Life and death in a medieval atmosphere.
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Animals↗
Biomedical subjects
C Borner
Publications and source records attributed to C Borner.
Apoptosis without caspases: an inefficient molecular guillotine?
Since the discovery that the cysteine protease CED-3 was essential for developmental death in the nematode C. elegans, the search has been on to identify homologous proteases governing mammalian apoptosis. Fourteen of these proteases, now called caspases, have been found to date, and studies with natural or chemical inhibitors, and more recently knock-out mice, confirmed the involvement of at least a subset of these proteases in various forms of mammalian apoptosis. However, there has been recent evidence that some apoptotic morphologies, such as cell shrinkage, membrane blebbing and nuclear condensation, are not blocked by caspase inhibitors and that the cells continue to die in a protracted and inefficient manner. This has led to the notion that caspases are not required for all aspects of apoptosis in mammals. Here we review the current knowledge about caspase-independent apoptosis, discuss the strengths and weaknesses of the reasoning that led to its proposition and provide insights into its possible regulation and physiological significance.
Animals↗
Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2.
Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.
Amino Acid Chloromethyl Ketones↗
The binding properties and biological activities of Bcl-2 and Bax in cells exposed to apoptotic stimuli.
The oncogene product Bcl-2 protects cells from apoptosis whereas its homolog Bax functions to kill cells. Several binding partners of Bcl-2 and Bax have been isolated, but none of them has yet provided clues as to exactly how Bcl-2 and Bax work. According to one view, Bcl-2 and Bax interact with survival and death effector molecules, respectively, and neutralize each other through heterodimerization. Alternatively, Bcl-2 requires Bax for death protection, and additional proteins bind to the heterodimer to regulate its activity. Here we used a co-immunoprecipitation strategy to distinguish between these two possibilities. We show that the Bcl-2-Bax heterodimer is maintained, and no other protein associates stably in detectable amounts with Bcl-2, Bax, or the heterodimer in anti-Bcl-2 and anti-Bax immunoprecipitates from normal cells and cells exposed to apoptotic stimuli. Analysis of cells expressing various levels of Bcl-2 and Bax, however, revealed that the degree of protection against apoptosis does not correlate with the number of Bcl-2-Bax heterodimers but the amount of Bcl-2 that is free of Bax. In addition, the survival activity of Bcl-2 is unaffected when Bax expression is ablated by an antisense strategy. Our findings suggest that the Bcl-2-Bax heterodimer is a negative regulator of death protection, and that Bcl-2 requires neither Bax nor major, stable interactions with other cellular proteins to exert its survival function. We therefore propose that Bcl-2 acts as an enzyme (capturing substrates in a transient way), as a homodi- or multimer, or through the interaction with non-proteaceous targets (lipids, ions).
Animals↗
Defects in the ubiquitin pathway induce caspase-independent apoptosis blocked by Bcl-2.
Apoptosis requires the activation of caspases (formerly interleukin 1beta-converting enzyme-like proteases), in particular those related to the caspase-3/7/6 subfamily. Recent data, however, revealed that, although caspase-specific inhibitors delay apoptosis, they are often incapable of preventing it. To obtain evidence for caspase-independent steps of apoptosis, we artificially created a high amount of short-lived or aberrant proteins by blocking the ubiquitin degradation pathway. A temperature-sensitive defect in the ubiquitin-activating enzyme E1 induced apoptosis independent of the activation of caspase-3 and -6 and the cleavage of their respective substrates poly(ADP-ribose) polymerase and lamin A. In addition, neither the caspase 3/7-specific inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone nor the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone were capable of blocking this type of cell death. By contrast, Bcl-2 overexpression effectively protected cells from apoptosis induced by a defect in the E1 enzyme at the nonpermissive temperature. Bcl-2 acted downstream of the accumulation of short-lived or aberrant proteins because it did not prevent the overexpression of the short-lived proteins p53, p27(kip1), and cyclins D1 and B1 under conditions of decreased ubiquitination. These results suggest the existence of short-lived proteins that may serve the role of caspase-independent effectors of apoptosis and attractive targets of the death-protective action of Bcl-2.
