Bcl-2 Gene Family Research Articles

FIP200 targets Ago2 and mitochondria in naive T cells to control tumor immunity

Abstract Naive T cells are poorly studied in cancer patients. Here, we report that naïve T cells are susceptible to apoptosis and lost in human and mouse cancer. Accompanied with this feature is a selective loss of autophagy component, FIP200 in naïve T cells in tumor bearing hosts. FIP200-deficient T cells functionally resemble their counterparts in tumor bearing hosts and mediate poor anti-tumor immunity in vivo. Mechanistically, loss of FIP200 disables the balance between pro- and anti-apoptotic Bcl-2 family members via elevating argonaute (Ago) 2 ubiquitination and subsequently eliminating microRNA1198-5p expression and consequently lifting microRNA1198-5p-mediated repression on pro-apoptotic gene Bak1 expression. In vivo rebalance of the Bcl-2 family genes via enforcing Bcl-2 expression partially rescues FIP200-deficient T cell apoptosis. In parallel, specific FIP200 deficiency causes potent mitochondria activation and reactive oxygen species (ROS) release in T cells. In vivo ROS blockade prevents FIP200-deficient T cell apoptosis. Thus, tumor may trigger naïve T cell apoptosis via targeting its FIP200 to escape anti-tumor immunity.

Dietary restriction suppresses apoptotic cell death, promotes Bcl-2 and Bcl-xl mRNA expression and increases the Bcl-2/Bax protein ratio in the rat cortex after cortical injury

Traumatic brain injury (TBI) is one of the leading causes of death and disability in humans.Subsequent pathological events occurring in the brain after TBI, referred to as secondary injury, continue to damage surrounding tissue resulting in substantial neuronal loss. Using an animal model of TBI we examined the effect of dietary restriction (DR) on the neuroapoptosis and Bcl-2 family genes as the main regulators of the intrinsic apoptotic pathway. Bcl-2, Bcl-xl and Bax mRNA and protein expression in the ipsilateral cortex of adult Wistar rats exposed to DR before TBI were studied from 2 to 28 days post injury. Our results showed that DR suppressed neuroapoptosis and promoted significant upregulation of antiapoptotic Bcl-2 and Bcl-xl mRNAs in the ipsilateral cortex following injury. Expression of the proapoptotic Bax gene increased in ad libitum (AL) fed rats but remained unchanged in rats exposed to DR. Although the expression of Bcl-2, Bcl-xl and Bax proteins was changed in a similar manner in both experimental groups, DR promoted a continuous increase in the Bcl-2:Bax protein ratio throughout the recovery period. Together with our previous finding that DR mediates inhibition of the extrinsic apoptotic pathway the present work reveals that modulation of the intrinsic pathway contributes to the beneficial effect of DR in brain injury. These findings provide new insight into the effects of DR on pro-survival signaling after injury, lending further support to its neuroprotective effect.

Response of the JAK-STAT pathway to mammalian hibernation in 13-lined ground squirrel striated muscle.

Over the course of the torpor-arousal cycle, hibernators must make behavioral, physiological, and molecular rearrangements in order to keep a very low metabolic rate and retain organ viability. 13-lined ground squirrels (Ictidomys tridecemlineatus) remain immobile during hibernation, and although the mechanisms of skeletal muscle survival are largely unknown, studies have shown minimal muscle loss in hibernating organisms. Additionally, the ground squirrel heart undergoes cold-stress, reversible cardiac hypertrophy, and ischemia-reperfusion without experiencing fatal impairment. This study examines the role of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway in the regulation of cell stress in cardiac and skeletal muscles, comparing euthermic and hibernating ground squirrels. Immunoblots showed a fivefold decrease in JAK3 expression during torpor in skeletal muscle, along with increases in STAT3 and 5 phosphorylation and suppressors of cytokine signaling-1 (SOCS1) protein levels. Immunoblots also showed coordinated increases in STAT1, 3 and 5 phosphorylation and STAT1 inhibitor protein expression in cardiac muscle during torpor. PCR analysis revealed that the activation of these pro-survival signaling cascades did not result in coordinate changes in downstream genes such as anti-apoptotic B-cell lymphoma-2 (Bcl-2) family gene expression. Overall, these results indicate activation of the JAK-STAT pathway in both cardiac and skeletal muscles, suggesting a response to cellular stress during hibernation.

