Sarit Larisch

High grade serous carcinoma (HGSC) is the most common subtype of ovarian cancer, and a very deadly disease, mostly due to lack of early detection methods. Wide spread disease at diagnosis has delayed the understanding that it often originates from the fallopian tube. This novel understanding has led to increased interest in fallopian tube developmental lineage markers, such as PAX8. In the adult fallopian tube, PAX8 is expressed in the fallopian tube secretory epithelial cell (FTSEC) and its expression is maintained through the process of FTSEC transformation Serous Tubal Intraepithelial Carcinoma (STIC) and to HGSC. We show that PAX8 has an essential pro-proliferative and anti-apoptotic role in HGSC. This role is mediated through direct transcriptional activation of mutated TP53, which often carries missense mutations that potentially lead to gain of function (GOF) oncogenic activities. Surprisingly, mutant p53 binds the p21 promoter and transcriptionally activates the expression o...

Purpose: XIAP [X-linked inhibitor of apoptosis (IAP) protein] is the best characterized mammalian caspase inhibitor. XIAP is frequently overexpressed in a variety of human tumors, and genetic inactivation of XIAP in mice protects against lymphoma. Therefore, XIAP is an attractive target for anticancer therapy. IAP antagonists based on a conserved IAP-binding motif (IBM), often referred to as “Smac-mimetics,” are currently being evaluated for cancer therapy in the clinic. ARTS (Sept4_i2) is a mitochondrial proapoptotic protein which promotes apoptosis by directly binding and inhibiting XIAP via a mechanism that is distinct from all other known IAP antagonists. Here, we investigated the ability of peptides derived from ARTS to antagonize XIAP and promote apoptosis in cancer cell lines.Experimental Design: The ability of synthetic peptides, derived from the C-terminus of ARTS, to bind to XIAP, stimulate XIAP degradation, and induce apoptosis was examined. We compared the response of several cancer cell lines to different ARTS-derived peptides. Pull-down assays were used to examine binding to XIAP, and apoptosis was evaluated using terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling, caspase activation, and Western blot analyses of caspase substrates.Results: The C-terminus of ARTS contains a unique sequence, termed ARTS-IBM (AIBM), which is important for binding to XIAP and cell killing. AIBM peptides can bind to XIAP-BIR3, penetrate cancer cells, reduce XIAP levels, and promote apoptosis.Conclusions: Short synthetic peptides derived from the C-terminus of ARTS are sufficient for binding to XIAP and can induce apoptosis in cancer cells. These results provide proof-of-concept for the feasibility of developing ARTS-based anticancer therapeutics. Clin Cancer Res; 18(9); 2569–78. ©2012 AACR.

Every cell in our body contains a “self-destruction” program. This cell death is a critical process allowing replacement of damaged cells with healthy ones to prevent wide range of diseases. When the cell’s death mechanism gets “stuck” and is not activated, cancer can result. In healthy cells there is a balanced system of proteins, some of which activate the normal death mechanism, and some of which inhibit this process. This is like the system of gas and brakes in a car. Researchers have found that cancer cells lack a protein, called ARTS, which is crucial for activating the cells’ death mechanism. The lack of ARTS causes cancer cells to escape death and become “immortal.” Small ARTS-like molecules have been discovered that can penetrate cancerous cells and reactivate the cell death program, effectively making the cancer cells “commit suicide.” We envision that these ARTS-like molecules will provide novel therapy for cancer.

A375 human melanoma cell cultures grown in the presence of TGF beta contained greatly reduced cell numbers and exhibited drastic alterations in cell morphology compared to the control cultures. Preincubation of the cells with the cytokine for only 18 h was sufficient to induce these changes irreversibly. Examination of TGF beta-treated cells in the electron microscope revealed large numbers of lipid-filled vacuoles in the cytoplasm, greatly contracted nuclei and some loss of the otherwise abundant microvilli. Thus TGF beta may have a direct toxic effect on the A375 melanoma cells.

