Bcl-2 family members and disease

Apoptosis plays an important role during development and in the maintenance of multicellular organisms. Bcl-2 family members affect cell death in either a positive or negative fashion. Although some redundancy exists between family members, expression of certain family members is important during development in an organ-specific manner. The founding family member bcl-2 tends to be highly expressed in the embryo and declines postnatally following differentiation and maturation. Altered expression of bcl-2, as well as other family members, has been observed in disease states potentially affecting treatment modalities. Here we examine the distribution and role death repressors bcl-2, bcl-x(L) and bcl-w as well as death effectors bax and bak play regulating apoptosis in a tissue-specific manner. Understanding the normal role of these proteins during embryogenesis and in the mature organ will give us important insight into what goes awry in various disease states.     

Developmental regulation of the bcl-2 family during spermatogenesis: insights into the sterility of bcl-w-/- male mice

Expression of bcl-w, a close relative of bcl-2 is essential for male fertility in mice. Although the initial wave of spermatogenesis in bcl-w -/- mice proceeds normally until 3-4 weeks of age, adults fail to produce sperm. To clarify why bcl-w is essential for adult but not juvenile spermatogenesis, we investigated the expression pattern of eight bcl-2 family members. We found that both the level and pattern of expression varied in different cell types during juvenile and adult spermatogenesis. Anti-apoptotic genes bcl-w, bcl-2 and bcl-xL were all expressed in spermatogonia during juvenile spermatogenesis, but only bcl-w was detected in spermatogonia of adult mice. A similar shift was evident in Sertoli cells. This developmental regulation may co-ordinate physiological germ cell apoptosis in wild type mice and account for the time of onset for pathological germ cell apoptosis in bcl-w -/- animals.     

Oxygen deprivation induced cell death: an update

Mammalian cells have multiple responses to low or zero oxygen concentrations. In the complete absence of oxygen, cells undergo cell death through apoptosis, and not necrosis. Apoptotic signaling during oxygen deprivation occurs through the release of cytochrome c and apaf-1 mediated caspase-9 activation. The upstream regulators of cytochrome c release are the Bcl-2 family members. Pro-apoptotic Bcl-2 family members such as bax or bak are clearly required to initiate cytochrome c/apaf-1/caspase-9 mediated cell death during oxygen deprivation. Here we review what is currently known oxygen deprivation induced cell death and speculate about initiating mechanisms resulting in the activation of pro-apoptotic Bcl-2 family members.     

The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues

Proapoptotic Bcl-2 family members have been proposed to play a central role in regulating apoptosis. However, mice lacking bax display limited phenotypic abnormalities. As presented here, bak(-/-) mice were found to be developmentally normal and reproductively fit and failed to develop any age-related disorders. However, when Bak-deficient mice were mated to Bax-deficient mice to create mice lacking both genes, the majority of bax(-/-)bak(-/-) animals died perinatally with fewer than 10% surviving into adulthood. bax(-/-)bak(-/-) mice displayed multiple developmental defects, including persistence of interdigital webs, an imperforate vaginal canal, and accumulation of excess cells within both the central nervous and hematopoietic systems. Thus, Bax and Bak have overlapping roles in the regulation of apoptosis during mammalian development and tissue homeostasis.     

Requirement of A1-a for bacillus Calmette-Guérin-mediated protection of macrophages against nitric oxide-induced apoptosis

The role of apoptosis in regulating the course of intracellular microbial infection is not well understood. We studied the relationship between apoptotic regulation and bacillus Calmette-Guérin (BCG) treatment in murine peritoneal exudate macrophages (PEM) and the J774 macrophage cell line. In both PEM and J774 cells, mRNA expression of the anti-apoptotic gene, A1, was selectively induced by BCG treatment as compared with other bcl2 family members (bcl-w, bcl-2, bcl-xl, bcl-xs, bax, bak, bad). In PEM, A1 expression was maximal by 8 h postinfection and was abrogated by the proteasomal inhibitor MG-132. The induction was independent of protein synthesis as well as the p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways and did not require live organism. Three genes encoding closely related isoforms of A1 were all expressed; however, the A1-a isoform displayed the greatest fold induction in PEM. BCG-induced A1 expression was associated with protection of host macrophages from NO-mediated apoptosis in both PEM and J774 cells. BCG-mediated protection was abrogated in PEM derived from A1-a(-/-) mice, indicating a requirement of A1-a for survival of inflammatory macrophages.     

