Protein Levels In Response Research Articles

Syntaxin-1a is a Direct Target of miR-29a in Insulin-producing β-Cells

Downregulation of proteins involved in the -exocytotic machinery has been implicated in the impairment of normal β-cell function in response to high glucose levels. Syntaxin-1a -(Stx-1a) is one of two t-SNAREs involved in insulin exocytosis and decreased expression of Stx-1a protein impairs glucose-stimulated insulin secretion (GSIS) in isolated rat pancreatic islets. In diabetic patients Stx-1a protein levels are reduced, but the mechanism of this suppression is unknown.MicroRNAs are small noncoding RNAs, which are important regulators of gene-expression at the post transcriptional level, partially binding to the 3'UTRs of their target gene transcripts either mediating transcript degradation or inhibiting translation. We have recently shown that miR-29a is upregulated in response to elevated glucose levels in β-cells and is involved in mediating the negative effect of high glucose levels on GSIS. Stx-1a has a predicted target site of miR-29a present in its 3' untranslated region. The objective of this study was to evaluate whether miR-29a targets Stx-1a directly to decrease mRNA and/or protein levels in response to glucose. Stx-1a mRNA and protein levels decreased in β-cells treated with increased glucose levels. Overexpression of miR-29a decreased Stx-1a mRNA and protein levels. Furthermore, miR-29a decreases the response of a luciferase reporter construct containing the predicted target site normally present in the Stx-1a gene. When 2 nucleotides are mutated in this target site, responsiveness to miR-29a disappears, confirming miR-29a binding to this sequence. Collectively, these data implicate miR-29a as a mediator of glucose-induced downregulation of Stx-1a in β-cells.

Proteomic responses of fruits to environmental stresses.

Fruits and vegetables are extremely susceptible to decay and easily lose commercial value after harvest. Different strategies have been developed to control postharvest decay and prevent quality deterioration during postharvest storage, including cold storage, controlled atmosphere (CA), and application of biotic and abiotic stimulus. In this review, mechanisms related to protein level responses of host side and pathogen side were characterized. Protein extraction protocols have been successfully developed for recalcitrant, low protein content fruit tissues. Comparative proteome profiling and functional analysis revealed that defense related proteins, energy metabolism, and antioxidant pathway played important roles in fruits in response to storage conditions and exogenous elicitor treatments. Secretome of pathogenic fungi has been well-investigated and the results indicated that hydrolytic enzymes were the key virulent factors for the pathogen infection. These protein level changes shed new light on interaction among fruits, pathogens, and environmental conditions. Potential postharvest strategies to reduce risk of fruit decay were further proposed based on currently available proteomic data.

Epigenetic Mechanisms in Psychiatry

Epigenetic Mechanisms in Psychiatry

MicroRNA-34a Modulates MDM4 Expression via a Target Site in the Open Reading Frame

BackgroundMDM4, also called MDMX or HDMX in humans, is an important negative regulator of the p53 tumor suppressor. MDM4 is overexpressed in about 17% of all cancers and more frequently in some types, such as colon cancer or retinoblastoma. MDM4 is known to be post-translationally regulated by MDM2-mediated ubiquitination to decrease its protein levels in response to genotoxic stress, resulting in accumulation and activation of p53. At the transcriptional level, MDM4 gene regulation has been less clearly understood. We have reported that DNA damage triggers loss of MDM4 mRNA and a concurrent increase in p53 activity. These experiments attempt to determine a mechanism for down-regulation of MDM4 mRNA.Methodology/Principal FindingsHere we report that MDM4 mRNA is a target of hsa-mir-34a (miR-34a). MDM4 mRNA contains a lengthy 3′ untranslated region; however, we find that it is a miR-34a site within the open reading frame (ORF) of exon 11 that is responsible for the repression. Overexpression of miR-34a, but not a mutant miR-34a, is sufficient to decrease MDM4 mRNA levels to an extent identical to those of known miR-34a target genes. Likewise, MDM4 protein levels are decreased by miR-34a overexpression. Inhibition of endogenous miR-34a increased expression of miR-34a target genes and MDM4. A portion of MDM4 exon 11 containing this 8mer-A1 miR-34a site fused to a luciferase reporter gene is sufficient to confer responsiveness, being inhibited by additional expression of exogenous mir-34a and activated by inhibition of miR-34a.Conclusions/SignificanceThese data establish a mechanism for the observed DNA damage-induced negative regulation of MDM4 and potentially provide a novel means to manipulate MDM4 expression without introducing DNA damage.

