Activation of the phosphatidylinositol 3-kinase/Akt pathway protects against interleukin-3 starvation but not DNA damage-induced apoptosis

Baf-3 cells are dependent on interleukin-3 (IL-3) for their survival and proliferation in culture. To identify anti-apoptotic pathways, we performed a retroviral-insertion mutagenesis on Baf-3 cells and selected mutants that have acquired a long term survival capacity. The phenotype of one mutant, which does not overexpress bcl-x and proliferates in the absence of IL-3, is described. We show that, in this mutant, Akt is constitutively activated leading to FKHRL1 phosphorylation and constitutive glycolytic activity. This pathway is necessary for the mutant to survive following IL-3 starvation but is not sufficient or necessary to protect cells from DNA damage-induced cell death. Indeed, inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in Baf-3 cells does not prevent the ability of IL-3 to protect cells against gamma-irradiation-induced DNA damage. This protective effect of IL-3 rather correlates with the expression of the anti-apoptotic Bcl-x protein. Taken together, these data demonstrate that the PI3K/Akt pathway is sufficient to protect cells from growth factor starvation-induced apoptosis but is not required for IL-3 inhibition of DNA damage-induced cell death.     

Bcl-x(L) complements Saccharomyces cerevisiae genes that facilitate the switch from glycolytic to oxidative metabolism

All eukaryotic organisms have mechanisms to adapt to changing metabolic conditions. The mammalian cell survival gene Bcl-x(L) enables cells to adapt to changes in cellular metabolism. To identify genes whose function can be substituted by Bcl-x(L) in a unicellular eukaryote, a genetic screen was performed using the yeast Saccharomyces cerevisiae. S. cerevisiae grows by anaerobic glycolysis when glucose is available, switching to oxidative phosphorylation when carbohydrate in the media becomes limiting (diauxic shift). Given that Bcl-x(L) appears to facilitate the switch from glycolytic to oxidative metabolism in mammalian cells, a library of yeast mutants was tested for the ability to efficiently undergo diauxic shift in the presence and absence of Bcl-x(L). Several mutants were identified that have a defect in growth when switched from a fermentable to a nonfermentable carbon source that is corrected by the expression of Bcl-x(L). These genes include the mitochondrial chaperonin TCM62, as well as previously uncharacterized genes. One of these uncharacterized genes, SVF1, promotes cell survival in mammalian cells in response to multiple apoptotic stimuli. The finding that TCM62 and the analogous human prohibitin gene also inhibit mammalian cell death following growth factor withdrawal implicates mitochondrial chaperones as regulators of apoptosis. Further characterization of the genes identified in this screen may enhance our understanding of Bcl-x(L) function in mammalian cells, and of cell survival pathways in general.     

Inactivation of caspase-8 on mitochondria of Bcl-xL-expressing MCF7-Fas cells: role for the bifunctional apoptosis regulator protein.

Apoptosis induction through CD95 (APO-1/Fas) critically depends on generation of active caspase-8 at the death-inducing signaling complex (DISC). Depending on the cell type, active caspase-8 either directly activates caspase-3 (type I cells) or relies on mitochondrial signal amplification (type II cells). In MCF7-Fas cells that are deficient for pro-caspase-3, even high amounts of caspase-8 produced at the DISC cannot directly activate downstream effector caspases without mitochondrial help. Overexpression of Bcl-x(L) in these cells renders them resistant to CD95-mediated apoptosis. However, activation of caspase-8 in control (vector) and Bcl-x(L) transfectants of MCF7-Fas cells proceeds with similar kinetics, resulting in a complete processing of cellular caspase-8. Most of the cytosolic caspase-8 substrates are not cleaved in the Bcl-x(L) protected cells, raising the question of how Bcl-x(L)-expressing MCF7-Fas cells survive large amounts of potentially cytotoxic caspase-8. We now demonstrate that active caspase-8 is initially generated at the DISC of both MCF7-Fas-Vec and MCF7-Fas-Bcl-x(L) cells and that the early steps of CD95 signaling such as caspase-8-dependent cleavage of DISC bound c-FLIP(L), caspase-8-dependent clustering, and internalization of CD95, as well as processing of pro-caspase-8 bound to mitochondria are very similar in both transfectants. However, events downstream of mitochondria, such as release of cytochrome c, only occur in the vector-transfected MCF7-Fas cells, and no in vivo caspase-8 activity can be detected in the Bcl-x(L)-expressing cells. Our data suggest that, in Bcl-x(L)-expressing MCF7-Fas cells, active caspase-8 is sequestered on the outer mitochondrial surface presumably by association with the protein "bifunctional apoptosis regulator" in a way that does not allow substrates to be cleaved, identifying a novel mechanism of regulation of apoptosis sensitivity by mitochondrial Bcl-x(L).     

