Apoptosis Repressor With Caspase Research Articles

Conversion of the death inhibitor ARC to a killer activates pancreatic β cell death in diabetes.

Loss of insulin-secreting pancreatic β cells through apoptosis contributes to the progression of type 2 diabetes, but underlying mechanisms remain elusive. Here, we identify a pathway in which the cell death inhibitor ARC paradoxically becomes a killer during diabetes. While cytoplasmic ARC maintains β cell viability and pancreatic architecture, a pool of ARC relocates to the nucleus to induce β cell apoptosis in humans with diabetes and several pathophysiologically distinct mouse models. β cell death results through the coordinate downregulation of serpins (serine protease inhibitors) not previously known to be synthesized and secreted by β cells. Loss of the serpin α1-antitrypsin from the extracellular space unleashes elastase, triggering the disruption of β cell anchorage and subsequent cell death. Administration of α1-antitrypsin to mice with diabetes prevents β cell death and metabolic abnormalities. These data uncover a pathway for β cell loss in type 2 diabetes and identify an FDA-approved drug that may impede progression of this syndrome.

ARC (apoptosis repressor with caspase recruitment domain) deficient mice develops severe knee joint histopathology but demonstrates reduced mechanical allodynia after surgically-induced osteoarthritis

ARC (apoptosis repressor with caspase recruitment domain) deficient mice develops severe knee joint histopathology but demonstrates reduced mechanical allodynia after surgically-induced osteoarthritis

Embryonic cardiomyocytes can orchestrate various cell protective mechanisms to survive mitochondrial stress

Whereas adult cardiomyocytes are highly susceptible to stress, cardiomyocytes in the prenatal heart appear to be rather resistant. To investigate how embryonic cardiomyocytes respond to metabolic stress in vivo, we utilized tissue mosaicism for mitochondrial dysfunction in 13.5dpc mouse hearts. The latter is based on inactivation of the X-linked gene encoding Holocytochrome c synthase (Hccs), which is essential for mitochondrial respiration. In heterozygous heart conditional Hccs knockout females (cHccs+/−) random X chromosomal inactivation results in a mosaic of healthy and HCCS deficient cells in the myocardium. Microarray RNA expression analyses identified genes involved in unfolded protein response (UPR) and programmed cell death as differentially expressed in cHccs+/− versus control embryonic hearts. Activation of the UPR is localized to HCCS deficient cardiomyocytes but does not involve ER stress pathways, suggesting that it is caused by defective mitochondria. Consistently, mitochondrial chaperones, such as HSP10 and HSP60, but not ER chaperones are induced in defective cells. Mitochondrial dysfunction can result in oxidative stress, but no evidence for excessive ROS (reactive oxygen species) production was observed in cHccs+/− hearts. Instead, the antioxidative proteins SOD2 and PRDX3 are induced, suggesting that ROS detoxification prevents oxidative damage in HCCS deficient cardiomyocytes. Mitochondrial dysfunction and unrestricted UPR can induce cell death, and we detected the initiation of upstream events of both intrinsic as well as extrinsic apoptosis in cHccs+/− hearts. Cell death is not executed, however, suggesting the activation of antiapoptotic mechanisms. Whereas most apoptosis inhibitors are either unchanged or downregulated in HCCS deficient cardiomyocytes, Bcl-2 and ARC (apoptosis repressor with caspase recruitment domain) are induced. Given that ARC can inhibit both apoptotic pathways as well as necrosis and attenuates UPR, we generated cHccs+/− embryos on an Arc knockout background (cHccs+/−,Arc−/−). Surprisingly, the absence of ARC does not induce cell death in embryonic or postnatal HCCS deficient cardiomyocytes and adult cHccs+/−,Arc−/− mice exhibit normal cardiac morphology and function. Taken together, our data demonstrate an impressive plasticity of embryonic cardiomyocytes to respond to metabolic stress, the loss of which might be involved in the high susceptibility of postnatal cardiomyocytes to cell death.

Abstract 2266: Investigating the role of the anti-apoptotic protein ARC in breast cancer cell DNA repair