Identification of novel alternative splice variants of the BCL2L12 gene in human cancer cells using next-generation sequencing methodology

The next-generation sequencing (NGS) technology has enabled genome-wide studies, providing massively parallel DNA sequencing. NGS applications constitute a revolution in molecular biology and genetics and have already paved new ways in cancer research. BCL2L12 is an apoptosis-related gene, previously cloned from members of our research group. Like most members of the BCL2 gene family, it is highly implicated in various types of cancer and hematological malignancies. In the present study, we used NGS to discover novel alternatively spliced variants of the apoptosis-related BCL2L12 gene in many human cancer cell lines, after 3′-RACE nested PCR. Extensive computational analysis uncovered new alternative splicing events and patterns, resulting in novel alternative transcripts of the BCL2L12 gene. PCR was then performed to validate NGS data and identify the derived novel transcripts of the BCL2L12 gene. Therefore, 50 novel BCL2L12 splice variants were discovered. Since BCL2L12 is involved in the apoptotic machinery, the quantification of distinct BCL2L12 transcripts in human samples may have clinical applications in different types of cancer.

The apoptotic effect of simvastatin via the upregulation of BIM in nonsmall cell lung cancer cells

ABSTRACTPurpose: Statins are known to have pleiotropic effects that induce cell death in certain cancer cells. BIM is a member of the bcl-2 gene family, which promotes apoptotic cell death. This study investigated the hypothesis that simvastatin has pro-apoptotic effects in epidermal growth factor receptor (EGFR)-mutated lung cancer cell lines via the upregulation of the expression of the BIM protein. Materials and methods: The cytotoxic effects of simvastatin on gefitinib-sensitive (HCC827, E716-A750del) and -resistant (H1975, T790M + L858R) nonsmall cell lung cancer (NSCLC) cells were compared. Cell proliferation and expression of apoptosis-related and EGFR downstream signaling proteins were evaluated. Expression of BIM was compared in H1975 cells after treatment with simvastatin or gefitinib. SiRNA-mediated BIM depletion was performed to confirm whether the cytotoxicity of simvastatin was mediated by the expression of BIM. Results: H1975 cells showed significantly reduced viability compared with HCC827 cells after treatment with simvastatin (2 μM) for 48 hours. In simvastatin-treated H1975 cells, expression of pro-apoptotic proteins was increased and the phosphorylation of ERK 1/2 (p-ERK 1/2) was reduced. Expression of BIM was suppressed by gefitinib (1 μM) treatment in H1975 cells, but it was significantly increased by treatment with simvastatin. BIM depletion by siRNA transfection enhanced the viability of H1975 cells that received simvastatin treatment and increased their expression of anti-apoptotic proteins. Conclusions: Simvastatin restored the expression of BIM to induce apoptotic cell death in NSCLC cells harboring an EGFR-resistant mutation. Our study suggests the potential utility of simvastatin as a BIM-targeted treatment for NSCLC.

Effect of sub-chronic intraperitoneal administration of aminoguanidine on the memory and hippocampal apoptosis-related genes in diabetic rats

Memory impairment is a common disorder in diabetes mellitus which is associated with hippocampal neuronal apoptosis. The present study was conducted to examine the effect of one-week intraperitoneal (ip), administration of aminoguanidine (AG) on passive avoidance learning (PAL) and Bcl-2 family gene expression in the hippocampus of rats. Sixty male rats were divided into ten groups: non-diabetic/diabetic animals with/without AG (50, 100, 200 and 400 mg/kg, ip) treatment for one week. PAL and Bcl-2 family genes were examined. AG (100 and 200 mg/kg) improved both memory and Bax, Bak, Bcl-2 and Bcl-xl deficiency significantly in diabetic rats. AG treatment also ameliorated the diabetes-induced changes in (Bcl-2+Bcl-xl)/(Bak+Bax) ratios considerably. These results propose that one-week ip administration of AG may recover the deficit cognition in diabetic rats via enhancing (Bcl-2+Bcl-xl)/(Bak+Bax) proportions (Tab. 2, Fig. 4, Ref. 55).

Knockdown of the putative Lifeguard homologue CG3814 in neurons of Drosophila melanogaster.

