Chlamydia trachomatis infection inhibits both Bax and Bak activation induced by staurosporine

We have previously shown that Chlamydia trachomatis inhibits host cell apoptosis and blocks mitochondrial cytochrome c release. We now report that activation of both Bax and Bak, two proapoptotic members of the Bcl-2 family that regulate mitochondrial cytochrome c release, was inhibited in chlamydia-infected cells. This observation has provided new information on the mechanisms of chlamydial antiapoptotic activity.     

Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane

The mechanism of Bax/Bak activation remains a central question in mitochondria-dependent apoptotic signaling. While it is established that all proapoptotic Bcl-2 homology 3 (BH3)-only proteins bind and neutralize the anti-apoptotic Bcl-2 family proteins, how this neutralization leads to Bax/Bak activation has been actively debated. Here, genome editing was used to generate cells deficient for all eight proapoptotic BH3-only proteins (OctaKO) and those that lack the entire Bcl-2 family (Bcl-2 allKO). Although the OctaKO cells were resistant to most apoptotic stimuli tested, they underwent Bax/Bak-dependent and p53/Rb-independent apoptosis efficiently when both Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins, were inactivated or eliminated. Strikingly, when expressed in the Bcl-2 allKO cells, both Bax and Bak spontaneously associated with the outer mitochondrial membrane (OMM) through their respective helix 9, and this association triggered their homo-oligomerization/activation. Together, these results strongly suggest that the OMM, not BH3-only proteins or p53/Rb, is the long-sought-after direct activator of Bax/Bak following BH3-only-mediated neutralization of anti-apoptotic Bcl-2 proteins.     

Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins

Commitment of cells to apoptosis is governed largely by the interaction between members of the Bcl-2 protein family. Its three subfamilies have distinct roles: The BH3-only proteins trigger apoptosis by binding via their BH3 domain to prosurvival relatives, while the proapoptotic Bax and Bak have an essential downstream role involving permeabilization of organellar membranes and induction of caspase activation. We have investigated the regulation of Bak and find that, in healthy cells, Bak associates with Mcl-1 and Bcl-x(L) but surprisingly not Bcl-2, Bcl-w, or A1. These interactions require the Bak BH3 domain, which is also necessary for Bak dimerization and killing activity. When cytotoxic signals activate BH3-only proteins that can engage both Mcl-1 and Bcl-x(L) (such as Noxa plus Bad), Bak is displaced and induces cell death. Accordingly, the BH3-only protein Noxa could bind to Mcl-1, displace Bak, and promote Mcl-1 degradation, but Bak-mediated cell death also required neutralization of Bcl-x(L) by other BH3-only proteins. The results indicate that Bak is held in check solely by Mcl-1 and Bcl-x(L) and induces apoptosis only if freed from both. The finding that different prosurvival proteins have selective roles has notable implications for the design of anti-cancer drugs that target the Bcl-2 family.     

Redox mechanisms of cytoprotection by Bcl-2

Bcl-2 is a multifunctional protein that protects against cell death induced by a wide variety of stimuli. The best characterized antiapoptotic Bcl-2 mechanism of action involves direct binding to proapoptotic proteins, e.g., Bax, inhibiting their ability to oligomerize and form pores in the mitochondrial outer membrane, through which soluble mitochondrial proapoptotic proteins, e.g., cytochrome c, are released into the cytosol. Bcl-2 also exerts antiapoptotic and antinecrotic effects that are mediated by its influence on cellular redox state and apparently independent of its interaction with proapoptotic proteins. Bcl-2 expression increases cell resistance to oxidants, augments the expression of intracellular defenses against reactive oxygen species, and may affect mitochondrial generation of superoxide radicals and hydrogen peroxide. This review focuses on the protective effects of Bcl-2 related to changes in mitochondrial redox capacity.     

BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak

The BH3-only proteins Bim and Bad bind to the antiapoptotic Bcl-2 proteins and induce apoptosis in wild-type cells and cells from either bax(-/-) or bak(-/-) animals. In contrast, constitutively active forms of Bim and Bad failed to induce apoptosis in bax(-/-)bak(-/-) cells. Expression of Bax restored susceptibility of the cells to Bim and Bad. In addition, Bax but not Bim or Bad sensitized the bax(-/-)bak(-/-) cells to a wide variety of cell death stimuli including UV irradiation, chemotherapeutic agents, and ER stress. These results suggest that neither activation of BH3-only proteins nor suppression of pro-survival Bcl-2 proteins is sufficient to kill cells in the absence of both Bax and Bak. Furthermore, whereas mouse embryo fibroblasts (MEF) expressing only Bax or Bak displayed resistance to transformation, bax(-/-)bak(-/-) MEF were nearly as prone to oncogenic transformation as p53(-/-) MEF. Thus, the function of either Bax or Bak appears required to initiate most forms of apoptosis and to suppress oncogenic transformation.     

A179L, a viral Bcl-2 homologue, targets the core Bcl-2 apoptotic machinery and its upstream BH3 activators with selective binding restrictions for Bid and Noxa

Several large DNA viruses encode Bcl-2 protein homologues involved in the regulation of the cellular apoptosis cascade. This regulation often involves the interaction of these viral proteins with diverse cellular Bcl-2 family members. We have identified the specific interactions of A179L, an African swine fever virus (ASFV) Bcl-2 homologue, with the active forms of the porcine BH3-only Bid protein (truncated Bid p13 and p15). Transient expression of ASFV A179L gene in Vero cells prevented apoptosis induced by these active forms of Bid protein. Interestingly, A179L protein was able to interact, also with the main core Bcl-2 proapoptotic proteins Bax and Bak, and with several BH3-only proteins with selective binding restrictions for full length Bid and Noxa. These results suggest a fine regulation for A179L action in the suppression of apoptosis in infected cells which is essential for efficient virus replication.     

Differentially expressed pro- and anti-apoptogenic genes in response to benzene exposure: Immunohistochemical localization of p53, Bag, Bad, Bax, Bcl-2, and Bcl-w in lung epithelia.

Benzene, a well-known human carcinogen, is a commonly used industrial chemical that evokes further toxicological concern because of its potential genotoxic risks as a constituent of petrol and the byproduct of combustion and cigarette smoke. The present study investigated the effects of benzene inhalation on the expression of pro- and antiapoptogenic genes in lung epithelia. Immunohistochemical expression was assessed for antiapoptotic Bcl-2 family proteins, including Bcl-2, Bcl-w, and Bag-1 as well as proapoptotic subfamily members with Bcl-2 homology (BH)1 1-3, namely Bax, those that consist of only the BH3 region, represented by Bad, and proapoptotic gene expression for p53. Rats exposed to benzene via inhalation (300 ppm) for 7 days showed a significant upregulation of proapoptotic gene expression for p53, Bax, and Bad as assessed by a semiquantitative segmental analysis of the lung epithelia, including bronchioles, terminal bronchioles, respiratory bronchioles, and alveoli. Bag-1, an antiapoptogenic gene, was also found to have significant upregulated expression in lung epithelia. Since the underlying mechanisms by which Bag-1 exerts its antiapoptogenic effects are not known, benzene may target the protein chaperones hsc70/hsp70, or RING finger protein associated with Bag-1 activity. Alternatively, the significant downregulation of Bcl-2 may have diminished the antiapoptotic synergism necessary for the effectiveness of Bag-1. Both Bcl-2 and Bcl-w were found to be significantly downregulated compared to the proapoptotic counterparts. These data support the role of benzene in activating proapoptogenic events that lead to the upregulation of gene expression that may provide a crucial defense mechanism within lung parenchyma to reduce mutation hazard and potential carcinogenic effects of benzene-initiated pathogenesis.     

