TNF-related apoptosis-inducing ligand (TRAIL) is known to selectively induce apoptosis in various tumour cells. However, downstream-signalling of TRAIL-receptor is not well defined. A functional genetic screening was performed to isolate genes interfering with TRAIL-induced apoptosis using cDNA retroviral library. Bcl-X(L) and FLIP were identified after DNA sequencing analysis of cDNA rescued from TRAIL-resistant clones. We found that increased expression of Bcl-X(L), but not Bcl-2, suppressed TRAIL-induced apoptosis in tumour cells. Western blot and immunohistochemical analyses showed that expression of Bcl-X(L), but not Bcl-2, was highly increased in human breast cancer tissues. Exposure of MDA-MB-231 breast tumour cells to TRAIL induced apoptosis accompanied by dissipation of mitochondrial membrane potential and enzymatic activation of caspase-3, -8, and -9. However, SK-BR-3 breast tumour cells exhibiting increased expression level of Bcl-X(L) were resistant to TRAIL, though upon exposure to TRAIL, caspase-8 and Bid were activated. Forced expression of Bcl-X(L), but not Bcl-2, desensitised TRAIL-sensitive MDA-MB-231 cells to TRAIL. Similar inhibitory effects were also observed in other tumour cells such as HeLa and Jurkat cells stably expressing Bcl-X(L), but not Bcl-2. These results are indicative of the crucial and distinct function of Bcl-X(L) and Bcl-2 in the modulation of TRAIL-induced apoptosis.

By making use of receptor-Ck positive lymphocytes (from normal human subjects) as well as receptor-Ck negative lymphocytes (from untreated chronic myeloid leukemic (CML) patients) as cellular models, we were able to show that receptor-Ck-dependent signalling is involved in the regulation of genes coding for Bcl-2 and cyclin D. Further, experiments directed to resolve the mechanism by which this receptor regulates these genes revealed that receptor-Ck, upon activation by cholesterol, initiates the cleavage of a 125 kDa cytoplasmic protein leading to the generation of a 47 kDa factor having specific affinity for genomic sterol regulatory element (SRE)/SRE-like sequence present in the promoter region of genes coding for Bcl-2 and cyclin D. Based upon these observations, we propose that the inability of leukemic cells to express receptor-Ck is responsible for the deregulated over-expression of genes coding for Bcl-2 and cyclin D and this phenomenon may be of importance in understanding leukemic haematopoiesis.

NR-13, Mcl-1, and BCL-X(L), are conserved anti-apoptotic proteins that belong to the anti-apoptotic Bcl-2 sub-family, which inhibits cell death by preventing mitochondrial membrane permeabilization (MMP). Given the anti-apoptotic functions of these proteins in vertebrates (e.g. human, mouse, and zebrafish) and the involvement of apoptotic regulation in immune responses, we studied the sequences of these genes and their transcript expression in Atlantic cod (Gadus morhua) during innate immune responses to viral and bacterial stimuli. Based on previously generated Atlantic cod expressed sequence tags (ESTs), we identified partial cDNA sequences of putative orthologues of Atlantic cod NR-13, Mcl-1, and Bcl-X, and obtained the full-length cDNA, genomic, and promoter region sequences for these genes. The analyses of Atlantic cod cDNA sequences, and comparisons of the cod deduced amino acid sequences to putative orthologues in other species, revealed the presence of highly conserved Bcl-2 homology (BH) and transmembrane (TM) domains in the Atlantic cod sequences. Analysis of gene structure revealed conserved intron/exon boundaries within the coding regions of human and Atlantic cod putative orthologues. We found that an intron/exon boundary immediately following the codon for the 8th residue (tryptophan) of the BH2 domain exists in all anti-apoptotic Bcl-2 sub-family genes regardless of vast evolutionary distance. We also identified a non-coding exon in the Atlantic cod NR-13-like gene, which appears to be absent in its putative mammalian orthologues. Quantitative reverse transcription-polymerase chain reaction (QPCR) was used to study constitutive gene expression in six tissues (blood, brain, gill, head kidney, pyloric caecum, and spleen) of non-stressed juvenile cod; NR-13 and Bcl-X2 were most highly expressed in gill, whereas Mcl-1 and Bcl-X1 were most highly expressed in blood. In cod challenged with intraperitoneal (IP) injections of the viral mimic polyriboinosinic polyribocytidylic acid (pIC), (1) NR-13 mRNA expression was significantly up-regulated (compared to both 0h pre-injection and timed saline injected controls) in spleen at 6h post-injection and in head kidney at both 6 and 24h post-injection (HPI), and (2) both Mcl-1 and Bcl-X2 were significantly up-regulated (compared to both 0h pre-injection and timed saline injected controls) in spleen at 6 HPI. QPCR was used to show that, in cod challenged with IP injections of formalin-killed, atypical Aeromonas salmonicida (ASAL), only NR-13 appeared to be responsive (significantly up-regulated in spleen at 6 HPI compared to 0h pre-injection controls). Interestingly, QPCR showed that saline injection had a mild (less than 3-fold) but significant inductive effect (compared to 0h pre-injection controls) on both NR-13 and Mcl-1 transcript expression in spleen at 2 HPI. Although we only obtained partial cDNA and genomic sequences for Bcl-X2, sufficient evidence was accumulated to show that two Bcl-X paralogues exist in Atlantic cod, possibly due to the teleost-specific genome duplication event. Promoter regions for NR-13, Mcl-1, and Bcl-X1 were obtained and analyzed for the first time in fish, and potential regulatory sites (e.g. putative NF-kappaB binding sites) that were found in the promoter regions of NR-13 and Mcl-1 may account for their transcriptional activation by pIC.

