Mitochondria in apoptosis and human disease

Apoptosis is a process of cell suicide whereby individual cells are destroyed while preserving the integrity and architecture of surrounding tissue. This targeted cell destruction is critical both in physiological contexts as well as pathological states. It seems increasingly evident that mitochondria play an important role in the regulation of programmed cell death via release of proapoptotic agents and/or disruption of cellular energy metabolism. The mechanisms of mitochondrial involvement are beginning to be elucidated, and may involve the participation of bcl-2 family members, reactive oxygen species, and caspases. As part of a central mechanism of amplification of the apoptotic signal, mitochondria may be an appropriate target for therapeutic agents designed to modulate apoptosis. This review focuses on recent advances in understanding mitochondrial mechanisms in apoptosis and the involvement of these pathways in human disease.     

Current concepts in Bcl-2 family member regulation of female germ cell development and survival

Since the cloning of the bcl-2 gene in 1985, considerable progress has been made in elucidating the function of Bcl-2 and related proteins in controlling apoptosis. Although much of this work initially relied on the ectopic expression of bcl-2 gene family members in cell lines in vitro, a number of genetically manipulated mice have been generated to better understand the in vivo significance of specific family members to organ development and homeostasis. Of the many tissues that exhibit apoptosis at some point during fetal or postnatal life, the female gonads arguably possess one of the highest and most protracted incidences of apoptosis, associated with development and maturation of the germ line. Moreover, female germ cells (oocytes) are, for as-yet poorly understood reasons, extremely vulnerable to a host of pathological insults, such as anti-cancer therapies, that ultimately cause premature ovarian failure and infertility due to accelerated oocyte death. Accordingly, efforts to understand the occurrence and regulation of apoptosis in the ovary are of considerable importance from both biological and clinical perspectives. This review will highlight what is known of apoptosis in the female gonads, and the role that Bcl-2 family members play in regulating this process.     

Cyclin dependent kinase 5 and its interacting proteins in cell death induced in vivo by cyclophosphamide in developing mouse embryos

Activation or inactivation of members of the cyclin-dependent kinase family is important during cell cycle progression. However, Cdk5, a member of this family that was originally identified because of its high structural homology to Cdc2, is activated during cell differentiation and cell death but not during cell cycle progression. We previously demonstrated a correlation between the up-regulation of Cdk5 protein and kinase activity and cell death during development and pathogenesis. We report here that cyclophosphamide (CP) induces massive apoptotic cell death in mouse embryos and that Cdk5 is expressed in apoptotic cells displaying fragmented DNA. During CP-induced cell death, Cdk5 protein expression is substantially increased as detected by immunohistochemistry but not by Western blot, while its mRNA level remains the same as control, and its kinase activity is markedly elevated. The up-regulation of Cdk5 during CP-induced cell death is not due to de novo protein synthesis. We also examined p35, a regulatory protein of Cdk5 in neuronal differentiation. Using a yeast two-hybrid system, we isolated p35, a neuronal differentiation specific protein, as a protein that interacts with Cdk5 in CP-treated embryos. p35 mRNA level does not change, but the protein expression of p25, a truncated form of p35, is elevated during cell death in vivo, as established here, as well as during cell death in vitro. Our results suggest a role for Cdk5 and its regulatory proteins during CP induced cell death. These results further support the view that Cdk5 and its regulation may be key players in the execution of cell death regardless of how the cell dies, whether through biological mechanisms, disease states such as Alzheimer's disease, or induction by CP.     

Pi 1 binding sites are negative regulators of bcl-2 expression in pre-B cells.

The bcl-2 gene is differentially regulated during B-cell development, with low-level expression in pre-B cells and higher-level expression in mature B cells. These changes correlate with susceptibility to cell death by apoptosis and suggest that the Bcl-2 protein may play a role in the control of cell death during B-cell development. We have identified two negative regulatory regions in the human bcl-2 5' flanking and 5' untranslated regions in pre-B cells; these regions have no significant function in mature B cells. Further investigation of these regions revealed two pre-B-cell-specific enhancer elements (pi 1 sites) in the 5' negative regulatory region and one in the 3' negative regulatory region. Mutational analysis confirmed that these three sites functioned as negative regulators of the bcl-2 promoter in the pre-B-cell line Nalm-6. Electrophoretic mobility shift assays with each of the three sites demonstrated a complex of identical mobility to that formed with the immunoglobulin heavy-chain enhancer pi 1 site. UV cross-linking experiments revealed that a protein with a molecular mass of 58 kDa bound to the three bcl-2 sites and to the immunoglobulin enhancer site. This protein reacted with an antibody against Ets family proteins. Constructs with the isolated pi 1 sites linked to the simian virus 40 promoter were used in transient transfection experiments in the pre-B-cell line. The bcl-2 sites decreased expression of the simian virus 40 promoter, while the immunoglobulin enhancer site increased its expression. The pi 1 sites in the bcl-2 gene may play a role in the developmental regulation of bcl-2 expression during B-cell differentiation.     