Bcl-2-regulated apoptosis: mechanism and therapeutic potential

Apoptosis is essential for tissue homeostasis, particularly in the hematopoietic compartment, where its impairment can elicit neoplastic or autoimmune diseases. Whether stressed cells live or die is largely determined by interplay between opposing members of the Bcl-2 protein family. Bcl-2 and its closest homologs promote cell survival, but two other factions promote apoptosis. The BH3-only proteins sense and relay stress signals, but commitment to apoptosis requires Bax or Bak. The BH3-only proteins appear to activate Bax and Bak indirectly, by engaging and neutralizing their pro-survival relatives, which otherwise constrain Bax and Bak from permeabilizing mitochondria. The Bcl-2 family may also regulate autophagy and mitochondrial fission/fusion. Its pro-survival members are attractive therapeutic targets in cancer and perhaps autoimmunity and viral infections.     

Acupuncture protected cerebral multi-infarction rats from memory impairment by regulating the expression of apoptosis related genes Bcl-2 and Bax in hippocampus

Vascular dementia (VaD) is the second most common cause of dementia in the world today. In this paper, we observed the effect of acupuncture on memory impairment, apoptosis and expression of Bcl-2 and Bax in hippocampus of cerebral multi-infarction rats. The results indicated that acupuncture significantly improved memory impairment induced by cerebral multi-infarction, as evaluated by shortened escape latency and increased swimming time in the target quadrant. Meanwhile, based on the observation in hippocampal CA1 region through methods of the terminal deoxynucleotidyl transferase nick end labeling (TUNEL), immunohistochemistry and in situ hybridization, acupuncture decreased the number of apoptotic cells and expression of the proapoptotic Bax gene, on the contrary, it increased expression of the antiapoptotic gene Bcl-2. The result of the research suggested that acupuncture can exert antiapoptotic effect through counter-regulating Bcl-2 and Bax gene expression.     

c-mip down-regulates NF-κB activity and promotes apoptosis in podocytes

The mechanisms of podocyte disorders in cases of idiopathic nephrotic syndrome (INS) are complex and remain incompletely elucidated. The abnormal regulation of NF-κB may play a key role in the pathophysiology of these podocyte diseases, but at present, NF-κB has not been thoroughly investigated. In this study, we report that induction of c-mip in podocytes of patients with INS is associated with a down-regulation of RelA, a potent antiapoptotic factor that belongs to the NF-κB family. Overexpression of c-mip in differentiated podocytes promotes apoptosis by inducing caspase-3 activity and up-regulating the proapoptotic protein Bax, whereas the overall levels of the antiapoptotic protein Bcl-2 was concomitantly decreased. The associated overexpression of RelA prevented the proapoptotic effects of c-mip. In addition, the targeted induction of c-mip in podocytes in vivo inhibited the expression of the RelA protein and increased the Bax/Bcl-2 ratio. The expression of both c-mip and active caspase-3 increased in focal and segmental glomerulosclerosis biopsies, and both proteins displayed a close spatial relationship. These results suggest that alterations in NF-κB activity might result from the up-regulation of c-mip and are likely to contribute to podocyte disorders in cases of INS.     

Broad degradation of proapoptotic proteins with the conserved Bcl-2 homology domain 3 during infection with Chlamydia trachomatis

Chlamydiae are obligate intracellular bacteria that can inhibit apoptosis of their host cell. As shown recently, this inhibition is in part explained by the proteolytic degradation of the proapoptotic Bcl-2 family members (BH3-only proteins) Bim, Puma, and Bad upon chlamydial infection. In this study, we further explore this antiapoptotic mechanism. In cells infected with a Chlamydia trachomatis L2 strain, Bim, Puma, and Bad were degraded with similar kinetics, and the degradation of all three was blocked by inhibition of the proteasome. Furthermore, the BH3-only proteins Bmf, Noxa, and tBid were also targeted by chlamydial infection. The constitutively expressed Bmf disappeared during infection. When Noxa was experimentally induced, the levels were also reduced by infection with C. trachomatis. In death-receptor-induced apoptosis, cleaved and activated tBid was degraded, and this destruction was also prevented by inhibition of the proteasome. These results show that chlamydial infection leads to a broad degradation of BH3-only proteins. This loss of proapoptotic factors can explain the almost general protection of infected cells against apoptotic stimuli.     

