Negative Regulator Of Apoptosis Research Articles

Cellular Inhibitor of Apoptosis Protein 1 ubiquitinates endonuclease G but does not affect endonuclease G-mediated cell death

Inhibitors of Apoptosis Proteins (IAPs) are evolutionarily well conserved and have been recognized as the key negative regulators of apoptosis. Recently, the role of IAPs as E3 ligases through the Ring domain was revealed. Using proteomic analysis to explore potential target proteins of DIAP1, we identified Drosophila Endonuclease G (dEndoG), which is known as an effector of caspase-independent cell death. In this study, we demonstrate that human EndoG interacts with IAPs, including human cellular Inhibitor of Apoptosis Protein 1 (cIAP1). EndoG was ubiquitinated by IAPs in vitro and in human cell lines. Interestingly, cIAP1 was capable of ubiquitinating EndoG in the presence of wild-type and mutant Ubiquitin, in which all lysines except K63 were mutated to arginine. cIAP1 expression did not change the half-life of EndoG and cIAP1 depletion did not alter its levels. Expression of dEndoG 54310, in which the mitochondrial localization sequence was deleted, led to cell death that could not be suppressed by DIAP1 in S2 cells. Moreover, EndoG-mediated cell death induced by oxidative stress in HeLa cells was not affected by cIAP1. Therefore, these results indicate that IAPs interact and ubiquitinate EndoG via K63-mediated isopeptide linkages without affecting EndoG levels and EndoG-mediated cell death, suggesting that EndoG ubiquitination by IAPs may serve as a regulatory signal independent of proteasomal degradation.

Abstract 65: Regulation of Cardiac Hypertrophy and Dilated Cardiomyopathy by CIP

Cardiac hypertrophy is one of the primary responses of the heart to pathophysiological stress. However, the mechanism of the transition from compensative hypertrophic growth to cardiac dilation is poor understood. Recently, we identified a cardiac-specific expressed gene CIP. The expression of CIP is unchanged in hypertrophic heart but significantly down-regulated in dilated hearts, suggesting CIP may play an important role in the transition from cardiac hypertrophy to dilated cardiomyopathy. We generated CIP knockout mice and found that CIP is dispensable for cardiac development. Interestingly, CIP-null mutant mice developed severe cardiac dilation 4 weeks after TAC (transverse aortic constriction) surgery, while control mice were still at the stage of compensative hypertrophic growth. Echocardiography and histological examinations showed that mutant hearts had enlarged chamber with thinner ventricle wall and decreased cardiac performance compared to controls. The expression of marker genes of cardiac disease, BNP and Myh7, was elevated. Consistently, deletion of CIP in Myh6-CnA transgenic mice result in premature death, displaying severe left ventricle dilation. Conversely, cardiac-specific CIP overexpression inhibited pressure overload-induced cardiac hypertrophy. CIP transgenic mice exhibit decreased ventricle weight/body weight ratio, decreased cardiomyocyte cross-section area and repressed expression of hypertrophic related marker genes. CIP overexpression also protected the heart from developing cardiac dilation and preserved the cardiac function after prolonged pressure overload. We performed unbiased microarray assay to document the transcriptome in CIP knockout and control mice which were subjected to pressure overload (TAC). The analysis of Gene Ontology term indicated the Negative Regulation of Apoptosis was down-regulated while the Collagen/Extracellular Structure Organization was up-regulated in CIP-null hearts under TAC condition. In summary, our studies established CIP as a key regulator of the transition from cardiac hypertrophy to dilated cardiomyopathy. The protective effect of CIP in cardiac remodeling indicates that CIP could become a therapeutic target for cardiac diseases.

Keratinocyte detachment-differentiation connection revisited, or anoikis-pityriasi nexus redux.

