Evolution of Bcl-2 homology motifs: homology versus homoplasy.

Bcl-2 family proteins regulate apoptosis in animals. This protein family includes several homologous proteins and a collection of other proteins lacking sequence similarity except for a Bcl-2 homology (BH)3 motif. Thus, membership in the Bcl-2 family requires only one of the four BH motifs. On this basis, a growing number of diverse BH3-only proteins are being reported. Although compelling cell biological and biophysical evidence validates many BH3-only proteins, claims of significant BH3 sequence similarity are often unfounded. Computational and phylogenetic analyses suggest that only some BH3 motifs arose by divergent evolution from a common ancestor (homology), whereas others arose by convergent evolution or random coincidence (homoplasy), challenging current assumptions about which proteins constitute the extended Bcl-2 family.

Targeting the anti-apoptotic Bcl-2 family members for the treatment of cancer.

Most cells express a variety of both anti-apoptotic and pro-apoptotic Bcl-2 proteins and the interaction within this family dictates whether a cell survives or dies. The dysregulation of the anti-anti-apoptotic Bcl-2 family members is one of the defining features of cancer cells in comparison to normal cells, and significantly contributes to the resistance of cancer cells to current treatment modalities. This anti-apoptotic subfamily of proteins is now a major target in the development of new methods to improve treatment outcomes for cancer patients. Several drugs directed at inhibiting Bcl-2 and related anti-apoptotic proteins have been developed with some showing considerable promise in the clinic. This Review presents the current knowledge of the role of the anti-apoptotic Bcl-2 family in cancer cells, as well as current and future perspectives on targeting this subfamily of proteins for therapeutic intervention in human malignancies. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

The role of Bcl-2 family proteins in chronic lymphocytic leukaemia.

Bcl-2 family proteins have long been implicated in the pathology of chronic lymphocytic leukaemia (CLL). Indeed, a number of these proteins have been shown to have prognostic importance in this disease. The precise ways in which these proteins impact upon CLL and the ways in which they are regulated remain incompletely resolved. However, significant advances have been recently made in our understanding of how these proteins are controlled by genetic, epigenetic and microenvironmental cues. Furthermore, major progress has been made in trying to target these proteins therapeutically. Here we review the current knowledge about this family of apoptosis-regulating proteins and how they impact upon drug resistance and disease progression. We also summarise evolution in the development of Bcl-2 family inhibitors for the treatment of CLL and other cancers.

Non-peptidic small molecule inhibitors against Bcl-2 for cancer therapy.

A critical regulator of the apoptotic machinery is the Bcl-2 family proteins whose over expression confers a protective effect on malignant cells against death signals of apoptosis. Cancer cells that are resistant to various anti-cancer drugs and treatment regimen are found to over express these Bcl-2 proteins such as Bcl-2, Bcl-X(L), Mcl-1, Bcl-w, and A1/Bfl1. In recent years there has been an exponential growth in the identification as well as synthesis of non-peptidic cell permeable small-molecule inhibitors (SMIs) of protein-protein interaction. The focus of this article is on inhibitors of anti-apoptotic protein Bcl-2. This review summarizes an up to date knowledge of the available SMIs, their mode of action as well as their current status in preclinical as well as clinical development.

Apoptosis-based treatment of glioblastomas with ABT-737, a novel small molecule inhibitor of Bcl-2 family proteins.

Defects in the apoptotic signaling cascades contribute to the poor therapeutic response of malignant gliomas. As glioblastomas are characterized by high expression levels of anti-apoptotic Bcl-2 family proteins, we studied the effects of the novel Bcl-2 inhibitor, ABT-737, on malignant glioma cells. ABT-737 treatment released the pro-apoptotic Bax protein from its binding partner Bcl-2 and potently induced apoptotic cell death in glioblastoma cells in vitro and in vivo. The local administration of ABT-737 prolonged the survival in an intracranial glioma xenograft model. Downregulation of Mcl-1 and overexpression of Bcl-2 sensitized the cells to ABT-737-mediated apoptosis. Moreover, ABT-737 potentiated the cytotoxicity of the chemotherapeutic drugs vincristine and etoposide, and of the death ligand TRAIL. As glioma stem cells may play a crucial role for the tumor progression and the resistance to treatment in glioblastomas, we investigated the effects of ABT-737 on the subpopulation of glioma cells exhibiting stem cell characteristics. Inhibition of proliferation and induction of apoptosis by ABT-737 were less efficient in glioma stem cells than in non-stem cell-like glioma cells. As the resistance of glioma stem cells was associated with high Mcl-1 expression levels, ABT-737 treatment combined with downregulation of Mcl-1 could represent a promising novel approach in glioblastoma treatment.

The anti-apoptotic Bcl-2 family member Mcl-1 promotes T lymphocyte survival at multiple stages.

