The potential of enzastaurin to enhance platelet aggregation and growth factor secretion: implications for cancer cell survival.

BACKGROUND: Enzastaurin is a protein kinase C (PKC)ß inhibitor with antiproliferative and proapoptotic effects that was in clinical development for the treatment of a variety of cancers. However, the primary endpoints in several clinical trials of enzastaurin were not met, and thrombosis was reported as an adverse effect in some trials. While investigating the role of PKC in regulating growth factor release from platelets, we found that, unlike other PKC inhibitors, enzastaurin may potentiate platelet aggregation. OBJECTIVE: To investigate the effects of enzastaurin on platelet aggregation, growth factor secretion from α-granules and cancer cell apoptosis in the presence of platelets. METHODS: Prostacyclin-washed platelets and platelet-rich plasma were isolated from the blood of healthy human volunteers. Platelet light-aggregometry was performed in the presence and absence of enzastaurin and acetylsalicylic acid (ASA). P-selectin was measured by flow cytometry, and vascular endothelial growth factor (VEGF) release was measured by ELISA. A549 lung carcinoma cells were treated with releasates from enzastaurin-titrated platelets. A cell death ELISA was performed to measure A549 apoptosis. RESULTS AND CONCLUSIONS: Enzastaurin (10(-8) -10(-6)  m) potentiated aggregation of prostacyclin-washed platelets and caused an increase in VEGF release from α-granules that, in turn, promoted cancer cell survival. In platelet-rich plasma, 10(-6)  m enzastaurin inhibited platelet aggregation, but not 10(-7)  m enzastaurin, which also failed to suppress VEGF secretion. ASA abrogated enzastaurin-potentiated washed-platelet aggregation and VEGF release. These findings indicate that, at high plasma protein-free drug concentrations, enzastaurin potentiates platelet aggregation and growth factor secretion, an effect that may counteract its anticancer activity. ASA nullifies this effect.

Apoptosis repressor with caspase recruitment domain is regulated by MAPK/PI3K and confers drug resistance and survival advantage to AML.

The apoptosis repressor with caspase recruitment domain (ARC) protein is known to suppress both intrinsic and extrinsic apoptosis. We previously reported that ARC expression is a strong, independent adverse prognostic factor in acute myeloid leukemia (AML). Here, we investigated the regulation and role of ARC in AML. ARC expression is upregulated in AML cells co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) and suppressed by inhibition of MAPK and PI3K signaling. AML patient samples with RAS mutations (N = 64) expressed significantly higher levels of ARC than samples without RAS mutations (N = 371) (P = 0.016). ARC overexpression protected and ARC knockdown sensitized AML cells to cytarabine and to agents that selectively induce intrinsic (ABT-737) or extrinsic (TNF-related apoptosis inducing ligand) apoptosis. NOD-SCID mice harboring ARC-overexpressing KG-1 cells had significantly shorter survival than mice injected with control cells (median 84 vs 111 days) and significantly fewer leukemia cells were present when NOD/SCID IL2Rγ null mice were injected with ARC knockdown as compared to control Molm13 cells (P = 0.005 and 0.03 at 2 and 3 weeks, respectively). Together, these findings demonstrate that MSCs regulate ARC in AML through activation of MAPK and PI3K signaling pathways. ARC confers drug resistance and survival advantage to AML in vitro and in vivo, suggesting ARC as a novel target in AML therapy.

Multilayer Architectures for Facial Action Unit Recognition.

In expression recognition and many other computer vision applications, the recognition performance is greatly improved by adding a layer of nonlinear texture filters between the raw input pixels and the classifier. The function of this layer is typically known as feature extraction. Popular filter types for this layer are Gabor energy filters (GEFs) and local binary patterns (LBPs). Recent work [1] suggests that adding a second layer of nonlinear filters on top of the first layer may be beneficial. However, it is unclear what is the best architecture of layers and selection of filters. In this paper, we present a thorough empirical analysis of the performance of single-layer and dual-layer texture-based approaches for action unit recognition. For the single hidden layer case, GEFs perform consistently better than LBPs, which may be due to their robustness to jitter and illumination noise as well as to their ability to encode texture at multiple resolutions. For dual-layer case, we confirm that, while small, the benefit of adding this second layer is reliable and consistent across data sets. Interestingly for this second layer, LBPs appear to perform better than GEFs.

