Bfk: a novel weakly proapoptotic member of the Bcl-2 protein family with a BH3 and a BH2 region

Proteins of the Bcl-2 family are critical regulators of apoptosis. Proapoptotic members, like Bax, contain three of the four Bcl-2 homology regions (BH1-3), while BH3-only proteins, like Bim, possess only the short BH3 motif. Database searches revealed Bfk, an unusual novel member of the Bcl-2 family that contains a BH2 and BH3 region but not BH1 or BH4. Bfk is thus most closely related to Bcl-G(L). It lacks a C-terminal membrane anchor and is cytosolic. Enforced expression of Bfk weakly promoted apoptosis and antagonized Bcl-2's prosurvival function. Like Bcl-G(L), Bfk did not bind to any Bcl-2 family members, even though its BH3 motif can mediate association with prosurvival proteins. Low amounts of Bfk were found in stomach, ovary, bone marrow and spleen, but its level in the mammary gland rose markedly during pregnancy, suggesting that Bfk may play a role in mammary development.     

Three novel Bid proteins generated by alternative splicing of the human Bid gene

Bid, a BH3-only Bcl-2 protein, is activated by proteolytic cleavage exposing the BH3 domain, which then induces apoptosis by interacting with pro-apoptotic Bcl-2 family proteins (e.g. Bax and Bak) at the mitochondrial surface. The arrangement of domains within Bid suggested that Bid function might be regulated in part by alternative splicing. We have determined the gene structure of human Bid and identified a number of novel exons. We have also demonstrated endogenous mRNA and protein expression for three novel isoforms of Bid, generated using these exons. Bid(S) contains the N-terminal regulatory domains of Bid without the BH3 domain; Bid(EL) corresponds to full-length Bid with additional N-terminal sequence; and Bid(ES) contains only the Bid sequence downstream of the BH3 domain. Expression of these isoforms is regulated during granulocyte maturation. In functional studies Bid(EL) induces apoptosis, whereas Bid(S) abrogates the pro-apoptotic effects of truncated Bid and inhibits Fas-mediated apoptosis. Bid(ES) induces apoptosis but is also able to partially inhibit the pro-apoptotic effects of truncated Bid. These three novel endogenously expressed isoforms of Bid are distinct in their expression, their cellular localization, and their effects upon cellular apoptosis. Differential expression of these novel Bid isoforms may regulate the function of Bid following cleavage and thus influence the fate of cells exposed to a range of pro-apoptotic stimuli.     

Apoptosis modulatory activities of transiently expressed Bcl-2: Roles in cytochrome c release and Bax regulation

Bcl-2 and Bcl-X_L are pro-survival members of the Bcl-2 family. These proteins have been shown to antagonize the pro-apoptotic activity of Bax and promote cell survival through blocking Bax translocation from the cytosol to mitochondria and by preventing the release of cytochrome c. However, it has been recently reported that transiently expressed Bcl-2 unexpectedly leads to significant cell toxicity. To study this intriguing phenomenon, we have carried out further analyses into the properties of transiently expressed Bcl-2. We found that various isoforms of human and different species of Bcl-2 were equally capable of inducing apoptosis. In addition, we discovered that transient expression of Bcl-2, unlike its pro-survival homolog Bcl-X_L, can lead to the release of cytochrome c from mitochondria and that the resulting cell death can be inhibited by caspase and calpain inhibitors. Moreover, we have shown that unlike the pro-apoptotic protein Bid, the toxicity associated with the transient expression of Bcl-2 occurs independent of the activity of the endogenous Bax. Finally, we found that in spite of its intrinsic toxicity, transiently expressed Bcl-2 is fully capable of blocking the ectopically expressed Bax from localizing to mitochondria. Taken together, these studies demonstrate that transiently expressed Bcl-2 displays opposing functional properties.     

