Susceptibility to p53 dependent apoptosis correlates with increased levels of Gas2 and Gas3 proteins

p53 dependent apoptosis is a critical regulator of tumorigenesis. In this paper we demonstrate that BALB/c cells transformed with a LT mutant perturbing pRb but not p53 functions (LT-2809) show unrestrained cell division under low serum condition which is actively counterbalanced by apoptosis. BALB/c cells transformed with a LT mutant perturbing p53 but not pRb functions (LT-K1), show similar unrestrained cell division but no evident signs of apoptosis when grown in low serum. Such apoptotic response of LT-2809 cells is characterised by increased expression of Gas2 which becomes proteolytically processed. Similarly Gas3 expression is markedly increased in LT-2809 cells with respect to LT-K1. Since both Gas2 and Gas3 have been previously associated with the apoptotic response at growth arrest, our observations suggest that they could also contribute to the regulation of cellular susceptibility to p53 dependent apoptosis.     

Bcl-2 targets protein phosphatase 1 alpha to Bad

The diverse forms of protein phosphatase 1 (PP1) in vivo result from the association of the catalytic subunit with different regulatory subunits. We recently have described that PP1alpha is a Ras-activated Bad phosphatase that regulates IL-2 deprivation-induced apoptosis. With the yeast two-hybrid system, GST fusion proteins, indirect immunofluorescence, and coimmunoprecipitation, we found that Bcl-2 interacts with PP1alpha and Bad. In contrast, Bad did not interact with 14-3-3 protein. Bcl-2 depletion decreased phosphatase activity and association of PP1alpha to Bad. Bcl-2 contains the RIVAF motif, analogous to the well characterized R/KXV/IXF consensus motif shared by most PP1-interacting proteins. This sequence is involved in the binding of Bcl-2 to PP1alpha. Disruption of Bcl-2/PP1alpha association strongly decreased Bcl-2 and Bad-associated phosphatase activity and formation of the trimolecular complex. These results suggest that Bcl-2 targets PP1alpha to Bad.     

The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis

Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H(2)O(2). The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H(2)O(2)-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H(2)O(2) treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H(2)O(2)-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H(2)O(2)-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H(2)O(2) activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.     

Bcl-xL/Bcl-2 coordinately regulates apoptosis, cell cycle arrest and cell cycle entry

Bcl-x(L) and Bcl-2 inhibit both apoptosis and proliferation. In investigating the relationship between these two functions of Bcl-x(L) and Bcl-2, an analysis of 24 Bcl-x(L) and Bcl-2 mutant alleles, including substitutions at residue Y28 previously reported to selectively abolish the cell cycle activity, showed that cell cycle delay and anti-apoptosis co-segregated in all cases. In determining whether Bcl-2 and Bcl-x(L) act in G(0) or G(1), forward scatter and pyronin Y fluorescence measurements indicated that Bcl-2 and Bcl-x(L) cells arrested more effectively in G(0) than controls, and were delayed in G(0)-G(1) transition. The cell cycle effects of Bcl-2 and Bcl-x(L) were reversed by Bad, a molecule that counters the survival function of Bcl-2 and Bcl-x(L). When control and Bcl-x(L) cells of equivalent size and pyronin Y fluorescence were compared, the kinetics of cell cycle entry were similar, demonstrating that the ability of Bcl-x(L) and Bcl-2 cells to enhance G(0) arrest contributes significantly to cell cycle delay. Our data suggest that cell cycle effects and increased survival both result from intrinsic functions of Bcl-2 and Bcl-x(L).     