Epidermis, a continuously self-renewing and differentiating organ, produces a protective stratum corneum that shields us from external chemical, physical and microbial threats. Epidermal differentiation is a multi-step process regulated by influences, some unknown, others insufficiently explored. Detachment of keratinocytes from the basement membrane is one such pro-differentiation stimulus. Here, we define the transcriptional changes during differentiation, especially those caused by detachment from the substratum. Using comprehensive transcriptional profiling, we revisited the effects of detachment as a differentiation signal to keratinocytes. We identified the genes regulated by detachment, the corresponding ontological categories and, using metaanalysis, compared the genes and categories to those regulated by other pro-differentiating stimuli. We identified 762 genes overexpressed in suspended keratinocyte, including known and novel differentiation markers, and 1427 in attached cells, including basal layer markers. Detachment induced epidermis development, cornification and desmosomal genes, but also innate immunity, proliferation inhibitors, transcription regulators and MAPKs; conversely the attached cells overexpressed cell cycle, anchoring, motility, splicing and mitochondrial genes, and both positive and negative regulators of apoptosis. Metaanalysis identified which detachment-regulated categories overlap with those induced by suprabasal location in vivo, by reaching confluency in vitro, and by inhibition of JUN kinases. Attached and in vivo basal cells shared overexpression of mitochondrial components. Interestingly, melanosome trafficking components were also overexpressed in the attached and in vivo basal keratinocytes. These results suggest that specific pro-differentiation signals induce specific features of the keratinization process, which are in vivo orchestrated into harmonious epidermal homeostasis.

Comprehensive analysis of the whole transcriptomes from two different pig breeds using RNA-Seq method.

Next-generation sequencing RNA-Seq technology is a powerful tool that creates new possibilities for whole-transcriptome analysis. In our study, the RNA-Seq method was applied to analyze global changes in transcriptome from muscle tissue (m. semimembranosus) in two pig breeds (Pietrain and Polish Landrace, PL). The breeds differ in terms of muscularity, growth rate and reproduction traits. Using three different approaches (deseq, cufflinks and edger) and taking into account the most restrictive criteria, 35 genes differentially expressed between Pietrain and PL pigs were identified. In both breeds, the most abundant were transcripts encoding ribosomal and cytoskeletal proteins (TPM3, TCAP, TMOD4, TPM2, TNNC1) and calcium-binding proteins involved in muscle contraction, calcium-mediated signaling or cation transport (CASQ1, MLC2V, SLC25A4, MYL3). In PL pigs, we identified up-regulation of several genes that play crucial roles in reproduction: female gamete generation (BDP1, PTPN21, USP9X), fertilization (EGFR) and embryonic development (CPEB4). In the Pietrain breed, only seven genes were over-expressed (CISH, SPP1, TUBA8, ATP6V1C2, IGKC, predicted LOC100510960 and LOC100626400), and they play important roles in, for example, negative regulation of apoptosis, immune response, cell-cell signaling, cell growth and migration as well as the metabolic process. The functions of the majority of selected genes were consistent with phenotypic variation in investigated breeds; thus, we proposed a new panel of candidate genes that can be associated with economically important pig traits.

Identification and characterization of suppressor mutants of spl11- mediated cell death in rice.

Lesion mimic mutants have been used to dissect programmed cell death (PCD) and defense-related pathways in plants. The rice lesion-mimic mutant spl11 exhibits race nonspecific resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae and the fungal pathogen Magnaporthe oryzae. Spl11 encodes an E3 ubiquitin ligase and is a negative regulator of PCD in rice. To study the regulation of Spl11-mediated PCD, we performed a genetic screen and identified three spl11 cell-death suppressor (sds) mutants. These suppressors were characterized for their resistance to X. oryzae pv. oryzae and M. oryzae and for their expression of defense-related genes. The suppression of the cell-death phenotypes was generally correlated with reduced expression of defense-related genes. When rice was challenged with avirulent isolates of M. oryzae, the disease phenotype was unaffected in the sds mutants, indicating that the suppression might be Spl11-mediated pathway specific and may only be involved in basal defense. In addition, we mapped one of the suppressor mutations to a 140-kb interval on the long arm of rice chromosome 1. Identification and characterization of these sds mutants should facilitate our efforts to elucidate the Spl11-mediated PCD pathway.