Akt maintains cell size and survival by increasing mTOR-dependent nutrient uptake

In multicellular organisms, constituent cells depend on extracellular signals for growth, proliferation, and survival. When cells are withdrawn from growth factors, they undergo apoptosis. Expression of constitutively active forms of the serine/threonine kinase Akt/PKB can prevent apoptosis upon growth factor withdrawal. Akt-mediated survival depends in part on the maintenance of glucose metabolism, suggesting that reduced glucose utilization contributes to growth factor withdrawal-induced death. However, it is unclear how restricting access to extracellular glucose alone would lead to the metabolic collapse observed after growth factor withdrawal. We report herein that growth factor withdrawal results in the loss of surface transporters for not only glucose but also amino acids, low-density lipoprotein, and iron. This coordinated decline in transporters and receptors for extracellular molecules creates a catabolic state characterized by atrophy and a decline in the mitochondrial membrane potential. Activated forms of Akt maintained these transporters on the cell surface in the absence of growth factor through an mTOR-dependent mechanism. The mTOR inhibitor rapamycin diminished Akt-mediated increases in cell size, mitochondrial membrane potential, and cell survival. These results suggest that growth factors control cellular growth and survival by regulating cellular access to extracellular nutrients in part by modulating the activity of Akt and mTOR.     

Bcl-2 Family Members and Functional Electron Transport Chain Regulate Oxygen Deprivation-Induced Cell Death

The mechanisms underlying cell death during oxygen deprivation are unknown. We report here a model for oxygen deprivation-induced apoptosis. The death observed during oxygen deprivation involves a decrease in the mitochondrial membrane potential, followed by the release of cytochrome c and the activation of caspase-9. Bcl-X(L) prevented oxygen deprivation-induced cell death by inhibiting the release of cytochrome c and caspase-9 activation. The ability of Bcl-X(L) to prevent cell death was dependent on allowing the import of glycolytic ATP into the mitochondria to generate an inner mitochondrial membrane potential through the F(1)F(0)-ATP synthase. In contrast, although activated Akt has been shown to inhibit apoptosis induced by a variety of apoptotic stimuli, it did not prevent cell death during oxygen deprivation. In addition to Bcl-X(L), cells devoid of mitochondrial DNA (rho degrees cells) that lack a functional electron transport chain were resistant to oxygen deprivation. Further, murine embryonic fibroblasts from bax(-/-) bak(-/-) mice did not die in response to oxygen deprivation. These data suggest that when subjected to oxygen deprivation, cells die as a result of an inability to maintain a mitochondrial membrane potential through the import of glycolytic ATP. Proapoptotic Bcl-2 family members and a functional electron transport chain are required to initiate cell death in response to oxygen deprivation.     