Bcl-2 and Bcl-xL play important roles in the crosstalk between autophagy and apoptosis

Autophagy and apoptosis play important roles in the development, cellular homeostasis and, especially, oncogenesis of mammals. They may be triggered by common upstream signals, resulting in combined autophagy and apoptosis. In other instances, they may be mutually exclusive. Recent studies have suggested possible molecular mechanisms for crosstalk between autophagy and apoptosis. Bcl-2 and Bcl-xL, the well-characterized apoptosis guards, appear to be important factors in autophagy, inhibiting Beclin 1-mediated autophagy by binding to Beclin 1. In addition, Beclin 1, Bcl-2 and Bcl-xL can cooperate with Atg5 or Ca(2+) to regulate both autophagy and apoptosis. Thus, Bcl-2 and Bcl-xL represent a molecular link between autophagy and apoptosis. Here, we discuss the possible roles of Bcl-2 and Bcl-xL in apoptosis and autophagy, and the crosstalk between them.     

Phospho-ser 15-p53 translocates into mitochondria and interacts with Bcl-2 and Bcl-xL in eugenol-induced apoptosis

Our previous studies demonstrated that antiallergic effects of herbs such as clove and Magnoliae Flos (MF) resulted from the induction of apoptosis in mast cells. We here examined whether the antiallergic activity was caused by eugenol (4-allyl-2-methoxyphenol) which was one of major ingredients in the essential oils or extracts of numerous plants including clove and Magnoliae Flos. RBL-2H3 cells were treated with eugenol, and DNA electrophoresis, Western blotting, immunocytochemistry, confocal microscopy and immunoprecipitation were conducted. Effect of eugenol was tested using a rat anaphylaxis model. RBL-2H3 cells treated with eugenol showed typical apoptotic manifestations and translocation of p53 into mitochondria. Antisense p53 partially prevented the induction of apoptosis. Noticeably, we observed that p53 translocated into mitochondria was phosphorylated on ser 15. Phospho-ser 15-p53 physically interacted with Bcl-2 and Bcl-xL in mitochondria and its translocation into mitochondria preceded cytochrome c release and mitochondrial membrane potential (MMP) reduction. We also depicted that the survival of animals even after administration of the fatal dose of compound 48/80 might result from the decreased number of mast cells by eugenol pretreatment. In conclusion, eugenol induces apoptosis in mast cells via translocation of phospho-ser 15-p53 into mitochondria.     

A chimeric protein induces tumor cell apoptosis by delivering the human Bcl-2 family BH3-only protein Bad

Deregulation of PI3K/Akt and Raf/Mek/Erk signal transduction cascades is one of the principal causes of neoplastic transformation. The inactivation of the proapoptotic protein Bad, upon phosphorylation by different kinases of these two pathways, may play an important role in different human malignancies. Therefore, we have expressed and purified a new chimeric protein, hGM-CSF-Bad, linking the human granulocyte-macrophage colony-stimulating factor to the N-terminus of the proapoptotic protein human Bad, to deliver Bad into tumor cells and induce apoptosis. Indeed, the human GM-CSF receptor is a good target because it is overexpressed on many leukemias and solid tumors and is not detectable on stem cells. We found that the chimeric protein binds the human GM-CSF receptor, is endocytosed, and appears to reach the cytosol via retrograde ER transport. After entering cells, the protein is able to induce apoptosis of human leukemia cells and human colon and gastric carcinoma cell lines (IC(50) values as low as 1 muM). We conclude that GM-CSF-Bad can overcome the inappropriate survival stimuli in transformed cells and restore the apoptotic pathway. The completely human sequence and the elevated selectivity for cancer cells could prevent immunogenicity and the nonspecific toxicity of targeted toxins in future clinical application of this fusion protein.     

