Bcl-2 family member Mcl-1 expression is reduced under hypoxia by the E3 ligase FBW7 contributing to BNIP3 induced cell death in glioma cells.

Mcl-1 is an anti-apoptotic Bcl-2 family member that is often over-expressed in the malignant brain tumor glioblastoma (GBM). It has been previously shown that epidermal growth factor receptors up-regulate Mcl-1 contributing to a cell survival response. Hypoxia is a poor prognostic marker in glioblastoma despite the fact that hypoxic regions have areas of necrosis. Hypoxic regions of GBM also highly express the pro-cell death Bcl-2 family member BNIP3, yet when BNIP3 is overexpressed in glioma cells, it induces cell death. The reasons for this discrepancy are unclear. Herein we have found that Mcl-1 expression is reduced under hypoxia due to degradation by the E3 ligase FBW7 leading to increased hypoxia induced cell death. This cell death is reduced by EGFR activation leading to increased Mcl-1 expression under hypoxia. Conversely, BNIP3 is over-expressed in hypoxia at times when Mcl-1 expression is decreased. Knocking down BNIP3 expression reduces hypoxia cell death and Mcl-1 expression effectively blocks BNIP3 induced cell death. Of significance, BNIP3 and Mcl-1 are co-localized under hypoxia in glioma cells. These results suggest that Mcl-1 can block the ability of BNIP3 to induce cell death under hypoxia in GBM tumors.

Identification of Hb Wayne and its effects on HbA1c measurement by 5 methods.

BACKGROUND: The World Health Organization and the American and Canadian Diabetes Associations approved HbA1c >6.5% as diagnostic for type 2 diabetes mellitus (T2DM). Hb variants and/or their chemically modified species can interfere with HbA1c measurements. We recently described a patient with Hb Wayne trait who was misdiagnosed with T2DM based on falsely elevated HbA1c. Hb Wayne is a clinically silent variant that exists as two isoforms: Hb Wayne I (Asn 139) and Hb Wayne II (Asp 139). METHODS: Hemoglobinopathy investigation was performed by HPLC (Bio-Rad VARIANT-II), alkaline and acid electrophoresis (Sebia Hydrasis2), capillary zone electrophoresis (Sebia CAPILLARYS2™) and DNA sequencing. HbA1c was measured by five methods. RESULTS: Hb Wayne eluted as two small fractions with retention times of 1.0 and 1.46min on the HPLC (Bio-Rad VARIANT-II). Alkaline gel and capillary electrophoresis showed two small bands migrating faster than HbA. Hb Wayne generated spuriously high results on the Bio-Rad VARIANT-II Turbo 2.0, no results on the Tosoh G8, and did not interfere with either the Sebia CAPILLARYS2™ or immunoassays from Roche (tinaquant) and Siemens (Bayer DCA2000+). Based on the Hb Wayne HPLC profile of 3 patients, an algorithm was developed to facilitate its detection, which identified 9 additional patients with Hb Wayne trait. CONCLUSIONS: We characterize Hb Wayne by chromatographic and electrophoretic techniques and show the effect of Hb Wayne on five common HbA1c methodologies. We developed a quality assurance tool to assist in detecting Hb Wayne trait during HbA1c analysis on the Bio-Rad VARIANT-II™ Turbo 2.0.

Truncated forms of BNIP3 act as dominant negatives inhibiting hypoxia-induced cell death.

BNIP3 (Bcl-2/adenovirus E1B Nineteen Kilodalton Interacting Protein) is a pro-cell death member of the Bcl-2 family of proteins. Its expression is induced by the transcription factor Hypoxia Inducible Factor-1 (HIF-1) under conditions of low oxygen (hypoxia) and is found over expressed in hypoxic regions of many tumors. When over expressed, BNIP3 induces cell death through induction of mitochondrial dysfunction that is dependent on the presence of BNIP3's TM domain. Herein, we have determined that the SkOv3 ovarian cancer cell line expresses a truncated BNIP3 protein, which results in the elimination of the transmembrane domain. Truncation that eliminates all four domains of BNIP3 protein also inhibits hypoxia-induced cell death in SkOv3, HEK293, U251 and MCF-7 cells. Three different mutations in a BNIP3 expression vector that lead to a truncated BNIP3 protein, lacking TM domain only, or lacking CD, BH3, and TM domains resulted in inhibition of hypoxia-induced cell death when transfected into HEK293 cells. We found that truncated BNIP3 failed to associate with the mitochondria and the truncated BNIP3 lacking all four domains can bind to wild type BNIP3. Taken together, truncation of BNIP3 could be a novel mechanism for cancer cells to avoid hypoxia-induced cell death mediated by BNIP3 over expression.