Growth factors can influence cell growth and survival through effects on glucose metabolism

Cells from multicellular organisms are dependent upon exogenous signals for survival, growth, and proliferation. The relationship among these three processes was examined using an interleukin-3 (IL-3)-dependent cell line. No fixed dose of IL-3 determined the threshold below which cells underwent apoptosis. Instead, increasing growth factor concentrations resulted in progressive shortening of the G(1) phase of the cell cycle and more rapid proliferative expansion. Increased growth factor concentrations also resulted in proportional increases in glycolytic rates. Paradoxically, cells growing in high concentrations of growth factor had an increased susceptibility to cell death upon growth factor withdrawal. This susceptibility correlated with the magnitude of the change in the glycolytic rate following growth factor withdrawal. To investigate whether changes in the availability of glycolytic products influence mitochondrion-initiated apoptosis, we artificially limited glycolysis by manipulating the glucose levels in the medium. Like growth factor withdrawal, glucose limitation resulted in Bax translocation, a decrease in mitochondrial membrane potential, and cytochrome c redistribution to the cytosol. In contrast, increasing cell autonomous glucose uptake by overexpression of Glut1 significantly delayed apoptosis following growth factor withdrawal. These data suggest that a primary function of growth factors is to regulate glucose uptake and metabolism and thus maintain mitochondrial homeostasis and enable anabolic pathways required for cell growth. Consistent with this hypothesis, expression of the three genes involved in glucose uptake and glycolytic commitment, those for Glut1, hexokinase 2, and phosphofructokinase 1, was found to rapidly decline to nearly undetectable levels following growth factor withdrawal.     

Effect of Bcl-x(L) overexpression on reactive oxygen species,intracellular calcium,and mitochondrial membrane potential following injury in astrocytes

Many studies have demonstrated the protective effects of Bcl-x(L) against both apoptotic and necrotic cell death, but the mode of action of Bcl-x(L) remains unclear. This work analyzed effects of Bcl-x(L) overexpression on cellular levels of reactive oxygen species (ROS), intracellular calcium ([Ca(2+)](i)), and mitochondrial membrane potential (DeltaPsi(m)) in cultured mouse primary astrocytes after exposure to glucose deprivation (GD) or hydrogen peroxide (H(2)O(2)). Upon exposure to GD or H(2)O(2), uninfected and Lac-Z-expressing astrocytes showed an immediate, rapid increase in ROS accumulation that was slowed and or reduced by Bcl-x(L). Changes in DeltaPsi(m) in response to the two insults differed. H(2)O(2) induced a decrease in DeltaPsi(m) that was initially greater in Bcl-x(L) cells, but then held stable. DeltaPsi(m) in control and Lac-Z-expressing cells initially declined more slowly, but after about 20 min showed rapid deterioration. Five hours of GD caused mitochondrial membrane hyperpolarization followed by a decrease in DeltaPsi(m,) which was not observed with Bcl-x(L) overexpression. Bcl-x(L) failed to inhibit the calcium dysregulation seen in control cells exposed to 400 microM H(2)O(2), but still improved cell survival. There was no increase in [Ca(2+)](i) with 5 h of GD. These data thus dissociate the effect of Bcl-x(L) on calcium homeostasis from effects on ROS, DeltaPsi(m,) and for H(2)O(2) exposure, cell survival.     

Serum-nutrient starvation induces cell death mediated by Bax and Puma that is counteracted by p21 and unmasked by Bcl-x(L) inhibition

The cyclin-dependent kinase inhibitor p21 (p21WAF1/Cip1) is a multifunctional protein known to promote cell cycle arrest and survival in response to p53-dependent and p53 independent stimuli. We herein investigated whether and how it might contribute to the survival of cancer cells that are in low-nutrient conditions during tumour growth, by culturing isogenic human colorectal cancer cell lines (HCT116) and breast cancer cell lines in a medium deprived in amino acids and serum. We show that such starvation enhances, independently from p53, the expression of p21 and that of the pro-apoptotic BH3-only protein Puma. Under these conditions, p21 prevents Puma and its downstream effector Bax from triggering the mitochondrial apoptotic pathway. This anti-apoptotic effect is exerted from the cytosol but it is unrelated to the ability of p21 to interfere with the effector caspase 3. The survival function of p21 is, however, overcome by RNA interference mediated Bcl-x(L) depletion, or by the pharmacological inhibitor ABT-737. Thus, an insufficient supply in nutrients may not have an overt effect on cancer cell viability due to p21 induction, but it primes these cells to die, and sensitizes them to the deleterious effects of Bcl-x(L) inhibitors regardless of their p53 status.     