Amino Acid Chloromethyl Ketones↗
Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c.
Following exposure of cells to stimuli that trigger programmed cell death (apoptosis), cytochrome c is rapidly released from mitochondria into the cytoplasm where it activates proteolytic molecules known as caspases that specifically cleave the amino-acid sequence DEVD and are crucial for the execution of apoptosis. The protein Bcl-2 interferes with this activation of caspases by preventing the release of cytochrome c. Here we study these molecular interactions during apoptosis induced by the protein Bax, a pro-apoptotic homologue of Bcl-2. We show that in cells transiently transfected with bax, Bax localizes to mitochondria and induces the release of cytochrome c, activation of caspase-3, membrane blebbing, nuclear fragmentation, and cell death. Caspase inhibitors do not affect Bax-induced cytochrome c release but block caspase-3 activation and nuclear fragmentation. Unexpectedly, Bcl-2 also fails to prevent Bax-induced cytochrome c release, although it co-localizes with Bax to mitochondria. Cells overexpressing both Bcl-2 and Bax show no signs of caspase activation and survive with significant amounts of cytochrome c in the cytoplasm. These findings indicate that Bcl-2 can interfere with Bax killing downstream of and independently of cytochrome c release.
Animals↗
Role of an acidic compartment in tumor-necrosis-factor-alpha-induced production of ceramide, activation of caspase-3 and apoptosis.
Tumor necrosis factor-alpha (TNF-alpha) apoptosis by recruiting a complex of cytosolic proteins at its plasma membrane receptor. Among them is caspase-8, an interleukin-1beta-converting enzyme (ICE)-like protease that initiates an amplified protease cascade to activate the cell-death machinery. The latter comprises at least caspase-3 and caspase-7, which execute cell death by cleaving numerous protein substrates, including poly(ADP-ribose) polymerase. In addition, TNF-alpha stimulates the production of ceramide, which also activates the death machinery. Whether the signaling pathways elicited by caspase-8 and ceramide proceed independently or intersect at a specific subcellular site is unknown. Using the lysosomotropic agent NH4Cl and the vesicularization inhibitor brefeldin A, we show here the convergence of TNF-alpha-induced death signaling on an acidic, subcellular compartment reminiscent of lysosomes. This compartment generates at least two signaling pathways that account for the caspase-3 activation and apoptosis induced by TNF-alpha, one involving ceramide and caspase-unrelated adapter molecules and another involving yet unknown lysosomal mediators. The apoptosis inhibitor Bcl-2 specifically acts on the ceramide-activated pathway to block caspase-3 activation and apoptosis. The latter result explains why Bcl-2 only partially blocks TNF-alpha-induced apoptosis.
Ammonium Chloride↗
Expression of calmodulin and calmodulin binding proteins in rat fibroblasts stably transfected with protein kinase C and oncogenes.
Molecular mechanisms leading to elevated calmodulin (CaM) expression in cancer have not yet been discovered. We have quantitated the levels of transcripts derived from all three CaM genes in a variety of the same origin rat fibroblasts transformed with oncogenes in combination with gene for protein kinase C using Northern blot analysis with three CaM gene specific cDNA probes. Five species of CaM mRNA were detected in all these cells. Surprisingly many of the investigated cell lines exhibited a decreased content of all CaM mRNAs as compared to control cells with CaMI and CaMII transcripts showing the most pronounced alterations. In contrast, CaM protein levels were increased in all these cell lines as determined by a radioimmunoassay. These results suggest that oncogenic up-regulation of CaM synthesis takes place posttranscriptionally. Several CaM binding proteins were found at different concentrations in the studied cell lines depending on the oncogenes used for transformation. However, CaM overexpression does not seem to affect the overall levels of CaM binding proteins.
Animals↗
Bcl-2 does not require Raf kinase activity for its death-protective function.