BNIP3 (Bcl-2 19 kDa interacting protein) acts as transcriptional repressor of apoptosis-inducing factor expression preventing cell death in human malignant gliomas.

The Bcl-2 19 kDa interacting protein (BNIP3) is a pro-cell-death BH3-only member of the Bcl-2 family. We previously found that BNIP3 is localized to the nucleus in the majority of glioblastoma multiforme (GBM) tumors and fails to induce cell death. Herein, we have discovered that nuclear BNIP3 binds to the promoter of the apoptosis-inducing factor (AIF) gene and represses its expression. BNIP3 associates with PTB-associating splicing factor (PSF) and HDAC1 (histone deacetylase 1) contributing to transcriptional repression of the AIF gene. This BNIP3-mediated reduction in AIF expression leads to decreased temozolomide-induced apoptosis in glioma cells. Furthermore, nuclear BNIP3 expression in GBMs correlates with decreased AIF expression. Together, we have discovered a novel transcriptional repression function for BNIP3 causing reduced AIF expression and increased resistance to apoptosis. Thus, nuclear BNIP3 may confer a survival advantage to glioma cells and explain, in part, why BNIP3 is expressed at high levels in solid tumors, especially GBM.

The pro-cell death Bcl-2 family member, BNIP3, is localized to the nucleus of human glial cells: Implications for glioblastoma multiforme tumor cell survival under hypoxia.

The Bcl-2 nineteen kilodalton interacting protein 3 (BNIP3) is a hypoxia-inducible proapoptotic member of the Bcl-2 family that induces cell death by associating with the mitochondria. Under normal conditions, BNIP3 is expressed in skeletal muscle and in the brain at low levels. In many human solid tumors, BNIP3 is upregulated in hypoxic regions but paradoxically, this BNIP3 expression fails to induce cell death. Herein, we have determined that BNIP3 is primarily localized to the nucleus of glial cells of the normal human brain, as well as in the malignant glioma cell line U251. Upon exposure of U251 cells to hypoxia, BNIP3 expression in the cytoplasm increases and localizes with the mitochondria, contributing to induction of cell death. In contrast, when BNIP3 is forcibly over expressed in the nucleus, it fails to induce cell death. Expression of N-terminal BNIP3 (lacking the transmembrane and conserved domains) in U251 cells blocks hypoxia-induced cell death acting as a dominant negative protein by binding to wild-type BNIP3 and blocking its association with the mitochondria. In glioblastoma multiforme (GBM) tumors, BNIP3 expression is increased in hypoxic regions of the tumor and is primarily localized to the nucleus in approximately 80% of tumors. Hence, BNIP3 is sequestered in the nucleus within the brain but under hypoxic conditions, BNIP3 becomes primarily cytoplasmic, promoting cell death. In GBMs, BNIP3 expression is increased but it remains sequestered in the nucleus in hypoxic regions, thereby blocking BNIP3's ability to associate with the mitochondria, providing tumor cells with a possible survival advantage.

Myogenic signaling by lithium in cardiomyoblasts is Akt independent but requires activation of the beta-catenin-Tcf/Lef pathway.

Rat H9c2 cardiomyoblasts can proliferate and maintain an undifferentiated state in the presence of serum. These cardiomyoblasts have been used as a cellular model to study myogenic differentiation after serum withdrawal. Here, we examined the effects of lithium, a known inhibitor of glycogen synthase kinase-3beta and activator of Wnt pathway in myogenic differentiation. We show that in the presence of serum, lithium induced the differentiation of H9c2 cells as measured by multinucleated myotube formation and expression of the muscle-specific proteins, myogenin and skeletal alpha-actin. This differentiation was preceded by nuclear accumulation of beta-catenin, which was associated with increased Tcf/Lef-dependent transcription. We also observed that lithium mediated the activation of phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target Akt. Inhibition of PI3-kinase by LY294002 and over-expression of dominant-negative PI3-kinase caused a marked reduction in beta-catenin levels. This inhibition was associated with decreased beta-catenin-Tcf/Lef-dependent transcription, lack of multinucleated myotube formation, and expression of the muscle-specific proteins. In contrast, expression of dominant-negative Akt failed to inhibit the effects of lithium. We conclude that the capacity of lithium to overcome the inhibitory effects of serum and to induce the differentiation of H9c2 cardiomyoblasts is mediated, in part, by the stabilization and nuclear translocation of beta-catenin in a PI3-kinase-dependent but Akt-independent manner. Once activated, beta-catenin then interacts with the Lef/Tcf complex to regulate expression of myogenic-inducing genes.