Lifeguard is an integral transmembrane protein that modulates FasL-mediated apoptosis by interfering with the activation of caspase 8. It is evolutionarily conserved, with homologues present in plants, nematodes, zebra fish, frog, chicken, mouse, monkey, and human. The Lifeguard homologue in Drosophila, CG3814, contains the Bax inhibitor-1 family motif of unknown function. Downregulation of Lifeguard disrupts cellular homeostasis and disease by sensitizing neurons to FasL-mediated apoptosis. We used bioinformatic analyses to identify CG3814, a putative homologue of Lifeguard, and knocked down CG3814/LFG expression under the control of the Dopa decarboxylase (Ddc-Gal4) transgene in Drosophila melanogaster neurons to investigate whether it possesses neuroprotective activity. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons resulted in a shortened lifespan and impaired locomotor ability, phenotypes that are strongly associated with the degeneration and loss of dopaminergic neurons. Lifeguard interacts with anti-apoptotic Bcl-2 proteins and possibly pro-apoptotic proteins to exert its neuroprotective function. The co-expression of Buffy, the sole anti-apoptotic Bcl-2 gene family member in Drosophila, and CG3814/LFG by stable inducible RNA interference, suppresses the shortened lifespan and the premature age-dependent loss in climbing ability. Suppression of CG3814/LFG in the Drosophila eye reduces the number of ommatidia and increases disruption of the ommatidial array. Overexpression of Buffy, along with the knockdown of CG3814/LFG, counteracts the eye phenotypes. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons in Drosophila diminishes its neuroprotective ability and results in a shortened lifespan and loss of climbing ability, phenotypes that are improved upon overexpression of the pro-survival Buffy.

Modulation of Bcl-x Alternative Splicing Induces Apoptosis of Human Hepatic Stellate Cells.

Liver fibrosis is a major cause of morbidity and mortality worldwide due to chronic viral hepatitis and, more recently, from fatty liver diseases. Activation and proliferation of hepatic stellate cells (HSCs) represent a key aspect of fibrogenesis and are associated with progressive reduction of HSC apoptosis. Bcl-x, an antiapoptotic member of Bcl-2 gene family, plays a role in apoptosis regulation in mammalian cells. Through alternative splicing, the Bcl-x gene yields two major protein isoforms with opposing functions, antiapoptotic Bcl-xL and proapoptotic Bcl-xS. This study aimed to investigate the role of Bcl-x and its alternate splicing in HSC apoptosis. The results indicated that the expression of Bcl-xL was dramatically higher than Bcl-2 in activated human HSCs. The relative expression of Bcl-xL over Bcl-xS increased gradually when HSCs were activated in cell culture, which was consistent with the increase in apoptosis resistance of activated HSCs. Redirection of Bcl-x splicing by an antisense oligonucleotide from the antiapoptotic isoform to the proapoptotic isoform induced death of HSCs without other apoptosis stimuli. We conclude that Bcl-x plays a role in regulation of HSC apoptosis and modulation of Bcl-x alternative splicing may become a novel molecular therapy for liver fibrosis.

Biomarker and Ex Vivo Sensitivity Analysis of Venetoclax (ABT-199/GDC-0199) in Combination with Bortezomib and Dexamethasone in Relapsed/Refractory Multiple Myeloma