Potential role of microRNA: Identification and functional analysis of microRNA in corticospinal tract after unilateral lesions of the medullary pyramid

Corticospinal tract is mainly descending tracts by dominating voluntary movement of the limbs and fine movement of distal limb, especially in mammals. Distal axonal degeneration is called anterograde degeneration. Proximal end is connected to the neuron cell body, whereas retrograde degeneration is very slight with the possibility of regeneration. MicroRNAs (miRNAs) are a short non-coding RNAs that regulate gene expression at the post-transcriptional level by binding with the 3′ untranslated region of target mRNAs. In order to understand the mechanism of underlying gene alteration in the rostral and caudal, respectively, after the corticospinal tract injury, we analyzed rostral and caudal mRNA and miRNA, respectively, using microRNA and mRNA profiles. We combined the predicted targets of miRNA with differentially expressed mRNA for selecting intersection gene. To predict the function miRNAs, GO and KEGG enrichment analysis were performed to find genes associated with change of rostral and caudal, respectively. The bioinformatics analysis indicated that changes in miRNA and target mRNA expression affected rostral regeneration, including negative regulation of apoptosis, positive regulation of cell proliferation, cell adhesion, oligodendrocyte development etc. It also affected caudal degeneration, including induction of apoptosis, down-regulating nervous system development, immune response etc. The current results illustrated that corticospinal tract injury produces a wide range changes of miRNAs, whereas mRNA also showed significantly change which affects key biological processes after injury in rostral and caudal.

Novel RNA oligonucleotide improves liver function and inhibits liver carcinogenesis in vivo.

Hepatocellular carcinoma (HCC) occurs predominantly in patients with liver cirrhosis. Here we show an innovative RNA-based targeted approach to enhance endogenous albumin production while reducing liver tumor burden. We designed short-activating RNAs (saRNA) to enhance expression of C/EBPα (CCAAT/enhancer-binding protein-α), a transcriptional regulator and activator of albumin gene expression. Increased levels of both C/EBPα and albumin mRNA in addition to a 3-fold increase in albumin secretion and 50% decrease in cell proliferation was observed in C/EBPα-saRNA transfected HepG2 cells. Intravenous injection of C/EBPα-saRNA in a cirrhotic rat model with multifocal liver tumors increased circulating serum albumin by over 30%, showing evidence of improved liver function. Tumor burden decreased by 80% (P = 0.003) with a 40% reduction in a marker of preneoplastic transformation. Since C/EBPα has known antiproliferative activities by way of retinoblastoma, p21, and cyclins, we used messenger RNA (mRNA) expression liver cancer-specific microarray in C/EBPα-saRNA-transfected HepG2 cells to confirm down-regulation of genes strongly enriched for negative regulation of apoptosis, angiogenesis, and metastasis. Up-regulated genes were enriched for tumor suppressors and positive regulators of cell differentiation. A quantitative polymerase chain reaction (PCR) and western blot analysis of C/EBPα-saRNA-transfected cells suggested that in addition to the known antiproliferative targets of C/EBPα, we also observed suppression of interleukin (IL)6R, c-Myc, and reduced STAT3 phosphorylation. A novel injectable saRNA-oligonucleotide that enhances C/EBPα expression successfully reduces tumor burden and simultaneously improves liver function in a clinically relevant liver cirrhosis/HCC model.

ATF6 upregulates XBP1S and inhibits ER stress-mediated apoptosis in osteoarthritis cartilage

As we previously reported, transcription factor XBP1S enhances BMP2-induced chondrocyte differentiation and acts as a positive mediator of chondrocyte hypertrophy. The purpose of this study was to determine (1) whether XBP1S influences ER stress-mediated apoptosis in osteoarthritis (OA); (2) whether ATF6 regulates IRE1/XBP1 signal pathway in OA cartilage; (3) what are the associated molecules affecting apoptosis in osteoarthritis and the molecular events underlying this process. Herein, we examined and found that ER stress-associated molecules were activated in OA patients, specifically XBP1S splice and expression were increased markedly by TNF-α and IL-1β treatments. Transcription factor ATF6 can specifically bind to the promoter of XBP1 gene and enhance the expression of XBP1S spliced by IRE1α in osteoarthritis cartilage. Furthermore, siXBP1S can enhance ER stress-mediated apoptosis and main matrix degradation in osteoarthritis. Whereas AdXBP1S can inhibit ER stress-mediated apoptosis and TNFα induced nitrite production in OA cartilage. In a word, our observations demonstrate the importance of XBP1S in osteoarthritis. ATF6 and IRE1α can regulate endogenous XBP1S gene expression synergistically in OA cartilage. More significantly, XBP1S was a negative regulator of apoptosis in osteoarthritis by affecting caspase 3, caspase 9, caspase 12, p-JNK1, and CHOP.