Cellular signaling pathways affect the function of ribonucleotide reductase mRNA binding proteins: mRNA stabilization, drug resistance, and malignancy (Review).

Ribonucleotide reductase is an enzyme that is essential for DNA synthesis and repair. It is composed of 2 dimeric proteins called R1 and R2 that are both necessary for enzymatic activity that reduces ribonucleotides to deoxyribonucleotides. This is the rate-limiting reaction that provides a supply of precursors for DNA synthesis therefore it is essential for cell proliferation. The importance of understanding the complex regulation of ribonucleotide reductase is emphasized by observations that mechanisms controlling its expression and activity may be altered during malignant cell proliferation which leads to drug resistance, making it a useful target to develop chemotherapeutic compounds in the treatment of cancer. Expression studies with the R1 and R2 genes have provided evidence for a direct role for the components of ribonucleotide reductase in determining malignant potential. Ribonucleotide reductase is regulated by transcriptional activation of gene expression and post-transcriptional mechanisms that alter mRNA message stability. Post-transcriptional regulation of mRNA turnover plays an important role in modulating mRNA steady state levels and therefore directly influences gene expression. The 3'-untranslated region (UTR) of R1 and R2 messages contain sequences that are important in regulating gene expression through changes in message stability. Studies have found that mRNA message stability is mediated by growth factors, cytokines and tumor promoters. Several studies have elucidated signal transduction pathways of tumor promoters, TGF-beta and oxidation/reduction agents. This report reviews how knowledge of these signaling pathways is revealing new insights into how ribonucleotide reductase mRNA binding proteins are important in regulating cellular proliferation, drug resistance and malignancy.

Late Simian virus 40 transcription factor is a target of the phosphoinositide 3-kinase/Akt pathway in anti-apoptotic Alzheimer's amyloid precursor protein signalling.

The association of familial Alzheimer's disease (FAD) with mutations in Alzheimer's amyloid precursor protein (APP) suggests important functions for APP in the central nervous system. Mutations in APP impair its function to confer resistance to apoptosis in cells under stress, and this may contribute to neurodegeneration in Alzheimer's disease (AD) brain, but the mechanisms involved are unknown. We examined the role of the late Simian virus 40 transcription factor (LSF), in anti-apoptotic APP pathways. We show that in APP-deficient B103 cells, expression of wild-type human APP (hAPPwt), but not of FAD-mutant APP, inhibited staurosporine (STS)-induced apoptosis. This inhibition was further enhanced by expression of LSFwt, although LSFwt alone was not sufficient to inhibit STS-induced apoptosis. In contrast, expression of dominant-negative LSF led to a marked increase in STS-induced cell death that was significantly blocked by hAPPwt. These effects of APP were accompanied by LSF nuclear translocation and dependent gene transcription. The activation of LSF is dependent on the expression of hAPPwt and is inhibited by the expression of dominant-negative forms of either phosphoinositide 3-kinase or Akt. These results demonstrate that LSF activation is required for the neuroprotective effects of APP via phosphoinositide 3-kinase/Akt signalling. Alterations in this pathway by aberrations in APP and/or LSF could promote neuronal loss in AD brain, due to secondary insults. Thus a link is established between APP and LSF and AD.

Anti-apoptotic wild-type Alzheimer amyloid precursor protein signaling involves the p38 mitogen-activated protein kinase/MEF2 pathway.

