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Aim: Aggravated neuronal loss, caused mainly by neuronal apoptosis, is observed in the brain of patients with Alzheimer's disease (AD) and animal models of AD. A truncated form of Dual‐specific and tyrosine phosphorylation‐regulated protein kinase 1A (Dyrk1A) plays a vital role in AD pathogenesis. Downregulation of anti‐apoptotic Bcl‐xL is tightly correlated with neuronal loss in AD. However, the molecular regulation of neuronal apoptosis and Bcl‐x expression by Dyrk1A in AD remains largely elusive. Here, we aimed to explore the role and molecular mechanism of Dyrk1A in apoptosis. Methods: Cell Counting Kit‐8 (CCK8), flow cytometry, and TdT‐mediated dUTP Nick‐End Labeling (TUNEL) were used to check apoptosis. The cells, transfected with Dyrk1A or/and ASF with Bcl‐x minigene, were used to assay Bcl‐x expression by RT‐PCR and Western blots. Co‐immunoprecipitation, autoradiography, and immunofluorescence were conducted to check the interaction of ASF and Dyrk1A. Gene set enrichment analysis (GSEA) of apoptosis‐related genes was performed in mice overexpressing Dyrk1A (TgDyrk1A) and AD model 5xFAD mice. Results: Dyrk1A promoted Bcl‐xS expression and apoptosis. Splicing factor ASF promoted Bcl‐x exon 2b inclusion, leading to increased Bcl‐xL expression. Dyrk1A suppressed ASF‐mediated Bcl‐x exon 2b inclusion via phosphorylation. The C‐terminus deletion of Dyrk1A facilitated its binding and kinase activity to ASF. Moreover, Dyrk1a1–483 further suppressed the ASF‐mediated Bcl‐x exon 2b inclusion and aggravated apoptosis. The truncated Dyrk1A, increased Bcl‐xS, and enrichment of apoptosis‐related genes was observed in the brain of 5xFAD mice. Conclusions: We speculate that increased Dyrk1A and truncated Dyrk1A may aggravate neuronal apoptosis by decreasing the ratio of Bcl‐xL/Bcl‐xS via phosphorylating ASF in AD. [ABSTRACT FROM AUTHOR]

Abstract Bcl-x pre-mRNA splicing serves as a typical example to study the impact of alternative splicing in the modulation of cell death. Dysregulation of Bcl-x apoptotic isoforms caused by precarious equilibrium splicing is implicated in genesis and development of multiple human diseases, especially cancers. Exploring the mechanism of Bcl-x splicing and regulation has provided insight into the development of drugs that could contribute to sensitivity of cancer cells to death. On this basis, we review the multiple splicing patterns and structural characteristics of Bcl-x. Additionally, we outline the cis-regulatory elements, trans-acting factors as well as epigenetic modifications involved in the splicing regulation of Bcl-x. Furthermore, this review highlights aberrant splicing of Bcl-x involved in apoptosis evade, autophagy, metastasis, and therapy resistance of various cancer cells. Last, emphasis is given to the clinical role of targeting Bcl-x splicing correction in human cancer based on the splice-switching oligonucleotides, small molecular modulators and BH3 mimetics. Thus, it is highlighting significance of aberrant splicing isoforms of Bcl-x as targets for cancer therapy.

Bcl-x pre-mRNA splicing serves as a typical example to study the impact of alternative splicing in the modulation of cell death. Dysregulation of Bcl-x apoptotic isoforms caused by precarious equilibrium splicing is implicated in genesis and development of multiple human diseases, especially cancers. Exploring the mechanism of Bcl-x splicing and regulation has provided insight into the development of drugs that could contribute to sensitivity of cancer cells to death. On this basis, we review the multiple splicing patterns and structural characteristics of Bcl-x. Additionally, we outline the cis-regulatory elements, trans-acting factors as well as epigenetic modifications involved in the splicing regulation of Bcl-x. Furthermore, this review highlights aberrant splicing of Bcl-x involved in apoptosis evade, autophagy, metastasis, and therapy resistance of various cancer cells. Last, emphasis is given to the clinical role of targeting Bcl-x splicing correction in human cancer based on the splice-switching oligonucleotides, small molecular modulators and BH3 mimetics. Thus, it is highlighting significance of aberrant splicing isoforms of Bcl-x as targets for cancer therapy. [ABSTRACT FROM AUTHOR]

Alternative splicing is deregulated in cancer and alternatively spliced products can be linked to cancer hallmarks. Targeting alternative splicing could offer novel effective cancer treatments. We investigated the effects of the crude extract of a South African medicinal plant, Cotyledon orbiculata, on cell survival of colon (HCT116) and esophageal (OE33 and KYSE70) cancer cell lines. Using RNASeq, we discovered that the extract interfered with mRNA regulatory pathways. The extract caused hnRNPA2B1 to splice from the hnRNPB1 to the hnRNPA2 isoform, resulting in a switch in the BCL2L1 gene from Bcl-xL to Bcl-xS causing activation of caspase-3-cleavage and apoptosis. Similar splicing effects were induced by the known anti-cancer splicing modulator pladienolide B. Knockdown of hnRNPB1 using siRNA resulted in decreased cell viability and increased caspase-3-cleavage, and over-expression of hnRNPB1 prevented the effect of C. orbiculata extract on apoptosis and cell survival. The effect of the hnRNPA2/B1 splicing switch by the C. orbiculata extract increased hnRNPA2B1 binding to Bcl-xl/s, BCL2, MDM2, cMYC, CD44, CDK6, and cJUN mRNA. These findings suggest that apoptosis in HCT116, OE33, and KYSE cancer cells is controlled by switched splicing of hnRNPA2B1 and BCL2L1, providing evidence that hnRNPB1 regulates apoptosis. Inhibiting this splicing could have therapeutic potential for colon and esophageal cancers. Targeting hnRNPA2B1 splicing in colon cancer regulates splicing of BCL2L1 to induce apoptosis. This approach could be a useful therapeutic strategy to induce apoptosis and restrain cancer cell proliferation and tumor progression. Here, we found that the extract of Cotyledon orbiculata, a South African medicinal plant, had an anti-proliferative effect in cancer cells, mediated by apoptosis induced by alternative splicing of hnRNPA2B1 and BCL2L1.