Abstract According to the American Cancer Society, more than 90% of breast cancer patients who succumb to the condition die as a result of the cancer metastasizing to other areas of the body. Our lab has previously shown that the presence of a chemoresistant invasive subpopulation of breast carcinoma cells is correlated with the prevalence of metastasis. Additionally, we have hypothesized that chemoresistance and survival of these cancer cells which eventually leads to relapse and metastasis following initial treatment with chemotherapeutic agents is achieved by enhanced DNA repair capability in this population. We reported that an anti-apoptotic protein ARC (apoptosis repressor with caspase recruitment domain) is over expressed in the invasive mammary cancer cells that are resistant to standard chemotherapeutic agents. Further, we have shown that ARC over expressing cells are resistant to chemotherapy both in vivo and in vitro. In this study, we examined cellular DNA repair signaling pathways in order to delineate the role of ARC in the pathogenesis of breast cancer. Utilizing cell lines where ARC was either knocked down or overexpressed, we investigated potential correlations of ARC with DNA repair capability by means of the single cell gel electrophoresis assay (comet assay). The results of these experiments indicated that over expression of ARC leads to increased cellular DNA repair while knocking it down leads to decreased cellular DNA repair. In order to determine the molecular basis of the role of ARC in the DNA repair pathways, we have performed qRT-PCR based gene expression analysis on ARC modified cell lines using techniques previously established in our lab. The results of these experiments indicated that increased levels of ARC are correlated with upregulation of key genes in single stranded DNA repair pathways such as the base excision and nucleotide excision repair pathways. We are working to identify the specific domain of ARC that serves as its effector in the DNA repair pathways. In particular, we are looking at the caspase recruitment domain (CARD) of ARC which is known to be key for ARC`s function in the apoptotic pathways. Once this domain is identified, it may serve as a potential drug target, the silencing of which could result in the disruption of key pathways required for invasive breast cancer cell survival. Such therapy may facilitate the ablation of chemoresistant breast cancer cell populations, prevent metastasis and tumor relapse, and increase long term patient survival. Citation Format: Eli Grunblatt, Evanka Madan, Sweta Roy, Sumanta Goswami. Investigating the role of the anti-apoptotic protein ARC in breast cancer cell DNA repair. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2266. doi:10.1158/1538-7445.AM2014-2266

ARC Is Regulated by MAPK and PI3K and Confers Drug Resistance and Survival Advantage to AML in vitro and in vivo

Abstract Abstract 893 ARC (Apoptosis repressor with caspase recruitment domain) is a unique antiapoptotic protein that has been shown to suppress the activation of both intrinsic and extrinsic apoptosis. We previously reported that ARC is one of the most potent adverse prognostic factors in AML and that high ARC protein expression predicted shorter survival and poor clinical outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report how ARC is regulated and its role in inhibition of AML apoptosis and in cell survival. We provide evidence that ARC expression is regulated by MAPK and PI3K signaling. Inhibition of MAPK and PI3K pathways decreased ARC mRNA and protein levels in AML cells. ARC expression in AML cells is upregulated in co-cultures with bone marrow-derived mesenchymal stromal cells (MSCs) and the upregulation is suppressed in the presence of MAPK or PI3K inhibitors. To investigate the role of ARC in apoptosis resistance in AML, we generated stable ARC overexpressing (O/E) KG-1 and stable ARC knock down (K/D) OCI-AML3 and Molm13 cells and treated them with Ara-C and agents selectively inducing intrinsic (ABT-737) or extrinsic (TRAIL) apoptosis. We found that ARC O/E cells are more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL-induced apoptosis: EC50s of Ara-C, ABT-737, or TRAIL treatment at 48 hours for ARC O/E KG-1 and control cells were 1.5 ± 0.1 μM vs. 83.5 ± 4.6 nM, 2.2 ± 0.2 μM vs. 60.2 ± 3.1 nM, or 0.97 ± 0.03 μg/mL vs. 0.17 ± 0.08 μg/mL, respectively and for ARC K/D OCI-AML3 and control cells were 0.33 ± 0.02 μM vs. 3.4 ± 0.2 μM, 0.24 ± 0.01 μM vs. 1.3 ± 0.1 μM, or 0.13 ± 0.09 μg/mL vs. 0.36 ± 0.03 μg/mL, respectively. Bone marrow microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from various therapeutic agent-induced apoptosis. Leukemia cells were co-cultured with MSCs in vitro study to mimic the in vivo condition. ARC was found to be highly expressed in MSCs and stable ARC K/D MSCs were generated. AML cell lines and primary patient samples were co-cultured with ARC K/D or control MSCs and treated with Ara-C, ABT-737, or TRAIL. Interestingly, ARC K/D MSCs lost their protective activity for leukemia cells treated with these agents. EC50s for OCI-AML3 cells co-cultured with ARC K/D or control MSCs for 48 hours treated with Ara-C, ABT-737, or TRAIL were 1.0 ± 0.04 μM vs. 4.5 ± 0.2 μM, 0.15 ± 0.06 μM vs. 0.53 ± 0.02 μM, or 1.4 ± 0.8 μg/mL vs. 8.1 ± 0.3 μg/mL, respectively. In addition, ARC O/E KG-1 cells grew faster and ARC K/D OCI-AML3 and Molm13 cells and ARC K/D MSCs grew slower than their respective controls. We then injected KG-1 cells into mice and found that NOD-SCID mice harboring ARC O/E KG-1 had significantly shorter survival than mice injected with the vector control KG-1 (median 84 vs. 111 days) as shown in the figure. Download : Download high-res image (24KB) Download : Download full-size image Collectively, results demonstrate that ARC plays critical roles in AML. ARC is regulated by MSCs through various signaling pathways in AML cells, protects leukemia cells from apoptosis induced by chemotherapy and by agents selectively inducing intrinsic and extrinsic apoptosis. ARC regulates leukemia cell growth in vitro and in vivo. The results suggest that ARC is a potential target for AML therapy. In addition, targeting ARC in MSCs suppresses microenvironmental protection of AML cells. Disclosures: No relevant conflicts of interest to declare.

