Repression Of Anti-apoptotic Genes Research Articles

KDM2B, an H3K36-specific demethylase, regulates apoptotic response of GBM cells to TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells. TRAIL resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at the epigenetic level is not understood. In this study, we investigated novel epigenetic mechanisms regulating TRAIL response in glioblastoma multiforme (GBM) cells by a short-hairpin RNA loss-of-function screen. We interrogated 48 genes in DNA and histone modification pathways and identified KDM2B, an H3K36-specific demethylase, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity, the activation of caspase-8, -3 and -7 and PARP cleavage. KDM2B knockdown also accelerated the apoptosis, as revealed by live-cell imaging experiments. To decipher the downstream molecular pathways regulated by KDM2B, levels of apoptosis-related genes were examined by RNA-sequencing upon KDM2B loss, which revealed derepression of proapoptotic genes Harakiri (HRK), caspase-7 and death receptor 4 (DR4) and repression of antiapoptotic genes. The apoptosis phenotype was partly dependent on HRK upregulation, as HRK knockdown significantly abrogated the sensitization. KDM2B-silenced tumors exhibited slower growth in vivo. Taken together, our findings suggest a novel mechanism, where the key apoptosis components are under epigenetic control of KDM2B in GBM cells.

EPIG-01THE FUNCTION OF CHROMATIN-MODIFYING ENZYMES IN GBM CELL APOPTOSIS

Emerging evidence suggests that apoptotic resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at an epigenetic level is not understood. In this study, we aimed to identify novel epigenetic mechanisms regulating the response of Glioblastoma Multiforme (GBM) cells to a pro-apoptotic ligand and an emerging therapeutic candidate, TRAIL. To this end, we conducted a short-hairpin RNA (shRNA) screen to assess the function of chromatin modifying-enzymes in TRAIL-induced apoptosis. We employed 2-3 shRNAs/gene to interrogate the role of 47 genes in DNA and histone modification pathways. Accordingly, we tested shRNAs targeting DNA-methyltransferases (DNMTs), Histone-Methyltransferases (HMTs), Histone demethylases (HDMs), Methyl-DNA binding proteins, and Polycomb complex members (PrC). Upon infection with individual shRNAs, U87MG cells were tested for their TRAIL response using cell viability assays. As a result, we identified KDM2B, an H3K36-specific HDM, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity and accelerated the apoptosis process, as revealed by live cell imaging experiments. In order to decipher the downstream molecular pathways regulated by KDM2B, we examined expression levels of apoptosis-related genes by RNA-sequencing and quantitative PCR, and observed de-repression of pro-apoptotic genes and repression of anti-apoptotic genes upon KDM2B silencing. We also observed attenuated tumor growth upon KDM2B silencing in vivo. Taken together, our findings suggest a novel mechanism where the key apoptosis components are under epigenetic control of KDM2B in GBM cells. Therefore, identifying the direct targets of KDM2B might provide a new understanding of apoptosis response in GBMs and aid in the design of successful pro-apoptotic therapies.

Abstract 4675: GR activation modifies ER transcriptional activity and results in decreased estrogen-mediated breast cancer cell proliferation

