is an important target of PKC .^13,14,15,16 However, overexpression of PKC did not change the cell proliferation rate in REH cells. This finding was consistent with the observation that PKC overexpression did not stimulate MAPK activity in these cells. In fact, REH cells or REH/PKC transfectants did not demonstrate any active ERK kinase unless bryo was introduced. REH/PKC transfectant cells have been shown to display enhanced chemoresistance when pretreated with bryo.⁹ The IC₅₀ for 24 h treatment of etoposide for the two representative clones used were >10 M (compared to 1 M for parental REH cells), while the IC₅₀ for 24 h treatment of etoposide for REH/PKC transfectant cells pretreated with bryo were >25 M.⁹ It is very likely that maximal chemoresistance arises in REH cells when both PKC and ERK kinases are active as Bcl2 kinases. Still, PKC can act as a direct physiologic kinase since Bcl2 is phosphorylated in REH PKC transfectants in the absence of ERK activity.

A number of enzymes have emerged as Bcl2 kinases.²⁸ Bcl2 kinases such as PKC promote cell survival via phosphorylation of Bcl2 at serine 70.^9,11 On the other hand, a number of stress kinases (eg JNK) promote multi-site phosphorylation of Bcl2 that results in inactivation of Bcl2 antiapoptotic function and the promotion of cell death.^45,46,47 After the discovery of PKC as a Bcl2 kinase,⁹ the ERK kinases were also found to activate Bcl2 and promote cell survival.³⁰ Since PKC can regulate the ERK kinases via Raf-1,^13,14,15,16 this raised the possibility that PKC may regulate Bcl2 indirectly via ERK. In this report, however, results show that MAPK is not involved in the protection of parental REH cell line or REH cells overexpressing PKC when treated with antineoplastic drug. In addition, blocking Bcl2 function using the Bcl2 ligand drug HA14-1 inhibited the protective effect of PKC overexpression in REH cells. This finding indicates that Bcl2 may be necessary in the protection of REH cells from drug-induced apoptosis by PKC .

While it is well documented that PP2A can negatively regulate PKC function,^35,36 it is not clear if a reciprocal regulatory pathway exists. It is known that phosphorylation can mediate PP2A function; however, at present only tyrosine sites have been identified as targets to negatively regulate PP2A function.^37,38 While serine phosphorylation of a PP2A regulatory subunit (B56 ) by the dsRNA-dependent protein kinase (PKR) has been established, this phosphorylation event promotes PP2A function.³⁹ Thus, the question remained if PKC can negatively regulate PP2A. Such a relationship is particularly important, considering that Bcl2 appears to be necessary for PKC -mediated cytoprotection in REH cells. Bcl2 phosphorylation is a dynamic process involving both Bcl2 kinases and phosphatases.^9,28,48 Interestingly, both PKC and Bcl2 are dephosphorylated by PP2A during stress stimuli, thus suggesting a complex level of regulation of the entire Bcl2 pathway during apoptosis.^25,26 Whether a similar but opposite regulatory mechanism for the Bcl2 pathway during survival conditions exists is currently unknown. Such a novel mechanism may exist, since the overexpression of PKC in the REH cells results in the suppression of PP2A activity while promoting chemoresistance. Though the mechanism how PKC may negatively regulate PP2A is not clear, one potential mechanism involves expression of a PP2A regulatory B subunit. Overexpression of PKC downregulated expression of the B56 regulatory subunit, while expression of the PP2A A and C subunits remained unchanged. Interestingly, the B56 regulatory subunit has been identified as a component of the Bcl2 phosphatase.²⁴ While the exact mechanism of PKC regulation of the PP2A is yet to be determined, the elucidation of the underlying mechanism would be of considerable interest in designing future chemotherapeutic agents that target signaling pathways.

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Acknowledgements

This work was supported by the National Institute of Health Grant CA30715-01 (PPR).