Effects of Hypoxia Exposure on Hepatic Cytochrome P450 1A (CYP1A) Expression in Atlantic Croaker: Molecular Mechanisms of CYP1A Down-Regulation

Hypoxia-inducible factor-α (HIF-α) and cytochrome P450 1A (CYP1A) are biomarkers of environmental exposure to hypoxia and organic xenobiotic chemicals that act through the aryl hydrocarbon receptor, respectively. Many aquatic environments heavily contaminated with organic chemicals, such as harbors, are also hypoxic. Recently, we and other scientists reported HIF-α genes are upregulated by hypoxia exposure in aquatic organisms, but the molecular mechanisms of hypoxia regulation of CYP1A expression have not been investigated in teleost fishes. As a first step in understanding the molecular mechanisms of hypoxia modulation of CYP1A expression in fish, we characterized CYP1A cDNA from croaker liver. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 to 4 weeks) caused significant decreases in hepatic CYP1A mRNA and protein levels compared to CYP1A levels in fish held in normoxic conditions. In vivo studies showed that the nitric oxide (NO)-donor, S-nitroso-N-acetyl-DL-penicillamine, significantly decreased CYP1A expression in croaker livers, whereas the competitive inhibitor of NO synthase (NOS), Nω-nitro-L-arginine methyl ester, restored CYP1A mRNA and protein levels in hypoxia-exposed (1.7 mg DO/L for 4 weeks) fish. In vivo hypoxia exposure also markedly increased interleukin-1β (IL-1β, a cytokine), HIF-2α mRNA and endothelial NOS (eNOS) protein levels in croaker livers. Pharmacological treatment with vitamin E, an antioxidant, lowered the IL-1β, HIF-2α mRNA and eNOS protein levels in hypoxia-exposed fish and completely reversed the down-regulation of hepatic CYP1A mRNA and protein levels in response to hypoxia exposure. These results suggest that hypoxia-induced down-regulation of CYP1A is due to alterations of NO and oxidant status, and cellular IL-1β and HIF-α levels. Moreover, the present study provides the first evidence of a role for antioxidants in hepatic eNOS and IL-1β regulation in aquatic vertebrates during hypoxic stress.

WS9.3 Contribution of the Th17 pathway in lung inflammatory response in F508del-CFTR mice

Objectives: The exuberant lung inflammatory response in CF has long been recognized as a neutrophil-dominated process. More recently, attention has been drawn to the contribution of other immune cell populations into the progressive lung tissue damage in CF e.g. macrophages. However, little is known about the role played by lymphocytes. The recent observation of increased levels of the proinflammatory IL-17, secreted by T helper 17 (Th17) cells, in the bronchoalveolar lavage (BAL) of CF patients undergoing pulmonary exacerbation, raised the assumption that Th17 pathway may play a central role in CF lung disease. This work aimed at investigating the contribution of Th-17 in lung inflammatory responses in CF mice model. Methods: We characterized, by qRT-PCR and ELISA, the in-vivo molecular inflammatory environment in lungs of normal and homozygous F508del-CFTR mice in basal conditions and after endotracheal instillation of lipopolysaccharide from Pseudomonas aeruginosa (LPS). We showed here that basal mRNA expression levels of IL-17A, IL-17F and IL-22 were barely detected in lungs from CF and non-CF mice, with, as expected, no detectable levels of the corresponding interleukins in BAL. Interestingly, IL-17A, IL-17F and IL-22 mRNA after LPS instillation were found to be higher in the CF group than in the normal group. These data were in agreement with those monitored at the protein level, as IL-17A and IL-22 responses were detected in BAL from CF but barely from normal. Th-17-related cytokines (IL-1b, IL-6, IL-23, GM-CSF) were similarly induced by LPS in both genotypes. Conclusion: Our results showed that responses to LPS of Th-17-related factors are exaggerated in CF mice.

Abstract 343: Induction of Apoptosis in Human Macrophages by 7-ketocholesterol and Iron Ascorbate