Abstract Background Homoharringtonine (HHT) is a natural alkaloid with potent antitumor activity, but its precise mechanism of action is still poorly understood. Methods We examined the effect of HHT on alternative splicing of Bcl-x and Caspase 9 in various cells using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The mechanism of HHT-affected alternative splicing in these cells was investigated by treatment with protein phosphatase inhibitors and overexpression of a protein phosphatase. Results Treatment with HHT downregulated the levels of anti-apoptotic Bcl-xL and Caspase 9b mRNA with a concomitant increase in the mRNA levels of pro-apoptotic Bcl-xS and Caspase 9a in a dose- and time-dependent manner. Calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), significantly inhibited the effects of HHT on the alternative splicing of Bcl-x and Caspase 9, in contrast to okadaic acid, a specific inhibitor of PP2A. Overexpression of PP1 resulted in a decrease in the ratio of Bcl-xL/xS and an increase in the ratio of Caspase 9a/9b. Moreover, the effects of HHT on Bcl-x and Caspase 9 splicing were enhanced in response to PP1 overexpression. These results suggest that HHT-induced alternative splicing of Bcl-x and Caspase 9 is dependent on PP1 activation. In addition, overexpression of PP1 could induce apoptosis and sensitize MCF7 cells to apoptosis induced by HHT. Conclusion Homoharringtonine regulates the alternative splicing of Bcl-x and Caspase 9 through a PP1-dependent mechanism. Our study reveals a novel mechanism underlying the antitumor activities of HHT.

Background: Verrucous carcinoma (VC) should be considered a distinct clinicopathologic entity different from the more common oral squamous cell carcinoma (OSCC) because of its unique biological behavior. Best way to understand the behavior of these carcinomas is to study them by means of molecular methods, especially in tumor progression tests and Bcl-X is an important antiapoptotic member of the Bcl-2 family and is one of the newest and most useful markers to determine the aggressiveness of many carcinomas. The relationship between this Bcl-X protein and carcinomatous behavior toward it is not studied extensively, which we attempted to evaluate using immunohistochemical analysis in selected carcinomas of the head and neck region. Method: We studied Bcl-X protein expression in sections of thirty OSCC and ten VC samples and correlated this with tumor differentiation. Results: There was a significant difference in cytoplasmic staining of Bcl-X expression with statistical analysis (P < 0.005) for VC and OSCC when compared as a group. No significance was seen among the different histological grades of OSCC and when compared with VC individually. Conclusion: The significant result between OSCC and VC suggests that their biologic course is comparable and can be helpful in differentiating them with each other for establishment of a better treatment protocol.

Alternative splicing is deregulated in cancer and alternatively spliced products can be linked to cancer hallmarks. Targeting alternative splicing could offer novel effective cancer treatments. We investigated the effects of the crude extract of a South African medicinal plant, Cotyledon orbiculata , on cell survival of colon (HCT116) and esophageal (OE33 and KYSE70) cancer cell lines. Using RNASeq, we discovered that the extract interfered with mRNA regulatory pathways. The extract caused hnRNPA2B1 to splice from the hnRNPB1 to the hnRNPA2 isoform, resulting in a switch in the BCL2L1 gene from Bcl-xL to Bcl-xS causing activation of caspase-3-cleavage and apoptosis. Similar splicing effects were induced by the known anti-cancer splicing modulator pladienolide B. Knockdown of hnRNPB1 using siRNA resulted in decreased cell viability and increased caspase-3-cleavage, and over-expression of hnRNPB1 prevented the effect of C. orbiculata extract on apoptosis and cell survival. The effect of the hnRNPA2/B1 splicing switch by the C. orbiculata extract increased hnRNPA2B1 binding to Bcl-xl/s, BCL2, MDM2, cMYC, CD44, CDK6, and cJUN mRNA. These findings suggest that apoptosis in HCT116, OE33, and KYSE cancer cells is controlled by switched splicing of hnRNPA2B1 and BCL2L1, providing evidence that hnRNPB1 regulates apoptosis. Inhibiting this splicing could have therapeutic potential for colon and esophageal cancers. Targeting hnRNPA2B1 splicing in colon cancer regulates splicing of BCL2L1 to induce apoptosis. This approach could be a useful therapeutic strategy to induce apoptosis and restrain cancer cell proliferation and tumor progression. Here, we found that the extract of Cotyledon orbiculata , a South African medicinal plant, had an anti-proliferative effect in cancer cells, mediated by apoptosis induced by alternative splicing of hnRNPA2B1 and BCL2L1. [ABSTRACT FROM AUTHOR]

Apoptosis plays a vital role in cell homeostasis during development and disease. Bcl-x, a member of the Bcl-2 family of proteins, is a mitochondrial transmembrane protein that functions to regulate the intrinsic apoptosis pathway. An alternative splicing (AS) event in exon 2 of Bcl-x results in two isoforms of Bcl-x with antagonistic effects on cell survival: Bcl-xL (long isoform), which is anti-apoptotic, and Bcl-xS (short isoform), which is pro-apoptotic. Bcl-xL is the most abundant Bcl-x protein and functions to inhibit apoptosis by a number of different mechanisms including inhibition of Bax. In contrast, Bcl-xS can directly bind to and inhibit the anti-apoptotic Bcl-xL and Bcl-2 proteins, resulting in the release of the pro-apoptotic Bak. There are multiple splice factors and signaling pathways that influence the Bcl-xL/Bcl-xS splicing ratio, including serine/arginine-rich (SR) proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), transcription factors, and cytokines. Dysregulation of the AS of Bcl-x has been implicated in cancer and diabetes. In cancer, the upregulation of Bcl-xL expression in tumor cells can result in resistance to chemotherapeutic agents. On the other hand, dysregulation of Bcl-x AS to promote Bcl-xS expression has been shown to be detrimental to pancreatic β-cells in diabetes, resulting in β-cell apoptosis. Therefore, manipulation of the splice factor, transcription factor, and signaling pathways that modulate this splicing event is fast emerging as a therapeutic avenue in the treatment of cancer and diabetes.