HIV-specific CD8+ T cells exhibit markedly reduced levels of Bcl-2 and Bcl-xL

Human immunodeficiency virus-specific CD8(+) T cells are highly sensitive to spontaneous and CD95/Fas-induced apoptosis, and this sensitivity may impair their ability to control HIV infection. To elucidate the mechanism behind this sensitivity, in this study we examined the levels of antiapoptotic molecules Bcl-2 and Bcl-x(L) in HIV-specific CD8(+) T cells from HIV-infected individuals. Bcl-2 expression was markedly decreased in HIV-specific CD8(+) T cells compared with CMV-specific and total CD8(+) T cells from HIV-infected individuals as well as total CD8(+) T cells from healthy donors. CD8(+) T cell Bcl-2 levels inversely correlated with spontaneous and CD95/Fas-induced apoptosis of CD8(+) T cells from HIV-infected individuals. HIV-specific CD8(+) T cells also had significantly lower levels of Bcl-x(L) compared with CMV-specific CD8(+) T cells. Finally, IL-15 induces both Bcl-2 and Bcl-x(L) expression in HIV-specific and total CD8(+) T cells, and this correlated with apoptosis inhibition and increased survival in both short- and long-term cultures. Our data indicate that reduced Bcl-2 and Bcl-x(L) may play an important role in the increased sensitivity to apoptosis of HIV-specific CD8(+) T cells and suggest a possible mechanism by which IL-15 increases their survival.     

Praf2 is a novel Bcl-xL/Bcl-2 interacting protein with the ability to modulate survival of cancer cells

Increased expression of Bcl-xL in cancer has been shown to confer resistance to a broad range of apoptotic stimuli and to modulate a number of other aspects of cellular physiology, including energy metabolism, cell cycle, autophagy, mitochondrial fission/fusion and cellular adhesion. However, only few of these activities have a mechanistic explanation. Here we used Tandem Affinity purification to identify novel Bcl-xL interacting proteins that could explain the pleiotropic effects of Bcl-xL overexpression. Among the several proteins co-purifying with Bcl-xL, we focused on Praf2, a protein with a predicted role in trafficking. The interaction of Praf2 with Bcl-xL was found to be dependent on the transmembrane domain of Bcl-xL. We found that Bcl-2 also interacts with Praf2 and that Bcl-xL and Bcl-2 can interact also with Arl6IP5, an homologue of Praf2. Interestingly, overexpression of Praf2 results in the translocation of Bax to mitochondria and the induction of apoptotic cell death. Praf2 dependent cell death is prevented by the co-transfection of Bcl-xL but not by its transmembrane domain deleted mutant. Accordingly, knock-down of Praf2 increases clonogenicity of U2OS cells following etoposide treatment by reducing cell death. In conclusion a screen for Bcl-xL-interacting membrane proteins let us identify a novel proapoptotic protein whose activity is strongly counteracted exclusively by membrane targeted Bcl-xL.     

HSpin1, a transmembrane protein interacting with Bcl-2/Bcl-xL,induces a caspase-independent autophagic cell death

The Drosophila spinster (spin) gene product is required for programmed cell death in the nervous and reproductive systems. We have identified a human homologue of the Drosophila spin gene product (HSpin1). HSpin1 bound to Bcl-2 and apoptosis regulator Bcl-X (Bcl-xL), but not to proapoptotic members such as Bcl-2-associated X protein and Bcl-2 homologous antagonist killer, in cells treated with TNF-alpha. Exogenous expression of HSpin1 resulted in the cell death without inducing a release of cytochrome c from mitochondria. Overexpression of Bcl-xL inhibited the HSpin1-induced cell death. Interestingly, a necrosis inhibitor, pyrrolidine dithiocarbomate, but not the pancaspase inhibitors, carbobenzoxy-VAD-fluoromethyl ketone and p35, blocked the HSpin1-induced cell death. HSpin1-induced cell death increases autophagic vacuole and mature form of cathepsin D, suggesting a novel caspase-independent cell death, which is link to autophagy.     

Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells

The pro-apoptotic protein, Bax, has been reported to translocate from cytosol to mitochondria following exposure of cells to apoptotic stresses including cytokine withdrawal and treatment with glucocorticoids and cytotoxic drugs. These observations, coupled with reports showing that Bax causes the release of mitochondrial cytochrome c, implicate Bax as a central mediator of the apoptotic process. In this report we demonstrate by subcellular fractionation a significant shift in Bax localization from cytosol to cellular membranes in two human tumor cell lines exposed to staurosporine or etoposide. Immunofluorescence studies confirmed that Bax specifically relocalized to the mitochondria. This redistribution of Bax occurred in concert with, or just prior to, proteolytic processing of procaspase-3, activation of DEVD-specific cleavage activity and degradation of poly(ADP-ribose) polymerase. However, Bax membrane translocation was independent of caspase activity as determined using the broad-range caspase inhibitor z-VAD-fmk. High level overexpression of the anti-apoptotic protein Bcl-2 prevented Bax redistribution to the mitochondria, caspase activation and apoptosis following exposure to staurosporine or etoposide. These data confirm the role of Bax in mitochondrial cytochrome c release, and indicate that prevention of Bax translocation to the mitochondrial membrane represents a novel mechanism by which Bcl-2 inhibits drug-induced apoptosis.     

Bcl-2 family members and apoptosis, taken to heart

Loss of myocardial cells via apoptosis has been observed in many cardiovascular diseases and has been shown to contribute to the initiation and progression of heart failure. The Bcl-2 family members are important regulators of the mitochondrial pathway of apoptosis. These proteins decide whether the mitochondria should initiate the cell death program and release proapoptotic factors such as cytochrome c. The Bcl-2 proteins consist of anti- and proapoptotic members and play a key role in regulating apoptosis in the myocardium. The antiapoptotic proteins have been demonstrated to protect against various cardiac pathologies, whereas the antiapoptotic proteins have been reported to contribute to heart disease. This review summarizes the current understanding of the role of Bcl-2 proteins in the heart. cardiovascular disease; cytochrome c; protein; mitochondria     

Decrease in intracellular superoxide sensitizes Bcl-2-overexpressing tumor cells to receptor and drug-induced apoptosis independent of the mitochondria

At least two mechanisms of early cytosolic acidification during apoptotic signaling have been described, one that involves caspase 8 activation downstream of receptor ligation and another dependent on mitochondria-derived hydrogen peroxide during merocil-induced apoptosis. Here, we show that Bcl-2 inhibits both mechanisms of acidification. Moreover, Bcl-2 overexpression resulted in a slightly elevated constitutive level of superoxide anion and pH in CEM leukemia cells. Interestingly, decreasing intracellular superoxide concentration with an inhibitor of the beta-nicotinamide adenine dinucleotide phosphate oxidase or by transient transfection with a dominant-negative form of the guanosine triphosphate-binding protein Rac1 resulted in a significant increase in the sensitivity of CEM/Bcl-2 cells to CD95- or merocil-induced apoptosis. This increase in sensitivity was a direct result of a significant increase in caspase 8 activation and caspase 8-dependent acidification in the absence of caspase 9 activity or cytochrome c release. These findings suggest a mechanism of switching from mitochondria-dependent to mitochondria-independent death signaling in the same cell, provided the intracellular milieu is permissive for upstream caspase 8 activation, and could have implications for favorably tailoring tumor cells for drug treatment even when the mitochondrial pathway is compromised by Bcl-2.     

The Us3 protein kinase of herpes simplex virus 1 blocks apoptosis and induces phosporylation of the Bcl-2 family member Bad

Viruses have evolved different strategies to interfere with apoptotic pathways in order to halt cellular responses to infection. The herpes simplex virus 1 (HSV-1) Us3 open-reading frame encodes a serine/threonine protein kinase that participates in the inhibition of apoptosis induced by virus infection and other stress agents. Previous studies have shown that Us3 counteracts the virus-induced activation of caspase-3 by acting at a premitochondrial stage. Using stable transfectants that express Us3 under the control of constitutive or inducible promoters we demonstrate that apoptosis induced by treatment with anti-Fas antibody and sorbitol is blocked when Us3 is expressed at levels comparable to those achieved during virus infection. Expression of Us3 correlated with phosphorylation of Bad, a BH3-only proapoptotic Bcl-2 family member that is also a target for growth factor-induced cellular kinases. Bad was phosphorylated by Us3 in in vitro kination assays. These results point to a strategy for viral inhibition of apoptosis based on functional inactivation of a critical component of the cellular death machinery.     