Epidermal growth factor inhibition of c-Myc-mediated apoptosis through Akt and Erk involves Bcl-xL upregulation in mammary epithelial cells

In earlier studies, we and others have established that activation of EGFR can promote survival in association with upregulation of Bcl-x(L). However, the mechanism responsible for upregulation of Bcl-x(L) is unknown. For the current studies we have chosen pro-apoptotic, c-Myc-overexpressing murine mammary epithelial cells (MMECs) derived from MMTV-c-Myc transgenic mouse tumors. We now demonstrate that EGFR activation promotes survival through Akt and Erk1/2. Blockade of EGFR kinase activity and the PI3-K/Akt and MEK/Erk pathways with pharmacological inhibitors resulted in a significant induction of cellular apoptosis, paralleled by a downregulation of both Akt and Erk1/2 proteins. Consistent with a survival-promoting role of Akt, we observed that constitutively activated Akt (Myr-Akt) inhibited apoptosis of pro-apoptotic, c-Myc-overexpressing cells following the inhibition of EGFR tyrosine kinase activity. In addressing possible downstream effectors of EGFR through activated Akt, we detected significant upregulation of Bcl-x(L) protein, suggesting this pro-survival protein is a target of Akt in MMECs. By using pharmacological inhibitors of PI3-K/Akt and MEK/Erk together with dominant-negative Akt and Erk1 we observed the decrease in Bcl-x(L) protein. Our findings may be of importance for understanding the emerging role of Bcl-x(L) as a potential marker of poor prognosis in breast cancer.     

Akt/Protein Kinase B Inhibits Cell Death by Preventing the Release of Cytochrome c from Mitochondria

Growth factors signaling through the phosphoinositide 3-kinase/Akt pathway promote cell survival. The mechanism by which the serine/threonine kinase Akt prevents cell death remains unclear. We have previously shown that Akt inhibits the activity of DEVD-targeted caspases without changing the steady-state levels of Bcl-2 and Bcl-x(L). Here we show that Akt inhibits apoptosis and the processing of procaspases to their active forms by delaying mitochondrial changes in a caspase-independent manner. Akt activation is sufficient to inhibit the release of cytochrome c from mitochondria and the alterations in the inner mitochondrial membrane potential. However, Akt cannot inhibit apoptosis induced by microinjection of cytochrome c. We also demonstrated that Akt inhibits apoptosis and cytochrome c release induced by several proapoptotic Bcl-2 family members. Taken together, our results show that Akt promotes cell survival by intervening in the apoptosis cascade before cytochrome c release and caspase activation via a mechanism that is distinct from Bad phosphorylation.     

Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis

The Bcl-2 family of proteins are involved in regulating the redox state of cells. However, the mode of action of Bcl-2 proteins remains unclear. This work analyzed the effects of Bcl-x(L) on the cellular redox state after treatment with tumor necrosis factor alpha (TNF-alpha) or exogenous oxidants. We show that in cells that undergo TNF-alpha-induced apoptosis, TNF-alpha induces a partial decrease in mitochondrial membrane potential (DeltaPsi(m)) followed by high levels of reactive oxygen species (ROS). ROS scavengers delay the progression of mitochondrial depolarization and apoptotic cell death. This indicates that ROS are important mediators of mitochondrial depolarization. However, ROS scavengers fail to prevent the initial TNF-alpha-induced decrease in DeltaPsi(m). In contrast, expression of Bcl-x(L) prevents both the initial decrease in DeltaPsi(m) following TNF-alpha treatment and the subsequent induction of ROS. Bcl-x(L) itself does not act as a ROS scavenger. In addition, Bcl-x(L) does not block the initial decrease in DeltaPsi(m) following treatment with the oxidant hydrogen peroxide. However, unlike control-transfected cells, Bcl-x(L)-expressing cells can recover their mitochondrial membrane potential following the initial drop in DeltaPsi(m) induced by hydrogen peroxide. These data suggest that Bcl-x(L) plays a regulatory role in controlling the membrane potential of and ROS production by mitochondria rather than acting as a direct antioxidant.     