Apoptosis Repressor with Caspase Recruitment Domain (ARC) Increases AML Cell Migration and Adhesion in Vitro and in Vivo by Regulating Leukemia-Stroma Interactions.

Abstract 2626Hematopoietic cells express a wide range of adhesion molecules and bone marrow (BM) stroma cells produce their corresponding ligands. Through these ligand-receptor pairs, hematopoietic cells interact with their BM microenvironment. The same system is hijacked by AML and often adhesion molecules in leukemia cells and/or their ligands in stroma cells are upregulated, promote leukemia-stroma interactions, and protect leukemia cells from therapeutic agents. Understanding the underlying mechanisms is critical for therapeutic strategies aimed at disrupting leukemia-stroma interactions. For example, pharmacological blockage of the CXCR4-SDF1a axis has been shown to result in chemosensitization of AML cells in vitro, in vivo and in clinical trials (Zeng Z et al., Blood 2009; Uy GL et al., Blood 2012; Andreeff M et al., ASCO 2012).ARC (Apoptosis repressor with caspase recruitment domain) is an antiapoptotic protein. We recently determined ARC expression in samples from 511 newly diagnosed AML patients by reverse-phase protein array and reported that ARC is one of the strongest adverse prognostic markers in AML (Carter BZ et al., Blood 2011). In the same sample set, we also probed the expression of more than 200 additional proteins enabling us to correlate ARC expression with the expressions of other proteins. Surprisingly, ARC expression was correlated with multiple proteins involved in cell adhesion and migration. We generated stable ARC overexpressing (O/E) KG-1 cells, ARC knock down (K/D) OCI-AML3 and Molm13 cells, and ARC K/D BM derived mesenchymal stroma cells (MSCs) to investigate ARC’s roles in leukemia-stroma interactions. Expression levels of FAK, integrinb3, fibronectin, and VLA4 were increased in ARC O/E and decreased in ARC K/D cells, compared to controls. CXCR4 mRNA and cell surface CXCR4 protein were higher in ARC O/E KG-1 (3.80- and 1.53-fold, P<0.01; respectively) and lower in ARC K/D cells (OCI-AML3: 0.43- and 0.70-fold, P=0.01, and Molm13: 0.46- and 0.74-fold, P=0.02, respectively), while levels of SDF1a mRNA as well as secreted SDF-1a protein determined by ELISA (P=0.013) were lower in ARC K/D as compared to control MSCs. Interestingly, MSCs co-cultured with AML cells significantly increased SDF1a secretion and this increase was greatly diminished when ARC was knocked down in MSCs.Migration of leukemic cells towards MSCs was significantly higher for ARC O/E and lower for ARC K/D AML cells (P<0.001) and migration of leukemic cells to ARC K/D MSCs was significantly diminished compared to control MSCs for both, OCI-AML3 (P<0.001) and primary AML cells (18.1±0.8% vs. 32.8±0.5%, n=4, P<0.001). In addition, adhesion to MSCs and to VCAM1 coated plates was increased for ARC O/E and decreased for ARC K/D AML cells (P<0.01). Furthermore, adhesion to ARC K/D MSCs was decreased compared to control MSCs for AML cells for both cell lines (KG-1, 43.6±1.2% vs. 58.2±1.7%; OCI-AML3, 34.8±1.0% vs. 48.7±0.7%; Molm13, 9.2±2.2% vs. 16.8±2.7%) and primary patient samples (52.1±1.6% vs. 66.3±6.4%, P=0.02 and 42.1±5.0% vs. 59.6±7.4%, P=0.03).The role of ARC in leukemia-stroma interactions was further assessed using a novel human extramedullary BM model in mice recently developed by our group (Chen Y et al., Blood 2012). Luciferase/GFP Molm13 cells were injected into Nod/ScidIL2Rg−/− mice after extramedullary human BM containing either ARC K/D or control MSCs was established in the flanks of mice. Significantly fewer AML cells engrafted in the human extramedullary BM developed from ARC K/D than from control MSCs as determined by immunohistochemistry staining for human CD45+ cells and quantitative image analysis (P<0.01).In conclusion, we here demonstrate that antiapoptotic ARC regulates migration and adhesion of leukemia cells to BM stroma in vitro and in vivo via activation of multiple mechanisms, not only in AML cells but also in MSCs, thus enhancing its anti-apoptotic activity. Given the fact that both intrinsic apoptosis resistance and extrinsic environmental factors contribute to drug resistance and relapse in AML, ARC may play a central role in AML and be an excellent therapeutic target: ARC inactivation may disrupt leukemia-stroma interactions and increase leukemia cell sensitivity to chemotherapy by increasing susceptibility to apoptosis. We speculate that ARC, a CARD containing protein, acts at least in part via activating NF-kB signaling, which is known to be regulated by CARD proteins. Disclosures:No relevant conflicts of interest to declare.