MEK inhibition enhances ABT-737-induced leukemia cell apoptosis via prevention of ERK-activated MCL-1 induction and modulation of MCL-1/BIM complex.

Recently, strategies for acute myeloid leukemia (AML) therapy have been developed that target anti-apoptotic BCL2 family members using BH3-mimetic drugs such as ABT-737. Though effective against BCL2 and BCL-X(L), ABT-737 poorly inhibits MCL-1. Here we report that, unexpectedly, ABT-737 induces activation of the extracellular receptor activated kinase and induction of MCL-1 in AML cells. MEK inhibitors such as PD0325901 and CI-1040 have been used successfully to suppress MCL-1. We report that PD0325901 blocked ABT-737-induced MCL-1 expression, and when combined with ABT-737 resulted in potent synergistic killing of AML-derived cell lines, primary AML blast and CD34+38-123+ progenitor/stem cells. Finally, we tested the combination of ABT-737 and CI-1040 in a murine xenograft model using MOLM-13 human leukemia cells.Whereas control mice and CI-1040-treated mice exhibited progressive leukemia growth, ABT-737, and to a significantly greater extent, ABT-737+CI-1040 exerted major anti-leukemia activity. Collectively, results demonstrated unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3-mimetic ABT-737 and mitogen-activated protein kinase signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces its expression. However, concomitant inhibition by BH3 mimetics and MEK inhibitors could abrogate this effect and may be developed into a novel and effective therapeutic strategy for patients with AML.

Low expression of PP2A regulatory subunit B55α is associated with T308 phosphorylation of AKT and shorter complete remission duration in acute myeloid leukemia patients.

The regulation of protein kinase B (AKT) is a dynamic process that depends on the balance between phosphorylation by upstream kinases for activation and inactivation by dephosphorylation by protein phosphatases. Phosphorylated AKT is commonly found in acute myeloid leukemia (AML) and confers an unfavorable prognosis. Understanding the relative importance of upstream kinases and AKT phosphatase in the activation of AKT is relevant for the therapeutic targeting of this signaling axis in AML. The B55α subunit of protein phosphatase 2A (PP2A) has been implicated in AKT dephosphorylation, but its role in regulating AKT in AML is unknown. We examined B55α protein expression in blast cells derived from 511 AML patients using reverse phase protein analysis. B55α protein expression was lower in AML cells compared with normal CD34+ cells. B55α protein levels negatively correlated with threonine 308 phosphorylation levels. Low levels of B55α were associated with shorter complete remission duration, demonstrating that decreased expression is an adverse prognostic factor in AML. These findings suggest that decreased B55α expression in AML is at least partially responsible for increased AKT signaling in AML and suggests that therapeutic targeting of PP2A could counteract this.

Roles of the Ras/Raf/MEK/ERK pathway in leukemia therapy.

The Ras/Raf/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway is often implicated in sensitivity and resistance to leukemia therapy. Dysregulated signaling through the Ras/Raf/MEK/ERK pathway is often the result of genetic alterations in critical components in this pathway as well as mutations at upstream growth factor receptors. Unrestricted leukemia proliferation and decreased sensitivity to apoptotic-inducing agents and chemoresistance are typically associated with activation of pro-survival pathways. Mutations in this pathway and upstream signaling molecules can alter sensitivity to small molecule inhibitors targeting components of this cascade as well as to inhibitors targeting other key pathways (for example, phosphatidylinositol 3 kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome 10 (PTEN)/Akt/mammalian target of rapamycin (mTOR)) activated in leukemia. Similarly, PI3K mutations can result in resistance to inhibitors targeting the Ras/Raf/MEK/ERK pathway, indicating important interaction points between the pathways (cross-talk). Furthermore, the Ras/Raf/MEK/ERK pathway can be activated by chemotherapeutic drugs commonly used in leukemia therapy. This review discusses the mechanisms by which abnormal expression of the Ras/Raf/MEK/ERK pathway can contribute to drug resistance as well as resistance to targeted leukemia therapy. Controlling the expression of this pathway could improve leukemia therapy and ameliorate human health.