Retinoblastoma protein regulates the crosstalk between autophagy and apoptosis, and favors glioblastoma resistance to etoposide.

Glioblastomas (GBMs) are devastating tumors of the central nervous system, with a poor prognosis of 1-year survival. This results from a high resistance of GBM tumor cells to current therapeutic options, including etoposide (VP-16). Understanding resistance mechanisms may thus open new therapeutic avenues. VP-16 is a topoisomerase inhibitor that causes replication fork stalling and, ultimately, the formation of DNA double-strand breaks and apoptotic cell death. Autophagy has been identified as a VP-16 treatment resistance mechanism in tumor cells. Retinoblastoma protein (RB) is a classical tumor suppressor owing to its role in G1/S cell cycle checkpoint, but recent data have shown RB participation in many other cellular functions, including, counterintuitively, negative regulation of apoptosis. As GBMs usually display an amplification of the EGFR signaling involving the RB protein pathway, we questioned whether RB might be involved in mechanisms of resistance of GBM cells to VP-16. We observed that RB silencing increased VP-16-induced DNA double-strand breaks and p53 activation. Moreover, RB knockdown increased VP-16-induced apoptosis in GBM cell lines and cancer stem cells, the latter being now recognized essential to resistance to treatments and recurrence. We also showed that VP-16 treatment induced autophagy, and that RB silencing impaired this process by inhibiting the fusion of autophagosomes with lysosomes. Taken together, our data suggest that RB silencing causes a blockage on the VP-16-induced autophagic flux, which is followed by apoptosis in GBM cell lines and in cancer stem cells. Therefore, we show here, for the first time, that RB represents a molecular link between autophagy and apoptosis, and a resistance marker in GBM, a discovery with potential importance for anticancer treatment.

Cucumber BAX inhibitor-1, a conserved cell death suppressor and a negative programmed cell death regulator under cold stress

Programmed cell death (PCD) is a genetically controlled and conserved process in eukaryotes during development as well as in response to pathogens and other stresses. BAX inhibitor-1 (BI-1) has been implicated as an anti-PCD factor which is highly conserved in plants. Sequence of putative cucumber BI-1 protein exhibited 77.7 % identity and 91.2 % positive value with the homologue Blast BI-1 protein of Arabidopsis thaliana (AtBI-1). This highly homologous protein to the AtBI-1 protein was named CsBI-1. This protein contains an open reading frame (ORF) of 250 amino acids with a BAX inhibitor domain and five transmembrane regions conserved among members of the BI-1 family. Primers designed by the cDNA of CsBI-1gene were used for further sequencing. Cell death in cold-stored cucumber developed concomitantly with increased expression of the CsBI-1 gene and reached maximum at day 6. However, cell death accelerated significantly after 9 d when sharp decrease of the CsBI-1 expression occurred. After warming to 20 °C, expression of the CsBI-1 gene was the highest at day 3, decreased afterwards, and the lowest expression was detected at day 9 when PCD obviously appeared. The overall results indicate that CsBI-1 is cucumber homologue of Arabidopsis thaliana AtBI-1 gene. CsBI-1 is a conserved cell death suppressor induced by cold stress and a negative regulator of PCD.

Abstract 993: Upregulation of B-cell activation genes and negative regulators of apoptosis determines resistance to bendamustine in chronic lymphocytic leukemia cells.