It has been widely accepted that the oncogene product bcl-2 protects mammalian cells from programmed cell death (apoptosis). The molecules and signalling pathways upon which bcl-2 acts are, however, still ill-defined. Recently, bcl-2 was shown to interact with c-raf-1 in vitro. Furthermore, an active form of c-raf-1 delayed apoptosis induced by trophic factor deprivation and enhanced the death-suppressive function of bcl-2 when co-expressed. This has led to the hypothesis that bcl-2 communicates cell-death protection via a raf-dependent signal transduction pathway. Here we show, by various immunological and biochemical methods, that bcl-2 does not stably associate with c-raf-1 in cellular extracts prepared from fibroblasts before or after treatment with agents that induce apoptosis. Unexpectedly, bcl-2 function is entirely maintained, if not improved, when raf-dependent signalling is experimentally abrogated. In fact, bcl-2 allows the stable overexpression of a kinase-defective dominant-negative raf mutant that usually interferes with cell viability and/or proliferation. Our results indicate that bcl-2 does not require c-raf-1 kinase activity and an associated mitogen-activated protein kinase signalling pathway for its survival function. This property may be exploited to dissect cellular events that are dependent or independent of c-raf-1 kinase activity.
Animals↗
Overxpression of Bcl-2 inhibits UVA-mediated immediate apoptosiinrat 6 fibroblasts: evidence for the involvement of Bcl-2 as an antioxidant.
We examined the effect of broad spectrum UVA (320-380 nm) and UVB (290-320 nm) radiation on the induction of apoptosis in the rat 6 fibroblast cell line (R6). UVA, but not UVB, induces apoptosis in this cell line. The morphological changes and DNA ladders associated with apoptosis occurred within the first 4 h after UVA irradiation, a phenomenon referred to as "immediate" apoptosis. From previous studies, it is known that Bcl-2 inhibits most types of apoptotic cell death. Overexpression of mouse Bcl-2 in the R6 fibroblasts inhibited the UVA-induced immediate apoptosis. The induction of the heme oxygenase 1 (HO-1) gene by UVA is a general response to oxidative stress. As a marker of oxidative stress, we monitored the effect of Bcl-2 overexpression on the level of HO-1 mRNA accumulation after UVA irradiation. The results showed that the overexpression of Bcl-2 in the R6 fibroblasts strongly reduces the level of HO-1 induction from 12.5- to 4.9-fold. We propose that Bcl-2 expression inhibits UVA-induced immediate apoptosis via an antioxidant pathway, suppressing either the generation or effects of specific UVA-mediated reactive oxygen species.
Animals↗
The role of protein kinase C in signal transduction, growth control and lipid metabolism.
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Animals↗
Diminished cell proliferation associated with the death-protective activity of Bcl-2.
The oncogene product Bcl-2 effectively spares cells from programmed cell death (apoptosis). The molecular mechanism underlying this death-protective activity has, however, remained enigmatic. Here we show that induction of Bcl-2 expression is consistently associated with a retardation of mammalian cell proliferation due to a prolongation of the G1 phase of the cell cycle. Whereas cells lacking Bcl-2 expression die from any point of the cell cycle in response to apoptotic agents, Bcl-2-overexpressing cells accumulate in the G0/G1 phase and are protected from cell death. Co-expression of Bax, a negative regulator of Bcl-2, reverts both the cell death protective and proliferation retarding activities of Bcl-2. Moreover, a Bcl-2 mutant defective in death protection does not affect cell division. These findings indicate that Bcl-2 contributes to cell survival by diminishing the rate of cell proliferation.
Animals↗
Bcl-2 interrupts the ceramide-mediated pathway of cell death.