L'invention concerne des methodes pour traiter ou pour reduire un cas de maladie, de trouble, ou un symptome chez un patient. La maladie, le trouble ou le symptome susmentionne comprenne: l'apoptose deregulee d'un neurone. La methode de l'invention consiste a mettre en contact le patient avec un compose ou une composition qui modifie une activite ou une expression d'une proteine ARTS (proteine associee a l'apoptose dans la voie de signalisation TGF-s). Cette invention concerne egalement des methodes pour determiner une predisposition ou un risque, chez un patient, par rapport a la maladie, le trouble ou au symptome susmentionne, ainsi que des methodes pour identifier un compose ou une composition utile pour un traitement prophylactique ou therapeutique de la schizophrenie, ces methodes consistant a detecter une activite ou une expression de la proteine ARTS ou a determiner une sequence d'un gene codant une proteine ARTS.

<p>A. Western blot (WB) analysis shows a strong increase in the expression levels of ARTS upon treatment of SH-SY5Y cells with the apoptotic inducer staurosporine (STS). This elevation in levels of ARTS is associated with an increase in caspase-3 activation. GAPDH was used as a loading control. B. A significant increase in levels of ARTS is seen in response to treatment with etoposide inducing apoptosis in SH-SY5Y cells. This strong up-regulation of ARTS is associated with a corresponding increase in expression of the apoptotic marker H2AX. Actin was used as a loading control. C. Endogenous ARTS binds to XIAP in SH-SY5Y cells. Immunoprecipitation assay (IP) with monoclonal anti ARTS antibody was performed on lysates from SH-SY5Y cells. Mouse IgG served as control for co-precipitation of the specific antigen. DI. A representative picture (viewed with x40 objective) showing immunofluorescence (IF) staining using anti-ARTS antibody in <i>Substantia nigra pars compacta</i> (SNpc) of rat brain treated with 6-hydroxy dopamine (6-OHDA) as compared to control injected with saline. 6-OHDA was injected into the left medial forebrain bundle of these rats, from where it is transported to the SN. A significant increase in expression of ARTS is shown in these cells (red). DAPI staining of nuclei is shown in blue. Scale bar represents 50 µm. DII. Co-localization of ARTS (red) and cleaved caspase-3 (green) is seen in a representative rat brain section of 6-OHDA treated rat viewed using x20 objective. Scale bar represents 100 µm. DIII, DIV. Percent of ARTS/cleaved caspase-3 and ARTS/TUNEL double positive cells in SNpc of 6-OHDA treated rats compared to saline injected controls. These pictures demonstrate the levels of ARTS among apoptotic neurons. Counts were done in sections from brains after one, three and seven days following injection of 6-OHDA or saline. Figures present mean ± SEM of the ratio between the percent of double positive cells in 6-OHDA brains relative to the average percent of double positive cells in saline treated brains, at each time point. A significant increase in percentage of ARTS/caspase-3 and ARTS/TUNEL double positive SN neurons is seen 24 hours after injection of 6-OHDA as compared to controls (significance levels were calculated from data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038837#pone.0038837.s003" target="_blank">Table S1</a>; **P<0.01, ***P<0.001 for difference from same day control by ANOVA with Bonferroni post hoc test). This suggests that ARTS may play an important role in promoting susceptibility to 6-OHDA-induced apoptosis in SN neurons.</p

<p><b>A.</b> Immunofluorescence (IF) assay was performed on SH-SY5Y cells, which were transiently co-transfected with AU5-ARTS (red) and 1myc-Parkin (green) and treated with 1.5 µM STS. These IF results reveal co-localization of ARTS and Parkin in untreated cells, with increased co-localization as early as 30 min after apoptotic induction. <b>B.</b> Working model for the role of ARTS in healthy Parkin expressing cells, and in cells containing mutated or dysfunctional Parkin. We propose that in healthy brain cells which contain intact Parkin, the levels of ARTS are kept low through constant down regulation by Parkin. Upon initiation of a stress or apoptotic stimuli, recruitment of Parkin to dysfunctional mitochondria occurs as well as translocation of ARTS to the cytosol at the early stages of apoptosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038837#pone.0038837-Edison2" target="_blank">[40]</a>. This may allow the binding and degradation of ARTS by Parkin and help maintain low levels of ARTS. This down-regulation of ARTS is expected to promote cell survival by preventing the inhibition of XIAP and caspase activation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038837#pone.0038837-Gottfried1" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038837#pone.0038837-Edison2" target="_blank">[40]</a>. According to our model, in brain cells containing mutated or dysfunctional Parkin, Parkin can no longer degrade ARTS. This leads to accumulation of ARTS and initiation of apoptosis through de-repression of caspases by antagonizing XIAP. This first wave of caspases leads to Mitochondrial Outer membrane Permeabilization (MOMP) which results in release of mitochondrial factors residing in the inner membrane space (IMS) such as Cytochrome c and Smac/Diablo, causing amplified caspase activation and cell death <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038837#pone.0038837-Edison2" target="_blank">[40]</a>.</p