Introduction: Multiple myeloma (MM) tumor cell survival is largely dependent on a complex network of interactions between pro-survival (BCL-2, BCL-XL, MCL-1) and pro-death (BIM, NOXA, BID, BAD, BAK, BAX) BCL-2 family members. Venetoclax (VEN) is a potent, selective, orally bioavailable small-molecule BCL-2 inhibitor. Bortezomib (BTZ) is a proteasome inhibitor that can impede MCL-1 activity by increasing the MCL-1 antagonist NOXA. The addition of VEN has been shown to enhance the activity of BTZ in MM cell line and xenograft models. Chromosomal abnormalities (immunoglobulin heavy chain translocations, amplification, deletions) can also influence sensitivity of MM cells to treatment. Results presented herein describe retrospective biomarker analysis in a subset of dose-escalation patients (pts) from an ongoing phase 1b study of VEN in combination with BTZ and dexamethasone in relapsed/refractory (R/R) MM.Methods: Exploratory objectives included evaluation of chromosomal abnormalities, BCL-2 family member gene expression levels, and ex vivosensitivity of pt MM tumor cells to VEN and/or BTZ. Interphase FISH analysis was performed on CD138-positively selected bone marrow mononuclear cells (BMMC) using probes for t(11;14), t(4;14), deletion 17p (17p-), deletion 13q (13q-), and amplification of chromosomes 5, 9, or 15 (denoted as hyperdiploid [HY]). Absolute quantitation of BCL-2 family members (BCL-2, BCL-XL, MCL-1, BCL-2A1, BIM, BID, BAD, BAX, and NOXA) was performed on CD138-selected BMMCs collected at baseline using TaqMan probe-based droplet digital polymerase chain reaction. Ex vivosensitivity of BMMCs collected at baseline to increasing doses of VEN and/or BTZ was determined by flow cytometry using a combined analysis for the loss of CD138 surface expression and the alteration of cellular morphology (lower FCS). Correlations between biomarkers and median best percentage change in M-protein were examined by Spearman test. Correlations between biomarkers and median time on study (mToS) were examined by log-rank and Wilcoxon tests for binary biomarkers, and by risk ratio from a Cox proportional hazard model for continuous biomarkers. No correction for multiple testing was performed.Results: Forty-one pts were enrolled as of 06/15/2015. The biomarker study subgroups were similar to the overall pt population with respect to demographics. Of the 37/41 (90%) pts assessed by FISH, 5 were t(11;14), 3 were t(4;14), 20 were 13q-, 10 were 17p-, and 15 were HY. In pts with HY, median best percentage change in M-protein was -79.4% compared to +101.65% in non-HY, while mToS was 309 days compared to 29 days in non-HY (p <0.0001), consistent with literature regarding a positive prognostic effect in MM. No difference in efficacy was observed in other FISH subtypes. BCL-2 family gene expression levels were determined in 25/41 (61%) pts. BCL-2, but not BCL-XL or MCL-1 gene expression, correlated with M-protein response (Spearman -0.5116, p=0.0149) and with a trend for increased mToS (HR=0.42 [95% CI 0.13-1.0]). Responders (≥ partial response, n=13; assessed by International Myeloma Working Group criteria 2011) had higher BCL-2 levels than subjects with stable disease/progressive disease (n=12; p=0.015). Pts with high BCL-XL or high MCL-1 were found in both the responder and nonresponder populations. Of the 14 pt samples collected for ex vivo sensitivity, 8 had sufficient CD138-positive cells for VEN ± BTZ dose curve analysis. Four of 8 (50%) were sensitive to VEN alone (median lethal dose [LD50] ≤300 nM) while 4/8 (50%) were sensitive to BTZ alone (LD50 ≤5 nM). Seven of 8 (88%) were sensitive to the combination (LD50 ≤5 nM BTZ/300 nM VEN).Conclusions: VEN in combination with BTZ and dexamethasone is active in R/R MM. CD138-enriched BMMCs were obtained in sufficient numbers to enable downstream FISH analysis, BCL-2 family gene expression profiling, and ex vivo sensitivity evaluation. The exploratory biomarker analysis showed that higher BCL-2 gene expression levels at baseline significantly correlated with clinical responses to therapy, with a trend toward increased mToS. Additionally, MM cells treated ex vivo demonstrated greater sensitivity to the combination of VEN/BTZ than to the 2 single agents alone. Further investigation of this dataset continues, including analysis of on-treatment tumor samples in a subset of pts. DisclosuresRoss:AbbVie: Employment, Equity Ownership. Off Label Use: Venetoclax is an investigational drug that is not yet approved in this indication.. Chyla:AbbVie: Employment, Equity Ownership. Roberts-Rapp:AbbVie: Employment, Equity Ownership. Goswami:AbbVie: Employment, Equity Ownership. Yan:AbbVie: Employment, Equity Ownership. Huang:AbbVie: Employment, Equity Ownership. Bhathena:AbbVie: Employment, Equity Ownership. Enschede:AbbVie: Employment, Equity Ownership. Maciag:AbbVie: Employment, Equity Ownership. Humerickhouse:AbbVie: Employment, Equity Ownership. McKeegan:AbbVie: Employment, Equity Ownership.

Germline Genetic Variation of ASXL1 and BIM Predicts Response to Imatinib and Identifies a Subset of High Sokal Risk Patients with the Greatest Risk of Treatment Failure and Disease Progression