Macrophage migration inhibitory factor (MIF) interacts with Bim and inhibits Bim-mediated apoptosis

The pro-apoptotic Bcl-2 family member Bim acts as a sensor for apoptotic stimuli and initiates apoptosis through the mitochondrial pathway. To identify novel regulators of Bim, we employed the yeast two-hybrid system and isolated the human gene encoding macrophage migration inhibitory factor (MIF), a ubiquitously expressed proinflammatory mediator that has also been implicated in cell proliferation, the cell cycle and carcinogenesis. The interaction between MIF and Bim was confirmed by both in vitro and in vivo protein interaction assays. Intriguingly, protein complexes between MIF and the three major Bim isoforms (BimEL/BimL/BimS) could be detected in HEK293 and K562 cells, especially in cells undergoing apoptosis. Moreover, exogenous expression of MIF partially inhibited Bim-induced apoptosis in HEK293 cells. SiRNA-mediated knockdown of MIF increased apoptosis in K562 cells exposed to the chemical oxidant diamide. Endogenous MIF may regulate the pro-apoptotic activity of Bim and inhibit the release of cytochrome c from mitochondria.     

The fourth isoform of the adenine nucleotide translocator inhibits mitochondrial apoptosis in cancer cells

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1–4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.     

Transient expression of wild-type or mitochondrially targeted Bcl-2 induces apoptosis, whereas transient expression of endoplasmic reticulum-targeted Bcl-2 is protective against Bax-induced cell death

Bcl-2 protein family members function either to promote or inhibit programmed cell death. Bcl-2, typically an inhibitor of apoptosis, has also been demonstrated to have pro-apoptotic activity (Cheng, E. H., Kirsch, D. G., Clem, R. J., et al. (1997) Science 278, 1966-1968). The pro-apoptotic activity has been attributed to the cleavage of Bcl-2 by caspase-3, which converts Bcl-2 to a pro-apoptotic molecule. Bcl-2 is a membrane protein that is localized in the endoplasmic reticulum (ER) membrane, the outer mitochondrial membrane, and the nuclear envelope. Here, we demonstrate that transient expression of Bcl-2 at levels comparable to those found in stably transfected cells induces apoptosis in human embryonic kidney 293 cells and in the human breast cell line MDA-MB-468 cells. Furthermore, we have targeted Bcl-2 specifically to either the ER or the outer mitochondrial membrane to test whether induction of apoptosis by Bcl-2 is dependent upon its localization within either of these membranes. Our findings indicate that Bcl-2 specifically targeted to the mitochondria induces cell death, whereas Bcl-2 that is targeted to the ER does not. The expression of Bcl-2 does result in its cleavage to a 20-kDa protein; however, mutation of the caspase-3 cleavage site (D34A) does not inhibit its ability to induce cell death. Additionally, we find that transiently expressed ER-targeted Bcl-2 inhibits cell death induced by Bax overexpression. In conclusion, the ability of Bcl-2 to promote apoptosis is associated with its localization at the mitochondria. Furthermore, the ability of ER-targeted Bcl-2 to protect against Bax-induced apoptosis suggests that the ER localization of Bcl-2 may play an important role in its protective function.     

Candidate tumour suppressor Fau regulates apoptosis in human cells: An essential role for Bcl-G

FAU, which encodes a ubiquitin-like protein (termed FUBI) with ribosomal protein S30 as a carboxy-terminal extension, has recently been identified as a pro-apoptotic regulatory gene. This activity may be mediated by Bcl-G (a pro-apoptotic member of the Bcl-2 family) which can be covalently modified by FUBI. FAU gene expression has been shown to be down-regulated in human breast, prostate and ovarian tumours, and this down-regulation is strongly associated with poor prognosis in breast cancer. We demonstrate here that ectopic FAU expression increases basal apoptosis in human T-cell lines and 293T/17 cells, whereas it has only a transient stimulatory effect on ultraviolet-C (UVC)-induced apoptosis. Conversely, siRNA-mediated silencing of FAU gene expression has no effect on basal apoptosis, but attenuates UV-induced apoptosis. Importantly, prior knockdown of Bcl-G expression ablates the stimulation of basal apoptosis by FAU, consistent with an essential downstream role for Bcl-G, itself a candidate tumour suppressor, in mediating the apoptosis regulatory role of FAU. In 293T/17 cells, Bcl-G knockdown also attenuates UV-induced apoptosis, so that Bcl-G may constitute a common factor in the pathways by which both FAU and UV-irradiation induce apoptosis. UV irradiation increases Bcl-G mRNA levels, providing an explanation for the transient nature of the effect of ectopic FAU expression on UV-induced apoptosis. Since failure of apoptosis is fundamental to the development of many cancers, the pro-apoptotic activity of the Fau/Bcl-G pathway offers an attractive explanation for the putative tumour suppressor role of FAU.     