Attenuation of retinal vascular development and neovascularization during oxygen-induced ischemic retinopathy in Bcl-2-/- mice

Bcl-2 is a death repressor that protects cells from apoptosis mediated by a variety of stimuli. Bcl-2 expression is regulated by both pro- and anti-angiogenic factors; thus, it may play a central role during angiogenesis. However, the role of bcl-2 in vascular development and growth of new vessels requires further delineation. In this study, we investigated the physiological role of bcl-2 in development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR). Mice deficient in bcl-2 exhibited a significant decrease in retinal vascular density compared to wild-type mice. This was attributed to a decreased number of endothelial cells and pericytes in retinas from bcl-2-/- mice. We observed, in bcl-2-/- mice, delayed development of retinal vasculature and remodeling, and a significant decrease in the number of major arteries, which branch off from near the optic nerve. Interestingly, hyaloid vessel regression, an apoptosis-dependent process, was not affected in the absence of bcl-2. The retinal vasculature of bcl-2-/- mice exhibited a similar sensitivity to hyperoxia-mediated vessel obliteration compared to wild-type mice during OIR. However, the degree of ischemia-induced retinal neovascularization was significantly reduced in bcl-2-/- mice. These results suggest that expression of bcl-2 is required for appropriate development of retinal vasculature as well as its neovascularization during OIR.     

Expression of p21 and bcl-2 proteins and p53 mRNA in surgically resected preparations of non-small cell lung cancer (stage IIIA) after etoposide and cisplatin chemotherapy

Apoptosis which is also a called programmed cell death plays an important role during development, homeostasis and in many diseases such as cancer. Apoptosis is a genetically encoded cell death program defined by characteristic morphological and biochemical features. It is well recognized as a distinct pathologic mechanism in tumours responding to anticancer therapies. Many genes play an important role in this process. We evaluated an expression of the tumour supressor gene p53 and proteins p21 and bcl-2 in non-small cell lung cancer. We examined resected tumour tissues from 30 patients who received neoadjuvant chemotherapy. As a control we assessed tissues from patients treated without chemotherapy. Histological slides of the resected tumours were evaluated by TUNEL, in situ hybridisation and with immunoperoxidase staining procedure. The results were documented by photography. We examined the level of extinction using cytophotometry. In conclusion, preoperative chemotherapy induces apoptosis in cancer cells. The level of p53 correlates with the acceleration of TUNEL reaction. The loss of bcl-2 expression correlated with an increased apoptotic cell death. There was an increased p21 protein expression in the examined cancer tissues after chemotherapy.     

Bcl-2 family gene modulation during spontaneous apoptosis of B-chronic lymphocytic leukemia cells

Malignant cell accumulation in B-cell chronic lymphocytic leukemia (B-CLL) is primarily caused by defective apoptosis rather than increased proliferation. To further understand the role of Bcl-2 family members, known regulators of apoptosis, in the abnormal B-CLL survival, we have measured their mRNA levels in fresh B-CLL cells and in cultures undergoing spontaneous apoptosis. Using RNA protection assays we found constitutive expression of most bcl-2 members with high levels of bcl2, bcl-w, bad, bak, bax, and the bcl-2/bax ratio, compared to normal PBL. Spontaneous apoptosis of B-CLL cells by in vitro culture resulted in decreased bcl-2, bcl-w, bfl-1, mcl-1, bak, bax, and bcl-2/bax expression. The pro-apoptotic member bik was only expressed in 5/19 cases and was not modulated during apoptosis, suggesting that bik is not involved in this process. Thus, several Bcl-2 family genes are regulated during B-CLL spontaneous apoptosis and their relative levels may contribute to in vivo progression of the disease.     