Bcl-2 and Bax mammalian regulators of apoptosis are functional in Drosophila

Studies of apoptosis in C. elegans have allowed the identification of three genes, ced-3, ced-4 and ced-9. Their products constitute the components of an induction pathway of apoptosis conserved in the nematode and mammals. In Drosophila, homologues have been found for CED-3, CED-4 and CED-9. CED-9 belongs to the Bcl-2 family which includes negative (Bcl-2) and positive (Bax) regulators of apoptosis. The recently discovered Bcl-2 family member named Drob-1 acts as a positive regulator of cell death. To address whether a Bcl-2 anti-apoptotic pathway exists in the fly, we studied the effects of expressing the mammalian genes bcl-2 in Drosophila. In embryos, expression of bcl-2 inhibits developmental and X-ray-induced apoptosis. Expressing bcl-2 or the pro-apoptotic mammalian bax in the developing eye and wing alters these structures, bcl-2 increasing the number of cells, while bax reduces the number of cells. In addition, the functional interaction between Bcl-2 and Bax is conserved. These results indicate that factors necessary for the activity of bcl-2 and bax are present in Drosophila. Therefore, a Bcl-2 pathway for inhibition of cell death may exist in the fly.     

Alterations in the cellular distribution of bcl-2, bcl-x and bax in the adult rat substantia nigra following striatal 6-hydroxydopamine lesions

The proteins of the bcl-2 family play an important role during apoptosis and may also regulate cell death in response to oxidative stress, which has been implicated in Parkinson's disease. In this study we examined the localization of the pro-apoptotic protein bax, and the anti-apoptotic proteins bcl-2 and bcl-x_L in the substantia nigra (SN) of the adult rat and their response to oxidative stress caused by striatal injections of 6-hydroxydopamine (6-OHDA). Our data show that bcl-2, bcl-x and bax proteins are present in the SN. Bcl-2 and bax are localized primarily in neurons including all those positive for tyrosine hydroxylase (TH). The intraneuronal distribution of bcl-2 and bax were different. Bcl-2 was diffuse throughout the cell while bax was localized in well-defined structures around the nucleus and within processes. Bcl-x staining in neurons was weak, though it was strongly expressed in GFAP-positive astrocytes. 6-OHDA injections, which resulted in loss of dopamine neurons between 7–14 days post-lesion, altered the distribution of bax, bcl-2 and bcl-x proteins in the SN. Bcl-2 and bax were decreased in the TH-positive cells of the SN from 3 to 14 days post-lesion and many TH-positive neurons were bcl-2 negative. Neuronal bcl-x was initially unchanged after lesion, but increased in astrocytes between 3–7 days post-lesion before the increase in GFAP immunoreactivity, which was detectable at days 10–14. While the neuronal distribution of bcl-2 and bcl-x does not change following lesion, bax became evenly distributed thought the soma. Morphological features of apoptosis, including TUNEL labeling and chromatin condensation was not observed. These data suggest that striatal 6-OHDA lesions do not result in classical apoptosis in the SN of the adult rat, even though there are changes in the content and distribution of members of the bcl-2 family of proteins.     

Sodium fluoride induces apoptosis and alters bcl-2 family protein expression in MC3T3-E1 osteoblastic cells

Chronic excessive fluoride intake is known to be toxic and can lead to fluorosis and bone pathologies. However, the cellular mechanisms underlying NaF-induced cytotoxicity in osteoblasts are not well understood. The objectives of this study were to determine the effects of fluoride treatment on MC3T3-E1 osteoblastic cell viability, cell cycle analysis, apoptosis and the expression levels of bcl-2 family members: bcl-2 and bax. MC3T3-E1 cells were treated with 10⁻⁵; 5 × 10⁻⁵; 10⁻⁴; 5 × 10⁻⁴ and 10⁻³ M NaF for up to 48 h. NaF was found to reduce cell viability in a temporal and concentration dependent manner and promote apoptosis even at low concentrations (10⁻⁵ M). This increased apoptosis was due to alterations in the expression of both pro-apoptotic bax and anti-apoptotic bcl-2. The net result was a decrease in the bcl-2/bax ratio which was found at both the mRNA and protein levels. Furthermore, we also noted that NaF-induced S-phase arrest during the cell cycle of MC3T3-E1 cells. These data suggest that fluoride-induced osteoblast apoptosis is mediated by direct effects of fluoride on the expression of bcl-2 family members.     