Apoptosis Repressor with Caspase Recruitment Domain Is Regulated by the cIAP1-NIK Axis and Confers Resistance to SMAC Mimetic Birinapant-Induced Cell Death in AML

Abstract 534The inhibitors of apoptosis (IAPs), including cIAP1, cIAP2, and XIAP are a family of anti-apoptotic proteins that play important roles in regulating cell survival. SMAC, a mitochondrial protein, is a natural cellular inhibitor of IAPs. SMAC mimetics, mimicking the IAP-binding site in the N-terminal AVPI peptide sequence of SMAC, are a new class of anticancer agents that degrade cIAPs and suppress XIAP activity. ARC (Apoptosis repressor with caspase recruitment domain) is an anti-apoptotic protein that inhibits the activation of caspase-8. We previously reported that the SMAC mimetic birinapant (TL32711; Tetralogic Pharmaceuticals, Malvern, PA) degrades cIAP1 and promotes apoptosis via the death receptor/caspase-8-mediated extrinsic pathway in primary AML cells and in AML cell lines in the presence of death receptor ligands (Carter BZ et al., ASH 2011). High ARC levels also predict adverse outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report that birinapant-induced reduction in cIAP1 is accompanied by increased ARC levels.cIAPs are known E3 ligases for NF-κB-inducing kinase (NIK), an upstream kinase of non-canonical NF-κB. SMAC mimetics, including birinapant cleave cIAPs, leading to stabilization of NIK and activation of non-canonical NF-κB signaling and its downstream targets. To determine whether ARC is regulated via the cIAP1-NIK axis, we knocked down NIK in OCI-AML3 and Molm13 cells by siRNAs and found that inhibition of NIK decreased ARC RNA and protein levels in these cells and suppressed birinapant-induced increases of ARC, suggesting that ARC is regulated via the cIAP1/NIK/NF-κB cascade. We determined levels of ARC and cIAP1 by reverse-phase protein array in 511 samples obtained from patients with newly diagnosed AML and found that cIAP1 and ARC were inversely correlated (R = −0.225, P< 0.0001) further supporting the negative regulation of ARC by cIAP1 in primary AML samples.Data indicate that birinapant induces caspase-8-mediated cell death, but increases levels of ARC in AML cells which inhibits caspase-8 activation, suggesting that ARC is a resistance factor for birinapant-induced cell death. To further investigate this mechanism, we generated stable ARC-knock down (K/D) OCI-AML3 and Molm13 cells and stable ARC-overexpressing (O/E) KG-1 cells and treated these cells with birinapant or birinapant plus TNFα. We found what ARC-K/D OCI-AML3 and Molm13 cells were more sensitive and ARC-O/E KG-1 cells were more resistant to birinapant- or birinapant plus TNFα-induced apoptosis than their control cells. We reported previously that demethylating agents can enhance birinapant-induced apoptosis induction in AML cells. Examination of NIK and ARC levels in decitabine or 5-azacytidine treated AML cells showed that the demethylating agents indeed decreased NIK and ARC protein levels.Leukemia cells are in close contact with the bone marrow (BM) microenvironment in vivo that protects them from cell death induced by various therapeutic agents. Leukemia cells were co-cultured with BM-derived mesenchymal stromal cells (MSCs) in vitro to mimic in vivo conditions. We found that birinapant decreased cIAP1 and increased ARC levels also in MSCs co-cultured with AML cells. We generated stable ARC-K/D MSCs and treated KG-1, OCI-AML3, and Molm13 cells co-cultured with ARC-K/D or vector control MSCs with birinapant plus TNFα and primary AML patient samples co-cultured with ARC-K/D or vector control MSCs with birinapant. ARC-K/D MSCs provided AML cells with less protection than control MSCs against birinapant plus TNFα- or birinapant-induced apoptosis.Collectively, data demonstrate that ARC is regulated via the cIAP1/NIK signaling pathway and is a resistance factor for SMAC mimetic birinapant-induced cell death. ARC K/D sensitizes AML cells to SMAC mimetic-induced cell death and also suppresses MSC-mediated protection of AML cells against drug-induced apoptosis. Disclosures:No relevant conflicts of interest to declare.