Tissue-specific Bcl-2 protein partners in apoptosis: An ovarian paradigm

Apoptosis is an essential physiological process by which multicellular organisms eliminate superfluous cells. An expanding family of Bcl-2 proteins plays a pivotal role in the decision step of apoptosis, and the differential expression of Bcl-2 members and their binding proteins allows the regulation of apoptosis in a tissue-specific manner mediated by diverse extra- and intracellular signals. The Bcl-2 proteins can be divided into three subgroups: 1) antiapoptotic proteins with multiple Bcl-2 homology (BH) domains and a transmembrane region, 2) proapoptotic proteins with the same structure but missing the BH4 domain, and 3) proapoptotic ligands with only the BH3 domain. In the mammalian ovary, a high rate of follicular cell apoptosis continues during reproductive life. With the use of the yeast two-hybrid system, the characterization of ovarian Bcl-2 genes serves as a paradigm to understand apoptosis regulation in a tissue-specific manner. We identified Mcl-1 as the main ovarian antiapoptotic Bcl-2 protein, the novel Bok (Bcl-2-related ovarian killer) as the proapoptotic protein, as well as BOD (Bcl-2-related ovarian death agonist) and BAD as the proapoptotic ligands. The activity of the proapoptotic ligand BAD is regulated by upstream follicle survival factors through its binding to constitutively expressed 14-3-3 or hormone-induced P11. In contrast, the channel-forming Mcl-1 and Bok regulate cytochrome c release and, together with the recently discovered Diva/Boo, control downstream apoptosis-activating factor (Apaf)-1 homologs and caspases. Elucidation of the role of Bcl-2 members and their interacting proteins in the tissue-specific regulation of apoptosis could facilitate an understanding of normal physiology and allow the development of new therapeutic approaches for pathological states.     

The structure of a Bcl-xL/Bim fragment complex: implications for Bim function

After antigen-driven expansion, the majority of T cells involved in an immune response die rapidly by apoptosis dependent on the Bcl-2 related proteins, Bim and Bax or Bak. The details of how these proteins are activated and interact are still unclear. The crystal structure of mouse Bcl-x(L) bound to a long helical fragment of Bim indicates that the structure of Bim is very different from proteins with a Bcl-2-like fold and may leave the BH3 region of Bim constitutively exposed. Based on the structural homology between Bcl-x(L) and Bax, we predicted that binding of Bim to Bax would require displacement of the Bax penultimate alpha helix. Consistent with this prediction, truncation of this short helix was required for Bim/Bax interaction and led to spontaneous activation of Bax. Our results suggest a way in which both Bim and Bax/Bak might be required for activated T cell apoptosis.     

The expression of Bcl-2 family proteins (Bcl-2, Bcl-x, Bax, Bak and Bim) in human lymphocytes

Bcl-2 family proteins regulate programmed cell death, and may play an important role in the selection of lymphocytes. We investigated the expression of Bcl-2, Bcl-x, Bax, Bak and Bim in human lymphocytes using flow-cytometry. Bcl-2 was down-regulated in CD4(+)8(+) (DP) thymocytes and CD19(+)38(+) tonsillar lymphocytes (GC B cells). Among DP thymocytes, cells co-expressing CD69 up-regulated Bcl-2, suggesting that the role of Bcl-2 is promoting survival of positively selected DP cells. Unexpectedly, the expression level of Bcl-x was higher in DP cells than in Single Positive (SP) cells and in CD69(+) DP thymocytes it was lower than in CD69(+) DP thymocytes. Expression of Bim was low in DP thymocytes but high in a subset of GC B cells. Bim and Bax were expressed more highly in SP than in DP thymocytes. Among peripheral blood lymphocytes (PBL), CD8(+) T cells expressed an approximately ten-fold higher level of Bcl-x than CD4(+) T cells while both subsets expressed similar levels of Bcl-2. Bak expression was low and Bim expression was absent in PBL. These results suggest that not only Bcl-2 but other members of the Bcl-2 family are involved in T cell development in the thymus and affinity maturation of B cells in the germinal center.     