Involvement of p53 and Bcl-2 family proteins in regulating programmed cell death and proliferation in human embryogenesis

Homeostasis and development in vertebrates are regulated by cell proliferation, differentiation and death. Permeability of mitochondrial membranes, a decisive feature of apoptosis, is regulated by Bcl-2 family regulators. Protein p53 is able to reduce bcl-2 and promote bax expression. This study focused on the immunohistochemical detection of the expression levels of Bcl-2 family regulators (anti-apoptotic Bcl-2 and Bcl-XL, pro-apoptotic Bcl-Xs and Bax), p53, and PCNA as a marker of proliferation, together with the evaluation of the level of apoptosis in human embryos (anlage of limbs, axial skeleton, metanephros, and intestine). Expression of observed proteins was assessed by a three-step immunohistochemistry and evidenced by the double-staining technique. Apoptosis was detected by the TUNEL technique. This study provided circumstantial evidence of the exclusive role of Bcl-2 and Bcl-XL proteins in the inhibition of apoptosis - only rarely were the Bcl-2/ Bcl-XL positive cells stained by TUNEL. The role of pro-apoptotic members of Bcl-2 family remains ambiguous, as TUNEL positive cells are both Bax/Bcl-Xs positive and negative. This study provided substantial evidence that expression patterns of observed proteins are neither fully explainable by "rheostat" theory, nor are the findings obtained from animal model tissue or cell culture commonly applicable to human embryos.     

Natural diterpenoid compound elevates expression of Bim protein, which interacts with antiapoptotic protein Bcl-2, converting it to proapoptotic Bax-like molecule

Overwhelming evidence indicates that Bax and Bak are indispensable for mediating cytochrome c release from mitochondria during apoptosis. Here we report a Bax/Bak-independent mechanism of cytochrome c release and apoptosis. We identified a natural diterpenoid compound that induced apoptosis in bax/bak double knock-out murine embryonic fibroblasts and substantially reduced the tumor growth from these cells implanted in mice. Treatment with the compound significantly increased expression of Bim, which migrated to mitochondria, altering the conformation of and forming oligomers with resident Bcl-2 to induce cytochrome c release and caspase activation. Importantly, purified Bim and Bcl-2 proteins cooperated to permeabilize a model mitochondrial outer membrane; this was accompanied by oligomerization of these proteins and deep embedding of Bcl-2 in the membrane. Therefore, the diterpenoid compound induces a structural and functional conversion of Bcl-2 through Bim to permeabilize the mitochondrial outer membrane, thereby inducing apoptosis independently of Bax and Bak. Because Bcl-2 family proteins play important roles in cancer development and relapse, this novel cell death mechanism can be explored for developing more effective anticancer therapeutics.     

Bcl-2: bax and bcl-2: Bcl-x ratios by image cytometric quantitation of immunohistochemical expression in ovarian carcinoma: correlation with prognosis

Bcl-2 is a proto-oncogene which is involved in prolonging cell survival by inhibiting programmed cell death. Bax and bcl-x are members of the bcl-2 family; when overexpressed, they can counteract the ability of bcl-2 to inhibit apoptosis. This suggests a model in which the ratios of bcl-2 to bax and bcl-x can be used to determine response to therapy and prognosis. The expression of bcl-2, bax and bcl-x was studied in 50 ovarian carcinomas. The percentage of positive area immunostained (PPA) in the nucleus and cytoplasm of each ovarian carcinoma was quantitated in 15 high power fields by image cytometry. The ratios were obtained by dividing the PPA of bcl-2 by the PPA of bax and bcl-x. 17 of 50 ovarian carcinomas (34%) stained positively for bcl-2, 39 for bax (78%) and 47 for bcl-x (94%). Although there is no significant statistical correlation between expression of bcl-2, bax or bcl-x and grade (P = 0.15; P = 0. 47; P = 0.56), stage (P = 0.71; P = 0.6; P = 0.42), and overall or disease-free survival (P = 0.26; P = 0.55; P = 0.16), increased bcl-2 expression was demonstrated in patients with shortened overall and disease-free survival. Also, increased expression of bax and bcl-x was associated with increased overall and disease-free survival. Bcl-2:bax and bcl-2:bcl-x ratios less than 1 are associated with survival advantage, although not statistically significant (P = 0.83; P = 0.93). Image cytometric measurement of bcl-2, bax, and bcl-x expression is feasible. There is a tendency for their expression to correlate with prognosis in ovarian carcinomas.     