802 Overcoming Imatinib Resistance by Targeting XIAP and ARC (Apoptosis Repressor with Caspase Recruitment Domain) in Blast Crisis CML Cells

802 Overcoming Imatinib Resistance by Targeting XIAP and ARC (Apoptosis Repressor with Caspase Recruitment Domain) in Blast Crisis CML Cells

Expression of ARC (apoptosis repressor with caspase recruitment domain), an antiapoptotic protein, is strongly prognostic in AML

AbstractRegulators of apoptosis in acute myeloid leukemia (AML) have been extensively studied and are considered excellent therapeutic targets. Apoptosis repressor with caspase recruitment domain (ARC), an antiapoptotic protein originally found to be involved in apoptosis of cardiac cells, was recently demonstrated to be overexpressed in several solid tumors. To assess its importance in AML, we profiled ARC expression in 511 newly diagnosed AML patients using a validated robust reverse-phase protein array and correlated ARC levels with clinical outcomes. ARC was variably expressed in samples from patients with AML. ARC level was not associated with cytogenetic groups or with FLT-3 mutation status. However, patients with low or medium ARC protein levels had significantly better outcomes than those with high ARC levels: longer overall survival (median, 53.9 or 61.6 vs 38.9 weeks, P = .0015) and longer remission duration (median, 97.6 or 44.7 vs 31.1 weeks, P = .0007). Multivariate analysis indicated that ARC was a statistically significant independent predictor of survival in AML (P = .00013). Inhibition of ARC promoted apoptosis and sensitized cytosine arabinoside-induced apoptosis in OCI-AML3 cells. These results suggest that ARC expression levels are highly prognostic in AML and that ARC is a potential therapeutic target in AML.

Targeting XIAP and ARC (apoptosis repressor with caspase recruitment domain) Overcomes Imatinib Resitance In Blast Crisis CML Cells.

AbstractAbstract 3415The advent of imatinib, a Bcr-Abl tyrosine kinase inhibitor (TKI) has revolutionized the treatment of patients with CML. Development of resistance and limited activity in blast crisis (BC) CML are evolving problems facing this therapy. We found that XIAP, a potent caspase inhibitor, is highly expressed in CML cells, in both, cell lines and patient samples. Treatment with imatinib deceased XIAP levels in imatinib-sensitive KBM5 but much less so in imatinib-resistant KBM5STI571 cells (harboring T315I mutation) suggesting that XIAP expression in CML is regulated at least in part via Bcr-Abl and that targeting XIAP may promote cell death in CML cells by circumventing imatinib resistance. To test this, we treated BC CML cells with XIAP antisense oligonucleotide (ASO) and with SMAC mimetic ABT-10 and found that inhibition of XIAP induced apoptotic cell death with similar efficacy in KBM5 cells and KBM5STI517 cells (EC50=6.3±0.3 μM and 8.4±0.4 μM at 48 hours, respectively for ABT-10). However, we noted that inhibition of XIAP by ASO induced the expression, in both KBM5 and KBM5STI571 cells, of apoptosis repressor with caspase recruitment domain (ARC) in both mRNA and protein levels but not the expression of Bcl-2 protein. ARC is a unique antiapoptotic protein. It acts through inhibiting caspases and antagonizing the activity and function of p53 and Bax. Therefore, its induction may antagonize the effect of XIAP downregulation. Indeed, inhibition of both XIAP and ARC by ASO induced significantly more cell death than inhibiting either protein alone in both KBM5 and KBM5STI cells. Furthermore, we demonstrated that XIAP inhibition induced-apoptosis was enhanced by imatinib in KBM5, but not in KBM5STI cells. Interestingly, inhibition of Bcr-Abl tyrosine kinase by imatinib not only decreased XIAP, but also suppressed ARC levels in KBM5 but had minimal effects on the levels of these proteins in KBM5STI571 cells and enforced expression of the Bcr-Abl p185 fusion protein (in HL-60 cells) greatly increased both XIAP and ARC levels. This induction was inhibited by imatinib suggesting that ARC is also a downstream target of Bcr-Abl tyrosine kinase. Therefore, imatinib enhancing XIAP inhibition induced-apoptosis in KBM5, not KBM5STI cells can be explained at least in part by its ability to decrease XIAP and ARC levels.In conclusion, XIAP is highly expressed in CML cells and upregulated by Bcr-Abl. Targeting XIAP promotes death of BC and TKI resistant CML cells. Results suggest that XIAP is a potential target in BC and TKI resistant CML cells and that XIAP inhibition-induced apoptosis is enhanced by imatinib in TKI sensitive cells and by ARC inhibition independent of cellular responses to TKIs. Inhibition of XIAP and ARC as a novel therapeutic strategy in CML warrants further investigation. Disclosures:Koller:Isis Pharmaceuticals: Employment.