▪Background: Scoring systems at CML diagnosis, such as Sokal risk, provide important response prediction for imatinib (IM) treated patients (pts). Specific treatment policies have been suggested for high risk pts to optimize otherwise inferior outcomes. However, responses among pts with high risk are heterogeneous and new biomarkers are required to facilitate rational selection of optimal therapy. Biological factors, such as germline genetic variation, may play a role in therapy response dynamics. We aimed to identify predictive biomarkers of response to IM at CML diagnosis to aid selection of front line therapy for optimal treatment outcomes.Methods: Targeted amplicon sequencing using a custom Ion AmpliSeq panel and the Ion Proton was performed for 35 genes: 10 BCL2 family genes involved in TKI initiated apoptosis (including BIM, BAD and BCL2); 5 drug metabolism genes; and 20 genes implicated in hematologic malignancies (including ASXL1 and TET2). Genotypes were determined for 200 candidate single nucleotide variants (SNPs) for 528 front line IM and 83 front line NIL treated pts. For the IM pts, baseline variables were assessed for association with outcome: Sokal risk, age, gender, assigned IM dose (400, 600 or 800 mg); and genotype.Results: SNPs significantly associated with outcome in univariate analyses were assessed in multivariate models with the other baseline variables. The Sokal risk, ASXL1 rs4911231 and BIM rs686952 SNPs were independent predictors of 12 mo MMR, 48 mo MR4, MR4.5 and failure free survival (FFS, loss of any response, death, progression to AP/BC). For the ASXL1 SNP, the homozygous T genotype (155/508 evaluable pts, 30%), and for the BIM SNP, the A allele (249/507 evaluable pts, 49%) were associated with superior outcomes.We explored the additive effect of combining the genotypes of the ASXL1 and BIM SNPs on outcome. Three risk groups were readily identified (defined in Fig): Good (16% of evaluable pts), Average (46%) and Poor (37%). There were significant differences in the cumulative incidence of 3 mo EMR, 12 mo MMR, and 48 mo MR4, MR4.5 and FFS, as stratified by these SNP risk groups in IM treated pts (Table and Fig A). No significant association was found for progression to AP/BC or survival for any baseline variable.To examine the predictive power of SNP genotype within the high Sokal risk group, high risk pts were stratified by SNP genotype group. Significant differences were observed for EMR, MMR, MR4, MR4.5 and FFS (Table and Figure B), demonstrating the ability of the SNP genotype within high Sokal risk pts to predict response. Moreover, high Sokal risk pts harboring a poor risk SNP genotype had a significantly higher risk of progression to AP/BC vs high Sokal risk pts with an average/good risk genotype, 12% vs 2% (P =.03).The impact of SNP genotype risk on achieving 12 mo MMR was examined in the 83 pts treated with frontline NIL (median 24 mo follow up). In contrast to the significant difference observed for IM pts, there was no significant difference for NIL pts: 75% vs 73% vs 64% for good, average and poor risk, respectively, P =.34, suggesting the poor risk conferred by genotype may be abrogated by more potent TKI.Conclusion: Our data suggest inherent genetic variation contributes to the heterogeneity of response to IM. An intronic SNP in BIM, a key initiator of TKI induced apoptosis, and a synonymous SNP in ASXL1 exon 12, a region commonly mutated in hematologic cancers, were strong biomarkers of IM response. The mechanism by which these SNPs affect response awaits further clinical and experimental evaluation. Among pts with high Sokal risk, the genotype of these 2 SNPs delineated response and identified a good risk subgroup where more potent TKI may not be required for optimal outcomes. Assessment of genetic variation at diagnosis may contribute to a prognostic score that will allow for optimization of therapy. [Display omitted] [Display omitted] DisclosuresYeung:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grant international meeting, Research Funding. Hughes:Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Branford:BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Maintenance of hematopoietic stem cell integrity and regulation of leukemogenesis by p53 and its coactivator Aspp1

Hematopoietic stem cells (HSCs) are predominantly in a quiescent state, thereby avoiding depletion due to various stresses. However, quiescent HSCs are vulnerable to mutagenesis due to low-fidelity DNA repair. The mechanism by which HSCs avoid mutation accumulation remains to be elucidated. HSCs are normally resistant to apoptosis because of their abundant expressions of pro-survival Bcl-2 family genes. In contrast, p53 is activated in HSCs in response to DNA damage. We have recently shown that pro-apoptotic Bcl-2 signals are activated through p53 preferentially in HSCs with damaged DNA. Aspp1, an apoptosis-stimulating protein of p53, is highly expressed in HSCs and coordinates with p53 to maintain the genomic soundness of the HSC pool. In this review, we will summarize apoptosis regulation and the roles of p53 in HSCs, and introduce our findings showing coordinated regulations of HSC self-renewal, DNA damage tolerance and hematological malignancies by Aspp1 and p53.

Metabolomic Analyses of Brain Tissue in Sepsis Induced by Cecal Ligation Reveal Specific Redox Alterations--Protective Effects of the Oxygen Radical Scavenger Edaravone.