Exposure of myeloid dendritic cells to exogenous or endogenous IL-10 during maturation determines their longevity

Dendritic cells (DC) are essential for the initiation of primary adaptive immune responses, and their functionality is strongly down-modulated by IL-10. Both innate and adaptive immune signals trigger the up-regulation of antiapoptotic Bcl-2 family members to facilitate the survival of DCs after maturation. However, whether IL-10 alters the expression of apoptotic-related genes in maturing DCs has not been determined. In this study, we demonstrate that spontaneous apoptosis rapidly occurred in myeloid DCs exposed to exogenous IL-10 upon maturation. Microarray analysis indicates that IL-10 suppressed the induction of three antiapoptotic genes, bcl-2, bcl-x, and bfl-1, which was coincident with the increased sensitivity of mature DCs to spontaneous apoptosis. IL-10 markedly inhibited the accumulation of steady state Bcl-2 message and protein in myeloid DCs activated through TLRs or TNFR family members, whereas exogenous IL-10 affected Bcl-x(L) expression in a moderate manner. In contrast, bcl-2 expression of plasmacytoid DCs was less sensitive to the effects of IL-10. We further show that autocrine IL-10 significantly limited the longevity of myeloid DCs and altered the expression kinetics of Bcl-2 but not Bcl-x(L) in maturing DCs. We conclude that the degree of IL-10 exposure and/or the level of endogenous IL-10 production upon myeloid DC maturation play a critical role in determining DC longevity. This regulatory mechanism of IL-10 is associated with the dynamic control of antiapoptotic Bcl-2 proteins.     

Bcl-2 expression modulates cell adhesion and migration promoting branching of ureteric bud cells

Bcl-2 is the founding member of a family of proteins that influence apoptosis. During kidney development bcl-2 not only acts as a survival factor, but may also impact cell adhesive mechanisms and by extension branching morphogenesis. The interrelationship between cell adhesion, migration and apoptosis, important during development, is poorly understood. Here we examined the impact lack of bcl-2, an inhibitor of apoptosis, has on ureteric bud (UB) cell adhesion, migration, and branching morphogenesis. Bcl-2 -/- UB cells demonstrated increased cell migration, increased cell invasion and decreased adhesion to vitronectin and fibronectin compared with wild-type cells. Bcl-2 +/+ UB cells readily branched in collagen gel and Matrigel while bcl-2 -/- UB cells did not undergo significant branching in either matrix. Re-expression of bcl-2 in bcl-2 -/- UB cells restored their ability to undergo branching morphogenesis in Matrigel. Consistent with our in vitro data, we show that in the absence of bcl-2, embryonic kidneys undergo decreased UB branching. We observed decreased numbers of UB branch points, UB branch tips and a decreased distance to the first UB branch point in the absence of bcl-2. The alterations in bcl-2 -/- UB cell adhesion and migration was also associated with a significant alteration in expression of a number of extracellular matrix proteins. Bcl-2 -/- UB cells exhibited increased fibronectin expression and decreased thrombospondin-1 and osteopontin expression. Taken together, these data suggest that bcl-2 is required for the proper regulation of cell adhesive and migratory mechanisms, perhaps through modulation of the cellular microenvironment.     

Surface expression of Bcl-2 in chronic lymphocytic leukemia and other B-cell leukemias and lymphomas without a breakpoint t(14;18)

Since its discovery in follicular lymphoma cells at the breakpoint t(14;18), Bcl-2 has been studied extensively in many basic and clinical science settings. Bcl-2 can locate as an integral mitochondrial membrane component, where its primary role is to block apoptosis by maintaining membrane integrity. Here we show that Bcl-2 also can position on the outer cell surface membrane of B cells from patients with chronic lymphocytic leukemia (B-CLL) and certain other leukemias that do not classically possess the chromosomal breakpoint t(14;18). Although low levels of Bcl-2 can be detected on the surface membrane of apparently healthy leukemic and normal B cells, expression of Bcl-2 correlates best with spontaneous or induced apoptosis. Notably, upon induction of apoptosis, B-CLL cells were much more efficient in upregulating surface Bcl-2 than normal B cells. It is not clear if this surface membrane expression is a passive consequence of the apoptotic process or an active attempt by the B cell to abort cell death by stabilizing the plasma membrane.     

