[Show abstract][Hide abstract] ABSTRACT: Low mRNA levels of pro-apoptotic protein BOK in WT and BAX/BAK DKO cells. WT and BAX/BAK DKO cells were treated with 10 mg/ml Tm or left untreated. DKO cells were also pre-incubated for 2 h in cell culture media containing 0.5% serum. After 6 h of treatment, bok mRNA levels were assessed by real time PCR. As control, the levels of bok mRNA expression were also monitored in mRNA extracts from mouse ovary.
[Show abstract][Hide abstract] ABSTRACT: Serum withdrawal recovers the susceptibility of BAX and BAK DKO cells to ER stress-induced cell death. WT MEFs were treated with 10 mg/ml Tm, 10 mM Thap or 20 mM brefeldin A in cells grown in regular cell culture media or in media containing 0.5% serum. Mild serum withdrawal was performed 2 h before the addition of ER stress agents. After 24 h of treatment, cell viability was analyzed with the MTS assay. Results are representative of two independent experiments performed in triplicate. Mean and standard deviations are presented of a representative experiment.
[Show abstract][Hide abstract] ABSTRACT: Effect of Cyclosporine A in cell death in WT and BAX and BAK DKO cells exposed to ER stress. (A) BAX/BAK WT cells were or not pre-treated with CsA 10 mM for 30 min. Then, cells were incubated with 0.25, 1 and 2,5 mg/mL Tm for 24 h. Cell viability was analyzed by PI staining and FACS analysis. Data is representative of three independent experiments. Mean and standard deviation are presented. Significant differences were obtained between indicated control and experimental groups using Student’s t-test (** p<0.01). (B) BAX/BAK DKO cells were pre-treated with cell culture media containing 0.5% serum for 2 h in the presence or absence of 1 mM of CsA. Then, cells were exposed to Tm or Thap for 24 h. Cell viability was analyzed by PI staining and FACS analysis. Data is representative of two independent experiments. Mean is presented.
[Show abstract][Hide abstract] ABSTRACT: BCL-XL in the control of cytocrome c redistribution and BH3-only protein expression and efficiency of retroviral transduction. BAX/BAK DKO MEFs that stably transduced with retroviruses expressing human BCL-XL or empty vector (Mock) cells were exposed to 20 µg/ml Tm in the presence of cell culture media containing 10% serum or pre-treated with media containing 0.5% serum. After 10 h, cells were stained with Mitotracker orange (red), fixed and cytochrome C (Cyt C, green) distribution visualized by indirect immuofluorescence. Images were acquired by confocal microscopy. Then, quantification of the Mandeŕs colocalization coefficient MTO/CytC (fraction of mitrotracker orange signal in co-localizing with cytochrome C signal) or CytC/MTO (fraction of the cytochrome C co-localizing mitotracker orange) were analyzed. Data represent the average and standard deviation of four independent experiments. One-way ANOVA test was used to analyze statistical significance (*: p<0.05; **: p<0.01; ***: p<0.01). (B) The expression of the HA-tagged BH3-only proteins was analyzed by Western blot after transfection of 293T packaging cells with pMIG-GFP retroviral vectors together with packaging vector to produce retroviruses employed in Figure 4F. Red arrowheads indicate the protein bands corresponding to each BH3-only protein. A HA-BAX expression vector was also used as control. (B) The percentage of retroviral transduction efficiency was assessed in WT and BAX/BAK DKO cells by monitoring the bicistronic expression of GFP by FACS analysis. Results are representative of two independent experiments performed in duplicated. Means are presented of a representative experiment.
[Show abstract][Hide abstract] ABSTRACT: Cell death in BAX and BAK DKO cells is independent of CypD. (A) BAX/BAK DKO cells were stably transduced with lentiviral vectors expressing an shRNA against cypD mRNA (shCypD) or control shRNA against the luciferase mRNA (shLuc). Then, cell viability was analyzed after treatment with 10 mM Thap in cells grown in regular cell culture conditions or pre-incubated for 2 h in media containing 0.5% serum. Untreated cells (NT) are also presented. After 24 h cell death was monitored by PI staining and FACS analysis. (B) WT and BAX/BAK/CypD TKO cells were treated with 10 mM Thap under regular cell growing conditions or in cells pre-treated with media containing 0.5% serum for 2 h. After 24 h, cell death was analyzed by PI staining and FACS analysis. Mean and standard deviation are presented of three determinations.