Background: Homoharringtonine (HHT) is a natural alkaloid with potent antitumor activity, but its precise mechanism of action is still poorly understood. Methods: We examined the effect of HHT on alternative splicing of Bcl-x and Caspase 9 in various cells using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The mechanism of HHT-affected alternative splicing in these cells was investigated by treatment with protein phosphatase inhibitors and overexpression of a protein phosphatase. Results: Treatment with HHT downregulated the levels of anti-apoptotic Bcl-xL and Caspase 9b mRNA with a concomitant increase in the mRNA levels of pro-apoptotic Bcl-xS and Caspase 9a in a dose- and time-dependent manner. Calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), significantly inhibited the effects of HHT on the alternative splicing of Bcl-x and Caspase 9, in contrast to okadaic acid, a specific inhibitor of PP2A. Overexpression of PP1 resulted in a decrease in the ratio of Bcl-xL/xS and an increase in the ratio of Caspase 9a/9b. Moreover, the effects of HHT on Bcl-x and Caspase 9 splicing were enhanced in response to PP1 overexpression. These results suggest that HHT-induced alternative splicing of Bcl-x and Caspase 9 is dependent on PP1 activation. In addition, overexpression of PP1 could induce apoptosis and sensitize MCF7 cells to apoptosis induced by HHT. Conclusion: Homoharringtonine regulates the alternative splicing of Bcl-x and Caspase 9 through a PP1-dependent mechanism. Our study reveals a novel mechanism underlying the antitumor activities of HHT. [ABSTRACT FROM AUTHOR]

Background: Verrucous carcinoma (VC) should be considered a distinct clinicopathologic entity different from the more common oral squamous cell carcinoma (OSCC) because of its unique biological behavior. Best way to understand the behavior of these carcinomas is to study them by means of molecular methods, especially in tumor progression tests and Bcl-X is an important antiapoptotic member of the Bcl-2 family and is one of the newest and most useful markers to determine the aggressiveness of many carcinomas. The relationship between this Bcl-X protein and carcinomatous behavior toward it is not studied extensively, which we attempted to evaluate using immunohistochemical analysis in selected carcinomas of the head and neck region.Method: We studied Bcl-X protein expression in sections of thirty OSCC and ten VC samples and correlated this with tumor differentiation.Results: There was a significant difference in cytoplasmic staining of Bcl-X expression with statistical analysis (P < 0.005) for VC and OSCC when compared as a group. No significance was seen among the different histological grades of OSCC and when compared with VC individually.Conclusion: The significant result between OSCC and VC suggests that their biologic course is comparable and can be helpful in differentiating them with each other for establishment of a better treatment protocol. [ABSTRACT FROM AUTHOR]

Objectives: Since its recognition as a physiologic process associated with tumor, among molecular mechanisms involved in tumor progression, defects in regulation of apoptosis have generated an accelerating volume of research that has sought to elucidate the role of programed cell death in pathogenesis and treatment of various tumors. Therefore, this study was performed to understand better the diverse biological profile of epithelial odontogenic tumors with the help of immunohistochemical expression of Bcl-X protein. Materials and Methods: We studied Bcl-X protein expression in 45 cases of epithelial odontogenic tumors which included 15 cases each of ameloblastomas, keratocystic odontogenic tumor (KCOT) and adenomatoid odontogenic tumor (AOT) and correlated the expression with their growth pattern. Results: Cytoplasmic staining of Bcl-X revealed overexpression in ameloblastoma when compared to KCOT and AOT. Percentage of positive cells showed a statistically significant difference, P = 0.007 between ameloblastoma and KCOT, whereas P < 0.001 between ameloblastoma and AOT. However, no significance was observed between KCOT and AOT (P = 0.132). Conclusion: The present study supports the fact that epithelial odontogenic tumors show diverse growth profiles. An increased Bcl-X expression was seen in ameloblastoma compared to KCOT and least expression in case of AOT which could be indicative of more aggressive biological behavior and increased cell survival activity of ameloblastoma than KCOT and AOT. This signifies the diagnostic relevance of this biomarker and also could be a possible regulator of the proliferative compartment by contributing in tumor progression and cytodifferentiation of epithelial odontogenic tumors. [ABSTRACT FROM AUTHOR]

The maternal and paternal genomes play different roles in mammalian brains as a result of genomic imprinting, an epigenetic regulation leading to differential expression of the parental alleles of some genes. Here we investigate genomic imprinting in the cerebellum using a newly developed Bayesian statistical model that provides unprecedented transcript-level resolution. We uncover 160 imprinted transcripts, including 41 novel and independently validated imprinted genes. Strikingly, many genes exhibit parentally biased—rather than monoallelic—expression, with different magnitudes according to age, organ, and brain region. Developmental changes in parental bias and overall gene expression are strongly correlated, suggesting combined roles in regulating gene dosage. Finally, brain-specific deletion of the paternal, but not maternal, allele of the paternally-biased Bcl-x, (Bcl2l1) results in loss of specific neuron types, supporting the functional significance of parental biases. These findings reveal the remarkable complexity of genomic imprinting, with important implications for understanding the normal and diseased brain.

Context: Tobacco smoking generates a tremendous amount of free radicals that induce oxidative stress (OS) in diabetics (pancreatic islet cells are defective).Salacia oblongaWall. (Celastraceae) is a proven antioxidant and antidiabetic plant whose mechanism of action is yet to be explored. Objective: The present study focuses on the protective ability ofS. oblongain tobacco smoke-induced oxidatively stressed pancreatic β-cell line. Materials and methods: The RINm5f cell line was exposed to tobacco smoke concentrate (TSC) (0.5–10%, 24 h), plant extract (1–75 µg/ml, 3 h), and their combinations. Cell viability was determined through MTT assay. Microscopic analysis was carried out in unstained and nonyl acridine orange-stained cells. The effect of toxic doses of TSC on DNA integrity was analyzed through DNA fragmentation assay. The TSC-induced nitric oxide generation was determined spectrophototmetrically. The expression of anti-apoptotic protein Bcl-X under the above treatment conditions was carried out through RT-PCR. Results: The LD50dose for TSC was found to be 1% TSC.Salacia oblongaextracts (10 and 15 µg/ml) were found to be optimum safe doses that significantly increased cell viability and decreased the nitric oxide production in TSC-treated cells. Pre-treatment with plant extract suppressed apoptosis through probable increase in the expression of anti-apoptotic protein Bcl-X in TSC-treated cells. Thus, the overall efficiency of plant extract in recovering cellular damage was proven. Discussion and conclusion: The results suggest that TSC-induced cellular alterations are related to rise in nitric oxide and Bcl-X mRNA expression and propose thatS. oblongamay confer significant cytoprotection against OS-mediated injury in β-cells. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma (KS) is the most common tumor affecting AIDS patients with over 20% of these patients afflicted by this disease. Previous studies have demonstrated that KS tumor cells predominantly express the prosurvival protein Bcl-XL compared with Bcl-2. In the current study, we have used an adenoviral vector that expresses Bcl-XS, a functional inhibitor of Bcl-XL, to study the significance of Bcl-XL expression in the KS cell line (SLK) or KS primary cultures. The results demonstrate that 75% to 80% of SLK or KS primary cells were killed by the Bcl-XS containing adenovirus whereas KS cells infected with control adenovirus showed no significant cell death or growth inhibition. Overexpression of Bcl-XL, but not Bcl-2, in SILK cells attenuated apoptosis induced by adenovirus Bcl-XS. Immunoprecipitation experiments revealed that adenoviral Bcl-XS associated with Bcl-XL, but not with Bcl-2. Mutational analysis showed that the α2 helical region of Bcl-XS containing the BH3 motif was critical for killing activity and interaction with Bcl-XL. These results suggest that Bcl-XS is a direct killer and Bcl-XL may act by interacting with and sequestering Bcl-XS. These studies also suggest that targeting Bcl-XL may be of therapeutic benefit for the treatment of tumors that are characterized by inappropriate expression of Bcl-XL.