Ceramide, a product of sphingomyelin turn-over, has been proposed as a novel lipid second messenger with specific roles in mediating antiproliferative responses including apoptosis and cell cycle arrest. In this study, we examine the relationship between the ceramide-mediated pathway of growth suppression and the bcl-2 protooncogene. In ALL-697 leukemia cells, the addition of the chemotherapeutic agent vincristine resulted in a time-dependent growth suppression characterized by marked apoptosis. The effects of vincristine on cell death were preceded by a prolonged and sustained accumulation of endogenous ceramide levels reaching -10.4 pmol ceramide/nmol phospholipids at 12 hr following the addition of vincristine--an increase of 220% over vehicle-treated cells. Overexpression of bcl-2 resulted in near total protection of cell death in response to vincristine. However, the ceramide response to vincristine was not modulated by overexpression of bcl-2, indicating that bcl-2 does not interfere with ceramide formation. Overexpression of bcl-2 prevented apoptosis in response to ceramide, suggesting that bcl-2 acts at a point downstream of ceramide. On the other hand, bcl-2 did not interfere with the ability of ceramide to activate the retinoblastoma gene product or to induce cell cycle arrest, suggesting that the effects of ceramide on cell cycle arrest can be dissociated from the effects on apoptosis. These studies suggest that ceramide and bcl-2 partake in a common pathway of cell regulation. The results also cast ceramide as a gauge of cell injury rather than an "executor" of cell death with clearly dissociable biological outcomes of its action depending on downstream factors.
Antineoplastic Agents, Phytogenic↗
Bcl-2 overexpression blocks activation of the death protease CPP32/Yama/apopain.
The C. elegans gene product ced-9 inhibits programmed cell death by negatively regulating the death-mediating protease ced-3. The mammalian homolog of ced-9 is the oncoprotein Bcl-2. Overexpression of Bcl-2 spares mammalian and nematodal cells from dying and prevents ectopic cell death in ced-9 loss-of-function mutants. Although Bcl-2 has been shown to act as an antioxidant under certain conditions, additional functions have emerged from studies under low oxygen pressure. Here we show that Bcl-2 overexpression impairs activation of the interleukin-1beta converting enzyme-related death protease CPP32/Yama/apopain, the mammalian homolog of ced-3. When U937 monocytes undergo programmed cell death in response to tumor necrosis factor alpha, the inactive CPP32 precursor is cleaved into its active forms. As a consequence poly(ADP ribose) polymerase, a major substrate of CPP32, is faithfully cleaved into a 85 kD fragment. Bcl-2 overexpressing cells are protected from tumor necrosis factor alpha-induced death and display neither CPP32 maturation nor PARP cleavage. The inhibitory effect of Bcl-2 on CPP32 activation is indirect since no physical interaction between the two proteins could be detected. These results indicate that Bcl-2 neutralizes an unknown cellular activator of CPP32 to save cells from programmed cell death.
Animals↗
Two closely related isoforms of protein kinase C produce reciprocal effects on the growth of rat fibroblasts. Possible molecular mechanisms.
We have previously reported that two closely related protein kinase C (PKC) isoforms, PKC alpha and PKC beta I, had divergent effects on the growth and transformation of the same parental R6 rat embryo fibroblast cell line (Housey, G. M., Johnson, M. D., Hsiao, W.-L. W. O'Brian, C. A., Murphey, J. P., Kirschmeier, P., and Weinstein, I. B. (1988) Cell 52, 343-354; Borner, C., Filipuzzi, I., Weinstein, I. B., and Imber, R. (1991) Nature 353, 78-80). Whereas cells that overexpress PKC beta I lost anchorage dependence, grew to higher saturation densities, and generated small tumors when injected into nude mice, none of these properties were seen with cells that overexpress PKC alpha. In fact, the latter cells grew even slower and to lower saturation densities as compared to control cells. Here we investigate possible molecular mechanisms underlying the reciprocal effects of PKC alpha and PKC beta I. Overexpression of both isoforms enhanced 12-O-tetradecanoyl phorbol-13 acetate-induced expression of the growth regulatory genes c-jun, c-myc, and collagenase and enhanced feedback inhibition of epidermal growth factor receptor binding and cellular levels of diacylglycerol. However, the cells overexpressing PKC beta I differed from those overexpressing PKC alpha by displaying a decreased requirement for growth factors and by the production of a mitogenic factor. Thus, the basis for enhanced growth and transformation of cells overexpressing PKC beta I may be the establishment of an autocrine growth factor loop. These findings may be relevant to the roles of specific isoforms of PKC in carcinogenesis and tumor growth.