[Show abstract][Hide abstract] ABSTRACT: Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L) overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.
[Show abstract][Hide abstract] ABSTRACT: Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes. However, the cellular mechanisms underlying these effects are incompletely understood. The lysosomal degradation pathway, autophagy, is an intracellular recycling system that functions during basal conditions in organelle and protein quality control. During stress, increased levels of autophagy permit cells to adapt to changing nutritional and energy demands through protein catabolism. Moreover, in animal models, autophagy protects against diseases such as cancer, neurodegenerative disorders, infections, inflammatory diseases, ageing and insulin resistance. Here we show that acute exercise induces autophagy in skeletal and cardiac muscle of fed mice. To investigate the role of exercise-mediated autophagy in vivo, we generated mutant mice that show normal levels of basal autophagy but are deficient in stimulus (exercise- or starvation)-induced autophagy. These mice (termed BCL2 AAA mice) contain knock-in mutations in BCL2 phosphorylation sites (Thr69Ala, Ser70Ala and Ser84Ala) that prevent stimulus-induced disruption of the BCL2-beclin-1 complex and autophagy activation. BCL2 AAA mice show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise- (and starvation)-induced autophagy in vivo, and autophagy induction may contribute to the beneficial metabolic effects of exercise.
[Show abstract][Hide abstract] ABSTRACT: The threonine endopeptidase Taspase1 has a critical role in cancer cell proliferation and apoptosis. In this study, we developed and evaluated small molecule inhibitors of Taspase1 as a new candidate class of therapeutic modalities. Genetic deletion of Taspase1 in the mouse produced no overt deficiencies, suggesting the possibility of a wide therapeutic index for use of Taspase1 inhibitors in cancers. We defined the peptidyl motifs recognized by Taspase1 and conducted a cell-based dual-fluorescent proteolytic screen of the National Cancer Institute diversity library to identify Taspase1 inhibitors (TASPIN). On the basis of secondary and tertiary screens the 4-[(4-arsonophenyl)methyl]phenyl] arsonic acid NSC48300 was determined to be the most specific active compound. Structure-activity relationship studies indicated a crucial role for the arsenic acid moiety in mediating Taspase1 inhibition. Additional fluorescence resonance energy transfer-based kinetic analysis characterized NSC48300 as a reversible, noncompetitive inhibitor of Taspase1 (K(i) = 4.22 μmol/L). In the MMTV-neu mouse model of breast cancer and the U251 xenograft model of brain cancer, NSC48300 produced effective tumor growth inhibition. Our results offer an initial preclinical proof-of-concept to develop TASPINs for cancer therapy.
[Show abstract][Hide abstract] ABSTRACT: Acute exposure to ionizing radiation can cause lethal damage to the gastrointestinal (GI) tract, a condition called the GI
syndrome. Whether the target cells affected by radiation to cause the GI syndrome are derived from the epithelium or endothelium
and whether the target cells die by apoptosis or other mechanisms are controversial issues. Studying mouse models, we found
that selective deletion of the proapoptotic genes Bak1 and Bax from the GI epithelium or from endothelial cells did not protect mice from developing the GI syndrome after sub–total-body
gamma irradiation. In contrast, selective deletion of p53 from the GI epithelium, but not from endothelial cells, sensitized
irradiated mice to the GI syndrome. Transgenic mice overexpressing p53 in all tissues were protected from the GI syndrome
after irradiation. These results suggest that the GI syndrome is caused by the death of GI epithelial cells and that these
epithelial cells die by a mechanism that is regulated by p53 but independent of apoptosis.
[Show abstract][Hide abstract] ABSTRACT: The proapoptotic proteins BAX and BAK constitute the mitochondrial apoptotic gateway that executes cellular demise after integrating death signals. The lethal BAK is kept in check by voltage-dependent anion channel 2 (VDAC2), a mammalian-restricted VDAC isoform. Here, we provide evidence showing a critical role for the VADC2-BAK complex in determining thymocyte survival in vivo. Genetic depletion of Vdac2 in the thymus resulted in excessive cell death and hypersensitivity to diverse death stimuli including engagement of the T cell receptor. These phenotypes were completely rescued by the concurrent deletion of Bak but not that of Bax. Thus, the VDAC2-BAK axis provides a mechanism that governs the homeostasis of thymocytes. Our study reveals a sophisticated built-in rheostat that likely fine-tunes immune competence to balance autoimmunity and immunodeficiency.