Bcl-2 proteins are of vital importance in regulation of apoptosis, and are involved in a number of biological processes such as carcinogenesis and immune responses. Bcl-2 genes have been well studied in mammals, while they are not well investigated in teleost fish including channel catfish, the major aquaculture species in the United States. In this study, we identified 34 bcl-2 genes from the channel catfish genome, and verified their identities by conducting phylogenetic and syntenic analyses. The expression profiles of the bcl-2 genes in response to bacterial infections (Edwardsiella ictaluri and Flavobacterium columnare) and hypoxia stress were determined by performing meta-analysis using the existing RNA-Seq datasets. Differential expressions of bcl-2 genes were observed after bacterial infections and hypoxia treatment, including 22 bcl-2 genes after E. ictaluri infection, 22 bcl-2 genes after F. columnare infection, and 19 bcl-2 genes after hypoxia stress. Overall, the expression of the pro-apoptotic bcl-2 genes were repressed after bacterial infection and hypoxia stress, indicating that bcl-2 genes are potentially involved in the stress response by reducing cell apoptosis. Some bcl-2 genes, such as bcl2b, mcl1a, bmf1, and bnip3, showed different expression pattern during the E. ictaluri and F. columnare infection, suggesting the difference in the pathogenicity of diseases. This work presented the first systematic identification and annotation of bcl-2 genes in catfish, providing essential genomic resources for further immune and physiological studies.

The genes Bcl-2 and Bax play important roles in apoptosis. Many studies have shown that formalin has a strong deleterious effect on male fertility and can induce apoptosis. L-carnitine has been reported to potentially reverse the negative effects of formalin, leading to improved spermatogenesis. In this study, we examined the levels of expression of Bcl-2 and Bax in mice treated with formalin and L-carnitine.

The pathogenesis of esophagus carcinoma involves a cascade process consisting of multiple factors and accumulation of gene mutations. It is known that vascular endothelial growth factor (VEGF) mainly regulates de novo vascular formation while B-cell lymphoma-2 (BCL-2) gene exerts a tumor-suppressing effect. The prominent expression of VEGFA and BCL-2 genes, along with the most famous tumor-suppressor gene, TP53, raise the possibly of gene interaction. This study therefore investigated the effect and correlation of TP53, BCL-2, and VEGFA genes on cell proliferation and apoptosis of esophagus carcinoma.

FAM3B/PANDER is a novel cytokine-like protein that induces apoptosis in insulin-secreting beta-cells. Since in silico data revealed that FAM3B can be expressed in prostate tumors, we evaluated the putative role of this cytokine in prostate tumor progression.

During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.