Bcl-2 and Bcl-XL belong to a family of proteins overexpressed in a variety of human cancers which inhibit apoptosis in response to a number of stimuli including chemotherapeutic agents and ionizing radiation. To better understand the role of these polypeptides in modulating the response of cancer cells to ionizing radiation we used cell lines that were engineered to overexpress the two polypeptides. Although Bcl-2 and Bcl-XL overexpression resulted in inhibition of radiation induced apoptosis, it did not result in enhanced clonogenic survival. Consistent with this was the observation that Bcl-2 and Bcl-XL protected cells from DNA fragmentation, loss of mitochondrial membrane potential, and caspase activation for up to 72 hours after irradiation. Beyond 72 hours, there was a rapid loss in the ability of Bcl-2 and Bcl-XL to inhibit these markers of apoptosis. When Bcl-XL was analyzed at 72 hours after irradiation and beyond, a rapid accumulation of a 16-kDa form of Bcl-XL was observed. To test the hypothesis that cleavage of the 29-kDa form of Bcl-XL by caspases to a 16-kDa polypeptide results in its inability to inhibit apoptosis beyond 72 hours, we constructed a cell line that overexpressed a caspase-resistant form of Bcl-XL (Bcl-XL-Δloop). Cells overexpressing Bcl-XL-Δloop were resistant to apoptosis beyond 72 hours after irradiation and did not contain the 16-kDa form at these time points. In addition, Bcl-XL-Δloop overexpression resulted in enhanced clonogenic survival compared with control or Bcl-XL overexpressing cells. These results provide a molecular basis for the observation that expression of Bcl-2 or Bcl-XL is not a prognostic marker of tumor response to cancer therapy.

Alternative splicing ( AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL- X mRNA. This, in turn, results in the selection of the proximal 5′ splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL- XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL- XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival. [ABSTRACT FROM AUTHOR]

The Bcl-2 family of proteins are well-characterized regulators of the intrinsic apoptotic pathway. Proteins within this family can be classified as either prosurvival or prodeath members and the balance between them present at the mitochondrial membrane is what determines if the cell lives or dies. Specific interactions among Bcl-2 family proteins play a crucial role in regulating programmed cell death. Structural studies have established a conserved interaction pattern among Bcl-2 family members. This interaction is mediated by the binding of the hydrophobic face of the amphipathic α-helical BH3 domain into a conserved hydrophobic groove on the prosurvival partners. It has been reported that an increase in the helical content of BH3 mimetic peptides considerably improves the binding affinity. In this context, this work states for designing peptides derived from the BH3 domain of the proapoptotic protein Bak by substitution of some non-interacting residues by the helical inducing residue Aib. Different synthetic peptides preserving BakBH3 relevant interactions were proposed and simulated presenting a better predicted binding energy and higher helical content than the wild type Bak peptide. [ABSTRACT FROM AUTHOR]

Anti-apoptotic Bcl-2-family members not only neutralize pro-apoptotic proteins but also directly regulate intracellular Ca signaling from the endoplasmic reticulum (ER), critically controlling cellular health, survival, and death initiation. Furthermore, distinct Bcl-2-family members may selectively regulate inositol 1,4,5-trisphosphate receptor (IPR): Bcl-2 likely acts as an endogenous inhibitor of the IPR, preventing pro-apoptotic Ca transients, while Bcl-X likely acts as an endogenous IPR-sensitizing protein promoting pro-survival Ca oscillations. Furthermore, distinct functional domains in Bcl-2 and Bcl-X may underlie the divergence in IPR regulation. The Bcl-2 homology (BH) 4 domain, which targets the central modulatory domain of the IPR, is likely to be Bcl-2's determining factor. In contrast, the hydrophobic cleft targets the C-terminal Ca-channel tail and might be more crucial for Bcl-X's function. Furthermore, one amino acid critically different in the sequence of Bcl-2's and Bcl-X's BH4 domains underpins their selective effect on Ca signaling and distinct biological properties of Bcl-2 versus Bcl-X. This difference is evolutionary conserved across five classes of vertebrates and may represent a fundamental divergence in their biological function. Moreover, these insights open novel avenues to selectively suppress malignant Bcl-2 function in cancer cells by targeting its BH4 domain, while maintaining essential Bcl-X functions in normal cells. Thus, IPR-derived molecules that mimic the BH4 domain's binding site on the IPR may function synergistically with BH3-mimetic molecules selectivity suppressing Bcl-2's proto-oncogenic activity. Finally, a more general role for the BH4 domain on IPRs, rather than solely anti-apoptotic, may not be excluded as part of a complex network of molecular interactions. [ABSTRACT FROM AUTHOR]

Cattle persistently infected with a noncytopathic Bovine viral diarrhea virus (BVDV) are at risk of developing fatal “mucosal disease” (MD). The authors investigated the role of various apoptosis pathways in the pathogenesis of lesions in animals suffering from MD. Therefore, they compared the expression of caspase-3, caspase-8, caspase-9, and Bcl-2L1 (Bcl-x) in tissues of 6 BVDV-free control animals, 7 persistently infected (PI) animals that showed no signs of MD (non-MD PI animals), and 11 animals with MD and correlated the staining with the localization of mucosal lesions. Caspase-3 and -9 staining were markedly stronger in MD cases and were associated with mucosal lesions, even though non-MD PI animals and negative controls also expressed caspase-9. Conversely, caspase-8 was not elevated in any of the animals analyzed. Interestingly, Bcl-x also colocalized with mucosal lesions in the MD cases. However, Bcl-x was similarly expressed in tissues from all 3 groups, and thus, its role in apoptosis needs to be clarified. This study clearly illustrates ex vivo that the activation of the intrinsic, but not the extrinsic, apoptosis pathway is a key element in the pathogenesis of MD lesions observed in cattle persistently infected with BVDV. However, whether direct induction of apoptosis in infected cells or indirect effects induced by the virus are responsible for the lesions observed remains to be established. [ABSTRACT FROM PUBLISHER]