Abstract Chronic lymphocytic leukemia (CLL), the most common leukemia in Western countries, is characterized by the accumulation of monoclonal CD5+ B cells in the blood, bone marrow, lymph nodes, spleen and other lymphoid organs. Bendamustine hydrochloride, a cytotoxic agent comprising structural features of both an alkylating drug and a purine nucleoside analog, was recently approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of CLL and indolent B cell non-Hodgkin's lymphoma (NHL). Although several recent clinical trials have documented the activity of bendamustine in CLL, the mechanisms of bendamustine resistance in CLL cells have not been yet elucidated. We analyzed the ex vivo cytotoxicity of bendamustine in cells from 48 CLL cases by incubating them with bendamustine 25 μM for 24 hours. Cell death was assessed by annexinV-PI labeling and cases were classified as bendamustine-resistant (cytotoxicity below 30%) or bendamustine-sensitive (cytotoxicity over 30%) cases. We found that sensitivity of CLL primary cells correlated with the mutational status of IGHV gene, being the IGHV-unmutated cases significantly more resistant to bendamustine (p<0.015) than those cases with mutated IGHV. Although the number of cases analyzed was low, no correlation with the mutational status of NOTCH1 or SF3B1 was observed. We compared the gene expression profiling of bendamustine-resistant and bendamustine-sensitive groups by performing a high-throughput analysis of basal gene expression in CLL cells with the U219 Affymetrix array. After a supervised analysis using the Rank Products method and a FDR below 0.001, we observed 393 up-regulated genes and 415 down-regulated genes in the bendamustine-resistant group. Annotation of the differentially expressed genes identified some well-known markers of prognosis in CLL, together with genes related to apoptosis regulation and B cell activation. One of the genes with a highest positive fold-change and most relevant according to its role in cell cycle progression, apoptosis and transformation was C-MYC. These results suggest that overexpression of genes related to proliferation and B-cell activation could be involved in CLL resistance to bendamustine therapy, which is known to be clinically more effective in indolent than in aggressive lymphomas. Further studies will assess whether this set of overexpressed genes might be useful as biomarkers to predict the sensitivity to bendamustine and open the door to new combinatorial strategies that might overcome resistance to bendamustine in CLL patients within the context of tailored medicine Citation Format: Eriong Lee-Verges, Arnau Montraveta, Magda Pinyol, Pedro Jares, Cristina Arimany-Nardi, Marta Aymerich, Neus Villamor, Marçal Pastor-Anglada, Armando Lopez-Guillermo, Patricia Perez-Galan, Gael Roue, Elias Campo, Dolors Colomer. Upregulation of B-cell activation genes and negative regulators of apoptosis determines resistance to bendamustine in chronic lymphocytic leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 993. doi:10.1158/1538-7445.AM2013-993

MADD knock-down enhances doxorubicin and TRAIL induced apoptosis in breast cancer cells.

The Map kinase Activating Death Domain containing protein (MADD) isoform of the IG20 gene is over-expressed in different types of cancer tissues and cell lines and it functions as a negative regulator of apoptosis. Therefore, we speculated that MADD might be over-expressed in human breast cancer tissues and that MADD knock-down might synergize with chemotherapeutic or TRAIL-induced apoptosis of breast cancer cells. Analyses of breast tissue microarrays revealed over-expression of MADD in ductal and invasive carcinomas relative to benign tissues. MADD knockdown resulted in enhanced spontaneous apoptosis in human breast cancer cell lines. Moreover, MADD knockdown followed by treatment with TRAIL or doxorubicin resulted in increased cell death compared to either treatment alone. Enhanced cell death was found to be secondary to increased caspase-8 activation. These data indicate that strategies to decrease MADD expression or function in breast cancer may be utilized to increase tumor cell sensitivity to TRAIL and doxorubicin induced apoptosis.

Ptaquiloside reduces NK cell activities by enhancing metallothionein expression, which is prevented by selenium

Pteridium aquilinum, one of the most important poisonous plants in the world, is known to be carcinogenic to animals and humans. Moreover, our previous studies showed that the immunosuppressive effects of ptaquiloside, its main toxic agent, were prevented by selenium in mouse natural killer (NK) cells. We also verified that this immunosuppression facilitated development of cancer. Here, we performed gene expression microarray analysis in splenic NK cells from mice treated for 14 days with ptaquiloside (5.3mg/kg) and/or selenium (1.3mg/kg) to identify gene transcripts altered by ptaquiloside that could be linked to the immunosuppression and that would be prevented by selenium. Transcriptome analysis of ptaquiloside samples revealed that 872 transcripts were expressed differentially (fold change>2 and p<0.05), including 77 up-regulated and 795 down-regulated transcripts. Gene ontology analysis mapped these up-regulated transcripts to three main biological processes (cellular ion homeostasis, negative regulation of apoptosis and regulation of transcription). Considering the immunosuppressive effect of ptaquiloside, we hypothesized that two genes involved in cellular ion homeostasis, metallothionein 1 (Mt1) and metallothionein 2 (Mt2), could be implicated because Mt1 and Mt2 are responsible for zinc homeostasis, and a reduction of free intracellular zinc impairs NK functions. We confirm these hypotheses and show increased expression of metallothionein in splenic NK cells and reduction in free intracellular zinc following treatment with ptaquiloside that were completely prevented by selenium co-treatment. These findings could help avoid the higher susceptibility to cancer that is induced by P. aquilinum-mediated immunosuppressive effects.