<p>. Parkin promotes degradation of ARTS through the Ubiquitin Proteasome System (UPS). COS-7 cells co-transfected with 1myc-Parkin and AU5-ARTS were treated with the proteasome inhibitor, MG132. Expression levels of ARTS were significantly decreased in the presence of Parkin, and were restored upon addition of MG132. <b>AII.</b> Densitometry analysis of WB results shown in AI. Protein levels were normalized to actin. Graph presents mean ± SEM of the densitometry values relative to actin levels. <b>B.</b> Parkin serves as an E3-ligase for ARTS. COS-7 cells co-transfected with 1myc-Parkin, AU5-ARTS and HA-8xubiquitin were subjected to in vivo ubiquitination assay followed by immunoprecipitation (IP) with either monoclonal anti-ARTS antibody or anti-Sept4_i1 antibody. Non-transfected cells and cells transfected with only 1myc-Parkin or AU5-ARTS served as controls. ARTS is ubiquitinated in the presence of Parkin. <b>C.</b> Parkin binds both ARTS and Sept4_i1. COS-7 cells were co-transfected with 1myc-Parkin together with Flag-Sept4_i1 or AU5-ARTS. Lysates were subjected to IP with anti-Parkin antibody followed by WB analysis. <b>D.</b> COS-7 cells treated with the proteasome inhibitor, MG132 were co-transfected with 1myc-Parkin or mutant Parkin T240R together with 6myc-ARTS or Flag -Sept4_i1. Immunoprecipitation was done with either anti-ARTS or anti-Sept4_i1 antibody followed by <i>In vivo</i> ubiquitination assay. Western blot analysis was performed with anti-ubiquitin antibody (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038837#s2" target="_blank">methods</a>). Parkin selectively ubiquitinates ARTS but not Sept4_i1. Mutant T240R Parkin lacking E3-ligase activity, demonstrates markedly decreased ability to ubiquitinate ARTS as compared to wt Parkin. <b>E</b>. COS-7 cells were transiently transfected with ARTS, Parkin, and the mutant Parkin T240R with compromised E3-ligase activity. The levels of cleaved caspase-3 (cCasp3) representing rate of apoptosis were visualized using western blot analyses. Co-transfection of COS-7 cells with ARTS and Parkin resulted in ARTS degradation and apoptotic suppression, as visualized by the absence of cleaved caspase-3 (cCasp3). In contrast, co-transfection with ARTS and mutant Parkin T240R restored apoptosis in these cells.</p

XIAP (Gottfried et al., 2004). IAPs comprise a family of important negative regulators of apoptosis that can di-Septins comprise a family of proteins whose overall rectly inhibit active caspases, the key executioners ofstructure has been conserved from yeast to Drosophila apoptosis (reviewed in Salvesen and Abrams, 2004).to humans. Septins are GTPases related to the Ras su-Furthermore, ARTS appears to be a tumor suppressor in acute lymphoblastic leukemia (ALL): ARTS, but not*Correspondence:

Apoptosis is a cell suicide process that is essential for development, tissue homeostasis and human health. Impaired apoptosis is associated with a variety of human diseases, including neurodegenerative disorders, autoimmunity and cancer. As the levels of pro- and anti-apoptotic proteins can determine the life or death of cells, tight regulation of these proteins is critical. The ubiquitin proteasome system (UPS) is essential for maintaining protein turnover, which can either trigger or inhibit apoptosis. In this review, we will describe the E3 ligases that regulate the levels of pro- and anti-apoptotic proteins and assisting proteins that regulate the levels of these E3 ligases. We will provide examples of apoptotic cell death modulations using the UPS, determined by positive and negative feedback loop reactions. Specifically, we will review how the stability of p53, Bcl-2 family members and IAPs (Inhibitor of Apoptosis proteins) are regulated upon initiation of apoptosis. As incre...