Elevated cell death in human preimplantation embryos is one of the cellular events compromising pregnancy rates after assisted reproductive technology treatments. We therefore explored the molecular pathways regulating cell death at the blastocyst stage in human embryos cultured in vitro. Owing to limited availability of human embryos, these pathways were further characterized in mouse blastocysts. Gene expression studies revealed a positive correlation between the cell death index and the expression of Bcl-x transcript. Cell death activation in human blastocysts was accompanied by changes in Bcl-x splicing, favoring production of Bcl-xS, an activator of cell death. Expression of Bcl-xS was detected in a subset of human blastocysts that show particular clustering in dying and/or dead cells. Altering the Bcl-xL/ Bcl-xS ratio in mouse embryos, in antisense experiments, confirmed that upregulation of Bcl-xS, with concomitant downregulation of Bcl-xL, compromised developmental potential and committed a subset of cells to undergoing cell death. This was accompanied by increased accumulation of reactive oxygen species levels without any impact on mtDNA content. In addition, altered Bcl-x splicing in favor of Bcl-xS was stimulated by culture in HTF medium or by addition of excessive glucose, leading to compromised embryo development. Thus, we conclude that inappropriate culture conditions affect Bcl-x isoform expression, contributing to compromised preimplantation embryo development. [ABSTRACT FROM AUTHOR]

B-cell chronic lymphocytic leukaemia (B-CLL) is characterized by clonal growth and accumulation of mature lymphoid cells due to disturbance in genetically regulated form of cell death called apoptosis. The intrinsic mechanism of apoptosis is controlled by Bcl-2 family proteins. Purine nucleoside analogues induce the apoptosis in cells in a state of quiescence. The aim of the study was to assess expression of selected Bcl-2 family proteins in neoplastic infiltration in bone marrow in patients with B-CLL treated with nucleoside analogues. The study comprised examination of bone marrow obtained routinely by trephine biopsy from 18 patients with B-CLL diagnosed before administration of purine nucleoside analogues treatment and after its completion. Expression of Bcl-2, Bcl-x and Bax proteins was examined. Lymphoid cells in bone marrow were present in all patients before administration of treatment. After treatment in two patients bone marrow was infiltrated in diffuse pattern, whereas other patients presented nodular pattern of infiltration. The difference between stage of infiltration before and after treatment was statistically significant (p<0.002). High percentage of infiltration ceils with positive anti Bcl-2 reaction from 42.0% in one patient to 85.33±3.06% in four patients before treatment was observed. After treatment percentage of infiltration cells with positive anti Bcl-2 antibody reaction was from 33.0±18.38% in two patients to 99.0% in one patient. Positive correlation between stage of infiltration and expression of Bcl-2 protein was confirmed before and after treatment. Such correlations were not observed in case of Bax and Bcl-x. Strong staining of immunohistochemical reaction of cells in lymphoid infiltration with Bcl-2 antibody was confirmed. There was a difference between Bcl-/Bax ratio before and after treatment, immunohistochemical assessment of expression of Bcl-2 family proteins in cells of lymphoid infiltration in bone marrow of patients with CLL is an important method in detection of minimal residual disease (MRD) after treatment. [ABSTRACT FROM AUTHOR]

Introduction: Reed-Sternberg cells are typical for Hodgkin's lymphoma. They are transformed post-germinative B cells that not achieve successful immunoglobulin gene rearrangement, and therefore predetermined to apoptosis. Several mechanisms, including latent Epstein Barr virus infection (EBV), enable survival of Hodgkin's lymphoma neoplastic cells. Goal of this study is correlation of Hodgkin's lymphoma EBV status with expression of apoptosis associated proteins in neoplastic cells. Association of Hodgkin's lymphoma with EBV was determined by chromogenic hybridization in situ detection of EBER1 and EBER2. Material and Methods: Presence of apoptosis associated proteins: bcl-2, bcl-X, bax and p53, were determined immunohistochemicaly in neoplastic cells of 39 EBV positive, and 42 EBV negative Hodgkin's lymphomas. Results: Bcl-2 protein was detected in neoplastic cells in 22.2%, Bcl-X in 87.7%, Bax in 80.2% and p53 in 67.8% of cases. In 44.4% cases simultaneous presence of Bcl-X, Bax and p53 protein was detected. Bcl-X is significantly more produced in mixed cellularity, and Bcl-X and Bax in nodular sclerosis. No significant difference was found in production of all four proteins between EBV positive and negative Hodgkin's lymphoma. Median intensity of p53, Bcl-2, Bcl-X and Bax staining of complete examined group, EBV positive and EBV negative group show significant difference, except between Bcl-X and Bax. Discussion: Survival capacity of Hodgkin's lymphoma neoplastic cells depends on activity of numerous regulators of programmed cell death. Expression of bcl-2, bcl-X, bax and p53 proteins is not different in neoplastic cells of EBV positive and EBV negative Hodgkin's lymphoma. [ABSTRACT FROM AUTHOR]

Toxicity of the typical antipsychotic haloperidol (HAL) comprises an apoptotic component that we link to pro-apoptotic Bcl-XS in PC12 preneuronal and N2a neuroblastoma cells. The mitochondrial translocation of Bcl-XS and its interaction with the pore-forming voltage-dependent anion channel (VDAC) correlates with the redistribution of cytochrome c and the cleavage of Poly(ADP-ribose) polymerase. Haloperidol-induced apoptosis is mediated by the sigma2 (σ2) receptor system and does not involve the expected antagonism of the dopamine D2 receptor, nor is it influenced by Vitamin E- or p53/Bax-mediated events. Pathological relevance is demonstrated by the cytotoxic synergism between HAL and the Alzheimer disease-related peptide β-amyloid(1–40), which correlates with Bcl-XS expression and its interaction with VDAC, and with cytosolic cytochrome c translocation. These data provide for a unique apoptotic mechanism that could underscore the clinical risks associated with HAL, particularly following chronic regimens or in the elderly.The Pharmacogenomics Journal (2006) 6, 279–288. doi:10.1038/sj.tpj.6500373; published online 7 February 2006 [ABSTRACT FROM AUTHOR]

Conditionally BCL-xL-overexpressing LNT-229 Tet-On glioma cell clones were generated to investigate whether the ‘antiapoptosis phenotype’ and the ‘motility phenotype’ mediated by BCL-2 family proteins in glioma cells could be separated. BCL-xL induction led to an immediate and concentration-dependent protection of LNT-229 cells from apoptosis. BCL-xL induction for up to 3 days did not result in altered invasiveness. In contrast, long-term BCL-xL induction for 21 days resulted in increased transforming growth factor-β2 expression, and in metalloproteinase-2 and -14 dependent, but integrin independent, increased invasiveness. Withdrawal of doxycycline (Dox) abolished the protection from apoptosis whereas the ‘invasion phenotype’ remained stable. Dox stimulation of BCL-xL-inducible LNT-229 cells conferred infiltrative growth to BCL-xL-positive glioma cells in vivo and reduced the survival of tumor-bearing mice. These data allow to dissect a direct antiapoptotic action of BCL-xL from an indirect effect, presumably mediated by altered gene expression, which modifies tumor cell invasiveness in vitro and in vivo.Cell Death and Differentiation (2006) 13, 1156–1169. doi:10.1038/sj.cdd.4401786; published online 28 October 2005 [ABSTRACT FROM AUTHOR]