Smac Mimetic SM-164 Potentiates APO2L/TRAIL- and Doxorubicin-Mediated Anticancer Activity in Human Hepatocellular Carcinoma Cells

BackgroundThe members of inhibitor of apoptosis proteins (IAPs) family are key negative regulators of apoptosis. Overexpression of IAPs are found in hepatocellular carcinoma (HCC), and can contribute to chemotherapy resistance and recurrence of HCC. Small-molecule Second mitochondria-derived activator of caspases (Smac) mimetics have recently emerged as novel anticancer drugs through targeting IAPs. The specific aims of this study were to 1) examine the anticancer activity of Smac mimetics as a single agent and in combination with chemotherapy in HCC cells, and 2) investigate the mechanism of anticancer action of Smac mimetics.MethodsFour HCC cell lines, including SMMC-7721, BEL-7402, HepG2 and Hep3B, and 12 primary HCC cells were used in this study. Smac mimetic SM-164 was used to treat HCC cells. Cell viability, cell death induction and clonal formation assays were used to evaluate the anticancer activity. Western blotting analysis and a pancaspase inhibitor were used to investigate the mechanisms.ResultsAlthough SM-164 induced complete cIAP-1 degradation, it displayed weak inhibitory effects on the viability of HCC cells. Nevertheless, SM-164 considerably potentiated Apo2 ligand or TNF-related apoptosis-inducing ligand (APO2L/TRAIL)- and Doxorubicin-mediated anticancer activity in HCC cells. Mechanistic studies demonstrated that SM-164 in combination with chemotherapeutic agents resulted in enhanced activation of caspases-9, -3 and cleavage of poly ADP-ribose polymerase (PARP), and also led to decreased AKT activation.ConclusionsSmac mimetics can enhance chemotherapeutic-mediated anticancer activity by enhancing apoptosis signaling and suppressing survival signaling in HCC cells. This study suggests Smac mimetics are potential therapeutic agents for HCC.

Disruption of the interaction between PMCA2 and calcineurin triggers apoptosis and enhances paclitaxel-induced cytotoxicity in breast cancer cells

Cancer is caused by defects in the signalling mechanisms that govern cell proliferation and apoptosis. It is well known that calcium-dependent signalling pathways play a critical role in cell regulation. A tight control of calcium homeostasis by transporters and channel proteins is required to assure a proper functioning of the calcium-sensitive signal transduction pathways that regulate cell growth and apoptosis. The plasma membrane calcium ATPase 2 (PMCA2) has been recently identified as a negative regulator of apoptosis that can play a significant role in cancer progression by conferring cells resistance to apoptosis. We have previously reported an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells. Here, we demonstrate that disruption of the PMCA2/calcineurin interaction in a variety of human breast cancer cells results in activation of the calcineurin/NFAT pathway, upregulation in the expression of the pro-apoptotic protein Fas Ligand and in a concomitant loss of cell viability. Reduction in cell viability is the consequence of an increase in cell apoptosis. Impairment of the PMCA2/calcineurin interaction enhances paclitaxel-mediated cytotoxicity of breast tumoral cells. Our results suggest that therapeutic modulation of the PMCA2/calcineurin interaction might have important clinical applications to improve current treatments for breast cancer patients.