Targeting the translational apparatus to improve leukemia therapy: roles of the PI3K/PTEN/Akt/mTOR pathway.

It has become apparent that regulation of protein translation is an important determinant in controlling cell growth and leukemic transformation. The phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome ten (PTEN)/Akt/mammalian target of rapamycin (mTOR) pathway is often implicated in sensitivity and resistance to therapy. Dysregulated signaling through the PI3K/PTEN/Akt/mTOR pathway is often the result of genetic alterations in critical components in this pathway as well as mutations at upstream growth factor receptors. Furthermore, this pathway is activated by autocrine transformation mechanisms. PTEN is a critical tumor suppressor gene and its dysregulation results in the activation of Akt. PTEN is often mutated, silenced and is often haploinsufficient. The mTOR complex1 (mTORC1) regulates the assembly of the eukaryotic initiation factor4F complex, which is critical for the translation of mRNAs that are important for cell growth, prevention of apoptosis and transformation. These mRNAs have long 5'-untranslated regions that are G+C rich, rendering them difficult to translate. Elevated mTORC1 activity promotes the translation of these mRNAs via the phosphorylation of 4E-BP1. mTORC1 is a target of rapamycin and novel active-site inhibitors that directly target the TOR kinase activity. Although rapamycin and novel rapalogs are usually cytostatic and not cytotoxic for leukemic cells, novel inhibitors that target the kinase activities of PI3K and mTOR may prove more effective for leukemia therapy.

Novel role of HDAC inhibitors in AML1/ETO AML cells: activation of apoptosis and phagocytosis through induction of annexin A1.

The chimeric fusion protein AML1-ETO, created by the t(8;21) translocation, recruits histone deacetylase (HDAC) to AML1-dependent promoters, resulting in transcriptional repression of the target genes. We analyzed the transcriptional changes in t(8;21) Kasumi-1 AML cells in response to the HDAC inhibitors, depsipeptide (FK228) and suberoylanilide hydroxamic acid (SAHA), which induced marked growth inhibition and apoptosis. Using cDNA array, annexin A1 (ANXA1) was identified as one of the FK228-induced genes. Induction of ANXA1 mRNA was associated with histone acetylation in ANXA1 promoter and reversal of the HDAC-dependent suppression of C/EBPalpha by AML1-ETO with direct recruitment of C/EBPalpha to ANXA1 promoter. This led to increase in the N-terminal cleaved isoform of ANXA1 protein and accumulation of ANXA1 on cell membrane. Neutralization with anti-ANXA1 antibody or gene silencing with ANXA1 siRNA inhibited FK228-induced apoptosis, suggesting that the upregulation of endogenous ANXA1 promotes cell death. FK228-induced ANXA1 expression was associated with massive increase in cell attachment and engulfment of Kasumi-1 cells by human THP-1-derived macrophages, which was completely abrogated with ANXA1 knockdown via siRNA transfection or ANXA1 neutralization. These findings identify a novel mechanism of action of HDAC inhibitors, which induce the expression and externalization of ANXA1 in leukemic cells, which in turn mediates the phagocytic clearance of apoptotic cells by macrophages.

The novel triterpenoid CDDO-Me suppresses MAPK pathways and promotes p38 activation in acute myeloid leukemia cells.

