Svitlana Kurinna

Ceramide promotes apoptosis in lung cancer-derived A549 cells by a mechanism involving c-Jun NH2-terminal kinase

Ceramide regulates diverse signaling pathways involving cell senescence, the cell cycle, and apoptosis. Ceramide is known to potently activate a number of stress-regulated enzymes, including the c-Jun NH2-terminal kinase (JNK). Although ceramide promotes apoptosis in human lung cancer-derived A549 cells, a role for JNK in this process is unknown. Here, we report that ceramide promotes apoptosis in A549 cells by a mechanism involving JNK. The JNK inhibitor SP600125 proved effective at protecting cells from the lethal effects of ceramide. To understand which JNK-mediated pathway may be involved, a number of JNK target proteins were examined, including the transcription factor, c-Jun, and the apoptotic regulatory proteins Bcl-XL and Bim. A549 cells exhibited basal levels of phosphorylated c-Jun in nuclear fractions, revealing that active c-Jun is present in these cells. Ceramide was found to inhibit c-Jun phosphorylation, suggesting that JNK-mediated phosphorylation of c-Jun is not likely involved in ceramide-induced apoptosis. Ceramide did not promote Bcl-XL phosphorylation. On the other hand, ceramide promoted phosphorylation of Bim and induced translocation of active JNK from the nucleus to the cytoplasm and mitochondrial fraction. Ceramide-mediated changes in localization of JNK were consistent with the observed changes in phosphorylation status of c-Jun and Bim. Furthermore, ceramide promoted Bim translocation to the mitochondria. Mitochondrial localization of Bim has been shown recently to promote apoptosis. These results suggest that JNK may participate in ceramide-induced apoptosis in A549 cells by a mechanism involving Bim.

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 α mediates chemoresistance in acute lymphoblastic leukemia through effects on Bcl2 phosphorylation

Overexpression of protein kinase C α (PKC α) 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 α was found not to affect cell proliferation, cell cycle, or activation of mitogen-activated protein kinases. The failure of PKC α overexpression to activate non-Bcl2 survival pathways suggested that PKC α-mediated chemoresistance requires Bcl2. Supporting this notion, REH/PKC α 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 αs ability to protect REH cells from etoposide. These findings suggested that Bcl2 is necessary for the protective function of PKC α in REH cells. Since Bcl2 phosphorylation status is negatively regulated by protein phosphatase 2A (PP2A) and PP2A regulates PKC α, we investigated whether PKC α can conversely regulate PP2A. Overexpression of PKC α 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 α subunit). The ability of PKC α to target both Bcl2 and the Bcl2 phosphatase represents a novel mechanism for chemoresistance.

Inhibition of mitochondrial metabolism by methyl-2-cyano-3,12-dioxooleana-1,9-diene-28-oate induces apoptotic or autophagic cell death in chronic myeloid leukemia cells

The initial success of the first synthetic bcr-abl kinase inhibitor imatinib has been dampened by the emergence of imatinib-resistant disease in blast crisis chronic myeloid leukemia. Here, we report that the novel triterpenoid methyl-2-cyano-3,12-dioxooleana-1,9-diene-28-oate (CDDO-Me) potently induced cytotoxicity in imatinib-resistant KBM5 cells expressing the T315I mutation of bcr-abl (24-h EC50, 540 nmol/L). In long-term culture, CDDO-Me abrogated the growth of human parental KBM5 and KBM5-STI cells with 96-h IC50 of 205 and 221 nmol/L, respectively. In addition, CDDO-Me rapidly decreased the viability of murine lymphoid Ba/F3 cells expressing wild-type p210 as well as the imatinib-resistant E255K and T315I mutations of bcr-abl. The low-dose effects of CDDO-Me are associated with inhibition of mitochondrial oxygen consumption, whereas the cytotoxic effects appear to be mediated by a rapid and selective depletion of mitochondrial glutathione that accompanies the increased generation of reactive oxygen species and mitochondrial dysfunction. Interestingly, the mitochondriotoxic effects of CDDO-Me are followed by the rapid autophagocytosis of intracellular organelles or the externalization of phosphatidylserine in different cell types. We conclude that alterations in mitochondrial function by CDDO-Me can result in autophagy or apoptosis of chronic myeloid leukemia cells regardless of the mutational status of bcr-abl. CDDO-Me is in clinical trials and shows signs of clinical activity, with minimal side effects and complete lack of cardiotoxicity. Studies in leukemias are in preparation. [Mol Cancer Ther 2008;7(5):1130–9]

p53 regulates a mitotic transcription program and determines ploidy in normal mouse liver