Alzheimer amyloid precursor protein (APP) effectively protects against apoptosis in neuronal cells under stress, but the mechanisms of this anti-apoptotic effect remain largely unknown. Transcription factors act as critical molecular switches in promoting neuronal survival. The myocyte enhancer factor-2 (MEF2) is a transcription factor, and is known to be necessary for neurogenesis and activity-dependent neuronal survival. This study examined the possible role of MEF2 in the anti-apoptotic signaling pathways activated by APP. We report that expression of wild-type human APP (hAPPwt) but not familial Alzheimer's disease mutant APP (FAD-hAPPmut) in APP-deficient rat B103 cells led to a significant increase in the level of phosphorylated MEF2. This differential phosphorylation was dependent on enhanced activation of p38 mitogen-activated protein kinase (p38 MAPK). Also, expression of hAPPwt mediated an increase in MEF2 DNA binding affinity that correlated with p38 MAPK-dependent trans-activation of a MEF2-responsive reporter gene. Furthermore, over-expression of dominant negative MEF2 in hAPPwt-expressing cells enhanced staurosporine-induced apoptosis, in contrast MEF2wt enhanced the capacity of hAPPwt to confer resistance to apoptosis. Thus, MEF2 plays a critical role in APP-mediated signaling pathways that inhibit neuronal apoptosis. A model of anti-apoptotic APP signaling is proposed where APP mediates p38 MAPK-dependent phosphorylation and activation of MEF2. Once activated MEF2 regulates neuronal survival by stimulation of MEF2-dependent gene transcriptions. Alteration of this function by mutations in APP and aberrant APP processing could contribute to neuronal degeneration seen in AD.

Transcriptional activation and increase in expression of Alzheimer's beta-amyloid precursor protein gene is mediated by TGF-beta in normal human astrocytes.

The overexpression of the Alzheimer amyloid precursor protein (APP) and its subsequent proteolytic processing may be one of several factors contributing to amyloid beta-peptide (Abeta) deposition in plaques and microvasculature in Alzheimer's disease (AD) brain. Cytokines and growth factors can influence the expression of APP in response to brain injury, but the underlying mechanisms are largely unknown. We examined the mechanisms by which transforming growth factor-beta (TGF-beta) affects the expression of APP in normal human astrocytes. We report that, TGF-beta up-regulated the expression of APP at the transcription level as determined by nuclear run-on experiments. Transient transfection of astrocytes with APP gene promoter (-2832 bp) chloramphenicol acetyltransferase (CAT) reporter constructs led to increased reporter activity upon TGF-beta stimulation. This reporter activity was mainly attributed to the APP proximal domain (-488 bp). The increase in APP gene transcription was associated with significant accumulation of intracellular APP, APP carboxyl terminal derived fragments, and total secreted Abeta. In addition, we observed a significant increase in levels of TGF-beta in Abeta plaques and its immediate vicinity in AD-affected brain relative to controls. These results indicate that high levels of TGF-beta in the cortex, may serve to up-regulate APP synthesis in reactive astrocytes and indirectly contributes to Abeta deposition. Closely related processes may induce cerebrovascular pathology in AD brain.

Transforming growth factor-beta-induced transcription of the Alzheimer beta-amyloid precursor protein gene involves interaction between the CTCF-complex and Smads.

Transforming growth factor-beta-1 (TGF-beta), a key regulator of the brain responses to injury and inflammation, has been implicated in upregulating the expression of the Alzheimer amyloid precursor protein (APP) and Alzheimer's disease (AD) pathogenesis. However, little is known about the mechanisms underlying the effects of TGF-beta on APP expression. Analysis of APP promoter activity upstream of the chloramphenicol acetyltransferase reporter gene in normal human astrocytes (NHAs), revealed that the APP promoter binding beta (APBbeta) site (-93/-82) is responsive to TGF-beta. This site interacts with the zinc finger nuclear factor CTCF, involved in APP transcriptional activity. As determined by gel shift assay, there was no significant difference in the CTCF-APBbeta complex binding activity in the presence or absence of TGF-beta treatment of NHAs. To further investigate the contributions of the CTCF-complex and Smad proteins to the TGF-beta induced APP promoter activity, we examined the distribution of these factors and their DNA binding activity. Interestingly, upon TGF-beta treatment both Smads 3 and 4 were translocated to the nuclei in contrast to Smad 2, which was cytoplasmic. However, CTCF was predominantly localized in the nuclei irrespective of TGF-beta treatment. Gel super shift assay coupled with Western blot analysis showed that Smads 3 and 4 specifically associated with the CTCF-APBbeta complex. In addition, AD brain sections showed increased expression and nuclear localization of Smad 4, which correlated with higher levels of APP and TGF-beta. However, over expression of Smad 4 on its own was not sufficient to affect APP expression. These results demonstrate that TGF-beta activation of Smad protein complexes promotes transcription of the APP gene. Increased synthesis of APP may in part determine Abeta production and deposition in affected AD brain.