Pyrazole or 1,2-Diazole is a five-membered heteroaromatic ring with two nitrogen atoms which is widely used in pharmacological research and organic synthesis. Several natural and synthetic pyrazole derivatives possess anti-cancer potential and some of them have underwent clinical trials. In this aspect, an investigation into the efficiency of the pyrazole nucleus to inhibit the growth and progression of various cancer cell lines/ experimental tumours would help in giving a better clarity to the anti-cancer behaviour of pyrazole containing drugs. This paper investigates the efficiency of pyrazole against Dalton's Lymphoma Ascites (DLA) cell line. Pyrazole inhibited the growth of DLA cells in vitro by committing them towards apoptosis. In vitro results were consistent in DLA induced murine solid tumour in vivo systems. Drug-treatment improved survival, reduced tumour loads, stabilized body weights and improved the haematological and serum biochemical parameters of DLA solid tumour bearing mice, thereby improving their overall survivability. Drug administration contained the aggravation of solid tumour by targeted downregulation of Cyclin-D1 and Ki-67. In addition, the mRNA expression levels of anti-apoptotic genes, BCL-2 and BCL-XL were downregulated in solid tumours, corroborating the in vitro results that pyrazole encourage apoptotic cell death in DLA cells. The new findings establish pyrazole as a potential anti-cancer drug candidate. The results must encourage future investigations into the efficacy of the drug against various cancer types.

Bcl-X(L), a member of the Bcl-2 family, can inhibit many forms of programed cell death. The three-dimensional structure of Bcl-X(L) identified a 60 amino acid loop lacking defined structure. Although amino acid sequence within this region is not conserved among Bcl-2 family members, structural modeling suggested that Bcl-2 also contains a large unstructured region. Compared with the full-length protein, loop deletion mutants of Bcl-X(L) and Bcl-2 displayed an enhanced ability to inhibit apoptosis. Despite enhanced function, the deletion mutants did not have significant alterations in the ability to bind pro-apoptotic proteins such as Bax. The loop deletion mutant of Bcl-2 also displayed a qualitative difference in its ability to inhibit apoptosis. Full-length Bcl-2 was unable to prevent anti-IgM-induced cell death of the immature B cell line WEHI-231. In contrast, the Bcl-2 deletion mutant protected WEHI-231 cells from death. Substantial differences were observed in the ability of WEHI-231 cells to phosphorylate the deletion mutant of Bcl-2 compared with full-length Bcl-2. Bcl-2 phosphorylation was found to be dependent on the presence of an intact loop domain. These results suggest that the loop domain in Bcl-X(L) and Bcl-2 can suppress the anti-apoptotic function of these genes and may be a target for regulatory post-translational modifications.

The BCL-2 family is conserved in evolution and shares a BCL-2 homology domain. It promotes and inhibits apoptosis. It is also known that apoptosis has a major role in effective cancer treatment. Therefore, it is of interest to document information related to the BCL-2 family of proteins for analysis by prediction tools. Hence, insights from a prediction based comparative functional analysis of 108 genes in this family are documented.

Bcl-2-associated athanogene (BAG), a group of proteins evolutionarily conserved and functioned as co-chaperones in plants and animals, is involved in various cell activities and diverse physiological processes. However, the biological functions of this gene family in rice are largely unknown. In this study, we identified a total of six BAG members in rice. These genes were classified into two groups, OsBAG1, -2, -3, and -4 are in group I with a conserved ubiquitin-like structure and OsBAG5 and -6 are in group Ⅱ with a calmodulin-binding domain, in addition to a common BAG domain. The BAG genes exhibited diverse expression patterns, with OsBAG4 showing the highest expression level, followed by OsBAG1 and OsBAG3, and OsBAG6 preferentially expressed in the panicle, endosperm, and calli. The co-expression analysis and the hierarchical cluster analysis indicated that the OsBAG1 and OsBAG3 were co-expressed with primary cell wall-biosynthesizing genes, OsBAG4 was co-expressed with phytohormone and transcriptional factors, and OsBAG6 was co-expressed with disease and shock-associated genes. β-glucuronidase (GUS) staining further indicated that OsBAG3 is mainly involved in primary young tissues under both primary and secondary growth. In addition, the expression of the BAG genes under brown planthopper (BPH) feeding, N, P, and K deficiency, heat, drought and plant hormones treatments was investigated. Our results clearly showed that OsBAGs are multifunctional molecules as inferred by their protein structures, subcellular localizations, and expression profiles. BAGs in group I are mainly involved in plant development, whereas BAGs in group II are reactive in gene regulations and stress responses. Our results provide a solid basis for the further elucidation of the biological functions of plant BAG genes.