Overexpression of Bcl-x(L) in beta-cells prevents cell death but impairs mitochondrial signal for insulin secretion

To study effects of Bcl-x(L) in the pancreatic beta-cell, two transgenic lines were produced using different forms of the rat insulin promoter. Bcl-x(L) expression in beta-cells was increased 2- to 3-fold in founder (Fd) 1 and over 10-fold in Fd 2 compared with littermate controls. After exposure to thapsigargin (10 microM for 48 h), losses of cell viability in islets of Fd 1 and Fd 2 Bcl-x(L) transgenic mice were significantly lower than in islets of wild-type mice. Unexpectedly, severe glucose intolerance was observed in Fd 2 but not Fd 1 Bcl-x(L) mice. Pancreatic insulin content and islet morphology were not different from control in either transgenic line. However, Fd 2 Bcl-x(L) islets had impaired insulin secretory and intracellular free Ca(2+) ([Ca(2+)](i)) responses to glucose and KCl. Furthermore, insulin and [Ca(2+)](i) responses to pyruvate methyl ester (PME) were similarly reduced as glucose in Fd 2 Bcl-x(L) islets. Consistent with a mitochondrial defect, glucose oxidation, but not glycolysis, was significantly lower in Fd 2 Bcl-x(L) islets than in wild-type islets. Glucose-, PME-, and alpha-ketoisocaproate-induced hyperpolarization of mitochondrial membrane potential, NAD(P)H, and ATP production were also significantly reduced in Fd 2 Bcl-x(L) islets. Thus, although Bcl-x(L) promotes beta-cell survival, high levels of expression of Bcl-x(L) result in reduced glucose-induced insulin secretion and hyperglycemia due to a defect in mitochondrial nutrient metabolism and signaling for insulin secretion.     

AKT1 drives endothelial cell membrane asymmetry and microglial activation through Bcl-xL and caspase 1, 3, and 9

Protein kinase B (Akt1) holds a central role for cellular growth, development, and survival, but the cellular pathways of Akt1 that prevent inflammatory demise in the vascular system remain undefined. Employing a constitutively active form of Akt1 (myristoylated Akt1) in endothelial cells (ECs), we demonstrate that Akt1 not only modulates intrinsic pathways of EC injury that involve genomic DNA destruction, but also uniquely regulates extrinsic mechanisms of cellular inflammation mediated by phosphatidylserine exposure (PS) and microglial activation. Activation of Akt1 is necessary and sufficient to prevent apoptotic EC destruction, since inhibition of the phosphatidylinositide-3-kinase pathway as well as transfection of ECs with a dominant-negative Akt1 mutant abrogates vascular protection. Furthermore, we illustrate that control of microglial activation by Akt1 is directly dependent on the modulation of EC membrane PS exposure. Akt1 provides a novel capacity to foster EC survival through the prevention of cysteine protease degradation of Bcl-x(L) that is intimately linked to the specific inhibition of caspase 1-, 3-, and 9-like activities and the modulation of mitochondrial membrane potential and cytochrome c release. Our work elucidates the critical role of Akt1 during cellular inflammation and identifies new downstream targets of Akt1 that may offer therapeutic potential against vascular disease.     