Nerve Growth Factor Determines Survival and Death of PC12 Cells by Regulation of the bcl-x, bax, and caspase-3 Genes

We investigated the effects of nerve growth factor (NGF) and NGF withdrawal on expression of members of the bcl-2 family of genes and caspase-3 in PC12 cells. NGF regulated several members of the bcl-2 family and caspase-3 in a manner consistent with its effect on apoptosis in PC12 cells. Levels of bcl-xl, bcl-xs, and caspase-3 mRNAs were increased by NGF treatment. The increases in caspase-3 and bcl-xs levels should have disposed the cells toward apoptosis but were opposed by the simultaneous increase in bcl-xl level. NGF withdrawal resulted in abrupt down-regulation of bcl-xl and up-regulation of bax, favoring apoptosis. Forced expression of bcl-xl after NGF withdrawal was sufficient to prevent cell death. Cell death was rapid when NGF was withdrawn after 5 days of treatment but relatively slow when NGF was withdrawn after only 1 or 2 days of treatment. This was consistent with the reduced accumulation of caspase-3 mRNA with shorter NGF treatments. These results indicate that Bcl-xl, Bcl-xs, Bax, and caspase-3 are important regulators of apoptosis in PC12 cells. Furthermore, regulation of their mRNA levels is implicated in the signal transduction of NGF.     

Hypoxia sensitizes cells to nitric oxide-induced apoptosis

Nitric oxide (NO) can induce apoptosis in a variety of cell types. A non-toxic concentration of nitric oxide under normal oxygen conditions triggered cell death under hypoxic conditions (1.5% O(2)) in fibroblasts. Nitric oxide administered during hypoxia induced the release of cytochrome c, caspase-9 activation, and the loss of mitochondrial membrane potential followed by DNA fragmentation and lactate dehydrogenase release (markers of cell death). Bcl-X(L) protected cells from nitric oxide-induced apoptosis during hypoxia by preventing the release of cytochrome c, caspase-9 activation, and by maintaining a mitochondrial membrane potential. Murine embryonic fibroblasts from bax(-/-) bak(-/-) mice exposed to nitric oxide during hypoxia did not die, indicating that pro-apoptotic Bcl-2 family members are required for NO-induced apoptosis during hypoxia. The nitric oxide-induced cell death during hypoxia was independent of cGMP and peroxynitrite. Cells devoid of mitochondrial DNA (rho secondary-cells) lack a functional electron transport chain and were resistant to nitric oxide-induced cell death during hypoxia, suggesting that a functional electron transport chain is required for nitric oxide-induced apoptosis during hypoxia.     

Proapoptotic effects of antiestrogens, progestins and androgen in breast cancer cells

The promoting action of E2 in breast cancer cells has been, until now, mainly linked to its action on prolifieration. Because of the importance of an increase in apoptosis in breast cancer prevention, we have studied the possible effects of various antiestrogens, progestins and an androgen on its occurrence in three hormone-dependent breast cancer cell lines. The antiestrogens were, a triphenylethylene derivative, 4 hydroxytamoxifen(4OHTAM) and two steroidal antiestrogens, ICI182780 and RU58668. The progestins were Org2058, a pregnane derivative, tibolone (OrgOD14), a normethyltestosterone derivative and OrgOM38 (the Δ4 isomer of OrgOD14) and the androgen dihydrotestosterone (DHT). Apoptosis was studied in MCF-7, ZR75-1 and T47-D cells using morphological approaches and flow cytometry. The antiestrogens, the progestins and DHT were proapoptotic but to different potencies according to the cell line studied. Indeed, the ‘pure’ steroidal antiestrogens were more efficient than 4OHTam in increasing apoptosis. We have also studied the level of expression of some of the proteins involved in the regulation of apoptosis. Bcl-2 and bcx_L, two antiapoptotic members of the bcl-2 family proteins, were inhibited by the progestins and the antiestrogens. In contrast, the proapoptotic proteins, bax and bak seemed to be constitutively expressed. Thus, since the ratio of proapoptotic and antiapoptotic proteins determines apoptosis or cell survival, the hormone effects are operating by modulating the antiapoptotic regulators of the balance. These data demonstrate that antiestrogens, progestins, and androgens can promote apoptosis in breast cancer cells, an effect which could be of importance in the therapeutic prevention of breast cancer.