Development of novel therapeutic strategies is a continuing challenge for the treatment of acute myeloid leukemia (AML). The novel triterpenoid, C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid (CDDO-Me), induces apoptosis in myeloid leukemic cell lines and in primary AML samples. In this report, the effects of CDDO-Me on CD34(+) AML progenitor cells in vitro were examined. CDDO-Me induced apoptosis in all but one of ten AML samples. CDDO-Me is known to inhibit the activation of ERK1/2. In this series of primary AML samples, ERK was expressed and phosphorylated in all patient samples studied and CDDO-Me inhibited ERK phosphorylation in five of 10 samples. However, CDDO-Me induced apoptosis in four of five samples without decreasing pERK levels, suggesting that pERK is not the sole target of the compound. CDDO-Me induced phosphorylation of p38 in AML-derived U937 cells. Pretreatment of U937 cells with a p38 inhibitor protected cells from the cyto-toxic effects of CDDO-Me. These findings suggest a role for p38 in CDDO-Me-induced apoptosis. In preliminary studies, CDDO-Me induced p38 phosphorylation in seven of eight primary AML samples. These findings suggest that CDDO-Me treatment shifts cell signaling away from cyto-protective pathways and thus CDDO-Me may be effective for the treatment of AML.

PKC alpha mediates chemoresistance in acute lymphoblastic leukemia through effects on Bcl2 phosphorylation.

Overexpression of protein kinase C alpha (PKC alpha) promotes Bcl2 phosphorylation and chemoresistance in human acute leukemia cells. The contribution of non-Bcl2 mechanisms in this process is currently unknown. In this report, overexpression of PKC alpha was found not to affect cell proliferation, cell cycle, or activation of mitogen-activated protein kinases. The failure of PKC alpha overexpression to activate non-Bcl2 survival pathways suggested that PKC alpha-mediated chemoresistance requires Bcl2. Supporting this notion, REH/PKC alpha transfectants were found to be as sensitive to HA14-1 (a drug that targets Bcl2 function) as parental cells. In addition, HA14-1 abrogated PKC alpha's ability to protect REH cells from etoposide. These findings suggested that Bcl2 is necessary for the protective function of PKC alpha in REH cells. Since Bcl2 phosphorylation status is negatively regulated by protein phosphatase 2A (PP2A) and PP2A regulates PKC alpha, we investigated whether PKC alpha can conversely regulate PP2A. Overexpression of PKC alpha was found to suppress mitochondrial PP2A activity by a mechanism that, at least in part, involves suppressed expression of the regulatory subunit comprising the Bcl2 phosphatase (ie the PP2A/B56 alpha subunit). The ability of PKC alpha to target both Bcl2 and the Bcl2 phosphatase represents a novel mechanism for chemoresistance.

Requirement for SAPK-JNK signaling in the induction of apoptosis by ribosomal stress in REH lymphoid leukemia cells.

The present studies examined performance of SAPK cascades and apoptotic commitment following ribosomal trauma in REH lymphoid leukemia cells. Ribostatic insults included disruption of ribosomal activity by mechanistically dissimilar agents such as blasticidin-S (BCS) (which binds 28S-rRNA to block peptidyl bond formation), kasugamycin (KSM) (which binds 18S-rRNA to prevent translational initiation), and cycloheximide (CHX) (which blocks A-site to P-site translocation of peptidyl-tRNA). Exposure of REH cells to BCS elicited DNA degradation and apoptotic cytolysis. BCS stimulated JNK1/JNK2 and p38, and their shared targets c-Jun and ATF2. Inhibition of JNK1/JNK2 (but not of p38) antagonized blasticidin-induced apoptosis, whereas targeting alternative ribosomal sites with KSM or CHX limited translation, but failed to activate the SAPK cascade or initiate apoptosis. Our findings indicate that interference with 28S-rRNA by BCS initiates apoptosis in REH cells through recruitment of SAPK-JNK signaling. Disparities between the lethal actions of BCS, KSM, and CHX appear to reflect established differences in the subribosomal targets of these agents. We propose that the SAPK cascade comprises an essential mechanism for the transduction of specific lethal stress signals emanating from active ribosomes, and that interference with the 28S-rRNA, rather than the peptidyl transfer center of the large subunit, is critical to apoptotic commitment.

Ceramide regulates cellular homeostasis via diverse stress signaling pathways.