Animals↗
Regulation of Fas(Apo-1/CD95)- and perforin-mediated lytic pathways of primary cytotoxic T lymphocytes by the protooncogene bcl-2.
Cytotoxic T cells (CTL) induce cell death of their target cells either by the surface interaction between Fas ligand and Fas or by the release of perforin and granzymes. Both lytic pathways induce apoptosis yet it is not known whether identical or distinct apoptotic pathways are activated. The protooncogene bcl-2 is known to protect various hematopoietic cells from apoptosis induced by diverse agents. Here we show that overexpression of the Bcl-2 protein in the murine mastocytoma line P815 or in concanavalin A-activated splenocytes suppresses apoptotic cell death induced by allospecific primary cytotoxic T lymphocytes (CTL) in which only the Fas lytic pathway was functional. Bcl-2 also reduced target cell killing induced by CTL whose lytic activity was dependent on the perforin/granzyme pathway only. These data provide evidence that, in the target cells studied here, both perforin/granzyme and Fas apoptotic pathways are modulated by Bcl-2 and suggest that these two pathways converge at a step prior to Bcl-2 inhibition.
Animals↗
The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis.
Bcl-2 is a mitochondrial- and perinuclear-associated protein that prolongs the lifespan of a variety of cell types by interfering with programmed cell death (apoptosis). Bcl-2 seems to function in an antioxidant pathway, and it is believed that membrane attachment mediated by a COOH-terminal hydrophobic tail is required for its full activity. To identify critical regions in bcl-2 alpha for subcellular localization, activity, and/or interaction with other proteins, we created, by site-directed mutagenesis, various deletion, truncation, and point mutations. We show here that membrane attachment is not required for the survival activity of bcl-2 alpha. A truncation mutant of bcl-2 alpha lacking the last 33 amino acids (T3.1) including the hydrophobic COOH terminus shows full activity in blocking apoptosis of nerve growth factor-deprived sympathetic neurons or TNF-alpha-treated L929 fibroblasts. Confocal microscopy reveals that the T3 mutant departs into the extremities of neurites in neurons and filopodias in fibroblasts. Consistently, T3 is predominantly detected in the soluble fraction by Western blotting, and is not inserted into microsomes after in vitro transcription/translation. We further provide evidence for motifs (S-N and S-II) at the NH2 and COOH terminus of bcl-2, which are crucial for its activity.
Amino Acid Sequence↗
Suppression of mitogenic activity by stable expression of the regulatory domain of PKC beta.
The amino-terminal regulatory domain portion of each protein kinase C (PKC) family member (which in the case of PKC beta 1 includes the pseudosubstrate, C1, V1 and C2 domains) plays an important role in regulating the kinase activity of the carboxyl-terminal catalytic domain. To examine the possibility that this regulatory domain region (designated 'PAT') might have biological functions independent of the catalytic domain, we have developed derivatives of R6 cells which stably express a truncated PKC beta 1 cDNA that encodes the amino-terminal 317 amino acids, including the entire regulatory domain. These R6-plPAT cells express abundant amounts of a 38 kDa protein which binds a labeled phorbol ester, but lacks protein kinase activity. In contrast to the 79 kDa PKC beta 1 holoenzyme which, when overexpressed in R6 cells, is found mostly in the cytosol, the 38 kDa PAT protein is predominantly associated with the particulate subcellular fraction. Furthermore, the PAT protein fails to show down-regulation following treatment of R6-plPAT cells with 12-O-tetradecanoylphorbol-13-acetate (TPA). Evidence is also presented that TPA-stimulated growth is suppressed in R6-plPAT cells. These findings suggest that the PKC beta 1 regulatory domain could be involved in the suppression of mitogenic signaling.
Animals↗