Abstract Approximately 75% of breast cancers (BC) express estrogen receptor-alpha (ER). The proliferative and anti-apoptotic actions of estrogen signaling in ER+ BC are well-established. Our laboratory recently reported that approximately 40% of ER+ breast cancers express significant glucocorticoid receptor (GR) mRNA; a meta-analysis of early-stage ER+ BC gene expression unexpectedly found that patients with tumors expressing high GR had significantly improved relapse-free survival (p-value 7.8E-8). Consistent with the improved outcome observed in ER+/GR+ BC patients, our laboratory and others have reported that GR activation can alter ER-mediated transcription and also antagonize estradiol (E2)-mediated cell proliferation. We therefore hypothesized that GR activation directly antagonizes the expression of ER-mediated pro-proliferative genes. Recapitulating observations reported by others, we first performed cell cycle analysis and found that the synthetic glucocorticoid dexamethasone (Dex) decreased E2-mediated proliferation of MCF-7 BC cells. To understand how GR activation inhibits ER-mediated pro-proliferative activity in ER+ BC cells, we performed chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to examine global GR chromatin occupancy in MCF-7 cells in the presence or absence of ER activation (+/- E2 at 10 nM). Surprisingly, in GR ChIP experiments we observed that in the presence of Dex, E2 treatment resulted in significantly increased GR association with ER response elements (EREs). Likewise, ER ChIP experiments showed that in the presence of E2, Dex treatment significantly increased the association of ER with GR response elements (GREs). Using genome-wide gene expression array analyses of MCF-7 cells, the gene expression signatures resulting from Dex and E2 co-treatment compared to E2 treatment alone are being examined. In analyses done to date, we found that in the presence of E2, Dex treatment inhibits the induction of ER-mediated pro-proliferative genes related to the cell cycle such as CCND1 (Cyclin D1) and CDC6. Furthermore, microarray analysis suggested that E2-mediated ER activation represses pro-apoptotic CASP7 and BIK gene expression, while co-treatment with Dex inhibits this repression. Additionally, anti-GR ChIP-seq analysis showed GR enrichment at CASP7 regulatory regions upon co-treatment with Dex and E2. Together, these data suggest that GR expression and subsequent GR activation may inhibit ER-mediated pro-proliferative gene expression as well as inhibit ER-mediated repression of anti-apoptotic genes through direct GR-chromatin interaction. These experiments suggest that GR activation can be an important component modulating ER activity in ER+/GR+ BC. Citation Format: Diana C. West, Charles F. Pierce, Deng Pan, Kyle Hernandez, Masha Kocherginsky, Suzanne D. Conzen. GR activation modifies ER transcriptional activity and results in decreased estrogen-mediated breast cancer cell proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4675. doi:10.1158/1538-7445.AM2015-4675

CD25 Blockade protects T Cells from Activation‐induced Cell Death (AICD) via Maintenance of TOSO Expression

CD25 monoclonal antibody binding to the alpha-chain of the Interleukin-2 (IL-2) receptor, blocks high-affinity IL-2 binding, thereby preventing complete T-cell activation and being of ample importance in transplantation medicine and potentially the treatment of autoimmune disease. However, CD25 antibodies do not only block T-cell activation but also prevent activation-induced cell death (AICD) attributing a dual function to IL-2. In this study, the modulation of the genomic expression profile of human peripheral blood mononuclear cells (PBMC) with therapeutic concentrations of humanized anti-CD25 mAb was investigated. PBMC were stimulated with CD3 antibody OKT-3 together with recombinant IL-2 in the absence or presence of anti-CD25 mAb. RNA was extracted and subjected to microarray analysis on U133A microarrays (Affymetrix). Anti-CD25 treatment inhibited several genes typically expressed during T-cell activation including granzyme B, signalling lymphocyte activation molecule, family member 1 (SLAMF1), CD40-Ligand (CD40-L), IL-9 and interferon (IFN)-gamma. Interestingly, anti-CD25 mAb also blocked the expression of several genes important for susceptibility to apoptosis, such as death receptor 6 (DR6) or reversed IL-2-mediated repression of anti-apoptotic genes, such as Fas apoptotic inhibitory molecule 3 (FAIM3)/TOSO. Functional significance of DR6 and TOSO expression in IL-2-dependent T-cell activation was subsequently evaluated by RNA interference in AICD: While siRNA specifically directed against DR6 did not modulate FAS-L-mediated apoptosis induction in primary T cells, down-regulation of TOSO significantly increased susceptibility to apoptosis, emphasizing an important role for TOSO in IL-2-mediated AICD.

Impaired Expression of Peripheral Blood Apoptotic‐Related Gene Transcripts in Acute Multiple Sclerosis Relapse

Differential expression of apoptotic genes may influence the susceptibility of activated lymphocytes to expand and induce acute relapse and persistent inflammation in patients with relapsing-remitting multiple sclerosis (RRMS). The exact relationship between alterations in apoptotic-related gene expression and clinical disease activity has not been broadly evaluated. In this study we studied peripheral blood mononuclear cells (PBMCs) expression of pro- and antiapoptotic genes in RRMS patients during acute relapse in comparison to patients in remission. Using cDNA Affymetrix microarrays platform (U133A2 microarrays) we analyzed the gene expression profile of PBMC derived from 22 RRMS patients in acute relapse (15 females, mean age 34.6 +/- 1.8 years, disease duration 5.6 +/- 0.8 years) in comparison to 20 sex- and age-matched RRMS patients in remission. One thousand five hundred seventy-eight gene transcripts significantly differentiated acute multiple sclerosis (MS) relapse from remission. This characteristic gene expression signature was enriched by an apoptotic-related pathway. The 1578 gene transcripts that significantly differentiated acute relapse from remission were enriched by 55 apoptotic-related genes in that reflected different operating pathways during the acute phase of the disease. These genes mainly involved the caspase-dependent pathway and included overexpression of the negative regulator of FAS-induced apoptosis (TOSO) and the BCL2 antiapoptotic family members (BCL2, BCL2 AA) as well as downexpression of proapoptotic genes like BAX, apoptotic protease-activating factor 1 (APAF1) and caspases 1, 2, 8, 9. and 10. An additional group of antiapoptotic genes related to T cell receptor-mediated apoptosis was also found to be overexpressed in acute relapse and included TCR-binding CD3E antigen, antiapoptotic serine threonin kinase (AKT), and NF kappa B-associated genes like reticuloendotheliosis viral oncogene homolog A (RELA) and human T cell leukemia virus type I-binding protein (Tax1BP) known to inhibit tumor necrosis factor (TNF)-induced apoptosis. Our findings demonstrate impaired apoptotic mechanisms in peripheral lymphocytes from RRMS patients during acute relapse. This suggests that the inflammatory process in active disease is targeted by inhibition of proapoptotic and repression of antiapoptotic genes that allow prolonged abnormal immune responses.