The sphingolipid ceramide is an important second signal molecule that regulates diverse signaling pathways involving apoptosis, cell senescence, the cell cycle, and differentiation. For the most part, ceramide's effects are antagonistic to growth and survival. Interestingly, ceramide and the pro-growth agonist, diacylglycerol (DAG) appear to be regulated simultaneously but in opposite directions in the sphingomyelin cycle. While ceramide stimulates signal transduction pathways that are associated with cell death or at least are inhibitory to cell growth (eg stress-activated protein kinase, SAPK, pathways), DAG activates the classical and novel isoforms of the protein kinase C (PKC) family. These PKC isoforms are associated with cell growth and cell survival. Furthermore, DAG activation of PKC stimulates other signal transduction pathways that support cell proliferation (eg mitogen-activated protein kinase, MAPK, pathways). Thus, ceramide and DAG generation may serve to monitor cellular homeostasis by inducing pro-death or pro-growth pathways, respectively. The production of ceramide is emerging as a fixture of programmed cell death. Ceramide levels are elevated in response to diverse stress challenges including chemotherapeutic drug treatment, irradiation, or treatment with pro-death ligands such as tumor necrosis factor alpha, TNF alpha. Consistent with this notion, ceramide itself is a potent apoptogenic agent. Ceramide activates stress-activated protein kinases like c-jun N-terminal kinase (JNK) and thus affects transcription pathways involving c-jun. Ceramide activates protein phosphatases such as protein phosphatase 1 (PP1) and protein phosphatase 2 (PP2A). Ceramide activation of protein phosphatases has been shown to promote inactivation of a number of pro-growth cellular regulators including the kinases PKC alpha and Akt, Bcl2 and the retinoblastoma protein. A new role has recently emerged for ceramide in the regulation of protein synthesis. Ceramide-induced activation of double-stranded RNA-dependent protein kinase (PKR), a protein kinase important in anti-viral host defense mechanisms and recently implicated in cellular stress pathways, results in the inhibition of protein synthesis as a prelude to cell death. Taken together, these properties of ceramide suggest that this important second-signal molecule may have useful properties as an anti-neoplastic agent. Thus, strategies to promote ceramide metabolism or use of ceramide analogs directly may one day become useful in the treatment of diseases like leukemia.

Phosphorylation of Bcl2 and regulation of apoptosis.

Members of the Bcl2 family of proteins are important regulators of programmed cell death pathways with individual members that can suppress (eg Bcl2, Bcl-XL) or promote (eg Bax, Bad) apoptosis. While the mechanism(s) of Bcl2's anti-apoptotic function is not yet clear, introduction of Bcl2 into most eukaryotic cell types will protect the recipient cell from a wide variety of stress applications that lead to cell death. There are, however, physiologic situations in which Bcl2 expression apparently fails to protect cells from apoptosis (eg negative selection of thymocytes). Further, Bcl2 expression in patient tumor samples does not consistently correlate with a worse outcome or resistance to anticancer therapies. For example, patient response and survival following chemotherapy is independent of Bcl2 expression at least for pediatric patients with ALL. These findings indicate that simple expression of Bcl2 may not be enough to functionally protect cells from apoptosis. The finding that Bcl2 is post-translationally modified by phosphorylation suggests another level of regulation of function. Recent studies have shown that agonist-activated phosphorylation of Bcl2 at serine 70 (single site phosphorylation), a site within the flexible loop domain (FLD), is required for Bcl2's full and potent anti-apoptotic function, at least in murine IL-3-dependent myeloid cell lines. Several protein kinases have now been demonstrated to be physiologic Bcl2 kinases indicating the importance of this post-translational modification. Since Bcl2 phosphorylation has been found to be a dynamic process involving both a Bcl2 kinase(s) and phosphatase(s), a mechanism exists to rapidly and reversibly regulate Bcl2's activity and affect cell viability. In addition, multisite Bcl2 phosphorylation induced by anti-mitotic drugs like paclitaxel may inhibit Bcl2 indicating the potential wide range of functional consequences that this post-translational modification may have on function. While post-translational mechanisms other than phosphorylation may also regulate Bcl2's function (eg ubiquitination), this review will focus on the regulatory role for phosphorylation and discuss its potential clinical ramifications.