Expression of the Apoptosis-Effector Gene, Bax, Is Up-Regulated in Vulnerable Hippocampal CA1 Neurons Following Global Ischemia

Abstract: The observation that delayed death of CA1 neurons after global ischemia is inhibited by protein synthesis inhibitors suggests that the delayed death of these neurons is an active process that requires new gene expression. Delayed death in CA1 has some of the characteristics of apoptotic death; however, candidate proapoptotic proteins have not been identified in the CA1 after ischemia. We studied the expression of Bax protein and mRNA, a member of the bcl-2 family that is an effector of apoptotic cell death, after global ischemia in the four-vessel global ischemia model in the rat and compared these results with the expression of the antiapoptotic gene bcl-2 . Bax mRNA and protein are both expressed in CA1 before delayed death, whereas bcl-2 protein is not expressed. Bcl-2 protein expression, but not that of Bax, is increased in CA3, a region that is ischemic but less susceptible to ischemic injury. In the dentate gyrus, both Bax and bcl-2 proteins are expressed. The selective expression of Bax in CA1 supports the hypothesis that Bax could contribute to delayed neuronal death in these vulnerable neurons by an independent mechanism or by forming heterodimers with gene family members other than bcl-2.     

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.     

Molecular mechanisms of “detachment-induced apoptosis—Anoikis”

Apoptosis following loss of cell anchorage is of relevance for development, tissue homeostasis and disease. In the following the role of cell-matrix anchorage as well as cell-cell anchorage for cell survival and apoptosis is reviewed and the complex molecular mechanisms inducing and perpetuating detachment-induced apoptosis will be discussed with special emphasis on the role of caspases, p53, bcl-2 family members and the Fas signaling pathway.     

Regulation of caspase activation in apoptosis: implications for transformation and drug resistance

Recent developments in the apoptosis field have uncovered a family of cysteine proteases, the Caspases, that act as signalling components as well as effectors of the cell death machinery. Caspases are constitutively present as inactive precursors within most cells and undergo proteolytic processing in response to diverse death-inducing stimuli to initiate the death programme. Active caspases can process other caspases of the same type as well as process caspases further downstream in the pathway that ultimately leads to collapse of the cell. This cellular collapse is thought to occur as a consequence of caspase-mediated cleavage of a diverse array of cellular substrates. Regulation of entry into the death programme is controlled at a number of levels by members of the Bcl-2 family, as well as by other cell death regulatory proteins. Recent data has shed light upon the mechanism of action of these regulatory molecules and suggests that the point of caspase activation is a major checkpoint in the cell death programme. Because many transformed cell populations possess derangements in cell death-regulatory genes, such as bcl-2, such cells frequently exhibit elevated resistance to cytotoxic chemotherapy. Thus, a deeper understanding of how apoptosis is normally regulated has therapeutic implications for disease states where the normal controls on the cell death machinery have been subverted. This revised version was published online in August 2006 with corrections to the Cover Date.     

AP-1, but not NF-kappa B, is required for efficient steroid-triggered cell death in Drosophila

Extensive studies in vertebrate cells have assigned a central role to Rel/NF-kappa B and AP-1 family members in the control of apoptosis. We ask here whether parallel pathways might function in Drosophila by determining if Rel/NF-kappa B or AP-1 family members contribute to the steroid-triggered death of larval salivary glands during Drosophila metamorphosis. We show that two of the three Drosophila Rel/NF-kappa B genes are expressed in doomed salivary glands and that one family member, Dif, is induced in a stage-specific manner immediately before the onset of programmed cell death. Similarly, Djun is expressed for many hours before salivary gland cell death while Dfos is induced in a stage-specific manner, immediately before this tissue is destroyed. We show that null mutations in the three Drosophila Rel/NF-kappa B family members, either alone or in combination, have no apparent effect on this death response. In contrast, Dfos is required for the proper timing of larval salivary gland cell death as well as the proper induction of key death genes. This study demonstrates a role for AP-1 in the stage-specific steroid-triggered programmed cell death of larval tissues during Drosophila metamorphosis.     

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.     

Members of the bcl-2 and caspase families regulate nuclear degeneration during chick lens fibre differentiation.