T lymphocyte development and function are tightly regulated by the intrinsic death pathway through members of the Bcl-2 family. Genetic studies have demonstrated that the Bcl-2 family member Mcl-1 is an important anti-apoptotic protein in the development of multiple cell types including T lymphocytes. However, the expression pattern and anti-apoptotic roles of Mcl-1 in T lymphocytes at different developmental stages remain to be fully determined. In this study, we examined the expression pattern of Mcl-1 in different populations of T cells at the single-cell level and found that Mcl-1 protein is constitutively expressed in all T cell populations and up-regulated upon TCR stimulation. We then investigated the role of Mcl-1 in the survival of these different populations by conditionally deleting Mcl-1 at various T cell stages. Our results show that Mcl-1 is required for the survival of double-negative and single-positive thymocytes as well as naive and activated T cells. Furthermore, we demonstrate that Mcl-1 functions together with Bcl-xL to promote double-positive thymocyte survival. Thus, Mcl-1 is a critical anti-apoptotic factor for the survival of T cells at multiple stages in vivo.

Mechanisms involved in tissue-specific apopotosis regulated by glucocorticoids.

Physiological cell turnover is under the control of a sharp and dynamic balance of different homeostatic mechanisms such as the equilibrium between cell proliferation and cell death. These mechanisms play an important role in maintaining normal tissue function and architecture. It is well known that apoptosis is the prevalent mode of physiological cell loss in most tissues. Steroid hormones like glucocorticoids have been identified as key signals controlling cell turnover by modulating programmed cell death in a tissue- and cell-specific manner. In this sense, several reports have demonstrated that glucocorticoids are able to induce apoptosis in cells of the hematopoietic system such as monocytes, macrophages, and T lymphocytes. In contrast, they protect against apoptotic signals evoked by cytokines, cAMP, tumor suppressors, in glandular cells such as the mammary gland epithelia, endometrium, hepatocytes, ovarian follicular cells, and fibroblasts. Although several studies have provided significant information on hormone-dependent apoptosis in an specific tissue, a clearly defined pathway that mediates cell death in response to glucocorticoids in different cell types is still misunderstood. The scope of this review is held to those mechanisms by which glucocorticoids control apoptosis, emphasizing tissue-specific expression of genes that are involved in the apoptotic pathway.

Combined proteasome and Bcl-2 inhibition stimulates apoptosis and inhibits growth in EBV-transformed lymphocytes: a potential therapeutic approach to EBV-associated lymphoproliferative diseases.

OBJECTIVES: Epstein-Barr virus (EBV) transforms B-cells into immortalized lymphoblastoid cells (LCLs) by triggering signaling pathways that lead to activation of multiple transcription factors and anti-apoptotic proteins, including NF-kappaB and Bcl-2, respectively. Since proteasome inhibition suppresses NF-kappaB activity, we sought to determine whether the proteasome inhibitor, bortezomib, alone or in combination with Bcl-2 inhibition, has potential as a therapeutic strategy in EBV-driven B-cell neoplasms. METHODS: We evaluated the effects of bortezomib in LCLs in vitro, in the presence and absence of the small molecular inhibitor of Bcl-2, HA14-1, on proliferation, apoptosis, caspase activation, and expression of Bcl-2 family members, and in vivo in the severe combined immunodeficiency (SCID) model of EBV+ lymphoproliferative disease. RESULTS: Bortezomib inhibited proliferation, stimulated apoptosis, and activated caspases-3 and -9 in a dose-dependent manner in LCLs. In vivo, bortezomib completely abrogated development of EBV+ lymphoproliferative disease in LCL-bearing SCID mice. When HA14-1 was added to bortezomib in vitro, we observed a synergistic anti-proliferative effect and enhancement of apoptosis and caspase activation, including activation of caspase-8, in LCLs. These events were associated with modulation of expression of Bcl-2 family members towards a pro-apoptotic profile with translocation of cytochrome C from mitochondria to cytoplasm. CONCLUSIONS: These studies demonstrated that bortezomib mediates anti-tumor effects in EBV-associated lymphoproliferations both in vitro and in vivo, and that its anti-proliferative and apoptotic effects are synergistically enhanced in the presence of a Bcl-2 inhibitor. These findings support further investigation of bortezomib in EBV+ lymphoproliferative diseases, and suggest that bortezomib in combination with Bcl-2 antagonists represents a potential therapeutic strategy for EBV-driven B-cell neoplasms.

The role of HRK gene in human cancer.