Novel role for JNK as a stress-activated Bcl2 kinase.

Interleukin (IL)-3-induced Bcl2 phosphorylation at Ser(70) may be required for its full and potent antiapoptotic activity. However, in the absence of IL-3, increased expression of Bcl2 can also prolong cell survival. To determine how Bcl2 may be functionally phosphorylated following IL-3 withdrawal, a stress-activated Bcl2 kinase (SAK) was sought. Results indicate that anisomycin, a potent activator of the stress kinase JNK/SAPK, can induce Bcl2 phosphorylation at Ser(70) and that JNK1 can be latently activated following IL-3 withdrawal to mediate Bcl2 phosphorylation. JNK1 directly phosphorylates Bcl2 in vitro, co-localizes with Bcl2, and collaborates with Bcl-2 to mediate prolonged cell survival in the absence of IL-3 or following various stress applications. Dominant-negative (DN)-JNK1 can block both anisomycin and latent IL-3 withdrawal-induced Bcl2 phosphorylation (>90%) and potently enhances cell death. Furthermore, low dose okadaic acid (OA), a potent protein phosphatase 1 and 2A inhibitor, can activate the mitogen-activated protein kinases JNK1 and ERK1/2, but not p38 kinase, to induce Bcl2 phosphorylation and prolong cell survival in factor-deprived cells. Since PD98059, a specific MEK inhibitor, can only partially inhibit OA-induced Bcl2 phosphorylation but completely blocks OA-induced Bcl2 phosphorylation in cells expressing DN-JNK1, this supports the conclusion that OA may stimulate Bcl2 phosphorylation via a mechanism involving both JNK1 and ERK1/2. Collectively, these findings indicate a novel role for JNK1 as a SAK and may explain, at least in part, how functional phosphorylation of Bc12 can occur in the absence of growth factor.

Regulation of Bcl2 phosphorylation and potential significance for leukemic cell chemoresistance.

Although considered tightly linked, the linkage effectors for proliferation and antiapoptotic signaling pathways are not clear. Phosphorylation of Bcl2 at serine 70 is required for suppression of apoptosis in interleukin 3 (IL-3)-dependent myeloid cells deprived of IL-3 or treated with antileukemic drugs and can result from agonist activation of mitochondrial protein kinase C alpha (PKCalpha). However, we have recently found that high concentrations of staurosporine up to 1 microM: can only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKCalpha-mediated Bcl2 phosphorylation in vitro, indicating the existence of a non-PKC, staurosporine-resistant Bcl2 kinase (SRK). Although the RAF-1MEK-1-mitogen-activated protein kinase (MAPK) cascade is required for factor-dependent mitogenic signaling, a direct role in antiapoptosis signaling is not clear. In particular, the role of phosphorylation in the regulation of death substrates is not yet clear. Our findings indicate a potential role for the MEK/MAPK pathway in addition to PKC in antiapoptosis signaling, involving Bcl2 phosphorylation that features a role for extracellular signal-regulated kinase (ERK)1 and 2 as SRKs. These findings indicate a novel role for ERK1 and 2 as molecular links between proliferative and survival signaling and may, at least in part, explain the apparent paradox by which Bcl2 may suppress staurosporine-induced apoptosis. Although the effect of phosphorylation on Bcl2 function is not clear, effector molecules that regulate Bcl2 phosphorylation may have clinical significance in patients with acute myelogenous leukemia (AML) who express detectable levels of Bcl2. Preliminary findings suggest that expression of PKCalpha, ERK2, and Bax in leukemic blast cells from patients with AML, although individually not prognostic, appears to have potential clinical value in predicting chemoresistance and survival outcomes.

Ceramide regulates protein synthesis by a novel mechanism involving the cellular PKR activator RAX.