Pro- and antiapoptotic proteins of the large Bcl-2 family are critical regulators of apoptosis via the mitochondrial pathway. Whereas antiapoptotic proteins of the family share all four Bcl-2 homology domains (BH1–BH4), proapoptotic members may lack some of these domains, but all so far described proapoptotic Bcl-2 proteins enclose BH3. The bcl-x gene gives rise to several alternative splice products resulting in proteins with distinct functions as the antiapoptotic Bcl-xL and proapoptotic Bcl-xS. Here, we describe a novel Bcl-x splice product of 138 amino acids termed Bcl-xAK (Atypical Killer), which encloses the Bcl-2 homology domains BH2 and BH4 as well as the transmembrane domain, but lacks BH1 and BH3. Weak endogenous expression of Bcl-xAK was seen in melanoma and other tumor cells. Interestingly, its overexpression by applying a tetracycline-inducible expression system resulted in significant induction of apoptosis in melanoma cells, which occurred in synergism with drug-induced apoptosis. After exogenous overexpression, Bcl-xAK was localized both in mitochondrial and in cytosolic cell fractions. By these findings, a completely new class of Bcl-2-related proteins is introduced, which promotes apoptosis independently from the BH3 domain and implies additional, new mechanisms for apoptosis regulation in melanoma cells.Oncogene (2006) 25, 2160–2169. doi:10.1038/sj.onc.1209253; published online 14 November 2005 [ABSTRACT FROM AUTHOR]

Post-mortem studies conducted over the past 15 years suggest that apoptosis could play a role in the pathophysiology of bipolar disorder (BD) and, to a lesser degree, schizophrenia (SZ). To test this hypothesis, we have performed a post hoc analysis of an extant gene expression profiling database obtained from the hippocampus using a novel methodology with improved sensitivity. Consistent with the working hypothesis, BDs showed a marked upregulation of 19 out of 44 apoptosis genes; however, contrary to the hypothesis, the SZ group showed a downregulation of genes associated with apoptotic injury and death. These changes in the regulation of apoptosis genes were validated using quantitative RT–PCR. Additionally, antioxidant genes showed a marked downregulation in BDs, suggesting that accumulation of free radicals might occur in the setting of a previously reported decrease of the electron transport chain in this disorder. Overall, the changes seen in BDs and SZs do not appear to be related to exposure to either neuroleptics or mood stabilizers. We conclude that fundamental differences in the genetic regulation of apoptosis and antioxidant genes may help discriminate between the pathophysiology of BD and SZ and potentially point to new treatment strategies that are specific for each disorder.Molecular Psychiatry (2006) 11, 241–251. doi:10.1038/sj.mp.4001758; published online 15 November 2005 [ABSTRACT FROM AUTHOR]

Abstract: Since apoptosis appeared to be related to neurodegenerative processes, neuroprotection has been involved in investigation of therapeutic approaches focused upon pharmacological agents to prevent neuronal programmed cell death. In this regard, erythropoietin (Epo) seems to play a critical role. The present work was focused on the study of the Epo protective effect upon human neuroblastoma SH-SY5Y cells subjected to differentiation by staurosporine. Under this condition, profuse neurite outgrowth was accompanied by programmed cell death (35% of apoptotic cells by Hoechst assay, showing characteristic DNA ladder pattern). A previous treatment with recombinant human Epo (rHuEpo) increased the expression of the specific receptor for Epo while prevented apoptosis. Simultaneously, morphological changes in neurite elongation and interconnection induced by staurosporine were blocked by Epo. These Epo effects proved to be associated to the induction of Bcl-xL at the mRNA and protein levels (RT-PCR and Western blot after immunoprecipitation) and were mediated by activation of pathways inhibited by wortmannin. In conclusion, the fact that both events induced by staurosporine, cell apoptosis and differentiation, were prevented in SH-SY5Y cells previously exposed to rHuEpo suggests interrelated signaling pathways triggered by the Epo/EpoR interaction. [Copyright &y& Elsevier]

Ultraviolet (UV) light exposure is a common cause of epithelial-derived skin cancers, and the epidermal response to UV-light has been extensively studied using both mouse models and cultured human keratinocytes (KCs). Elimination of cells with UV-induced DNA damage via apoptosis provides a powerful mechanism to minimize retention or expansion of genetically abnormal cells. This cell editing function has largely been ascribed to the biological role of the p53 tumor suppressor gene, as mutations or deletions involving p53 have been linked to skin cancer development. Rather than introducing mutations, or using cells with complete loss of wild-type p53, we used an siRNA-based approach to knockdown, but not eliminate, p53 levels in primary cultures of human KCs followed by UV-irradiation. Surprisingly, when p53 levels were reduced by 50–80% the apoptosis induced by exposure to UV-light was accelerated and markedly enhanced (two- to three- fold) compared to control siRNA treated KCs. The p53 siRNA treated KCs were characterized by elevated E2F-1 levels accompanied by accelerated elimination of the Mcl-1 and Bcl-xL antiapoptotic proteins, as well as enhanced Bax oligomerization. Forced overexpression of either Mcl-1 or Bcl-xL reduced the UV-light enhanced apoptotic response in p53 siRNA treated KCs. We conclude that p53 not only can provide proapoptotic signals but also regulates a survival pathway influencing Mcl-1 and Bcl-xL levels. This overlooked survival function of p53 may explain previous paradoxical responses noted by investigators using p53 heterozygous and knockout mouse models, and opens up the possibility that not all liaisons within the cell involving p53 necessarily represent fatal attractions.Oncogene (2005) 24, 5299–5312. doi:10.1038/sj.onc.1208650; published online 6 June 2005 [ABSTRACT FROM AUTHOR]