Inflammation is the new paradigm for the development and progression of atherosclerotic lesions in coronary artery disease. Factors that increase oxidative stress and apoptosis of cells within the lesion contribute to sustaining the inflammatory response. It was hypothesized that redox-active iron and oxysterols, which have both been found in association with atherosclerotic plaques, contribute to oxidative stress and apoptosis by distinct, but overlapping signaling pathways. The human acute monocytic leukemia cell line THP-1 was used as a model system to study the roles of redox-active iron and 7-ketocholesterol in macrophage apoptosis. The ability of both reagents to induce apoptosis in THP-1 macrophages was demonstrated using flow cytometry. The kinetics of poly(ADP-ribose) polymerase (PARP) cleavage as measured by ELISA were also similar. qRT-PCR was used to measure mRNA levels of several pro- and anti-apoptotic genes. While 7-ketocholesterol increased the level of CHOP mRNA (a transcription factor in endoplasmic reticulum (ER)-initiated apoptosis), there was no similar increase in response to iron ascorbate treatment. Expression of other markers of ER stress, like the transcription factors ATF3 and CEBPbeta, was increased by both compounds, but the timing of changes in gene expression was different. TNFalpha regulates both apoptosis and inflammatory cytokine production. Macrophages are major producers of TNFalpha as well as being highly responsive to the cytokine. A comparison of the changes in TNFalpha mRNA and protein levels in response to iron ascorbate and 7-ketocholesterol suggested that while ERK1/2 and NFkB signaling are important in TNFalpha expression, differentially regulated post-transcriptional processes determine the release of TNFalpha by macrophages. These preliminary results suggest that the apoptotic pathways activated by oxysterols and redox-active iron may be different and that these compounds may have additive or synergistic effects on lesion progression.

Abundances of Iron-Binding Photosynthetic and Nitrogen-Fixing Proteins of Trichodesmium Both in Culture and In Situ from the North Atlantic

Marine cyanobacteria of the genus Trichodesmium occur throughout the oligotrophic tropical and subtropical oceans, where they can dominate the diazotrophic community in regions with high inputs of the trace metal iron (Fe). Iron is necessary for the functionality of enzymes involved in the processes of both photosynthesis and nitrogen fixation. We combined laboratory and field-based quantifications of the absolute concentrations of key enzymes involved in both photosynthesis and nitrogen fixation to determine how Trichodesmium allocates resources to these processes. We determined that protein level responses of Trichodesmium to iron-starvation involve down-regulation of the nitrogen fixation apparatus. In contrast, the photosynthetic apparatus is largely maintained, although re-arrangements do occur, including accumulation of the iron-stress-induced chlorophyll-binding protein IsiA. Data from natural populations of Trichodesmium spp. collected in the North Atlantic demonstrated a protein profile similar to iron-starved Trichodesmium in culture, suggestive of acclimation towards a minimal iron requirement even within an oceanic region receiving a high iron-flux. Estimates of cellular metabolic iron requirements are consistent with the availability of this trace metal playing a major role in restricting the biomass and activity of Trichodesmium throughout much of the subtropical ocean.

Maternal lipopolysaccharide alters the newborn oxidative stress and C-reactive protein levels in response to an inflammatory stress.

Maternal infection is associated with oxidative stress (OS) and inflammatory responses. We have previously shown that maternal exposure to lipopolysaccharide (LPS) at E18 alters the subsequent offspring immune response. As immune responses are mediated, in part, by OS, we sought to determine if maternal inflammation during pregnancy programs offspring OS and C-reactive protein (CRP) levels. Pregnant Sprague-Dawley rats received intraperitoneal (i.p.) injections of saline or LPS at 18 days' gestation (n = 4), and pups delivered spontaneously at term. At postnatal day 24, male and female offspring received i.p. injection of LPS. Serum lipid peroxides formation (PD) and CRP levels were determined before and at 4 h following the LPS injection. Pups of LPS-exposed dams had significantly higher basal OS (PD 29.4 ± 5.4 v. 10.1 ± 4.8 nmol/ml) compared with controls. In response to LPS, CRP levels (20.4 ± 2.8 v. 5.7 ± 1.0 ng/ml) were significantly higher among pups of LPS-exposed dams than controls. Prenatal maternal exposure to LPS increases baseline OS levels in neonates and CRP levels in response to LPS. These results suggest that maternal inflammation during the antenatal period may induce long-term sequelae in the offspring that may predispose to adult disease.

Su1862 MicroRNA-7 Targets the Oncogenic Function of YY1 and Suppresses Colon Cancer Cell Growth Through Regulating Wnt and p53 Pathways