Apoptosis regulators play one of the most critical roles in tumorigenesis, and an imbalance between cell proliferation and apoptosis may contribute to tumor progression. HRK was itself originally identified as a proapoptotic gene induced by diminished levels of cytokine in hematopoietic cells and cultured neurons and repressed by the expression of death-repressor proteins. A few analyses of HRK protein expression in primary central nervous system lymphomas have been performed, and little is known about the epigenetic or post-transcriptional mechanisms that may participate in HRK inactivation. Here we show the data on the 5'-CpG methylation status, loss of heterozygosity on 12q13.1 and its association with HRK expression in human malignancies, including prostate cancers, astrocytic tumors and primary central nervous system lymphomas. Aberrant methylation of CpG islands within the promoter is an epigenetic event largely responsible for the silencing of the HRK gene and subsequent low apoptotic counts in our series of malignancies. Inactivation of HRK apparently occurs in a substantial proportion of all tumor phenotypes and, as a potential proapoptotic gene, HRK may contribute to the development and progression of many human cancers.

BH3-only proteins and their roles in programmed cell death.

The Bcl-2 family of proteins controls the mitochondrial pathway to apoptosis. It consists of pro-survival and pro-apoptotic members, and their interactions decide whether apoptogenic factor confined to the mitochondrial intermembrane space can leak to the cytosol. Despite the intense efforts to understand the molecular mechanisms that lead to the permeabilization of the mitochondrial membrane, this particular issue remains a matter of intense controversy. It is well accepted that pro-apoptotic Bax and Bak are directly responsible for the damage to the mitochondria, but pro-survival family members prevent them from doing so. It is also accepted that stress signals activate selected Bcl-2 homology (BH)3-only proteins. But do these BH3-only proteins bind and activate Bax and Bak directly, or do they inhibit the pro-survival family members?

BH3-only proteins in rheumatoid arthritis: potential targets for therapeutic intervention.

Rheumatoid arthritis (RA) is a debilitating disease, resulting in the destruction of bone and cartilage, and in the permanent disfigurement of joints. Although the precise cause of RA is currently unresolved, it has become clear that the damaging effects are a result of the toxic milieu caused by an influx of inflammatory cells and the resulting heightened proinflammatory state within the joint. As the amount of literature suggesting that this preponderance of cells is a result of decreased local apoptosis in the joint continues to increase, in this review, we describe how Bcl-2 family pro-apoptotic BH3-only proteins, particularly Bim and Bid, could act to protect against the development of the disease. We also suggest a role for BH3-mimetic drugs as potential therapeutics in the treatment of RA.

Parthenolide sensitizes cells to X-ray-induced cell killing through inhibition of NF-kappaB and split-dose repair.

Human cancers have multiple alterations in cell signaling pathways that promote resistance to cytotoxic therapy such as X rays. Parthenolide is a sesquiterpene lactone that has been shown to inhibit several pro-survival cell signaling pathways, induce apoptosis, and enhance chemotherapy-induced cell killing. We investigated whether parthenolide would enhance X-ray-induced cell killing in radiation resistant, NF-kappaB-activated CGL1 cells. Treatment with 5 microM parthenolide for 48 to 72 h inhibited constitutive NF-kappaB binding and cell growth, reduced plating efficiency, and induced apoptosis through stabilization of p53 (TP53), induction of the pro-apoptosis protein BAX, and phosphorylation of BID. Parthenolide also enhanced radiation-induced cell killing, increasing the X-ray sensitivity of CGL1 cells by a dose modification factor of 1.6. Flow cytometry revealed that parthenolide reduced the percentage of X-ray-resistant S-phase cells due to induction of p21 waf1/cip1 (CDKN1A) and the onset of G1/S and G2/M blocks, but depletion of radioresistant S-phase cells does not explain the observed X-ray sensitization. Further studies demonstrated that the enhancement of X-ray-induced cell killing by parthenolide is due to inhibition of split-dose repair.

Mitochondrial protein import: a matter of death?

Mitochondria play a central role not only in energy generation but also for apoptosis. A key step in mitochondrial apoptosis is the release of mitochondrial proteins, most importantly cytochrome c. This release is orchestrated by the pro- and anti-apoptotic members of the Bcl-2 protein family. The functions of these Bcl-2 family members are clear in terms of order and of principle: the pro-apoptotic BH3-only protein group contains the triggers, which cause the activation of the effectors Bax and Bak, while the anti-apoptotic Bcl-2-like proteins prevent this activation. However, the molecular details are still insufficiently clear and the proposed models have certain gaps and are partly contradictory. We have recently presented evidence that targeting to mitochondria of at least one BH3-only protein is essential for its pro-apoptotic functions. Here we discuss how this mechanism might fit into and expand existing models and speculate about the potential implications of this finding.

Prognostic relevance of a novel semiquantitative classification of Bcl2 immunohistochemical expression in human infiltrating ductal carcinomas of the breast.