Recent studies have revealed that cell death stimuli can trigger programmed necrosis, necroptosis. Receptor-interacting serine-threonine kinase family RIP plays a crucial role in regulating the switch between apoptosis and necroptosis. Two studies now describe a novel RIP1 containing ~2 MDa 'Ripoptosome' complex assembled in the cytosol to mediate both apoptosis and necroptosis in response to genotoxic stress and TLR3 stimulation. Intriguingly, cIAPs and XIAP function as endogenous inhibitors of Ripoptosome by direct ubiquitination of its components.

Apoptosis related protein in TGF-β signaling pathway (ARTS/septin 4 isoform 2) hereforth referred to as ARTS, was originally found to promote apoptosis induced by TGF-β, but later was shown to promote apoptosis induced by a wide variety of apoptotic stimuli. In vivo and in vitro studies revealed that ARTS-induced apoptosis is mainly executed through direct binding and antagonizing XIAP. High levels of XIAP are found in many types of cancers and often correlate with poor prognosis. ARTS was shown to function as a tumor-suppressor protein in human patients and mouse-tumor models. In particular, Septin 4/ARTS-deficient mice have increased tumor susceptibility and contain increased numbers of stem cells (SCs) and progenitor cells, apparently owing to their resistance towards apoptosis. Based on these results we propose that loss of proapoptotic ARTS may act as the ‘first hit’ initiating tumorigenesis in two distinct ways. First, loss of ARTS-mediated apoptosis leads to increased numbers...

Apoptosis Related protein in TGF-β Signaling pathway (ARTS) was originally discovered in cells undergoing apoptosis in response to TGF-β, but ARTS also acts downstream of many other apoptotic stimuli. ARTS induces apoptosis by antagonizing the anti-apoptotic proteins XIAP and Bcl-2. Here, we identified the pro-apoptotic Sept4/ARTS gene as a p53-responsive target gene. Ectopic p53 and a variety of p53-inducing agents increased both mRNA and protein levels of ARTS, whereas ablation of p53 reduced ARTS expression in response to multiple stress conditions. Also, γ-irradiation induced p53-dependent ARTS expression in mice. Consistently, p53 binds to the responsive DNA element on the ARTS promoter and transcriptionally activated the promoter-driven expression of a luciferase reporter gene. Interestingly, ARTS binds to and sequesters p53 at mitochondria, enhancing the interaction of the latter with Bcl-XL. Ectopic ARTS markedly augments DNA damage stress- or Nutlin-3-triggered apoptosis, w...

Inhibitors of apoptosis (IAPs) are a family of proteins that regulate cell death and inflammation. XIAP (X-linked IAP) is the only family member that suppresses apoptosis by directly binding to and inhibiting caspases. On the other hand, cIAPs suppress the activation of the extrinsic apoptotic pathway by preventing the formation of pro-apoptotic signaling complexes. IAPs are negatively regulated by IAP-antagonist proteins such as Smac/Diablo and ARTS. ARTS can promote apoptosis by binding and degrading XIAP via the ubiquitin proteasome-system (UPS). Smac can induce the degradation of cIAPs but not XIAP. Many types of cancer overexpress IAPs, thus enabling tumor cells to evade apoptosis. Therefore, IAPs, and in particular XIAP, have become attractive targets for cancer therapy. In this review, we describe the differences in the mechanisms of action between Smac and ARTS, and we summarize efforts to develop cancer therapies based on mimicking Smac and ARTS. Several Smac-mimetic small ...

The International Conference on Cell Death in Cancer and Toxicology 2018 (February 20-22, 2018) provided an international forum for scientific collaborations across multiple disciplines in cancer, cell death, and toxicology. During the three-day symposium, researchers and clinicians shared recent advances in basic, clinical, and translational research in cancer. Several student poster abstracts were selected for platform talks and many young investigators participated in the meeting. Together, this highly interactive meeting showcased the rapid expansion in biomedical research in India and paved the way for future meetings on cell death and cancer throughout India.