oma, a significant proportion of patients either fail to achieve initial response or develop acquired resistance. Clinical trials using Herceptin for the treatment of esophageal and gastroesophageal adenocarcinomas are ongoing in several Western countries. Understanding mechanisms that could hamper the response to Herceptin could influence the therapeutic decision making options and the management of these patients. Methods and Results: The CellTiter cell viability, clonogenic survival, and Annexin V assays results indicated that stable or transient t-DARPP expression in OE19 cells significantly promoted cell survival and blocked apoptosis after treatment with 20 μg/ml Herceptin (p<0.01). The western blot results showed that t-DARPP sustained p-ERBB2 (Y1248) and p-AKT (S473) protein levels in response to Herceptin. Conversely, knockdown of endogenous t-DARPP in OE33 cells significantly decreased cell survival and p-ERBB2 (Y1248) and p-AKT (S473) protein levels in response to Herceptin (p<0.01). Western blot results showed that transient and stable tDARPP expression without Herceptin increased protein levels of ERBB2, p-ERBB2 (Y1248), and p-AKT (S473). We hypothesized that t-DARPP enhances ERBB2 protein stability, thus blocking ERBB2 receptor steady-state down-regulation. The cycloheximide-based chase assay results showed that stable expression of t-DARPP increased the half-life of ERBB2 protein to 43.6 h as opposed to 29.6 h in control cells. We next investigated the effect of t-DARPP on Herceptin binding to ERBB2 receptor. Using co-immunoprecipitation, we have also shown that t-DARPP interacts with ERBB2 protein, hence, interfering with Herceptin binding to ERBB2 receptor, blocking Herceptin-induced down-regulation of downstream signaling. We also developed a Herceptin-resistant OE19 cell model, generated after selection with Herceptin for six months. The acquired resistance cell model demonstrated upregulation of endogenous t-DARPP and recapitulated the signaling events of Herceptin resistance. Conclusions: Our data demonstrate, for the first time, that t-DARPP enhances AKT signaling and is a key contributor to Herceptin resistance in EAC. In addition, t-DARPP mediates resistance to Herceptin through blocking ERBB2 receptor drug accessibility in EAC. The studies using In Vivo models are ongoing.

Su1861 A Disintegrin-Like and Metalloprotease With Thrombospondin Type 1 Motif 9 is a Functional Tumor Suppressor in Gastric Cancer Through Inhibiting AKT/mTOR Pathway

oma, a significant proportion of patients either fail to achieve initial response or develop acquired resistance. Clinical trials using Herceptin for the treatment of esophageal and gastroesophageal adenocarcinomas are ongoing in several Western countries. Understanding mechanisms that could hamper the response to Herceptin could influence the therapeutic decision making options and the management of these patients. Methods and Results: The CellTiter cell viability, clonogenic survival, and Annexin V assays results indicated that stable or transient t-DARPP expression in OE19 cells significantly promoted cell survival and blocked apoptosis after treatment with 20 μg/ml Herceptin (p<0.01). The western blot results showed that t-DARPP sustained p-ERBB2 (Y1248) and p-AKT (S473) protein levels in response to Herceptin. Conversely, knockdown of endogenous t-DARPP in OE33 cells significantly decreased cell survival and p-ERBB2 (Y1248) and p-AKT (S473) protein levels in response to Herceptin (p<0.01). Western blot results showed that transient and stable tDARPP expression without Herceptin increased protein levels of ERBB2, p-ERBB2 (Y1248), and p-AKT (S473). We hypothesized that t-DARPP enhances ERBB2 protein stability, thus blocking ERBB2 receptor steady-state down-regulation. The cycloheximide-based chase assay results showed that stable expression of t-DARPP increased the half-life of ERBB2 protein to 43.6 h as opposed to 29.6 h in control cells. We next investigated the effect of t-DARPP on Herceptin binding to ERBB2 receptor. Using co-immunoprecipitation, we have also shown that t-DARPP interacts with ERBB2 protein, hence, interfering with Herceptin binding to ERBB2 receptor, blocking Herceptin-induced down-regulation of downstream signaling. We also developed a Herceptin-resistant OE19 cell model, generated after selection with Herceptin for six months. The acquired resistance cell model demonstrated upregulation of endogenous t-DARPP and recapitulated the signaling events of Herceptin resistance. Conclusions: Our data demonstrate, for the first time, that t-DARPP enhances AKT signaling and is a key contributor to Herceptin resistance in EAC. In addition, t-DARPP mediates resistance to Herceptin through blocking ERBB2 receptor drug accessibility in EAC. The studies using In Vivo models are ongoing.

Abstract 2820: Vorinostat abrogates ridaforolimus-induced activation of Akt in synovial sarcoma cells: A possible rationale for their synergism