The sphingolipid ceramide is an important second signal molecule and potent apoptotic agent. The production of ceramide is associated with virtually every known stress stimulus, and thus, generation of this sphingolipid has been suggested as a universal feature of apoptosis. Recent studies suggest that an important component of cell death following diverse stress stimuli (e.g. interleukin-3 withdrawal, sodium arsenite treatment, and peroxide treatment) is the activation of the double-stranded RNA-activable protein kinase, PKR, resulting in the inhibition of protein synthesis (Ito, T., Jagus, R., and May, W. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7455-7459). The recently discovered cellular PKR activator, RAX, is phosphorylated in association with PKR activation (Ito, T., Yang, M., and May, W. S. (1999) J. Biol. Chem. 274, 15427-15432). Since RAX is phosphorylated by an as yet undetermined SAPK and ceramide is a potent activator of SAPKs such as JNK, a role for ceramide in the activation of RAX might be possible. Results indicate that overexpression of exogenous RAX potentiates ceramide-induced killing. Furthermore, ceramide can potently inhibit protein synthesis. Since ceramide potently promotes RAX and eukaryotic initiation factor-2alpha phosphorylation, a possible role for ceramide in this process may involve the activation of PKR by RAX. Since 2-aminopurine, a serine/threonine kinase inhibitor that has previously been shown to inhibit PKR, blocks both the potentiation of ceramide killing by RAX and ceramide-induced inhibition of protein synthesis, ceramide appears to promote PKR activation, at least indirectly. Collectively, these findings suggest a novel role for ceramide in the regulation of protein synthesis and apoptosis.

BAX and PKCalpha modulate the prognostic impact of BCL2 expression in acute myelogenous leukemia.

Previously, we demonstrated that the level of BCL2 expression is prognostic in acute myelogenous leukemia (AML). High levels of BCL2 correlate with an adverse outcome when associated with favorable and intermediate prognosis cytogenetics (FIPC), whereas low levels portend an adverse outcome when associated with unfavorable cytogenetics (UC). Because BCL2 function can be modulated by dimerization with family members, like BAX, or by phosphorylation by protein kinase C alpha (PKCalpha), we hypothesize that the relative expression of these proteins in primary leukemic cells might alter the prognostic impact of BCL2 expression. We therefore measured BAX and PKCalpha protein levels in peripheral blood mononuclear cell lysates from 165 newly diagnosed AML patients and correlated the expression of these proteins with BCL2 expression, patient survival, and remission induction success. Expression levels of BAX and PKCalpha were normalized against a control cell line, K562. BAX and PKCalpha expression levels were heterogeneous and did not correlate with the percentage of blasts in the sample (R2 = 0.01 and <0.01). The median expression of both was similar across FAB groups but the range was greater for M4. A similar distribution of expression was observed in all cytogenetic groups, except that patients with inversion 16 demonstrated lower levels of BAX. Individually, neither PKCalpha nor BAX expression was prognostic of response to induction therapy or survival. A similar outcome was obtained when patients were stratified by cytogenetics into FIPC and UC groups. However, the ratio of either BCL2:BAX (B2:BX) or PKCalpha*B2:BX (PK*B2:BX) was highly prognostic. Patients with FIPC and a lower ratio (less than median) of either B2:BX or PK*B2:BX had a significantly higher remission induction rate (88 versus 69%, P = 0.04) and longer survival (median: 141 versus 80.5 weeks, P = 0.007) compared with those with ratios more than median. For patients with UC, values of either B2:BX or PK*B2:BX below the median had an inferior response rate to induction therapy (35 versus 78%, P = 0.0006) and inferior survival outcomes (median survival: 11 versus 53 weeks, P = 0.00002). Interestingly, FIPC and UC patients with antiapoptotic ratios (defined as B2:BX or PK*B2:BX more than median) had identical response rates and survival outcomes. In multivariate analyses, the compound variables of cytogenetics and B2:BX, or PK*B2:BX were independent predictors of survival. These results suggest that expression levels of proteins that affect the functional status of BCL2 modify the prognostic impact of BCL2 and suggest that the role of apoptosis in different cases of AML varies independently in the different cytogenetic subgroups.