The pathophysiology of sepsis-associated encephalopathy (SAE) is complex and remains incompletely elucidated. Dysregulated reactive oxygen species (ROS) production and mitochondrial-mediated necrotic-apoptotic pathway have been proposed as part of the pathogenesis. The present study aimed at analyzing the preventive effect of the free radical scavenger edaravone on sepsis-induced brain alterations. Sepsis was induced by cecal ligation and puncture (CLP) and the mice were divided into three groups-CLP vehicle (CLPV), CLP and edaravone (MCI-186, 3-methyl-1-phenyl-2-pyrazolin-5-one) (CLPE), and sham-operated (Sham). Mice in CLPV and CLPE were injected with saline or edaravone intraperitoneally at a dose of 10 mg/kg twice daily. The treatments were initiated 4 days prior to the surgical procedure. Mortality, histological changes, electron microscopy (EM), and expression of Bcl-2 family genes (Bcl-2 and Bax) were analyzed in selected brain regions. CLPE showed significant improvement in survival compared with CLPV 18 h postinduction of sepsis (P < 0.05). At the same time point, pathohistological analysis also showed marked reduction of neuronal cell death in both parietal cortex and hippocampus in the CLPE (P < 0.05). RT-PCR and immunoblotting directed at the Bcl-2 family revealed increased Bax mRNA levels in hippocampus at 12 h in CLPV as well as an increased Bax/Bcl-2 protein ratio, changes that were significantly suppressed in CLPE. In conclusion, our study suggests that sepsis induced by cecal ligation alters cerebral redox status and supports a proapoptotic phenotype. The free radical scavenger edavarone reduces mortality of septic mice and protects against sepsis-induced neuronal cell death.

Effect of chronic intraperitoneal aminoguanidine on memory and expression of Bcl-2 family genes in diabetic rats.

Long-term hyperglycemia associates with memory defects via hippocampal cells damaging. The aim of the present study was to examine the effect of 1 month of i.p. injections of AG on passive avoidance learning (PAL) and hippocampal apoptosis in rat. Eighty male rats were divided into 10 groups: control, nondiabetics and STZ-induced diabetics treated with AG (50, 100, 200, and 400 mg/kg, i.p.). PAL and the Bcl-2 family gene expressions were determined. Diabetes resulted in memory and Bcl-2 family gene expression deficits. AG (50 and 100 mg/kg) significantly improved the learning and Bcl-2, Bcl-xl, Bax, and Bak impairment in diabetic rats. However, negative effects were indicated by higher doses of the drug (200 and 400 mg/kg). Present study suggests that 1 month of i.p. injections of lower doses of AG, may improve the impaired cognitive tasks in STZ-induced diabetic rats possibly by modulating Bcl-2 family gene expressions.

Proteazom ve HDAC Inhibisyonunun Androjenden-Bagimsiz PC-3 Hucre Hattinda Bcl-2, Bcl-XL, Bim ve Bik Proteinlerinin Ifadelenme Duzeylerini Degistirmesi

Objective: Prostate cancer is the most common cancer observed and it is estimated to have caused 10% of all cancer-related deaths in men in western countries. Transition from androgen-dependent form to metastatic androgen-independent phase is a consequence of deranged apoptotic response by the cancer cells to therapy. Bcl-2 family members are important modulators of apoptotic response. In this study, we investigated the effects of proteasome and histone deacetylase (HDAC) inhibitor treatment on the expression changes in some Bcl-2 family genes at protein level. Methods: PC-3 cells were cultured in plate before being exposed to different concentrations of unaccompanied bortezomib and TSA as well as bortezomib – TSA combination. After the protein isolation from control and treated cells, whole cell lysate was used for determining Bcl-2, Bcl-XL, Bim and Bik proteins with western blotting method. Results: We observed that after the drug treatment, the expression levels of pro-apoptotic Bim and Bik proteins have increased and those of anti-apoptotic Bcl-2 and Bcl-XL proteins have decreased. Synergy between bortezomib and TSA induces apoptosis. Conclusion: Our results showed that application of low doses of bortezomib and TSA combination may be sufficient for apoptotic response. Our findings may come in handy for the advanced prostate cancer research.