BACKGROUND: Bcl2 is an important prognostic parameter in human breast cancer. However, the evaluation of Bcl2 expression by immunohistochemistry is carried out using arbitrary scoring criteria. In the present study, we evaluated the clinical relevance of a novel, semiquantitative classification of the Bcl2 immunostaining based on both the distribution and the intensity of the staining reaction. PATIENTS AND METHODS: The proposed classification was first validated in 69 breast cancer specimens by comparing the Bcl2 immunostaining with the Bcl2 messenger RNA (mRNA) levels evaluated by real-time RT-PCR. Since a highly significant association was found between protein and mRNA for Bcl2, the immunohistochemical scoring system was applied to 442 patients with infiltrating ductal carcinomas of the breast with long-term follow-up (median observation time 106 months). RESULTS: In the entire series, the Bcl2 variable was an independent predictor of clinical outcome, and its prognostic independence was maintained when lymph node-negative and -positive patients were considered separately. In this regard, of particular interest was the observation of a subgroup of node-negative breast cancer patients with a negative Bcl2 immunostaining, who had a very high probability of relapse or death (respectively about five and seven times greater than patients with a positive Bcl2 immunostaining). Moreover, the Bcl2 variable retained prognostic significance also in subgroups of patients treated with either adjuvant endocrine therapy or chemotherapy. CONCLUSIONS: Our results demonstrated that in breast cancer, Bcl2 protein expression parallels its mRNA level, and it has a highly significant and independent prognostic relevance.

20(S)-Ginsenoside Rg3 prevents endothelial cell apoptosis via inhibition of a mitochondrial caspase pathway.

Ginseng, refering to the roots of the species of the genus Panax ginseng, has been widely used in traditional oriental medicine for its wide spectrum of medicinal effects, such as anti-inflammatory, anti-tumorigenic, adaptogenic, and anti-aging activities. Many of its medicinal effects are attributed to the triterpene glycosides known as ginsenosides. In this study, we report a novel anti-apoptotic activity of 20(S)-ginsenoside Rg3 ((20S)Rg3) and its underlying molecular mechanism in human endothelial cells (ECs). ECs undergo apoptosis associated with increased LEHDase (caspase-9) and DEVDase (caspase-3) activity and DNA fragmentation after 24h of serum deprivation. These apoptotic markers were suppressed by the addition of (20S)Rg3. (20S)Rg3 increased the expression of Bax and conversely decreased Bcl-2. (20S)Rg3 potently induced a rapid and sustained Akt activation and Bad phosphorylation, resulting in the inhibition of mitochondrial cytochrome c release. These anti-apoptotic activities of (20S)Rg3 were significantly abrogated in cells expressing dominant negative Akt. Taken together, our results suggest that (20S)Rg3 prevents EC apoptosis via Akt-dependent inhibition of the mitochondrial apoptotic signaling pathway. The novel property of (20S)Rg3 may be valuable for developing new pharmaceutical means that will control unwanted endothelial cell death at the site of vascular injury.

WT1 influences apoptosis through transcriptional regulation of Bcl-2 family members.

WT1 is a zinc finger transcriptional regulatory protein that has been implicated in both normal development and in the generation of a variety of malignancies. Alternative splicing of the WT1 mRNA results in the expression of four major protein isoforms. There is increasing evidence that one of the functions of WT1 is the regulation of apoptosis, but there are conflicting reports about whether WT1 is pro- or anti-apoptotic. Careful review of the literature suggests that these apparent contradictions probably reflect cell lineage and isoform-specific differences in WT1 function. Expression of WT1 prevents programmed cell death in some cell types but promotes it in others, and different WT1 isoforms exert distinct effects as well. Investigation into the mechanisms by which WT1 regulates apoptosis has revealed that several bcl-2 family members are either direct or indirect WT1 target genes, including bcl-2 itself, the pro-apoptotic family members Bak and Bax, and the anti-apoptotic family member Bfl-1/A1. The specific bcl-2 family members regulated by WT1 expression vary by cell type and by isoform. Thus, a complete understanding of the role of WT1 in apoptosis will have to account for lineage- and isoform-specific effects of WT1 at both the cellular and molecular levels.

BCL-2 in the crosshairs: tipping the balance of life and death.

The discovery of B-cell lymphoma-2 (BCL-2) over 20 years ago revealed a new paradigm in cancer biology: the development and persistence of cancer can be driven by molecular roadblocks along the natural pathway to cell death. The subsequent identification of an expansive family of BCL-2 proteins provoked an intensive investigation of the interplay among these critical regulators of cell death. What emerged was a compelling tale of guardians and executioners, each participating in a molecular choreography that dictates cell fate. Ten years into the BCL-2 era, structural details defined how certain BCL-2 family proteins interact, and molecular targeting of the BCL-2 family has since become a pharmacological quest. Although many facets of BCL-2 family death signaling remain a mechanistic mystery, small molecules and peptides that effectively target BCL-2 are eliminating the roadblock to cell death, raising hopes for a medical breakthrough in cancer and other diseases of deregulated apoptosis.