Functions of p53 during mitosis reportedly include prevention of polyploidy and transmission of aberrant chromosomes. However, whether p53 plays these roles during genomic surveillance in vivo and, if so, whether this is done via direct or indirect means remain unknown. The ability of normal, mature hepatocytes to respond to stimuli, reenter the cell cycle, and regenerate liver mass offers an ideal setting to assess mitosis in vivo. In quiescent liver, normally high ploidy levels in adult mice increased with loss of p53. Following partial hepatectomy, p53−/− hepatocytes exhibited early entry into the cell cycle and prolonged proliferation with an increased number of polyploid mitoses. Ploidy levels increased during regeneration of both wild‐type (WT) and p53−/− hepatocytes, but only WT hepatocytes were able to dynamically resolve ploidy levels and return to normal by the end of regeneration. We identified multiple cell cycle and mitotic regulators, including Foxm1, Aurka, Lats2, Plk2, and Plk4, as directly regulated by chromatin interactions of p53 in vivo. Over a time course of regeneration, direct and indirect regulation of expression by p53 is mediated in a gene‐specific manner. Conclusion: Our results show that p53 plays a role in mitotic fidelity and ploidy resolution in hepatocytes of normal and regenerative liver. (HEPATOLOGY 2013)

Ceramide promotes apoptosis in chronic myelogenous leukemia-derived K562 cells by a mechanism involving caspase-8 and JNK

Ceramide is a sphingolipid that activates stress kinases such as p38 and c-JUN N-Terminal Kinase (JNK). Though Chronic Myelogenous Leukemia (CML) derived K562 cells resist killing by short chain C2-ceramide, we report here that longer chain C6-ceramide promotes apoptosis in these cells. C6-ceramide induces cleavage of Caspase-8 and Caspase-9, but only Caspase-8 is required for apoptosis. The sphingolipid killed CML derived KBM5 cells and, to a lesser extent, imatinib-resistant KBM5-STI cells suggesting that BCR-ABL can not completely block C6-ceramide-induced apoptosis but the kinase may regulate the process. BCR-ABL is known to suppress Protein Phosphatase 2A (PP2A) in CML cells. While C6-ceramide can activate PP2A in acute leukemia cells, the sphingolipid did not activate the phosphatase in K562 cells. C6-ceramide did not activate p38 kinase but did promote JNK activation and phosphorylation of JUN. Inhibition of JNK by pharmacological agent protected K562 cells from C6-ceramide suggesting that JNK plays an essential role in C6-ceramide mediated apoptosis. Furthermore, the sphingolipid promoted MCL-1 phosphorylation by a mechanism that, at least in part, involves JNK. The findings presented here suggest that Caspase-8, JNK, and perhaps MCL-1 may play important roles in regulating cell death and may represent new targets for therapeutic strategies for CML.

PKR regulates B56α-mediated BCL2 phosphatase activity in acute lymphoblastic leukemia-derived REH cells

Protein phosphatase 2A (PP2A) is a heterotrimer comprising catalytic, scaffold, and regulatory (B) subunits. There are at least 21 B subunit family members. Thus PP2A is actually a family of enzymes defined by which B subunit is used. The B56 family member B56α is a phosphoprotein that regulates dephosphorylation of BCL2. The stress kinase PKR has been shown to phosphorylate B56α at serine 28 in vitro, but it has been unclear how PKR might regulate the BCL2 phosphatase. In the present study, PKR regulation of B56α in REH cells was examined, because these cells exhibit robust BCL2 phosphatase activity. PKR was found to be basally active in REH cells as would be predicted if the kinase supports B56α-mediated dephosphorylation of BCL2. Suppression of PKR promoted BCL2 phosphorylation with concomitant loss of B56α phosphorylation at serine 28 and inhibition of mitochondrial PP2A activity. PKR supports stress signaling in REH cells, as suppression of PKR promoted chemoresistance to etoposide. Suppression of PKR promoted B56α proteolysis, which could be blocked by a proteasome inhibitor. However, the mechanism by which PKR supports B56α protein does not involve PKR-mediated phosphorylation of the B subunit at serine 28 but may involve eIF2α activation of AKT. Phosphorylation of serine 28 by PKR promotes mitochondrial localization of B56α, because wild-type but not mutant S28A B56α promoted mitochondrial PP2A activity. Cells expressing wild-type B56α but not S28A B56α were sensitized to etoposide. These results suggest that PKR regulates B56α-mediated PP2A signaling in REH cells.