Changes in the Expression of Antiapoptotic Protein Bcl-2 in the Rat Neocortex and Hippocampus in Different Hypobaric Hypoxia Regimes

Studies during the 1980s demonstrated delayed neuron death in hippocampal fi eld CA1 several days after ischemia [7]. Subsequent studies also produced similar fi ndings in other parts of the brain with high sensitivity to hypoxia/ ischemia – the neocortex, striatum, and cerebellum. This type of neuron damage was shown to be linked with the mechanism of induction of apoptosis (programmed cell death), which involve genes and killer proteins [8]. A central position in the regulation of the mechanisms of apoptosis is occupied by proteins of the bcl-2 gene family – the proapoptotic Bax and Bcl-xs and the antiapoptotic Bcl-2 and Bcl-xL [6, 9, 10]. Induction of apoptosis after harmful exposure to ischemia/hypoxia in susceptible areas of the brain is associated with increases in the content of proapoptotic and decreases in the content of antiapoptotic proteins. Studies in the early 1990s identifi ed another phenomenon – “ischemic/hypoxic brain tolerance,” induced before the harmful effects of moderate, so-called preconditioning (PC) ischemia/hypoxia, which to a signifi cant extent prevents neuron death in susceptible areas of the brain [1]. PC with moderate hypobaric hypoxia (MHH) was showed to be able to eliminate the structural-functional damage to hippocampal and neocortical neurons, and also to be able to change the ratio of the contents of proand antiapoptotic proteins within them induced by severe hypoxia (SH) [14, 15]. Different PC regimes (number of episodes, duration of episodes) have been found to have effects with different effi cacies on the intracellular protection mechanisms. Further studies seeking optimum PC regimes, inducing effective mobilization of protective mechanisms, especially those involved in regulating apoptosis, are needed [1]. The aim of the present work was to study the effects of various PC regimes using MHH on the expression of the antiapoptotic protein Bcl-2 in neocortical and hippocampal neurons during and after exposure to harmful SH. Changes in the Expression of Antiapoptotic Protein Bcl-2 in the Rat Neocortex and Hippocampus in Different Hypobaric Hypoxia Regimes

Hyperactivity and depression-like traits in Bax KO mice

The Bax gene is a member of the Bcl-2 gene family and its pro-apoptotic Bcl-associated X (Bax) protein is believed to be crucial in regulating apoptosis during neuronal development as well as following injury. With the advent of mouse genomics, mice lacking the pro-apoptotic Bax gene (Bax KO) have been extensively used to study how cell death helps to determine synaptic circuitry formation during neurodevelopment and disease. Surprisingly, in spite of its wide use and the association of programmed neuronal death with motor dysfunctions and depression, the effects of Bax deletion on mice spontaneous locomotor activity and depression-like traits are unknown. Here we examine the behavioral characteristics of Bax KO male mice using classical paradigms to evaluate spontaneous locomotor activity and depressive-like responses. In the open field, Bax KO animals exhibited greater locomotor activity than their control littermates. In the forced swimming test, Bax KO mice displayed greater immobility times, a behavior despair state, when compared to controls. Collectively, our findings corroborate the notion that a fine balance between cell survival and death early during development is critical for normal brain function later in life. Furthermore, it points out the importance of considering depressive-like and hyperactivity behavioral phenotypes when conducting neurodevelopmental and other studies using the Bax KO strain.

Vaccinia Virus Protein A49 Is an Unexpected Member of the B-cell Lymphoma (Bcl)-2 Protein Family

Vaccinia virus (VACV) encodes several proteins that inhibit activation of the proinflammatory transcription factor nuclear factor κB (NF-κB). VACV protein A49 prevents translocation of NF-κB to the nucleus by sequestering cellular β-TrCP, a protein required for the degradation of the inhibitor of κB. A49 does not share overall sequence similarity with any protein of known structure or function. We solved the crystal structure of A49 from VACV Western Reserve to 1.8 Å resolution and showed, surprisingly, that A49 has the same three-dimensional fold as Bcl-2 family proteins despite lacking identifiable sequence similarity. Whereas Bcl-2 family members characteristically modulate cellular apoptosis, A49 lacks a surface groove suitable for binding BH3 peptides and does not bind proapoptotic Bcl-2 family proteins Bax or Bak. The N-terminal 17 residues of A49 do not adopt a single well ordered conformation, consistent with their proposed role in binding β-TrCP. Whereas pairs of A49 molecules interact symmetrically via a large hydrophobic surface in crystallo, A49 does not dimerize in solution or in cells, and we propose that this hydrophobic interaction surface may mediate binding to a yet undefined cellular partner. A49 represents the eleventh VACV Bcl-2 family protein and, despite these proteins sharing very low sequence identity, structure-based phylogenetic analysis shows that all poxvirus Bcl-2 proteins are structurally more similar to each other than they are to any cellular or herpesvirus Bcl-2 proteins. This is consistent with duplication and diversification of a single BCL2 family gene acquired by an ancestral poxvirus.