Mitochondrial fission leads to Smac/DIABLO release quenched by ARC

Apoptosis plays a critical role for the development of a variety of cardiac diseases. Cardiomyocytes are enriched in mitochondria, while mitochondrial fission can regulate apoptosis. The molecular mechanism governing cardiomyocyte apoptosis remain to be fully elucidated. Our results showed that Smac/DIABLO is necessary for apoptosis in cardiomyocytes, and it is released from mitochondria into cytosol in response to apoptotic stimulation. Smac/DIABLO release is a consequence of mitochondrial fission mediated by dynamin-related protein-1 (Drp1). Upon release Smac/DIABLO binds to X-linked inhibitor of apoptosis protein (XIAP), resulting in the activation of caspase-9 and caspase-3. Their activation is a prerequisite for the initiation of apoptosis because the administration of z-LEHD-fmk and z-DQMD-fmk, two relatively specific inhibitors for caspase-9, and caspase-3, respectively, could significantly attenuate apoptosis. Smac/DIABLO release could not be blocked by these caspase inhibitors, indicating that it is an event upstream of caspase activation. ARC (apoptosis repressor with caspase recruitment domain), an abundantly expressed apoptotic repressor in cardiomyocytes, could inhibit mitochondrial fission and Smac/DIABLO release. Our data reveal that Smac/DIABLO is a target of ARC in counteracting apoptosis.

Induction of the Apoptosis Inhibitor ARC by Ras in Human Cancers

Inhibition of apoptosis is critical for carcinogenesis. ARC (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that antagonizes both intrinsic and extrinsic apoptosis pathways. Although normally expressed in striated myocytes and neurons, ARC is markedly induced in a variety of primary human epithelial cancers and renders cancer cells resistant to killing. The mechanisms that mediate the induction of ARC in cancer are unknown. Herein we demonstrate that increases in ARC abundance are stimulated by Ras through effects on transcription and protein stability. Overexpression of activated N-Ras or H-Ras in normal cells is sufficient to increase ARC mRNA and protein levels. Similarly, transgenic expression of activated H-Ras induces ARC in both the normal mammary epithelium and resulting tumors of intact mice. Conversely, knockdown of endogenous N-Ras in breast and colon cancer cells significantly reduces ARC mRNA and protein levels. The promoter of the Nol3 locus, encoding ARC, is activated by N-Ras and H-Ras in a MEK/ERK-dependent manner. Ras also stabilizes ARC protein by suppressing its polyubiquitination and subsequent proteasomal degradation. In addition to the effects of Ras on ARC abundance, ARC mediates Ras-induced cell survival and cell cycle progression. Thus, Ras induces ARC in epithelial cancers, and ARC plays a role in the oncogenic actions of Ras.

Kazrin F is involved in apoptosis and interacts with BAX and ARC

Kazrin has recently been identified as a functional protein that is involved in cell-cell junctions and in signal transduction. Here, we identified a new isoform, Kazrin F, which is 518 aa in length and has 97 aa unique at the N-terminus. Knockdown of Kazrin F using siRNA caused cell apoptosis and a marked decrease in cell viability measured by MTT and TUNEL assays. Co-immunoprecipitation analysis revealed that Kazrin F interacts with ARC (apoptosis repressor with caspase recruitment domain) and Bax (Bcl-2-associated X protein). Co-localization of Kazrin F with ARC and Bax in the cytoplasm was determined by immunofluorescence analysis. These results suggested that Kazrin F might play an important role in regulating cellular apoptosis by interacting with ARC and Bax.