Active Bax and Bak are functional holins

The mechanism of Bax/Bak-dependent mitochondrial outer membrane permeabilization (MOMP), a central apoptotic event primarily controlled by the Bcl-2 family proteins, remains not well understood. Here, we express active Bax/Bak in bacteria, the putative origin of mitochondria, and examine their functional similarities to the λ bacteriophage (λ) holin. As critical effectors for bacterial lysis, holin oligomers form membrane lesions, through which endolysin, a muralytic enzyme, escapes the cytoplasm to attack the cell wall at the end of the infection cycle. We found that active Bax/Bak, but not any other Bcl-2 family protein, displays holin behavior, causing bacterial lysis by releasing endolysin in an oligomerization-dependent manner. Strikingly, replacing the holin gene with active alleles of Bax/Bak results in plaque-forming phages. Furthermore, we provide evidence that active Bax produces large membrane holes, the size of which is controlled by structural elements of Bax. Notably, lysis by active Bax is inhibited by Bcl-xL, and the lysis activity of the wild-type Bax is stimulated by a BH3-only protein. Together, these results mechanistically link MOMP to holin-mediated hole formation in the bacterial plasma membrane.     

Differential expression of cell death regulators in response to thapsigargin and adriamycin in Bcl-2 transfected DU145 prostatic cancer cells

Functional overexpression of Bcl-2 has been reported to confer an anti-apoptotic potential in a variety of cell types. The role of Bcl-2 in epithelial cell-cycle control and in interactions with other cell-cycle regulators is not clearly understood. Its expression has been correlated with the hormono- and chemo-resistant phenotype in advanced prostate cancer. The aim of this study was to investigate the mechanisms through which Bcl-2 mediates increased cytotoxic chemoresistance by assessing alterations in the expression of cell death regulatory molecules. The DU145 human prostatic adenocarcinoma cell line was stably transfected with a Bcl-2 encoding expression plasmid. Two Bcl-2 transfectants, DKC9 and DKC11, were expanded for further study. The effects of Bcl-2 expression on cellular proliferation, cell death (+/- adriamycin or thapsigargin), and expression of cell-cycle/death regulators (p53, PCNA, Bax, Bak, Bcl-X(L)) were evaluated. Compared with controls, Bcl-2 transfectants showed no difference in the rate of proliferation, a decrease in p53 (approximately two-fold), an increase in Bax (approximately two-fold) and PCNA (approximately three-fold), and no change in the levels of Bcl-X(L) and Bak proteins. DKC9 and DKC11 also exhibited a significantly increased chemoresistance to adriamycin (0.0025-5 microM) and thapsigargin (0.0025-5 microM) compared with controls. In the presence of thapsigargin or adriamycin, levels of Bcl-2 and its heterodimeric partner Bax were elevated approximately two-fold with no change in Bak in Bcl-2 transfectants in contrast to controls, where Bak was increased (two-fold). This is the first study to demonstrate that Bcl-2 transfection modulates the expression of mutant p53, Bax, and PCNA in prostate cancer cells. Moreover, Bcl-2 overexpression conferred a significant cytotoxic chemoresistance and altered the balance of expression of death promoters (from Bak, a dominant death promoter in controls, to Bax) in response to thapsigargin and adriamycin.     

Immunohistochemical analysis of Bcl-2 family proteins in adenocarcinomas of the stomach.