Herpesviruses are known to acquire several genes from their hosts during evolution. We found that a significant proportion of virus homologues encoded by HSV-1, HSV-2, EBV and KSHV and their human counterparts contain G-quadruplex motifs in their promoters. We sought to understand the role of G-quadruplexes in the regulatory regions of viral Bcl-2 homologues encoded by KSHV (KS-Bcl-2) and EBV (BHRF1). We demonstrate that the KSHV KS-Bcl-2 and the EBV BHRF1 promoter G-quadruplex motifs (KSHV-GQ and EBV-GQ) form stable intramolecular G-quadruplexes. Ligand-mediated stabilization of KS-Bcl-2 and BHRF1 promoter G-quadruplexes significantly increased the promoter activity resulting in enhanced transcription of these viral Bcl-2 homologues. Mutations disrupting KSHV-GQ and EBV-GQ inhibit promoter activity and render the KS-Bcl-2 and the BHRF1 promoters non-responsive to G-quadruplex ligand. In contrast, promoter G-quadruplexes of human bcl-2 gene inhibit promoter activity. Further, KS-Bcl-2 and BHRF1 promoter G-quadruplexes augment RTA (a virus-encoded transcription factor)-mediated increase in viral bcl-2 promoter activity. In sum, this work highlights how human herpesviruses have evolved to exploit promoter G-quadruplexes to regulate virus homologues to counter their cellular counterparts.

Corals play a key role in ocean ecosystems and carbonate balance, but their molecular response to ocean acidification remains unclear. The only previous whole-transcriptome study (Moya et al. Molecular Ecology, 2012; 21, 2440) documented extensive disruption of gene expression, particularly of genes encoding skeletal organic matrix proteins, in juvenile corals (Acropora millepora) after short-term (3 d) exposure to elevated pCO2 . In this study, whole-transcriptome analysis was used to compare the effects of such 'acute' (3 d) exposure to elevated pCO2 with a longer ('prolonged'; 9 d) period of exposure beginning immediately post-fertilization. Far fewer genes were differentially expressed under the 9-d treatment, and although the transcriptome data implied wholesale disruption of metabolism and calcification genes in the acute treatment experiment, expression of most genes was at control levels after prolonged treatment. There was little overlap between the genes responding to the acute and prolonged treatments, but heat shock proteins (HSPs) and heat shock factors (HSFs) were over-represented amongst the genes responding to both treatments. Amongst these was an HSP70 gene previously shown to be involved in acclimation to thermal stress in a field population of another acroporid coral. The most obvious feature of the molecular response in the 9-d treatment experiment was the upregulation of five distinct Bcl-2 family members, the majority predicted to be anti-apoptotic. This suggests that an important component of the longer term response to elevated CO2 is suppression of apoptosis. It therefore appears that juvenile A. millepora have the capacity to rapidly acclimate to elevated pCO2 , a process mediated by upregulation of specific HSPs and a suite of Bcl-2 family members.

Cadmium is a potent toxicant and carcinogenic substance for human and experimental animals. The evidences indicate that cadmium induces aberrant gene expression, inhibition of DNA damage repair, and apoptosis. In this study, we investigated the effects of IP (intraperitoneal) injection of cadmium on mRNA levels expression of Bcl-2 and Bax genes in rat small intestine.