Repression of anti-apoptotic genes via AP-1 as a mechanism of apoptosis induction in ventricular cardiomyocytes

Nitric oxide (NO) is increased under several pathophysiological, mainly inflammatory processes in the heart and has been characterized as an inducer of apoptosis in cardiomyocytes. The transcription factor activating protein-1 (AP-1) has been identified as a mediator of NO-induced apoptosis. Genes that are regulated by AP-1 under apoptotic conditions have not been identified yet. Therefore, we performed a microarray analysis with subsequent real-time polymerase chain reaction (PCR) to identify genes regulated by AP-1 in NO-induced ventricular cardiomyocytes of rats and tested the functional role of these genes in apoptosis. Cardiomyocytes were transformed with AP-1 decoy oligonucleotides for inhibition of AP-1 activity. These, as well as non-transformed control cells, were stimulated with the NO donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 100 microM) for 2 h. Some of the genes with differential gene expression on microarrays were further analysed by real-time PCR. Genes that are induced by SNAP were not identified. However, four genes, pyridoxal kinase, heat shock protein 10 (Hsp10), antigen identified by monoclonal antibodies 4F2 (4F2) and myosin light chain 2, were downregulated by SNAP in presence of AP-1. Pyridoxal kinase, Hsp10 and 4F2 have anti-apoptotic effects in unstimulated cells because downregulation of their expression by antisense oligos induced apoptosis in cardiomyocytes. An involvement of these genes in NO-induced apoptosis could only be proven for pyridoxal kinase. In conclusion, using microarray technology, we identified three anti-apoptotic genes (Hsp10, 4F2 and pyridoxal kinase) in ventricular cardiomyocytes, which may help the cells to resist some apoptotic stimuli. The downregulation of these genes results in cardiomyocyte apoptosis. Prevention of their downregulation may protect cardiomyocytes against apoptotic stimuli, and this may be of therapeutic benefit.

Antiproliferative and apoptotic effects of zinc–citrate compound (CIZAR®) on human epithelial ovarian cancer cell line, OVCAR-3

Objective. Zinc inhibits the growth of several carcinoma cells through induction of cell cycle arrest and apoptosis. The intracellular concentration of zinc and its dynamic changes are critically important in cell biology. We investigated the effects of zinc–citrate compound (CIZAR®) on normal human ovarian epithelial cells (NOSE) and human epithelial ovarian cancer cell line, OVCAR-3. Methods. To investigate the potential effect of CIZAR® on cell growth and survival, cells were treated with different doses and exposed to different times. Intracellular concentration of zinc was measured by colorimetric assay. Mitochondrial aconitase activity was determined in cell extracts using aconitase assay. The flow cytometric assay, DNA laddering, and morphological analysis were done to investigate cytotoxic effects of CIZAR®. Molecular mechanism of cell death was investigated by p53, Bcl-xL, Bcl-2, Bax protein, activity of caspase-3 and -12, and activity of telomerase. Results. CIZAR®-induced zinc accumulation in OVCAR-3 cells was higher than that in NOSE cells. CIZAR® treatment resulted in a time- and dose-dependent decrease in cell number in OVCAR-3 cells in comparison with NOSE cells. M-aconitase activity was significantly decreased in OVCAR-3 cells within 4 h exposure to CIZAR® but relatively constant in NOSE cells. The flow cytometric assay, DNA laddering, and morphological analysis indicated apoptosis in OVCAR-3 cells but not in NOSE cells. CIZAR® increased the expression of p21 waf1 which is a part of p53-independent pathway and induced reduction of telomerase activity. CIZAR® reduced expression of Bcl-2 and Bcl-xL proteins but induced expression of Bax protein. CIZAR® induced apoptosis of OVCAR-3 cells by activation of caspase-12 and caspase-3 pathway. Conclusions. Exposure to CIZAR® induces apoptosis in OVCAR-3 cells which accumulate high intracellular levels of zinc, but not in NOSE cells, which do not accumulate high levels of zinc. CIZAR® prevents the proliferation of OVCAR-3 cells by inactivation of m-aconitase activity and induces apoptosis by induction of proapoptotic gene (Bax), repression of antiapoptotic genes (Bcl-2, Bcl-xL), and consequently activation of caspase-3. CIZAR® also induced activation of caspase-12. The CIZAR® will offer new window in prevention and treatment of epithelial ovarian cancer.