Combination Therapy with Gossypol Reveals Synergism against Gemcitabine Resistance in Cancer Cells with High BCL-2 Expression

Although gemcitabine is highly active in several cancer types, intrinsic and acquired drug resistance remains a major challenge. Overexpression of Bcl-2 has been associated with gemcitabine resistance. The aim of this study is to determine whether gossypol can overcome gemcitabine resistance in cell lines with high level of Bcl-2 expression in combination drug therapy. Our study demonstrated that in 10 cell lines derived from different cancers, high Bcl-2 baseline expression was observed in cell lines that were resistant to gemcitabine (GEM-R). Furthermore, synergistic effect of combination therapy was observed in gemcitabine-resistant (GEM-R) cell lines with high Bcl-2 expression, but not in a gemcitabine-sensitive (GEM-S) cell lines regardless of Bcl-2 expression. Gossypol treatment resulted in the decrease of anti-apoptotic genes such as Bcl-2 and Bcl-xl and an upregulation of the pro-apoptotic gene, Noxa. Furthermore, the addition of gossypol to gemcitabine resulted in lower expressions of anti-apoptotic genes compared to gemcitabine alone. Gene expression profiling in GEM-R and GEM-S cell lines suggest that anti-apoptotic genes such as pAkt and PI3KR2 may play important role in gemcitabine resistance, while pro-apoptotic Bcl-2 related genes (Bad, Caspase-6 and Calpain-1) may regulate synergistic interaction in combination therapy.     

Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G(1) arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G(1) arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.     

Estrogen withdrawal-induced human breast cancer tumour regression in nude mice is prevented by Bcl-2

We recently showed that estrogen induces expression of the anti-apoptotic protein, Bcl-2 in MCF-7 human breast cancer cells. Since estrogen-dependent breast tumours can regress following estrogen withdrawal, we hypothesized that stable Bcl-2 expression would prevent estrogen-withdrawal induced regression of MCF-7 tumours. We therefore established tumours in ovariectomized female nude mice implanted with an estrogen-release pellet using untransfected MCF-7 cells or MCF-7 cells stably transfected with a Bcl-2 cDNA sense or antisense expression vector. All tumours grew at similar rates indicating that Bcl-2 levels have no effect on tumour formation. After removal of the estrogen pellet, Bcl-2 antisense tumours and untransfected MCF-7 tumours regressed means of 49% and 52%, respectively, after estrogen pellet removal whereas Bcl-2 sense tumours were significantly stabilized. Regressing tumours displayed characteristics of apoptotic cells. These results show that Bcl-2 can prevent hormone-dependent breast tumour regression and are consistent with the notion that decreased Bcl-2 levels following estrogen withdrawal renders hormone-dependent breast tumour cells sensitive to apoptotic regression.     