Correlation of the expression of inflammatory factors with expression of apoptosis-related genes, B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax), in burned rats was investigated. Forty healthy Sprague-Dawley rats were selected and randomly divided into SHAM group (n=10), I° burn group (n=10), II° burn group (n=10) and III° burn group (n=10). Changes in tumor necrosis factor-α (TNF-α), Bax messenger ribonucleic acid (mRNA), Bcl-2 mRNA, Bax protein and Bcl-2 protein expression levels were detected. The correlation of TNF-α, Bax and Bcl-2 with the degree of burn in rats was observed, and the correlation of TNF-α with Bax and Bcl-2 was also analyzed. Moreover, Bax mRNA and Bcl-2 mRNA were detected via reverse transcription-quantitative polymerase chain reaction, and TNF-α, Bax protein and Bcl-2 protein were detected via enzyme-linked immunosorbent assay. In burn groups, TNF-α, Bax mRNA and Bax protein levels were significantly increased at each time point compared with those at the previous time point (P<0.05), but Bcl-2 mRNA and protein levels were significantly decreased compared with those at the previous time point (P<0.05). At the same time point, TNF-α, Bax mRNA, Bcl-2 mRNA, Bax protein and Bcl-2 protein expression levels had statistically significant differences between any given two groups (P<0.05). The TNF-α expression level was positively correlated with Bax expression levels and negatively correlated with Bcl-2 expression levels. Additionally, TNF-α, Bax mRNA and Bax protein had positive correlations with the degree of burn and time after burn, while Bcl-2 mRNA and Bcl-2 protein had negative correlations with the degree of burn and time after burn. Continuous monitoring of changes in the TNF-α level can be used as a means to evaluate the degree of burn and apoptosis, and to prevent the deepening of burn wounds, thus facilitating the early clinical evaluation of prognosis.

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.

Previous research suggested that a novel compound PNT2258 inhibits B-cell lymphoma 2 (BCL-2) transcription by DNA interference (DNAi) and demonstrated its activity in preclinical xenograft models and in a pilot Phase II clinical trial in non-Hodgkin's lymphoma (NHL). While the drug downregulates BCL-2 at the promoter, mRNA, and protein levels, there is a significant homology (13-16 bases) between PNT100 and a number of promoters of genes involved in cell cycle regulation and survival. In this study, we identify cyclin-dependent kinase-4 (CDK4) as an unintended target gene of PNT2258 and examine its relevance to NHL.

Malignant melanoma is one of the most difficult cancers to treat due to its resistance to chemotherapy. Despite recent successes with BRAF inhibitors and immune checkpoint inhibitors, many patients do not respond or become resistant to these drugs. Hence, alternative treatments are still required. Due to the importance of the BCL-2-regulated apoptosis pathway in cancer development and drug resistance, it is of interest to establish which proteins are most important for melanoma cell survival, though the outcomes of previous studies have been conflicting. To conclusively address this question, we tested a panel of established and early passage patient-derived cell lines against several BH3-mimetic drugs designed to target individual or subsets of pro-survival BCL-2 proteins, alone and in combination, in both 2D and 3D cell cultures. None of the drugs demonstrated significant activity as single agents, though combinations targeting MCL-1 plus BCL-XL, and to a lesser extent BCL-2, showed considerable synergistic killing activity that was elicited via both BAX and BAK. Genetic deletion of BFL-1 in cell lines that express it at relatively high levels only had minor impact on BH3-mimetic drug sensitivity, suggesting it is not a critical pro-survival protein in melanoma. Combinations of MCL-1 inhibitors with BRAF inhibitors also caused only minimal additional melanoma cell killing over each drug alone, whilst combinations with the proteasome inhibitor bortezomib was more effective in multiple cell lines. Our data show for the first time that therapies targeting specific combinations of BCL-2 pro-survival proteins, namely MCL-1 plus BCL-XL and MCL-1 plus BCL-2, could have significant benefit for the treatment of melanoma.

Increased apoptosis was recently found in the hypertrophied left ventricle of spontaneously hypertensive rats (SHRs). Although the available evidence suggests that apoptosis can be induced in cardiac cells by various insults including pressure overload, cardiac apoptosis appears to result from an exaggerated local production of angiotensin in adult SHRs. Altered expressions of Bcl associated X (Bax), Bcl-2, chemokine receptor (CCR)-2, monocyte chemoattractant protein (MCP)-1, transforming growth factor (TGF)-β1, phosphorylated extracellular signal-regulated kinases (PERK), and connexin 43 proteins, and kallikrein mRNA were investigated to explore the effects of losartan on the SHR model.