Parkinson's disease (PD) is a movement neurodegenerative disorder characterized by death of dopaminergic neurons in the substantia nigra pars compacta of the brain that leads to movement impairments including bradykinesia, resting tremor, postural instability and rigidity. Mutations in several genes have been associated with familial PD, such as parkin, pink, DJ-1, LRKK2 and α-synuclein. Lately, mutations in the GBA gene were recognized as a major cause for the development of PD.Mutations in the GBA gene, which encodes for lysosomal β-glucocerebrosidase (GCase), lead to Gaucher disease (GD), an autosomal recessive sphingolipidosis characterized by accumulation of glucosylceramide, mainly in monocyte-derived cells. It is a heterogeneous disease, with Type 1 patients that do not present any primary neurological signs, and Type 2 or Type 3 patients who suffer from a neurological disease. The propensity of type 1 GD patients and carriers of GD mutations to develop PD is significantl...

Here we describe a protein product of the human septin H5/PNUTL2/CDCrel2b gene, which we call ARTS (for apoptosis-related protein in the TGF-beta signalling pathway). ARTS is expressed in many cells and acts to enhance cell death induced by TGF-beta or, to a lesser extent, by other apoptotic agents. Unlike related septin gene products, ARTS is localized to mitochondria and translocates to the nucleus when apoptosis occurs. Mutation of the P-loop of ARTS abrogates its competence to activate caspase 3 and to induce apoptosis. Taken together, these observations expand the functional attributes of septins previously described as having roles in cytokinesis and cellular morphogenesis.

Experimental evidences suggest an important role for Fas receptor activation in a wide range of myocardial pathologies, which are associated with a variety of arrhythmias and mechanical dysfunction. Our recent studies have shown that Fas-mediated arrhythmias and mechanical disturbances of ventricular myocytes can be accounted for by activation of the phospholipase C-1,4,5-inositol triphosphate-intracellular calcium release (PLC-1,4,5-IP(3)-[Ca(2+)](i)) cascade, which is responsible for attenuating the transient outward current (I(to)) and augmenting the L-type Ca(2+) current (I(Ca,L)). We have also shown that whereas ventricular myocytes are resistant to Fas-mediated apoptosis, hypoxia predisposes myocytes to apoptosis induced by Fas activation by shifting the balance between pro-apoptotic and anti-apoptotic proteins towards the former. Since protein tyrosine phosphorylation has been shown to be involved in Fas signaling, we have hypothesized that inhibiting tyrosine kinases will attenuate Fas-mediated effects in ventricular myocytes in normoxic and hypoxic conditions. Therefore, we tested the effect of the tyrosine kinases inhibitors, genistein (50 micromol/l) and herbimycin A (50 microg/ml), on the abovementioned Fas-mediated effects in cultured neonatal rat ventricular myocytes (NRVM) and in freshly isolated adult murine ventricular myocytes. Fas receptor was activated by incubating NRVM with recombinant Fas ligand (rFasL, 50 ng/ml) and enhancing antibody (1 microg/ml), or by incubation of murine ventricular myocytes with the Fas-activating antibody Jo2 (10 microg/ml). The major findings were that genistein prevented Fas-mediated increase in 1,4,5-IP(3) production in NRVM (quantified by ion-exchange chromatography): 216 +/- 41 counts per minute (cpm) in control, 605 +/- 184 cpm in FasL-treated cardiomyocytes and 137 +/- 51 cpm in rFasL + genistein-treated cultures. Accordingly, genistein or herbimycin A abolished the diastolic [Ca(2+)](i)-rise (as measured by fura-2 fluorescence) and arrhythmogenic activity in both rat and murine ventricular myocytes, and the Fas-mediated changes in I(to) and I(Ca,L) in murine ventricular myocytes. Importantly, genistein attenuated Fas-mediated apoptosis in hypoxic (22 h in 1% O(2)) NRVM; the apoptotic ratios as measured by DAPI fluorescence assay were: 4.6 +/- 1.0% in control, 12.5 +/- 3.0% in rFasL and 7.3 +/- 1.6% in rFasL + genistein-treated NRVM. This prevention of apoptosis by tyrosine kinases blockade was accompanied by inhibition of hypoxia-induced increased Fas expression and decreased expression of the anti-apoptotic protein xIAP. In conclusion, our findings indicate that tyrosine phosphorylation is involved in Fas signaling in ventricular myocytes, and can, therefore, serve as a novel potential target for attenuating Fas-mediated dysfunction in normoxic and hypoxic myocardium.