甘氨鹅脱氧胆酸钠通过MAPK/ERK1/2介导Bcl-2在Ser70位点的磷酸化引起人肝细胞癌的抗药

B细胞淋巴瘤-2（Bcl-2）是一种重要的抗凋亡蛋白质,在多种人类肿瘤中普遍过表达。甘氨鹅脱氧胆酸钠（GCDA）与消化道肿瘤的发生发展密切相关,并能介导肝癌细胞对化疗药物的抵抗。本文旨在探讨在GCDA介导的人肝细胞癌（HCC）耐药性中Bcl-2的作用及其机制。本研究以肝癌细胞系为研究对象,Western印迹结果显示,Bcl-2在多种肝癌细胞系中均有表达。设计靶向Bcl-2的siRNA沉默HCC细胞系内源性Bcl-2的表达,发现Bcl-2沉默之后促进了化疗药物5-FU介导的HCC细胞凋亡。机制上,GCDA可介导Bcl-2在Ser70位点的磷酸化,而Ser70位点的磷酸化能够被PD98059（MAPK/ERK1/2抑制剂）所抑制。构建huBcl2-WT和huBcl2-S70A真核表达载体,脂质体转染HCC细胞系。用Annexin V-FITC/PI流式细胞术检测凋亡细胞。结果显示,huBcl2-WT过表达能抑制5 FU介导的凋亡,S70位点失活突变成A后,Bcl-2的过表达不能抑制5-FU介导的凋亡。本研究提示,GCDA通过MAPK/ERK1/2通路介导的Bcl-2 Ser70位点的磷酸化,在肝癌细胞的存活和抗药中发挥重要作用。抑制Bcl-2能够促进化疗药物5-FU介导的HCC细胞凋亡,该结果为治疗GCDA介导的耐药性肝癌提供新的思路。     

Activation of multidomain and BH3-only pro-apoptotic Bcl-2 family members in p53-defective cells

In the p53-deficient human B lymphoma Namalwa cell line that quickly undergoes apoptosis after DNA topoisomerase I inhibitor (camptothecin, CPT) treatment, we observed rapid and slight induction of the pro-apoptotic BH3-only Bik, Bim-EL, Bim-L and Bim-S proteins. In contrast, the expression levels of Bad and multidomain Bax-alpha and Bak remained mostly unchanged after CPT treatment. However, multiple pro-apoptotic proteins, including Bax-alpha, Bak, Bik, Bim-EL and Bim-L, translocated rapidly to the mitochondria after CPT treatment. Gel filtration chromatography experiments demonstrated that somes of the pro-apoptotic proteins assemble themselves into high molecular weight protein complexes. The protein composition of these oligomers was further analyzed by co-immunoprecipitation experiments performed on highly purified mitochondrial fractions, which revealed the formation of Bax/Bak, Bax/VDAC1, Bak/VDAC1, Bim/VDAC1 and Bim/Bcl-2 complexes after DNA damage induction. Thus, it appeared that induction, mitochondrial translocation and assembly in multimeric protein complexes of several pro-apoptotic members of the Bcl-2 family correlated with the rapid activation of apoptosis in a p53-independent pathway after CPT-mediated DNA strand breaks.     

Expression of Bcl—2 inhibited Fas—mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Apoptosis plays an important role in embryonic development,tissue remodeling,immune regulation and tumor regression.Two groups of molecules(Bcl-2 family and “Death factor” family) are involved in regulating apoptosis.In order to know about the effect of Bcl-2 on apoptosis induced by Fas,a typical member of “Death factor” family,the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells,a human hepatocellular carcinoma cell line which expresses endogenous Fas,but not FasL and Bcl-2.The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells.However,the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis.Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis.These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.     

甘氨鹅脱氧胆酸钠通过MAPK/ERK1/2介导Bcl-2在Ser70位点的磷酸化引起人肝细胞癌的抗药

B细胞淋巴瘤-2（Bcl-2）是一种重要的抗凋亡蛋白质,在多种人类肿瘤中普遍过表达。甘氨鹅脱氧胆酸钠（GCDA）与消化道肿瘤的发生发展密切相关,并能介导肝癌细胞对化疗药物的抵抗。本文旨在探讨在GCDA介导的人肝细胞癌（HCC）耐药性中Bcl-2的作用及其机制。本研究以肝癌细胞系为研究对象,Western印迹结果显示,Bcl-2在多种肝癌细胞系中均有表达。设计靶向Bcl-2的siRNA沉默HCC细胞系内源性Bcl-2的表达,发现Bcl-2沉默之后促进了化疗药物5-FU介导的HCC细胞凋亡。机制上,GCDA可介导Bcl-2在Ser70位点的磷酸化,而Ser70位点的磷酸化能够被PD98059（MAPK/ERK1/2抑制剂）所抑制。构建huBcl2-WT和huBcl2-S70A真核表达载体,脂质体转染HCC细胞系。用Annexin V-FITC/PI流式细胞术检测凋亡细胞。结果显示,huBcl2-WT过表达能抑制5 FU介导的凋亡,S70位点失活突变成A后,Bcl-2的过表达不能抑制5-FU介导的凋亡。本研究提示,GCDA通过MAPK/ERK1/2通路介导的Bcl-2 Ser70位点的磷酸化,在肝癌细胞的存活和抗药中发挥重要作用。抑制Bcl-2能够促进化疗药物5-FU介导的HCC细胞凋亡,该结果为治疗GCDA介导的耐药性肝癌提供新的思路。     