71 PATHOPHYSIOLOGICAL PATHWAYS IN ESSENTIAL HYPERTENSIVE KIDNEYS

Background: The kidney has long been considered to have an important role in hypertension etiology. Methods: Using transcriptomics we identified 60 mRNAs and 24 miRNAs differentially expressed in renal cortex and medulla between European hypertensive and normotensive subjects. Confirmation was by real-time quantitative PCR. Functional experiments were conducted. Bioinformatic analyses, involving gene ontology (GO) analysis and gene set test (GST), were used to identify disturbances in intracellular pathways. Results: The sodium transport regulator SIK1, APOE, NR4A1–3, and renin were overexpressed in hypertension. CD36 was under-expressed, as in the SHR. Bioinformatic predictions of miRNA target sites in several genes of particular interest, including renin, were confirmed by co-transfection experiments involving microRNA mimics in a human kidney cell line. By GST for cortex data, terms identified included renin-angiotensin system, electron transport chain, mitochondrial respiratory chain complex I, regulation of transcription. By GO terms included sodium ion transport, nitric oxide mediated signal transduction, circulatory system process, response to reactive oxygen species, signalling, urea cycle and apoptosis. In the medulla, GST revealed sodium ion transport, sodium ion binding, symporter activity, oxidation reduction, mitochondrial inner membrane, ATP binding, apical plasma membrane, transmembrane transport, transporter activity and regulation of transcription. GO analysis identified calcium-mediated signalling, regulation of transcription, developmental processes, negative regulation of apoptosis, regulation of catecholamine metabolic process, dephosphorylation, RNA metabolic process, inactivation of MAP kinase activity, cytokine receptor binding, spliceosome and endocytosis. Conclusions: These new findings illuminate mechanistic disturbances and should assist in understanding hypertension pathophysiology. Novel treatment strategies could ensue.

Molecular functions of metallothionein and its role in hematological malignancies

Metallothionein (MT) was reported to be a potential negative regulator of apoptosis, and various reports have suggested that it may play roles in carcinogenesis and drug resistance, in at least a portion of cancer cells. The author summarizes the current understanding of the molecular functions of MT for tumor cell growth and drug resistance. These activities are regulated through intracellular metal ion modulation and free radical scavenging. Compared with analyses of solid tumors, few studies have analyzed the roles of MT in hematological malignancies. This review mainly describes the functions of MT in hematopoietic cells. Furthermore, through expression analyses of leukemias and lymphomas, the roles of MT in the biology of these diseases are particularly focused upon.

Hypothermia produces rat liver proteomic changes as in hibernating mammals but decreases endoplasmic reticulum chaperones

Hypothermia is used in the clinic for protection of organs such as the brain against ischemic injury during aortic/complex congenital cardiac surgery or post-resuscitation encephalopathy. The principal mechanism of hypothermic protection is suppression of metabolism, however, the pleiotropic effects of cooling are incompletely understood. Here, we used a rat model system to evaluate metabolic changes induced by deep hypothermia. The hypothermia-induced changes were identified using fluorescence-based two-dimensional (2-D) difference gel electrophoresis (DIGE) and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry. Rats were randomly assigned to a normothermic control group (37°C, n=6) or hypothermia group (23°C, n=6) that received surface cooling for 3h. Liver tissue was excised for assessment. Functional profiling of differently expressed proteins was performed as an enrichment analysis of Gene Ontology (GO) terms and pathways. We found that the livers of anesthetized rats with deep hypothermia showed significant downregulation of proteins in the endoplasmic reticulum and mitochondria, and of those involved in ATP binding, amino acid metabolism and urea cycle, response to oxidative stress, anti-apoptosis, negative regulation of apoptosis. The changes in the proteome of the hypothermic rats showed similarities, except with regard to endoplasmic reticulum chaperones, to those identified elsewhere in mammals undergoing hibernation.

Abstract 5430: Analysis of genome-wide DNA methylation in CCRF-CEM cells confirms hypermethylation at reported epigenetic markers of T-cell lymphoblastic leukemia and identifies novel therapeutic targets with reduced methylation upon treatment with dietary indoles