The optical clarity of the lens is ensured by the programmed removal of nuclei and other organelles from the lens fibre cells during development. The morphology of the degenerating nuclei is similar to that observed during apoptosis and is accompanied by DNA fragmentation. Proteins encoded by the bcl-2 proto-oncogene family are important in either promoting or inhibiting apoptosis, and caspases are involved in downstream proteolytic events. Here, the expression of bcl-2 family members (bcl-2, bax, bad, and bcl-x(s/l)) and caspases-1, -2, -3, -4, and -6 was investigated through a range of stages of chick lens development using immunocytochemistry, Western blotting, and affinity labelling for caspases using biotinylated caspase inhibitors. Using differentiating lens epithelial cell cultures, it was demonstrated that the addition to cultures of synthetic peptide inhibitors of caspases -1, -2, -4, -6, and -9 brought about a 50-70% reduction in the number of degenerating nuclei per unit area of culture, as assessed by image analysis. These effects were comparable to those seen when general inhibitors of caspases were added to cultures. On the other hand, inhibitors of caspases-3 and -8 were not effective in significantly reducing the number of TUNEL-labelled nuclei. Expression of the caspase substrates poly(ADP-ribose) polymerase (PARP) and the 45-kDa subunit of DNA fragmentation factor (DFF 45) was also observed in the developing lens. Western blots of cultures to which caspase inhibitors were added revealed alterations in the PARP cleavage pattern, but not in that of DFF. These results demonstrate a role for members of the bcl-2 family and caspases in the degeneration of lens fibre cell nuclei during chick secondary lens fibre development and support the proposal that this process has many characteristics in common with apoptosis.     

Image cytometric bcl-2:bax and bcl-2:bcl-x ratios in invasive breast carcinoma: correlation with prognosis

BACKGROUND: The bcl-2 family of proteins are important regulators of apoptosis. Some of the members, such as bcl-2 and bcl-x(L), inhibit cell death, whereas others, such as bax and bcl-x(S), promote cell death. We evaluated the ratios of bcl-2:bax and bcl-2:bcl-x expression by image cytometry in invasive breast carcinoma to determine prognostic significance. DESIGN: Five-micron sections of formalin-fixed, paraffin-embedded tissue from 88 invasive breast carcinomas were immunostained using steam antigen retrieval, an avidin biotin-complex technique with automated stainer and primary antibodies against bcl-2 (1/160; Dako, Carpenteria, CA), bax (1/1,500; PharMingen, San Diego, CA), and bcl-x (1/1,500; PharMingen). Positive controls were tonsil (bcl-2) and normal breast (bax and bcl-x) tissue samples. Immunostain was measured in 15 high power fields as percentage positive area (PPA) in nuclei and cytoplasm using the CAS 200 image analyzer (Becton Dickinson, San Jose, CA). RESULTS: Median follow-up was 105 months (range 11-130). Significantly improved disease-free survival was found in patients with a bcl-2:bcl-x ratio > or = 1 by univariate and multivariate analyses. The bcl-2:bax ratio was not predictive of overall or disease-free survival. A significant difference in overall and disease-free survival was found between carcinomas with positive and negative bcl-2 expression by univariate analysis; by multivariate analysis, bcl-2 expression was an independent prognostic factor for disease-free survival. The 5-year survival rates were 77% and 50% in patients with bcl-2-positive and bcl-2-negative carcinomas, respectively. CONCLUSION: A bcl-2:bcl-x ratio > or = 1, assessed by image cytometry, is significantly associated with improved disease-free survival in patients with invasive breast carcinoma. Significantly increased overall and disease-free survival is associated with positive bcl-2 expression.     

Survivin: a target from brain cancer to neurodegenerative disease

Apoptosis is an important contributing factor during neuronal death in a variety of neurodegenerative disorders, including multiple sclerosis, Parkinson's disease and sciatic nerve injury. Whereas several clinical and preclinical studies have focused on the neuroprotective effects of caspase inhibitors, their clinical benefits are still unclear. Here, we discuss novel alternative strategies to protect neuronal cells from apoptotic death using members of the inhibitors of apoptosis (IAP) family. We specifically review the different roles of survivin, which is an important member of the IAP family that serves a dual role in the inhibition of apoptosis as well as a vital role in mitosis and cell division. Due to the various roles of survivin during cell division and apoptosis, targeting this protein illustrates a new therapeutic window for the treatment of neurodegenerative diseases.