Regulation of mitochondrial metabolism: yet another facet in the biology of the oncoprotein Bcl-2

The Bcl-2 (Bcl is B-cell lymphocytic-leukaemia proto-oncogene) family comprises two groups of proteins with distinct functional biology in cell-fate signalling. Bcl-2 protein was the first member to be discovered and associated with drug resistance in human lymphomas. Since then a host of other proteins such as Bcl-xL, Bcl-2A1 and Mcl-1 with similar anti-apoptotic functions have been identified. In contrast, the pro-apoptotic Bcl-2 proteins contain prototypic effector proteins such as Bax and Bak, and the BH3 (Bcl-2 homology)-only proteins comprising Bak, Bid, Bim, Puma and Noxa. A complex interplay between the association of pro-apoptotic and anti-apoptotic proteins with each other determines the sensitivity of cancer cells to drug-induced apoptosis. The canonical functional of Bcl-2 in terms of apoptosis inhibition is its ability to prevent mitochondrial permeabilization via inhibiting the translocation and oligomerization of pro-apoptotic proteins such as Bax; however, more recent evidence points to a novel mechanism of the anti-apoptotic activity of Bcl-2. Overexpression of Bcl-2 increases mitochondrial oxygen consumption and in doing so generates a slight pro-oxidant intracellular milieu, which promotes genomic instability and blocks death signalling. However, in the wake of overt oxidative stress, Bcl-2 regulates cellular redox status thereby preventing excessive build-up of ROS (reactive oxygen species), which is detrimental to cells and tissues. Taken together, the canonical and non-canonical activities of Bcl-2 imply a critical involvement of this protein in the processes of tumour initiation and progression. In the present paper we review these functionally distinct outcomes of Bcl-2 expression with implications for the chemotherapeutic management of cancers.     

Role of Bcl-2 family members in invertebrates

Proteins belonging to the Bcl-2 family function as regulators of 'life-or-death' decisions in response to various intrinsic and extrinsic stimuli. In mammals, cell death is controlled by pro- and anti-apoptotic members of the Bcl-2 family, which function upstream of the caspase cascade. Structural and functional homologues of the Bcl-2 family proteins also exist in lower eukaryotes, such as nematodes and flies. In nematodes, an anti-apoptotic Bcl-2 family protein, CED-9, functions as a potent cell death inhibitor, and a BH3-only protein, EGL-1, acts as an inhibitor of CED-9 to facilitate the spatio-temporal regulation of programmed cell death. On the other hand, the Drosophila genome encodes two Bcl-2 family proteins, Drob-1/Debcl/dBorg-1/dBok and Buffy/dBorg-2, both of which structurally belong to the pro-apoptotic group, despite abundant similarities in the cell death mechanisms between flies and vertebrates. Drob-1 acts as a pro-apoptotic factor in vitro and in vivo, and Buffy/dBorg-2 exhibits a weak anti-apoptotic function. The ancestral role of the Bcl-2 family protein may be pro-apoptotic, and the evolution of the functions of this family of proteins may be closely linked with the contribution of mitochondria to the cell death pathway.     