The super anti-apoptotic factor Bcl-xFNK constructed by disturbing intramolecular polar interactions in rat Bcl-xL

A powerful artificial anti-apoptotic factor will be useful for medical applications of the future therapies for many diseases by prolonging survival of sick cells. For constructing it, we designed the super anti-apoptotic factor by disturbing three intramolecular polar interactions among alpha-helix structures of Bcl-x(L). The resultant mutant Bcl-x(L), named Bcl-xFNK, was expected to make the pore-forming domain more mobile and flexible than the wild-type. When overexpressed in Jurkat cells, Bcl-xFNK was markedly more potent in prolonging survival following apoptosis-inducing treatment with a kind of cell death cytokines (anti-Fas), a protein kinase inhibitor (staurosporine), cell cycle inhibitors (TN-16, camptothecin, hydroxyurea, and trichostatin A), or oxidative stress (hydrogen peroxide and paraquat) than wild-type Bcl-x(L). Furthermore, the transfectants of bcl-xFNK became more resistant against a calcium ionophore and even a heat treatment than wild-type Bcl-x(L). In addition, Bcl-xFNK showed marked anti-apoptotic activity in Chinese hamster ovary and Jurkat cells deprived of serum. Thus, Bcl-xFNK may be the first mutant generated by site-directed mutagenesis of Bcl-x(L) with a gain-of-function phenotype. Interestingly, Bcl-xFNK was found to allow interleukin-3-dependent FDC-P1 to grow without interleukin-3, but not BaF/3. In Bcl-xFNK transfectants of FDC-P1 and Jurkat, the p42/p44 mitogen-activated protein kinase was activated by 2 to 5 times, but not in those of BaF/3 and Chinese hamster ovary. Bcl-xFNK might gain a new function to activate the mitogen-activated protein kinase in a cell-type specific manner. The findings of this study suggest that the central alpha5-alpha6 pore-forming region of anti-apoptotic factor Bcl-x(L) has a pivotal role in suppressing apoptosis.     

Lack of oxidative phosphorylation and low mitochondrial membrane potential decrease susceptibility to apoptosis and do not modulate the protective effect of Bcl-x(L) in osteosarcoma cells

We explored the role of low mitochondrial membrane potential (DeltaPsim) and the lack of oxidative phosphorylation in apoptosis by assessing the susceptibility of osteosarcoma cell lines with and without mitochondrial DNA to staurosporine-induced death. Our cells without mitochondrial DNA had low DeltaPsim and no functional oxidative phosphorylation. Contrary to our expectation, these cells were more resistant to staurosporine-induced death than were the parental cells. This reduced susceptibility was associated with decreased activation of caspase 3 but not with the mitochondrial permeability transition pore or cytochrome c release from the mitochondria. Apoptosis in both cell lines was associated with an increase in DeltaPsim. Bcl-x(L) could protect both cell types against caspase 3 activation and apoptosis by a mechanism that does not appear to be mediated by mitochondrial function or modulation of DeltaPsim. Nevertheless, we found that Bcl-x(L) expression can stimulate cell respiration in cells with mitochondrial DNA. Our results showed that the lack of functional oxidative phosphorylation and/or low mitochondrial membrane potential are associated with an antiapoptotic effect, possibly contributing to the development of some types of cancer. It also reinforces a model in which Bcl-x(L) can exert an antiapoptotic effect by stimulating oxidative phosphorylation and/or inhibiting caspase activation.     

RAD21L, a novel cohesin subunit implicated in linking homologous chromosomes in mammalian meiosis