Supernatants of lymphokine-activated killer (LAK) cells were highly cytotoxic for melanoma A375 cells. A high-molecular-weight fraction was isolated from such supernatants by gel filtration on an S-300 Sephacryl column (Fraction 1; Fr1). The cytotoxic activity in Fr1 was heat- and acid-resistant and was completely abolished by a rabbit antibody against TGF-beta. We conclude that Fr1 contains TGF-beta or a cross-reactive molecule, associated with a high-molecular-weight carrier.

Supernatants of human lymphokine-activated killer (LAK) cells grown in vitro were tested for cytotoxic activity against several mouse and human neoplastic cell lines. All LAK preparations tested (14/14) exhibited cytotoxic activity (40-90% killing of the target cells). Sephacryl S-300 Gel filtration experiments indicated that the biological activity of the LAK supernatant is associated with molecular moieties ranging from 800 kDa or more, to less than 10 kDa. The finding of strong cytotoxic activity in LAK supernatants against several tumor lines points to the possibility of employing soluble products of these cells, rather than the living cells themselves, for therapeutic purposes.

We showed in previous work that LAK cell supernatants contain a large molecular weight factor with toxic activity for A375 melanoma and other cell lines. The factor, Fr1, was identified tentatively as TGF beta-related, since its activity was abolished by anti-TGF beta serum. This relatedness is further confirmed in the present work, which demonstrates that, like TGF beta, Fr1 stimulates the release and deposition of fibronectin and induces morphological changes indistinguishable from those induced by TGF beta. The TGF beta derived from LAK cells, although associated with a large carrier molecule, is directly acting and does not dissociate from its carrier following gel filtration in acetic acid. Its carrier is different from alpha-2 macroglobulin. SDS-PAGE and immunoblotting showed that FR1 contains TGF beta complexed with large molecules (150 and > 200 kDa), which dissociate in reduced gels to molecules of 60-67 kDa. We interpret these data as showing that TGF beta secreted by LAK cells is, presumably, covalently linked to monomeric carrier molecules of approximately 60 kDa, which, in turn, are S-S bonded to form multimeric molecules of 150 kDa and > 200 kDa.

The murine septin4 gene (Sept4) has been implicated in diverse cellular functions, including cytokinesis, apoptosis, and tumor suppression. Here, we investigated the function of Sept4 proteins during mouse development by creating a targeted deletion of the Sept4 genomic locus. Sept4 mutant mice are viable but male sterile due to immotile and structurally defective sperm. During spermatogenesis, Sept4 proteins were essential for proper mitochondrial architecture and establishment of the annulus, a ring-like structure in the tail region of sperm. In addition, Sept4 mutant sperm showed defects in the elimination of residual cytoplasm during sperm maturation and had increased staining for the caspase inhibitor XIAP. This is consistent with a role of the proapoptotic Sept4 protein ARTS in promoting caspase-mediated removal of cytoplasm via inhibition of XIAP. Our results indicate that Sept4 proteins play distinct but evolutionarily conserved functions in different cellular compartments.

Since apoptosis is an important contributor to heart diseases in which ischemia and hypoxia are key elements, we tested the hypothesis that hypoxia predisposes neonatal rat ventricular myocytes (NRVM) to Fas-mediated apoptosis, by shifting the balance between antiapoptotic and proapoptotic proteins towards the latter. Normoxic or hypoxic (22 h, 1% O(2)) cultured NRVM were exposed to recombinant Fas L (rFasL) for 7 h, and apoptosis measured thereafter. Whereas in normoxic NRVM, rFasL did not cause apoptosis measured by the TUNEL assay (4.8+/-0.5% in control versus 4.5+/-0.9% in rFasL), in hypoxic cultures rFasL increased the background apoptosis level by 100%. That Fas was functional in normoxic NRVM, despite its inability to mediate apoptosis, was evidenced by the finding that Fas activation increased the diastolic [Ca(2+)](i) levels measured by Fura 2 fluorescence, and caused arrhythmias. In support of our working hypothesis, hypoxia increased Fas expression by 200% (measured by quantitative Western blot), and the expression of the proapoptotic proteins ARTS and FADD by 323 and 250%, respectively, and decreased the expression of the antiapoptotic proteins ARC and FLIP by 90 and 60%, respectively. By upregulating Fas expression and key proapoptotic proteins, and by downregulating antiapoptotic proteins, hypoxia predisposes ventricular myocytes to Fas-induced apoptosis.