Regulation of BNIP3 in normal and cancer cells

Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is a mitochondrial pro-apoptotic protein that has a single Bcl-2 homology 3 (BH3) domain and a COOH-terminal transmembrane (TM) domain. Al-though it belongs to the Bcl-2 family and can hetero-dimerize with Bcl-2, its pro-apoptotic activity is distinct from those of other members of the Bcl-2 family. For example, cell death mediated by BNIP3 is independent of caspases and shows several characteristics of necrosis. Furthermore, the TM domain, but not the BH3 domain, is required for dimerization, mitochondrial targeting and pro-apoptotic activity. BNIP3 plays an important role in hypoxia-induced death of normal and malignant cells. Its expression is markedly increased in the hypoxic regions of some solid tumors and appears to be regulated by hypoxia-inducible fac-tor (HIF), which binds to a site on the BNIP3 promoter. Silencing, followed by methylation, of the BNIP3 gene occurs in a significant proportion of can-cer cases, especially in pancreatic cancers. BNIP3 also has a role in the death of cardiac myocytes in ischemia. Further studies of BNIP3 should provide insight into hypoxic cell death and may contribute to im-proved treatment of cancers and cardiovascular diseases.     

BNIP3 heterodimerizes with Bcl-2/Bcl-X(L) and induces cell death independent of a Bcl-2 homology 3 (BH3) domain at both mitochondrial and nonmitochondrial sites

BNIP3 (formerly NIP3) is a pro-apoptotic, mitochondrial protein classified in the Bcl-2 family based on limited sequence homology to the Bcl-2 homology 3 (BH3) domain and COOH-terminal transmembrane (TM) domain. BNIP3 expressed in yeast and mammalian cells interacts with survival promoting proteins Bcl-2, Bcl-X(L), and CED-9. Typically, the BH3 domain of pro-apoptotic Bcl-2 homologues mediates Bcl-2/Bcl-X(L) heterodimerization and confers pro-apoptotic activity. Deletion mapping of BNIP3 excluded its BH3-like domain and identified the NH(2) terminus (residues 1-49) and TM domain as critical for Bcl-2 heterodimerization, and either region was sufficient for Bcl-X(L) interaction. Additionally, the removal of the BH3-like domain in BNIP3 did not diminish its killing activity. The TM domain of BNIP3 is critical for homodimerization, pro-apoptotic function, and mitochondrial targeting. Several TM domain mutants were found to disrupt SDS-resistant BNIP3 homodimerization but did not interfere with its killing activity or mitochondrial localization. Substitution of the BNIP3 TM domain with that of cytochrome b(5) directed protein expression to nonmitochondrial sites and still promoted apoptosis and heterodimerization with Bcl-2 and Bcl-X(L). We propose that BNIP3 represents a subfamily of Bcl-2-related proteins that functions without a typical BH3 domain to regulate apoptosis from both mitochondrial and nonmitochondrial sites by selective Bcl-2/Bcl-X(L) interactions.     

The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance

Three-dimensional (3D) cultures are a valuable platform to study acquired multicellular apoptotic resistance of cancer. We used spheroids of cell lines and actual tumor to study resistance to the proteasome inhibitor bortezomib in mesothelioma, a highly chemoresistant tumor. Spheroids from mesothelioma cell lines acquired resistance to bortezomib by failing to upregulate Noxa, a pro-apoptotic sensitizer BH3-only protein that acts by displacing Bim, a pro-apoptotic Bax/Bak-activator protein. Surprisingly, despite their resistance, spheroids also upregulated Bim and thereby acquired sensitivity to ABT-737, an inhibitor of anti-apoptotic Bcl-2 molecules. Analysis using BH3 profiling confirmed that spheroids acquired a dependence on anti-apoptotic Bcl-2 proteins and were ‘primed for death''. We then studied spheroids grown from actual mesothelioma. ABT-737 was active in spheroids grown from those tumors (5/7, ∼70%) with elevated levels of Bim. Using immunocytochemistry of tissue microarrays of 48 mesotheliomas, we found that most (33, 69%) expressed elevated Bim. In conclusion, mesothelioma cells in 3D alter the expression of Bcl-2 molecules, thereby acquiring both apoptotic resistance and sensitivity to Bcl-2 blockade. Mesothelioma tumors ex vivo also show sensitivity to Bcl-2 blockade that may depend on Bim, which is frequently elevated in mesothelioma. Therefore, mesothelioma, a highly resistant tumor, may have an intrinsic sensitivity to Bcl-2 blockade that can be exploited therapeutically.