No preview · Article · Feb 2009 · Science Signaling
[Show abstract][Hide abstract] ABSTRACT: The proapoptotic BCL-2 family member BAD resides in a glucokinase-containing complex that regulates glucose-driven mitochondrial respiration. Here, we present genetic evidence of a physiologic role for BAD in glucose-stimulated insulin secretion by beta cells. This novel function of BAD is specifically dependent upon the phosphorylation of its BH3 sequence, previously defined as an essential death domain. We highlight the pharmacologic relevance of phosphorylated BAD BH3 by using cell-permeable, hydrocarbon-stapled BAD BH3 helices that target glucokinase, restore glucose-driven mitochondrial respiration and correct the insulin secretory response in Bad-deficient islets. Our studies uncover an alternative target and function for the BAD BH3 domain and emphasize the therapeutic potential of phosphorylated BAD BH3 mimetics in selectively restoring beta cell function. Furthermore, we show that BAD regulates the physiologic adaptation of beta cell mass during high-fat feeding. Our findings provide genetic proof of the bifunctional activities of BAD in both beta cell survival and insulin secretion.
[Show abstract][Hide abstract] ABSTRACT: We investigated the mechanism by which B lymphocyte stimulator (BLyS)/BAFF, a tumor necrosis factor superfamily ligand, promotes B-cell survival and resistance to atrophy. BLyS stimulation activates 2 independent signaling pathways, Akt/mTOR and Pim 2, associated with cell growth and survival. BLyS blocks the cell volume loss (atrophy) that freshly isolated B cells normally undergo when maintained in vitro while concurrently increasing glycolytic activity and overall metabolism. This atrophy resistance requires Akt/mTOR. We used a genetic approach to resolve the contributions of Akt/mTOR and Pim kinase pathways to BLyS-mediated survival. Pim 2-deficient B cells are readily protected from death by BLyS stimulation, but this protection is completely abrogated by treatment with the mTOR inhibitor rapamycin. Furthermore, rapamycin treatment in vivo significantly reduces both follicular and marginal zone B cells in Pim-deficient but not healthy hosts. BLyS-dependent survival requires the antiapoptotic protein Mcl-1. Mcl-1 protein levels rise and fall in response to BLyS addition and withdrawal, respectively, and conditional deletion of the Mcl-1 gene renders B cells refractory to BLyS-mediated protection. Because BlyS is required for the normal homeostasis of all B cells, these data suggest a therapeutic strategy simultaneously inhibiting mTOR and Pim 2 could target pathogenic B cells.
[Show abstract][Hide abstract] ABSTRACT: Soft tissue sarcomas are mesenchymal tumors that are fatal in approximately one-third of patients. To explore mechanisms of sarcoma pathogenesis, we have generated a mouse model of soft tissue sarcoma. Intramuscular delivery of an adenovirus expressing Cre recombinase in mice with conditional mutations in Kras and Trp53 was sufficient to initiate high-grade sarcomas with myofibroblastic differentiation. Like human sarcomas, these tumors show a predilection for lung rather than lymph node metastasis. Using this model, we showed that a prototype handheld imaging device can identify residual tumor during intraoperative molecular imaging. Deletion of the Ink4a-Arf locus (Cdkn2a), but not Bak1 and Bax, could substitute for mutation of Trp53 in this model. Deletion of Bak1 and Bax, however, was able to substitute for mutation of Trp53 in the development of sinonasal adenocarcinoma. Therefore, the intrinsic pathway of apoptosis seems sufficient to mediate p53 tumor suppression in an epithelial cancer, but not in this model of soft tissue sarcoma.
[Show abstract][Hide abstract] ABSTRACT: Cytochrome c (CYT c) is a protein that employs the caspase recruitment domain (CARD)-containing proteins APAF-1 and CASP-9 to activate effectors CASP-3 and -7. By using affinity labeling techniques and mass spectrometry analysis, we show that histone H1.2 is a regulator of caspases upon UV irradiation. We demonstrated that histone H1.2 forms a protein complex with APAF-1, CASP-9 and CYT c upon UV irradiation. In cell-free systems, we show that histone H1.2 triggers activation of CASP-3 and -7 via APAF-1 and CASP-9. We therefore conclude that upon DNA damage histone H1.2 acts as a positive regulator of apoptosome formation.