Recent findings have uncovered a role for the Bcl-x gene in the survival of dopaminergic neurons. The exact nature of this role has been difficult to examine because of the embryonic lethality of Bcl-x gene disruption in mouse models. Here we report the generation of catecholaminergic cell-specific conditional Bcl-x gene knock-out mice using Cre- lox recombination technology. First we produced transgenic mice that express Cre recombinase from an exogenous rat tyrosine hydroxylase promoter (TH-Cre mice). These mice were crossed to Z/AP and Z/EG reporter mouse strains to verify catecholaminergic (TH- positive) cell-specific Cre expression. The TH-Cre mice then were mated to mice possessing the Bcl-x gene flanked by loxP sites, thereby producing offspring with Bcl-x deletion limited to catecholaminergic cells. The resulting mice are viable but have one-third fewer catecholaminergic neurons than do control animals. They demonstrate a deficiency in striatal dopamine and also tend to be smaller and have decreased brain mass when compared with controls. Surprisingly, surviving neurons were found that lacked Bcl-x immunoreactivity, thereby demonstrating that this gene is dispensable for the ongoing survival of a subpopulation of catecholaminergic cells. [ABSTRACT FROM AUTHOR]

BcI-2 family members either negatively or positively regulate the apoptotic threshold of cells. Bcl-xES (extra short), a novel Bcl-x member, possesses a unique combination of BH4 and BH2 domains as well as a COOH-terminal hydrophobic transmembrane anchor domain. Bcl-xES contains sequences of hydrophobic α-6 helices but lacks sequences of α-5 helices, suggesting that it does not have pore channel-forming activity but functions uniquely as a trapping protein. mRNA expression analysis by reverse transcriptase-polymerase chain reaction and RNase protection assay reveal that Bcl-xES is expressed in a variety of human cancer cell lines and human tumors, including bone marrow from patients with acute lymphoblastic leukemia. Bcl-xES expression is much less pronounced in some specimens of normal human tissues, including the breast, ovary, testis and lung. Stable, transfected human B lymphoma Namalwa variant cells expressing Bcl-xES were derived to investigate its role in apoptosis. Bcl-xES had a preventive effect on cell death induced by tumor necrosis factor-alpha and various concentrations of anticancer drugs, including camptothecin, etoposide and cisplatin. Its protective action on cell death was correlated with the inhibition of mitochondrial cytochrome c release and caspase activation. In a yeast two-hybrid system, Bcl-xES interacted with most Bcl-2 family members, including those containing only a BH3 domain, and with the Ced-4 homolog Apaf-1. Co-immunoprecipitation and gel filtration chromatography experiments suggest that Bcl-xES delays drug-induced apoptosis by disturbing the formation of Bax oligomers and preventing cytochrome c release, but also by interacting with Apaf-1 and inhibiting procaspase-9 activation, thus averting the apoptogenic proteolytic caspase cascade and cell death. [ABSTRACT FROM AUTHOR]

In recent years, significant progress has been made in understanding cardiomyocyte differentiation. However, little is known about the regulation of myocyte survival despite the fact that myocyte apoptosis is a leading cause of heart failure. Here we report that transcription factor GATA-4 is a survival factor for differentiated, postnatal cardiomyocytes and an upstream activator of the antiapoptotic gene Bcl-X. An early event in the cardiotoxic effect of the antitumor drug doxorubicin is GATA-4 depletion, which in turn causes cardiomyocyte apoptosis. Mouse heterozygotes for a null Gata4 allele have enhanced susceptibility to doxorubicin cardiotoxicity. Genetic or pharmacologic enhancement of GATA-4 prevents cardiomyocyte apoptosis and drug-induced cardiotoxicity. The results indicate that GATA-4 is an antiapoptotic factor required for the adaptive stress response of the adult heart. Modulation of survival/apoptosis genes by tissue-specific transcription factors may be a general paradigm that can be exploited effectively for cell-specific regulation of apoptosis in disease states. [ABSTRACT FROM AUTHOR]

Neuronal cell death is a key feature of both normal nervous system development and neuropathological conditions. The Bcl-2 family, via its regulation of both caspase-dependent and caspase-independent cell death pathways, is uniquely positioned to critically control neuronal cell survival. Targeted gene disruptions of specific bcl-2 family members and the generation of transgenic mice overexpressing anti- or pro-apoptotic Bcl-2 family members have confirmed the importance of the Bcl-2 family in the nervous system. Data from studies of human brain tissue and experimental animal models of neuropathological conditions support the hypothesis that the Bcl-2 family regulates cell death in the mature nervous system and suggest that pharmacological manipulation of Bcl-2 family action could prove beneficial in the treatment of human neurological conditions such as stroke and neurodegenerative diseases. [Copyright &y& Elsevier]

In germ cells, the function of which is to form the next generation, apoptotic cell death occurs during development, as in the case of somatic cells. In this study, we show that Bcl-x knockout heterozygous (Bcl-x+/−) mice exhibit severe defects in male germ cells during development. A substantial increase in apoptosis of male germ cells occurs at around embryonic day 13.5 (E13.5) in Bcl-x+/− embryos, leading to hypoplasia of postnatal testes and reduced fertility. On the other hand, female germ cells at the same stages do not show discernible differences between wild-type and Bcl-x+/− embryos. This phenotype of Bcl-x haploinsufficiency shows that regulation of apoptosis becomes different between the sexes at around the onset of sex differentiation. Through this study, we found that, in wild-type embryos, (1) apoptosis is much more frequent (approximately 10 times) in the male than in female germ cells, and (2) expression of Bcl-xL, but not that of Bax, is higher in female than in male germ cells, at around E13.5. Male fetal germ cells, cultured with gonadal somatic cells in vitro, showed higher frequencies of apoptosis than those cultured without gonadal somatic cells. On the other hand, in the absence of gonadal somatic cells, both male and female fetal germ cells in vitro showed similar frequencies of apoptosis to female fetal germ cells in vivo. Therefore, male germ cell apoptosis, of which the default pathway is similar to that of the female, is likely to be influenced by male gonadal environments. [Copyright &y& Elsevier]

The Bcl-2-related proteins Bcl-XL and Bcl-XS represent alternative splice products and exert opposite activities in the control of apoptosis, but their significance for melanoma is not yet clear. Applying the tetracycline-inducible expression system Tet-On, we found overexpression of Bcl-XS by itself sufficient to induce apoptosis in vitro in stably transfected human melanoma cell lines. Combination with proapoptotic agents such as etoposide, pamidronate, and ceramide resulted in additive proapoptotic effects, whereas Bcl-XL protected from apoptosis caused via CD95/Fas stimulation. In nude mice growth of melanoma xenotransplants derived from stably transfected cells was significantly reduced after induction of Bcl-XS by doxycycline. Our results indicate that Bcl-X proteins are of major importance for control of apoptosis in malignant melanoma. [Copyright &y& Elsevier]