NFκB-dependent Down-regulation of Tumor Necrosis Factor Receptor-associated Proteins Contributes to Interleukin-1-mediated Enhancement of Ultraviolet B-induced Apoptosis

Activation of the transcription factor nuclear factor-kappaB (NFkappaB) by inflammatory cytokines like tumor necrosis (TNF) factor and interleukin-1 (IL-1) is generally associated with the induction of antiapoptotic pathways. Therefore, NFkappaB inhibits both intrinsically and extrinsically induced apoptosis and thus is regarded to act universally in an antiapoptotic fashion. Accordingly, activation of NFkappaB by IL-1 was shown to result in reduction of death ligand-induced apoptosis via up-regulation of antiapoptotic inhibitor of apoptosis proteins (IAPs). In contrast, apoptosis induced by ultraviolet-B radiation (UVB) was shown to be enhanced in an NFkappaB-dependent manner, indicating that NFkappaB can also act in a proapoptotic fashion. This study investigates the molecular mechanisms underlying IL-1-mediated enhancement of UVB-induced apoptosis. We show that NFkappaB activation in costimulation with UVB treatment results in repression of antiapoptotic genes and consequently in down-regulation of the respective proteins, like c-IAP, FLICE-inhibitory protein (FLIP), and some members of the TNF receptor-associated (TRAF)2 protein family. In parallel, TNFalpha is released, leading to activation of signaling pathways mediated by TNF receptor-1 (TNF-R1). Although TNF is well known to induce both proapoptotic and antiapoptotic effects, the down-regulated levels of TRAF-1, -2, and -6 proteins by IL-1 plus UVB action leads to a shift toward promotion of the proapoptotic pathway. In concert with the down-regulation of IAPs and FLIP, TNF-R1 activation as an additional proapoptotic stimulus now results in significant enhancement of UVB-induced apoptosis. Taken together, elucidation of the molecular mechanisms underlying IL-1-mediated enhancement of UVB-induced apoptosis revealed that NFkappaB does not exclusively act in an antiapoptotic fashion but may also mediate proapoptotic effects.

The Effects of Phytohormones and 5-Azacytidine on Apoptosis in Etiolated Wheat Seedlings

The development of etiolated wheat (Triticum aestivum L.) seedlings is necessarily accompanied by apoptosis in their coleoptiles and first leaves. Internucleosome DNA fragmentation, which is characteristic of apoptosis, was detected in the coleoptile as soon as six days after germination. After eight days of germination, DNA fragmentation was clearly expressed in the coleoptile and was noticeable in the apical part of the first-leaf blade. Growing of intact seedlings or incubation of their shoots in the presence of such phytohormones as benzyladenine, gibberellin A3, fusicoccin C, and 2,4-D at the concentration of 10–5 M did not essentially affect DNA fragmentation in the coleoptile. As distinct from antioxidants, none of the phytohormones used prevented apoptosis in wheat seedlings. In contrast, ABA (10–5 M) and an ethylene producer, ethrel (2-chloroethylphosphonic acid, 10–2–10–3 M), stimulated sharply DNA fragmentation in the coleoptile. An inhibitor of DNA methylation, 5-azacytidine, was very efficient in the stimulation of DNA fragmentation in the coleoptiles of eight-day-old seedlings at its concentration of 100 μg/ml. Thus, some phytohormones can regulate apoptosis, and DNA methylation is involved in this process. Our results indicate that apoptosis activation by some phytohormones may be mediated by their regulation of DNA methylation/demethylation, which is responsible for the induction of genes encoding apoptogenic proteins and/or the repression of antiapoptotic genes.