Heat-induced apoptosis and gene expression in bovine mammary epithelial cells

The objective of this study was to identify the apoptosis and cell-defence response of bovine mammary epithelial cells under heat stress (HS). Cells were exposed to either 38°C or 42°C for 0.5, 1, 3, 5, 8, or 12 h, and the transcription of heat shock proteins (Hsps), Bcl-2 family, caspases and apoptosis-regulated genes were quantified by quantitative real-time polymerase chain reaction. Caspase-3, -7 and -8 were markedly upregulated by HS and the peak gene abundance appeared at 5 h. However, the same family numbers, caspase-6 and -9 were sustained downregulated in HS. The expression of anti-apoptotic gene Bcl-2, Bcl-2A and Mcl-1 increased sharply in HS but returned to pre-HS levels after 8 h. The pro-apoptotic genes: Bax, Bak and Bid were downregulated during HS. The striking changes of signalling factors of apoptosis: tumour necrosis factor receptor, p53, Apaf-1 was upregulated, and Fas was downregulated in HS. Stress proteins Hsp genes (hsp27, hsp70 and hsp90) were generally increased at 42°C and this was especially apparent for hsp70 transcription as it was increased 14-fold at 1 h. Simultaneously, HS induced cell apoptosis, and the peak of apoptosis rate appeared at 3 and 5 h, which were assessed by flow cytometry. Our results suggest that HS induces cell apoptosis, disturbs the normal biological activity, and aroused intracellular thermotolerance responses of bovine mammary epithelial cells.

Oestrous cycle-dependent expression of Fas and Bcl2 family gene products in normal canine endometrium.

During the oestrous cycle canine endometrium undergoes cyclical cellular proliferation, apoptosis and differentiation. To study the regulation of endometrial apoptosis and proliferation events the expression of apoptosis-related genes was analysed by real-time polymerase chain reaction and cellular expression of their proteins was identified through immunohistochemistry. Cellular apoptosis and proliferation events were detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL) and proliferation marker Ki67 immunostaining, respectively. The highest proliferative index was observed in the follicular phase (all endometrial cellular components) and at early dioestrus (basal glands). This was associated with a low apoptotic index and a strong expression of anti- (Bcl2) and pro-apoptotic proteins (Fas, FasL, Bax). Subsequently (Days 11-45 of dioestrus), basal glandular epithelium experienced the highest apoptotic index, coincidental with a decrease of Bcl2 expression and a low ratio of Bcl2/Bax transcription. An increase in the apoptotic index of crypts, stromal and endothelial cells was observed at late dioestrus and the beginning of anoestrus. These results indicate that pro- and anti-apoptotic proteins regulate the balance between cell proliferation and death in the canine endometrium during the oestrous cycle. High Bcl2 expression in both the follicular and early dioestrous phases stimulate glandular proliferation and prevent apoptosis but, in the non-pregnant uterus, a decrease in Bcl2 expression together with an increase in pro-apoptotic proteins induces apoptosis of basal glandular epithelium cells.

Mutated Ptpn11 alters leukemic stem cell frequency and reduces the sensitivity of acute myeloid leukemia cells to Mcl1 inhibition.

PTPN11 encodes the Shp2 non-receptor protein-tyrosine phosphatase implicated in several signaling pathways. Activating mutations in Shp2 are commonly associated with juvenile myelomonocytic leukemia (JMML) but are not as well defined in other neoplasms. Here we report that Shp2 mutations occur in human acute myeloid leukemia (AML) at a rate of 6.6% (6/91) in the ECOG E1900 dataset. We examined the role of mutated Shp2 in leukemias harboring MLL translocations which co-occur in human AML. The hyperactive Shp2E76K mutant, commonly observed in leukemia patients, significantly accelerated MLL-AF9 mediated leukemogenesis in vivo. Shp2E76K increased leukemic stem cell frequency and affords MLL-AF9 leukemic cells IL3 cytokine hypersensitivity. As Shp2 is reported to regulate anti-apoptotic genes, we investigated Bcl2, Bcl-xL and Mcl1 expression in MLL-AF9 leukemic cells with and without Shp2E76K. While the Bcl2 family of genes was upregulated in Shp2E76K cells, Mcl1 showed the highest upregulation in MLL-AF9 cells in response to Shp2E76K. Indeed, expression of Mcl1 in MLL-AF9 cells phenocopies expression of Shp2E76K suggesting Shp2 mutations cooperate through activation of anti-apoptotic genes. Finally, we show Shp2E76K mutations reduce sensitivity of AML cells to small molecule mediated Mcl1 inhibition suggesting reduced efficacy of drugs targeting MCL1 in patients with hyperactive Shp2.