ARC (apoptosis repressor with caspase recruitment domain) is a novel marker of human colon cancer

The ability of cells to escape apoptosis is critical for carcinogenesis as well as resistance to radiation and chemotherapy. ARC (Apoptosis Repressor with CARD (caspase recruitment domain)) is an unusual inhibitor of apoptosis in that it antagonizes both the extrinsic (death receptor) and intrinsic (mitochondrial/ER) apoptosis pathways. ARC is expressed predominantly in terminally differentiated cells such as cardiac and skeletal myocytes and neurons. Recently, however, the abundance of ARC was noted to be markedly increased in the epithelium of primary human breast cancers compared with benign breast tissue and to confer chemo- and radiation-resistance. Whether the induction of ARC is specific to breast cancer or a more general feature of neoplasia remains unknown. In this study, we assessed the abundance and subcellular localization of ARC in 21 human colon cancer cell lines and in 44 primary human colon adenocarcinomas and adjacent benign colonic tissue. ARC was present at high levels in most colon cancer cell lines and in almost all primary colon cancers compared with corresponding controls. Levels of ARC in the cytoplasm were increased in well, moderately, and poorly differentiated cancers compared with benign tissue, while levels of nuclear ARC were increased only in moderately differentiated tumors. Moreover, epithelial cancers of the ovary and cervix exhibited increased ARC abundance compared with controls. These results demonstrate that ARC is a novel marker of human colon cancer and suggest that it may be a feature of epithelial cancers.

Ubiquitination and Degradation of the Anti-apoptotic Protein ARC by MDM2

Current evidence shows that cardiomyocyte apoptosis plays a central role in the pathogenesis of myocardial disease and that reactive oxygen species is critically responsible for mediating cardiomyocyte apoptosis in both ischemia-reperfusion injury and dilated cardiomyopathy. ARC (Apoptosis Repressor with Caspase recruitment domain) is an anti-apoptotic protein that is found abundantly in terminally differentiated cells such as cardiomyocytes. The ARC knock-out mouse developed larger infarct in response to ischemia-reperfusion and transitioned more rapidly and severely to dilated cardiomyopathy following aortic constriction. In addition, ARC protein levels are decreased in human dilated cardiomyopathy and when cardiomyocytes are exposed to oxidative stress in vitro, but the mechanisms regulating ARC protein levels are not known. Here we show that degradation of ARC is dependent on the p53-induced ubiquitin E3 ligase, MDM2. Oxidative stress reduced ARC levels and up-regulated MDM2. MDM2 directly accelerated ARC protein turnover via ubiquitination and proteasomal-dependent degradation. This activity requires a functioning MDM2 ring finger domain because the MDM2(C464A) mutant was unable to direct ARC degradation. Furthermore, ARC degradation requires MDM2, because MDM2 knock-out fibroblasts showed defective ARC degradation that could be rescued by MDM2. Proteasomal inhibitors rescued both MDM2 and H(2)O(2)-induced degradation of ARC and inhibited cardiomyocyte apoptosis. Dilated cardiomyopathic hearts from mice that have undergone transverse aortic banding have increased MDM2 levels associated with decreased ARC levels. We conclude that MDM2 is a critical regulator of ARC levels in cardiomyocytes. Prevention of MDM2-induced degradation of ARC represents a potential therapeutic target to prevent cardiomyocyte apoptosis.

The Apoptosis Inhibitor ARC Undergoes Ubiquitin-Proteasomal-mediated Degradation in Response to Death Stimuli: IDENTIFICATION OF A DEGRADATION-RESISTANT MUTANT

Efficient induction of apoptosis requires not only the activation of death-promoting proteins but also the inactivation of inhibitors of cell death. ARC (apoptosis repressor with caspase recruitment domain) is an endogenous inhibitor of apoptosis that antagonizes both central apoptosis pathways. Despite its potent inhibition of cell death, cells that express abundant ARC eventually succumb. A possible explanation is that ARC protein levels decrease dramatically in response to death stimuli. The mechanisms that mediate decreases in ARC protein levels during apoptosis and whether these decreases initiate the subsequent cell death are not known. Here we show that endogenous ARC protein levels decrease in response to death stimuli in a variety of cell contexts as well as in a model of myocardial ischemia-reperfusion in intact mice. Decreases in ARC protein levels are not explained by alterations in the abundance of ARC transcripts. Rather, pulse-chase experiments show that decreases in steady state ARC protein levels during apoptosis result from marked destabilization of ARC protein. ARC protein destabilization, in turn, is mediated by the ubiquitin-proteasomal pathway, as mutation of ARC ubiquitin acceptor residues stabilizes ARC protein and preserves its steady state levels during apoptosis. In addition, this degradation-resistant ARC mutant exhibits improved cytoprotection. We conclude that decreases in ARC protein levels in response to death stimuli are mediated by increased ARC protein degradation via the ubiquitin-proteasomal pathway. Moreover, these data demonstrate that decreases in ARC protein levels are a trigger, and not merely a consequence, of the ensuing cell death.