Cascades of transcription regulation during liver regeneration

An increasing demand for new strategies in cancer prevention and regenerative medicine requires a better understanding of molecular mechanisms that control cell proliferation in tissue-specific manner. Regenerating liver is a unique model allowing use of biochemical, genetic, and engineering tools to uncover molecular mechanisms and improve treatment of hepatic cancers, liver failure, and fibrotic disease. Molecular mechanisms of liver regeneration involve extra- and intracellular factors to activate transcription of genes normally silenced in quiescent liver. While many upstream signaling pathways of the regenerating liver have been extensively studied, our knowledge of the downstream effectors, transcription factors (TFs), remains limited. This review describes consecutive engagement of pre-existing and de novo synthesized TFs, as cascades that regulate expression of growth-related and metabolic genes during liver regeneration after partial hepatectomy in mice. Several previously recognized regulators of regenerating liver are described in the light of recently identified co-activator and co-repressor complexes that interact with primary DNA-binding TFs. Published results of gene expression and chromatin immunoprecipitation analyses, as well as studies of transgenic mouse models, are used to emphasize new potential regulators of transcription during liver regeneration. Finally, a more detailed description of newly identified transcriptional regulators of liver regeneration illustrates the tightly regulated balance of proliferative and metabolic responses to partial hepatectomy.

Bcl2 phosphorylation and active PKC α are associated with poor survival in AML [13]

additional 203 bp PCR product. The second strategy consists of nested PCR, and subsequent restriction enzyme digestion with BsaXI (New England Biolabs, Hitchin, UK). In case of mutated DNA, the PCR product remains undigested, whereas unmutated DNA results in two fragments of 170 and 203 bp in size. None of the MDS/AML cases showed evidence of the JAK2 V617F mutation, regardless of the presence or absence of fibrosis. Interestingly, the mutation was detected in three of four cases (75%) diagnosed as MDS/MPD. In contrast to the MDS/AML with fibrosis, these four patients showed some clinical features of chronic myeloproliferative disease, including splenomegaly, leukocytosis and/or thrombocytosis, as well as, morphological features of CMPD in the trephine biopsy. One of the positive cases was classified as atypical chronic myelogenous leukemia negative for BCR/ABL fusion transcripts. In contrast to the results from Ohyashiki et al., our results inidicate that the JAK2 V617F mutation is exceedingly rare in bona fide MDS or de novo AML, regardless of the presence of fibrosis. Furthermore, the identification of the JAK2 mutation in MDS/MPD cases indicates that these cases are more closely related to classical CMPD. This interpretation is supported by other reports demonstrating the occurrence of the JAK2 mutation in MDS/MPD, such as chronic myelomonocytic leukemia, although the latter entity shows a lower incidence of JAK2 mutations than CMPD. We believe that the analysis of the JAK2 V617F mutation, in difficult-to-classify cases, will help to clarify the borderline between MDS, on one hand, and atypical CMPD and MDS/MPD on the other hand. The better understanding of these entities, potentially may lead in the near future to identify new therapeutic options for these patients.

Genome-Wide Location Analysis Reveals Distinct Transcriptional Circuitry by Paralogous Regulators Foxa1 and Foxa2

Gene duplication is a powerful driver of evolution. Newly duplicated genes acquire new roles that are relevant to fitness, or they will be lost over time. A potential path to functional relevance is mutation of the coding sequence leading to the acquisition of novel biochemical properties, as analyzed here for the highly homologous paralogs Foxa1 and Foxa2 transcriptional regulators. We determine by genome-wide location analysis (ChIP-Seq) that, although Foxa1 and Foxa2 share a large fraction of binding sites in the liver, each protein also occupies distinct regulatory elements in vivo. Foxa1-only sites are enriched for p53 binding sites and are frequently found near genes important to cell cycle regulation, while Foxa2-restricted sites show only a limited match to the forkhead consensus and are found in genes involved in steroid and lipid metabolism. Thus, Foxa1 and Foxa2, while redundant during development, have evolved divergent roles in the adult liver, ensuring the maintenance of both genes during evolution.