Mammalian Bcl-x(L) protein localizes to the outer mitochondrial membrane, where it inhibits apoptosis by binding Bax and inhibiting Bax-induced outer membrane permeabilization. Contrary to expectation, we found by electron microscopy and biochemical approaches that endogenous Bcl-x(L) also localized to inner mitochondrial cristae. Two-photon microscopy of cultured neurons revealed large fluctuations in inner mitochondrial membrane potential when Bcl-x(L) was genetically deleted or pharmacologically inhibited, indicating increased total ion flux into and out of mitochondria. Computational, biochemical, and genetic evidence indicated that Bcl-x(L) reduces futile ion flux across the inner mitochondrial membrane to prevent a wasteful drain on cellular resources, thereby preventing an energetic crisis during stress. Given that F(1)F(O)-ATP synthase directly affects mitochondrial membrane potential and having identified the mitochondrial ATP synthase β subunit in a screen for Bcl-x(L)-binding partners, we tested and found that Bcl-x(L) failed to protect β subunit-deficient yeast. Thus, by bolstering mitochondrial energetic capacity, Bcl-x(L) may contribute importantly to cell survival independently of other Bcl-2 family proteins.     

Upstream regulatory role for XIAP in receptor-mediated apoptosis

X-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of cell death that functions by suppressing caspases 3, 7, and 9. Here we describe the establishment of Jurkat-derived cell lines stably overexpressing either full-length XIAP or a truncation mutant of XIAP that can only inhibit caspase 9. Characterization of these cell lines revealed that following CD95 activation full-length XIAP supported both short- and long-term survival as well as proliferative capacity, in contrast to the truncation mutant but similar to Bcl-x(L). Full-length XIAP was also able to inhibit CD95-mediated caspase 3 processing and activation, the mitochondrial release of cytochrome c and Smac/DIABLO, and the loss of mitochondrial membrane potential, whereas the XIAP truncation mutant failed to prevent any of these cell death events. Finally, suppression of XIAP levels by RNA interference sensitized Bcl-x(L)-overexpressing cells to death receptor-induced apoptosis. These data demonstrate for the first time that full-length XIAP inhibits caspase activation required for mitochondrial amplification of death receptor signals and that, by acting upstream of mitochondrial activation, XIAP supports the long-term proliferative capacity of cells following CD95 stimulation.     

Transforming growth factor-beta 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells

Transforming growth factor-beta1 (TGF-beta1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-beta1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-beta1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-beta1 induced apoptosis. The proapoptotic effects of TGF-beta1 occur via a p53-independent pathway, since p53(null) FDCP-Mix demonstrated the same responses to TGF-beta1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-x(L) expression. In FDCP-Mix cells, neither IL-3 nor TGF-beta1 induced any change in Bcl-x(L) protein levels or the proapoptotic proteins Bad or Bax. However, TGF-beta1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-beta1-induced apoptosis but was incapable of inhibiting TGF-beta1-mediated growth arrest. We conclude that TGF-beta1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-x(L). The significance of these results for stem cell survival in bone marrow are discussed.     

Production of reactive oxygen species and loss of viability in yeast mitochondrial mutants: protective effect of Bcl-xL

The capacity of yeast cells to produce reactive oxygen species (ROS), both as a response to manipulation of mitochondrial functions and to growth conditions, was estimated and compared with the viability of the cells. The chronological ageing of yeast cells (growth to late-stationary phase) was accompanied by increased ROS accumulation and a significantly higher loss of viability in the mutants with impaired mitochondrial functions than in the parental strain. Under these conditions, the ectopic expression of mammalian Bcl-x(L), which is an anti-apoptotic protein, allowed cells to survive longer in stationary phase. The protective effect of Bcl-x(L) was more prominent in respiratory-competent cells that contained defects in mitochondrial ADP/ATP translocation, suggesting a model for Bcl-x(L) regulation of chronological ageing at the mitochondria. Yeast can also be triggered into apoptosis-like cell death, at conditions leading to the depletion of the intramitochondrial ATP pool, as a consequence of the parallel inhibition of mitochondrial respiration and ADP/ATP translocation. If respiratory-deficient (rho(0)) cells were used, no correlation between the numbers of ROS-producing cells and the viability loss in the population was observed, indicating that ROS production may be an accompanying event. The protective effect of Bcl-x(L) against death of these cells suggests a mitochondrial mechanism which is different from the antioxidant activity of Bcl-x(L).