姜黄素对晚期宫颈癌患者Bcl-xL、Bcl-2、EphA2及EphrinA1表达的影响

目的:探究姜黄素对宫颈癌晚期放疗患者Bcl-x L、Bcl-2、Eph A2与Ephrin A1表达的影响。方法:收集160例晚期宫颈癌放疗患者的宫颈癌石蜡标本,分为姜黄素组与对照组,均给予放射治疗,姜黄素组放疗期间加服姜黄素片剂,采用免疫组化等实验方法,观察两组病例标本Bcl-x L、Bcl-2、Eph A2与Ephrin A1的表达情况,比较两组病理标本化疗前后的凋亡细胞指数(AI)与微血管密度(MVD)。结果:姜黄素组的Bcl-x L、Bcl-2、Eph A2与Ephrin A1的表达阳性率分别为8.3%、43.33%、46.67%、15.0%,均显著低于对照组(P0.05);放疗后姜黄素组的AI显著高于对照组(P0.05),MVD显著低于对照组(P0.05)。结论:姜黄素可抑制Bcl-x L、Bcl-2的表达以促进肿瘤细胞凋亡,同时降低Eph A2与Ephrin A1的表达以减少肿瘤微血管形成,有显著的抗肿瘤与放疗增敏作用。     

Bcl-2 and Bcl-xL inhibit CD95-mediated apoptosis by preventing mitochondrial release of Smac/DIABLO and subsequent inactivation of X-linked inhibitor-of-apoptosis protein.

Bcl-2 and Bcl-x(L) are reported to inhibit CD95-mediated apoptosis in "type II" but not in "type I" cells. In the present studies, we found that stimulation of CD95 receptors, with either agonistic antibody or CD95 ligand, resulted in the activation of caspase-8, which in turn processed caspase-3 between its large and small subunits. However, in contrast to control cells, those overexpressing either Bcl-2 or Bcl-x(L) displayed a distinctive pattern of caspase-3 processing. Indeed, the resulting p20/p12 caspase-3 was not active and did not undergo normal autocatalytic processing to form p17/p12 caspase-3, because it was bound to and inhibited by endogenous X-linked inhibitor-of-apoptosis protein (XIAP). Importantly, Bcl-2 and Bcl-x(L) inhibited the release of both cytochrome c and Smac from mitochondria. However, since Smac alone was sufficient to promote caspase-3 activity in vitro by inactivating XIAP, we proposed the existence of a death receptor-induced, Smac-dependent and apoptosome-independent pathway. This type II pathway was subsequently reconstituted in vitro using purified recombinant proteins at endogenous concentrations. Thus, mitochondria and associated Bcl-2 and Bcl-x(L) proteins may play a functional role in death receptor-induced apoptosis by modulating the release of Smac. Our data strongly suggest that the relative ratios of XIAP (and other inhibitor-of-apoptosis proteins) to active caspase-3 and Smac may dictate, in part, whether a cell exhibits a type I or type II phenotype.     

Unknotting the roles of Bcl-2 and Bcl-xL in cell death

The antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL play important roles in inhibiting mitochondria-dependent extrinsic and intrinsic cell death pathways. It seems that these two proteins have distinct functions for inhibiting extrinsic and intrinsic cell death pathways. The overexpression of Bcl-2 is able to inhibit not only apoptotic cell death but also in part nonapoptotic cell death, which has the role of cell cycle arrest in the G1 phase, which may promote cellular senescence. The overexpression of Bcl-2 may also have the ability to enhance cell death in the interaction of Bcl-xL with other factors. The overexpression of Bcl-xL enhances autophagic cell death when apoptotic cell death is inhibited in Bax(-/-)/Bak(-/-) double knockout cells. This review discusses the previously unexplained aspects of Bcl-2 and Bcl-xL functions associated with cell death, for better understanding of their functions in the regulation.