Aberrant expression of bcl-gene products has been implicated in the development of non-melanoma skin cancers. Recently, altered expression of α -smooth muscle actin has been proposed as predictive of tumour behaviour in basal cell carcinomas. The purpose of this study was to compare the aggressive and non-aggressive basal cell carcinomas in terms of bcl-gene products and α -smooth muscle actin expression. Fifty excisional biopsy samples were studied by immunohistochemical technique for the differential expressions of bcl-2, bax, bcl-x and α -smooth muscle actin. Bcl-2, bcl-x and bax were expressed in 34 (68 % ), 38 (76 % ) and 41 (82 % ) specimens, respectively. Immunoreactivity for α -smooth muscle actin was noted both in tumour nests (64 % ) and within the stroma (54 % ). There was a significant difference between aggressive and non-aggressive basal cell carcinomas in terms of bcl-2 and stromal α -smooth muscle actin immunoreactivity. Non-aggressive basal cell carcinomas display a concordant expression of bcl-family proteins, whereas aggressive tumours reveal a discordant pattern. An increased expression of stromal α-smooth muscle actin with a concomitant decrease or loss of bcl-2 expression may be highly suggestive of aggressiveness in basal cell carcinoma. [ABSTRACT FROM AUTHOR]

Previous studies indicated that hepatitis C virus core protein influences cellular apoptosis. However, the precise mechanisms of the effects are not fully understood. Therefore, in this study, we examined the mechanisms of the effects on cell apoptosis by core protein, using transiently transfected and magnetically collected core-producing HepG2 cells. First, to elucidate the target site of core protein in the apoptotic pathway, we examined the activation of caspases after anti-Fas antibody stimulation. Core protein inhibited the apoptotic cascade downstream from caspase 8 and upstream from caspase 3. Next, to clarify more direct mechanisms of this effect, mRNA levels of several bcl-2-related genes were examined. An RNase protection assay showed that the mRNA of bcl-xl increased in the core-producing cells. We showed that this increase was mediated by the enhancement of bcl-x promoter activity by core protein through an extracellular-regulated kinase pathway. These results suggest that core protein inhibits apoptosis at the mitochondria level through augmentation of Bcl-x expression, resulting in an inhibition of caspase 3 activation. [Copyright &y& Elsevier]

Background: In inflammatory bowel disease (IBD), cytokine levels (such as interleukin-1 (IL-1)) are elevated. We have shown previously that IL-1 activates phospholipid signaling pathways in intestinal epithelial cells (IEC), leading to increased ceramide levels. Aim: To determine whether ceramide induces apoptosis in IEC. Methods: Apoptosis was evaluated by annexin-Vbinding or Hoechst nuclear staining. Levels of bcl-2, bcl-x, bax, p53 and p21 were determined by Western blotting, and cell cycle analysis was determined by flow cytometry. Results: IL-1 increased ceramide accumulation in a time-dependent and concentration-dependent manner with a peak response at 4 h, with [IL-1] = 30 ng/ml. Neither IL-1 nor ceramide induced apoptosis in IEC, but they increased bcl-2 levels and decreased bax and p21 levels without affecting bcl-x and p53 levels. They also caused a slight but significant increase in the G2/M phase. These data suggest a role for ceramide in IBD and suggest a possible mechanism for the enhanced tumorigenic activity in IBD patients. [ABSTRACT FROM AUTHOR]

Apaf1 has been described as the core of the apoptosome. Deficiency in murine Apaf1 leads to embryonic lethality with a phenotype affecting many aspects of developmental apoptosis. In the developing brain, Apaf1 is a death regulator of the neuronal founder cells. Combined intercrosses of mouse lines mutant for members of the mitochondrial death pathway are providing us with some clues about the relative regulation existing among neuronal cell populations. Apaf1-deficient embryos display an interesting phenotype in the inner ear and in limb development, which involves different caspase-dependent and -independent pathways. Moreover, APAF1 is mutated in human melanomas, and its depletion contributes to malignant transformation in a mouse model of cancer. This review has a double aim: the analysis of the alternatives taken by the embryo to bring into the suicidal program different cells at different stages, and the relevance of APAF1 in the onset and progression of cancer. [ABSTRACT FROM AUTHOR]

BCL-X is a member of the BCL-2 family. It regulates apoptosis and plays a critical role in hematological malignancies. It is well-known that >90% of human genes undergo alternative splicing. A total of 10 distinct splicing transcripts of the BCL-X gene have been identified, including transcript variants 1–9 and ABALON. Different transcripts from the same gene have different functions. The present review discusses the progress in understanding the different alternative splicing transcripts of BCL-X, including their characteristics, functions and expression patterns. The potential use of BCL-X in targeted therapies for hematological malignancies is also discussed. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-α and lipopolysaccharide induce apoptotic cell death in bovine glomerular endothelial cells. Background. The glomerular endothelial cell is a specialized microvascular cell type involved in the regulation of glomerular ultrafiltration. During gram-negative sepsis, glomerulonephritis, and acute renal failure, bacterial lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α) may cause severe cell damage. Our aim was to study and compare the direct effects of TNF-α and LPS on the induction of apoptosis in bovine glomerular endothelial cells. Methods. Primary bovine glomerular endothelial cells were stimulated with TNF-α or LPS, and apoptotic cell death was investigated by DNA fragmentation analysis, morphological studies, measurement of cytochrome c efflux and mitochondrial permeability transition, Bak, Bad, Bax, Bcl-2, Bcl-xL protein expression, and caspase-3–like protease activity. Results. TNF-α, as well as LPS, elicited apoptotic cell death both time and concentration dependently. Along with DNA ladder formation, we detected the formation of 50 kbp high molecular weight DNA fragments, nuclear condensation, and mitochondrial permeability transition. Concerning all parameters, LPS signaling proved to be more rapid than TNF-α. Mechanistically, TNF-α–induced cell death was preceded by an efflux of mitochondrial cytochrome c into the cytosol and, subsequently, by a marked increase in the proapoptotic protein Bak and a decrease in the anti-apoptotic Bcl-xL protein content. Comparable but more pronounced effects were seen with LPS. Later, caspase-3–like protease activity was first detectable after 10 hours and was continuously increased up to 24 hours in both TNF-α– and LPS-stimulated cells. Correspondingly, we detected an extended cleavage of the nuclear enzyme poly(ADP-ribose) polymerase. Caspase inhibitors Z-Asp-CH2 -DCB and Z-VAD-fmk... [ABSTRACT FROM AUTHOR]

Bcl-xL, a member of the Bcl-2 family, can inhibit many forms of programed cell death. The three-dimensional structure of Bcl-xL 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-xL 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-xL and Bcl-2 can suppress the anti-apoptotic function of these genes and may be a target for regulatory post-translational modifications. [ABSTRACT FROM AUTHOR]