Abstract Acute lymphoblastic leukemia/lymphoma (ALL) is the most common class of malignancies among children and cases of T-cell origin are less responsive to therapy than their B-cell counterparts. Cruciferous vegetables have been demonstrated to be chemoprotective in a number of cancer cell types due to their high content of indoles and isothiocyanates. Previously, we demonstrated that indole-3-carbinol (I3C) is an effective transplacental chemopreventive agent in a dibenzo[def, p]chrysene (DBC)-induced model of murine T-ALL. We further confirmed that treatment of human T-ALL cell lines with I3C and diindolylmethane (DIM), the primary product of I3C condensation, reduces cell proliferation, viability, and expression of cell-cycle proteins. Aberrant DNA methylation is a recognized event in the development of cancer and pharmacological demethylation of promoter-associated CpG islands is of therapeutic interest to restore expression of epigenetically silenced genes. We utilized genome-wide DNA methylation analysis to identify potential epigenetic therapeutic targets of I3C and DIM in a model of T-ALL. By differential methylation region (DMR) analysis with stringent bonferroni multiple testing correction, 1,699 DMR probes were identified which mapped to 298 unique transcripts. The differential degree of hypomethylation by I3C, compared with DIM, is consistent with previous findings that DIM is a more potent therapeutic in this model. I3C (60 µM) significantly reduced methylation in 53% of DMRs yet increased methylation in the remaining 173 gene-mapped DMRs. DIM treatment with the consumer available supplement formulation known as Bioresponse-DIM (BR-DIM; 5 µM), or with the pure crystalline form (C-DIM; 5 µM or 15 µM), reduced methylation in over 500 probes and more than 200 genes. Functional annotation of differentially hypomethylated genes revealed that homeobox genes and transcription factors were most commonly represented with negative regulators of apoptosis and Wnt/Frizzled families also among the top gene clusters. Many identified genes have been previously recognized as being frequently hypermethylated in hematological malignancies and our results support demethylation of these critical cancer survival pathways by dietary indoles derived from cruciferous vegetables. These and novel genes from our analysis are currently being validated and examined at the level of transcription. Supported by P01CA90890, P42ES016465, CA135523 and The Linus Pauling Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5430. doi:1538-7445.AM2012-5430

Gene Expression Analysis of Label-Retaining Pancreatic Adenocarcinoma Cells: Defining a New Class of Cancer Stem Cell

Introduction: A new paradigm of treatment for patients with pancreatic adenocarcinoma is needed given the dismal prognosis. Recent identification of cancer stem cells using cell surface markers has offered a new approach to therapy. We hypothesize that identification of cancer stem cells should capitalize on their unique functional properties such as self-renewal by asymmetric division. Label-retaining cells have been described to represent adult tissue stem cells and to self-renew by asymmetric division. Thus, we undertook this study to investigate whether pancreatic cancers contain label-retaining cancer cells (LRCC), and to investigate whether LRCC have a unique gene signature amenable to novel therapeutic approaches. Methods: Three pancreatic adenocarcinoma cell lines (PaTu8988T, BxPC-3, and Panc-1) were labeled with fluorescent nucleotide analogues (Cy5-dUTP) to isolate live LRCC. Labeled cells were sorted using FACS after one cell cycle to identify a Cy5+ population of cells which had incorporated the fluorescent nucleotide into their genome. the Cy5+ cells were placed back in culture and grown for a total of 8 cell cycles. FACS was then used to identify the Cy5+ cells within the expanded population, referred to as LRCC. Total RNA was isolated from the LRCC, non-LRCC (NLRCC) and normal cells. Following amplification and in vitro transcription, cRNA was loaded onto Illumina HumanHT-12 v4.0 Expression BeadChips. Microarray analysis was performed using Agilent GeneSpringGX software package, v11.5 (Agilent Technologies, Santa Clara, CA). Results: The proportion of cells retaining the fluorescent nucleotides was higher than mathematically expected in each group. Using FACS, the percent of LRCC isolated from the total cell mass ranged from 2.97%-5.02% compared to the expected 0.3% (p=0.02, Student's t-test). Cluster analysis of microarray data generated from LRCC, NLRCC and control cell RNA demonstrated that the NLRCC population was similar to the control cells and the LRCC population segregated separately. Gene expression analysis revealed a distinct subset of genes up-regulated > 2 fold change in the LRCC compared to the NLRCC. Canonical pathway analysis of the upregulated subsets of genes noted increased activity of the Wnt-β-catenin and Hedgehog pathway. Gene ontology analysis revealed increased expression of negative regulators of apoptosis in the LRCC population. Conclusions: We show that 3 pancreatic cancer cell lines contain LRCC. Based on the fact that LRCC are hypothesized to be putative stem cells, and we have demonstrated that pancreatic LRCC have a gene signature consistent with stem cells, we propose that pancreatic LRCC may represent a novel class of pancreatic cancer stem cells. Additionally, the unique gene signature of LRCC may provide novel targets for therapy specifically directed towards pancreatic cancer stem cells.