CK2 Inhibits Apoptosis and Changes Its Cellular Localization Following Ionizing Radiation

In this study, we show that CK2 (casein kinase II, CKII) participates in apoptotic responses following ionizing radiation (IR). Using HeLa human cervical carcinoma cells, we find that transfection of small interfering RNA against the CK2 alpha and/or alpha' catalytic subunits results in enhanced apoptosis following IR damage as measured by flow cytometry techniques, compared with a control small interfering RNA. Within 2 to 6 hours of IR, CK2 alpha partially localizes to perinuclear structures, whereas a marked nuclear localization of alpha' occurs. Treatment with a pan-caspase inhibitor or transfection of ARC (apoptosis repressor with caspase recruitment domain) suppresses the apoptotic response to IR in the CK2-reduced cells, indicating involvement of caspases. Additionally, we find that CK2 alpha and/or alpha' reduction affects cell cycle progression independent of IR damage in this human cell line. However, the G2-M checkpoint following IR is not affected in CK2 alpha- and/or alpha'-reduced cells. Thus, our data suggest that CK2 participates in inhibition of apoptosis and negatively regulates caspase activity following IR damage.

Characterization of ARC, apoptosis repressor interacting with CARD, in normal and dystrophin-deficient skeletal muscle.

Duchenne muscular dystrophy is an X-linked recessive disorder, primarily characterized by progressive muscle weakness and wasting. The disease results from the absence of dystrophin, however the precise molecular mechanisms leading to muscle pathology are poorly understood. Dystrophic muscles undergo increased oxidative stress and altered calcium homeostasis, which may contribute to myofiber loss by triggering both necrosis and apoptosis. Recent studies have identified ARC (apoptosis repressor with caspase recruitment domain) as an abundant protein in human muscle that can inhibit both hypoxia and caspase-8-induced apoptosis as well as protect cells from oxidative stress. To explore a potential role for ARC in protecting muscle fibers from dystrophic breakdown, we have cloned and characterized murine ARC and studied its expression in normal and dystrophic mouse muscle. ARC is expressed at high levels in striated muscle and displays fiber-type restricted expression patterns. ARC expression levels are normal in dystrophic mdx mice, although the intracellular localization pattern of ARC is slightly altered compared with normal muscles. Overexpression of ARC in transgenic mdx mice failed to alleviate the dystrophic pathology in skeletal muscles, suggesting that misregulation of the molecular pathways regulated by ARC does not significantly contribute to myofiber death.

The Apoptotic Regulatory Protein ARC (Apoptosis Repressor with Caspase Recruitment Domain) Prevents Oxidant Stress-mediated Cell Death by Preserving Mitochondrial Function

ARC is an apoptotic regulatory protein expressed almost exclusively in myogenic cells. It contains a caspase recruitment domain (CARD) through which it has been shown to block the activation of some initiator caspases. Because ARC also blocks caspase-independent events associated with apoptosis, such as hypoxia-induced cytochrome c release, we examined its role in cell death triggered by exposure to hydrogen peroxide (H(2)O(2)) in the myogenic cell line, H9c2. Cell death in this model was caspase-independent and characterized by dose-dependent reduction in ARC expression accompanied by disruption of the mitochondrial membrane potential (Delta psi(m)) and loss of plasma membrane integrity, typical of necrotic cell death. Ectopic expression of ARC prevented both H(2)O(2)-induced mitochondrial dysfunction and cell death without affecting the stress kinase response, suggesting that ARCs protective effects were downstream of early signaling events and not due to quenching of H(2)O(2). ARC was also effective in blocking H(2)O(2)-induced loss of membrane integrity and/or disruption of Delta psi(m) in two human cell lines in which it is not normally expressed. These results demonstrate that, in addition to its ability to block caspase-dependent and -independent events in apoptosis, ARC also prevents necrosis-like cell death via the preservation of mitochondrial function.

Endogenous and exogenous ARC in serum withdrawal mediated PC12 cell apoptosis: a new pro-apoptotic role for ARC.

The role of ARC (Apoptosis Repressor with Caspase Recruitment Domain) in PC12 cell serum withdrawal driven apoptosis was studied. A progressive and massive increase in ARC protein occurs during serum withdrawal that correlates with declining survival and processing of caspase-2, previously shown to associate with ARC. This accumulation of ARC occurs in a transcriptional and translational independent manner. Additionally, ARC is localized exclusively in the nucleus of PC12 cells. Furthermore, transfection of PC12 cells with hARC-Flag promotes death and fails to protect the cells from apoptosis by serum withdrawal. Therefore, ARC functions in a pro-apoptotic manner in PC12 cell serum withdrawal induced apoptosis.