The apoptosis-regulating proteins Bcl-2, Bax, Bcl-X, Bak, and Mcl-1 were examined by immunohistochemical methods in 48 archival specimens of adenocarcinoma of the stomach, and the results were correlated with tumor histology (intestinal versus diffuse pattern) and clinical stage (early- versus late-stage disease, ie, stages I and II versus stage III). Tumor cells containing immunostaining for the anti-apoptotic proteins Bcl-2, Bcl-X, and Mcl-1 were present in 26 (54%), 41 (85%), and 36 (75%) of the 48 cases evaluated, respectively, whereas immunopositivity for the pro-apoptotic proteins Bax and Bak was found in 44 (92%) and 42 (88%) specimens Comparisons of these immunostaining results with tumor histology revealed statistically significant differences for Bax (P = 0.03), Bcl-X (P = 0.003), and Mcl-1 (P = 0.005), which were all more frequently immunopositive for tumors with an intestinal than a diffuse histological pattern (chi 2 analysis). In addition, the percentage of immunopositive tumor cells was significantly higher for Bcl-X (62 +/- 6% versus 45 +/- 6%, mean +/- SE, P = 0.01) and for Mcl-1 (48 +/- 6% versus 30 +/- 6%; P = 0.04) in tumors with intestinal versus diffuse histology (unpaired t-test). In contrast, the percentage of Bcl-2-immunopositive tumor cells was higher in tumors with diffuse histology compared with intestinal (32 +/- 5% versus 12 +/- 5%; P = 0.01), whereas the percentages of Bax- and Bak-immunopositive tumor cells were not significantly different between these two histological types. In 34 specimens, residual normal gastric epithelial cells (foveolar cells) were present for direct comparisons of immunointensity with tumor cells. The immunointensity for the Bcl-2, Bcl-X, and Mcl-1 proteins was stronger in tumor cells compared with normal foveolar cells in 7 (21%), 15 (44%), and 8 (2.1%) of 34 cases, respectively, whereas the immunointensity of the proapoptotic proteins Bax and Bak was reduced compared with normal cells in 8 (24%) and 24 (71%) cases. Immunointensity, however, did not correlate with histology. clinical stage was not significantly associated with the presence or absence of immunopositive tumor cells, the percentage of immunopositive cells, or immunointensity. Taken together, these results establish for the first time that several Bcl-2 family proteins are expressed in gastric adenocarcinomas and suggest that the repertoire of these proteins may differ depending on the histological type. The findings therefore support the notion that the intestinal and diffuse types of gastric cancer arise at least in part through different mechanisms.     

Immunohistochemical assessment of apoptosis-associated proteins: p53, Bcl-xL, Bax and Bak in gastric cancer cells in correlation with clinical and pathomorphological factors

PurposeThe p53 protein as well as Bcl-2 family proteins such as Bax, Bak and Bcl-xL regulate apoptosis. The study objective was to analyze the expression of p53, Bak, Bcl-xL and Bax in gastric cancer and in healthy gastric mucosa.Material and MethodsThe study group consisted of 66 patients with gastric cancer, treated surgically in II Department of General and Gastroenterological Surgery, Medical University of Bialystok. The expression of the studied proteins was assessed using the immunohistochemical method.ResultsSignificant differences were found in the expressions of the studied proteins as compared to healthy gastric mucosa. The expressions of p53 and Bax were significantly higher (70%vs13% and 50%vs13%), whereas those of Bak and Bcl-xL significantly lower (18% vs 83% and 74% vs 97%) in cancer cells than in normal mucosa (p<0.001). Significant differences were also noted in the expressions of Bax and Bcl-xL in relation to histological type. In the intestinal type (Lauren I), the expressions of Bax and Bcl-xL were higher as compared to the diffuse type (Lauren II) (93% vs 43% and 91% vs 43%). Simultaneously, correlations were noted between changes in the expression of Bax vs Bcl-xL and Bak. High expression of Bax showed a positive correlation with reduced Bak and Bcl-xL (p<0.05). Moreover, positive expression of p53 caused poorer distant survival of patients (p<0.05).ConclusionOur study concluded that disturbances in the expression of p53, Bax, Bcl-xL and Bak proteins are associated with their involvement in the process of carcinogenesis in the stomach. It is suggesting that they might appeared in the early phase of carcinogenesis.