Abstract Background: Curative treatments for patients with metastatic synovial sarcoma (SS) do not exist and such patients have a poor prognosis. Previously, we demonstrated that the combination of the mTOR inhibitor ridaforolimus and the HDAC inhibitor vorinostat exhibited synergism in a variety of cell lines. Here, we explore whether the Akt pathway is potentially involved in mediating the synergistic effects of ridaforolimus and vorinostat. Methods: Two SS cell lines (HS-SY-II and SYO-I) were treated with the single agents or with combinations of ridaforolimus and vorinostat. After 72 hours, cell viability was measured using the cell proliferation assay (MTS). Combination Indices (CI) were calculated to determine whether each combination was synergistic, additive, or antagonistic. Western Blot analysis assessed alterations in total and phospho-Akt protein levels in response to drug treatment. Results: Ridaforolimus IC50 was 10.9nM in HS-SY-II and 23.1nM in SYO-I; vorinostat IC50 was 440nM in HS-SY-II and 561nM in SYO-I. CI were 0.28 and 0.63 in HS-SY-II and SYO-I, respectively, indicating synergism between the two agents. The ridaforolimus/vorinostat combination was assessed in other tumor types, including osteosarcoma (U2OS), metastatic melanoma (Stew1 and Stew2), pancreatic cancer (Panc1 and BxPC3), and lung cancer (A549). The combination was synergistic in all cell lines: CI ranged from 0.37 to 0.77, except in Panc1, where it was additive (CI=0.92). As previously observed with other mTOR inhibitors, ridaforolimus alone increased pAkt-ser473 levels in SS cell lines; this effect was abrogated by the addition of vorinostat. Conclusions: The combination of ridaforolimus and vorinostat demonstrates in vitro synergism in SS as well as in a variety of other tumor types. The addition of vorinostat prevented ridaforolimus-induced Akt activation, a possible mechanism of resistance to mTOR inhibition. Adding HDAC inhibition to mTOR inhibitors may be a route to circumvent Akt-mediated resistance to mTOR inhibitors. Our results also indicate that this combination may demonstrate broad anti-neoplastic activity. 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 2820. doi:1538-7445.AM2012-2820

Acclimation increases freezing stress response of Arabidopsis thaliana at proteome level

This study used 2DE to investigate how Arabidopsis thaliana modulates protein levels in response to freezing stress after sub-lethal exposure at −10°C, both in cold-acclimated and in non-acclimated plants. A map was implemented in which 62 spots, corresponding to 44 proteins, were identified. Twenty-two spots were modulated upon treatments, and the corresponding proteins proved to be related to photosynthesis, energy metabolism, and stress response. Proteins demonstrated differences between control and acclimation conditions. Most of the acclimation-responsive proteins were either not further modulated or they were down-modulated by freezing treatment, indicating that the levels reached during acclimation were sufficient to deal with freezing. Anabolic metabolism appeared to be down-regulated in favor of catabolic metabolism. Acclimated plants and plants submitted to freezing after acclimation showed greater reciprocal similarity in protein profiles than either showed when compared both to control plants and to plants frozen without acclimation. The response of non-acclimated plants was aimed at re-modulating photosynthetic apparatus activity, and at increasing the levels of proteins with antioxidant-, molecular chaperone-, or post-transcriptional regulative functions. These changes, even less effective than the acclimation strategy, might allow the injured plastids to minimize the production of non-useful metabolites and might counteract photosynthetic apparatus injuries.

Evidence that truncated TrkB isoform, TrkB-Shc can regulate phosphorylated TrkB protein levels

Tropomyosin receptor kinase B (TrkB) is best known as the receptor for brain-derived neurotrophic factor (BDNF). In humans, three major isoforms of TrkB, the full-length receptor (TrkB-TK+) and two C-terminal truncated receptors (TrkB-TK− and TrkB-Shc) are expressed in various tissues. In comparison to TrkB-TK+ and TrkB-TK−, TrkB-Shc is less well characterized. In this study, we analyzed the biological function of the TrkB-Shc receptor in response to exogenous BDNF treatment. In experiments transiently overexpressing TrkB-Shc in CHOK1 cells, we found that TrkB-Shc protein levels were rapidly decreased when cells were exposed to exogenous BDNF. When we assessed the functional impact of TrkB-Shc on TrkB-TK+ activity, we found that phosphorylated TrkB-TK+ protein levels were significantly decreased in the presence of TrkB-Shc and moreso following BDNF exposure. Interestingly, while the reduction of phosphorylated TrkB-TK+ protein was more pronounced in the presence of TrkB-Shc following BDNF exposure, the stability of TrkB-Shc protein itself was increased. Our findings suggest that cells may increase TrkB-Shc protein levels in response to exogenous BDNF exposure to regulate TrkB-TK+ activity by increasing degradation of activated receptor complexes as a means to prevent overactivation or inappropriate temporal and spatial activation of BDNF/TrkB-TK+ signaling.

RhoB is associated with the anti-angiogenic effects of celiac patient transglutaminase 2-targeted autoantibodies

Celiac patient-derived anti-transglutaminase 2 (TG2) antibodies disturb several steps in angiogenesis, but the detailed molecular basis is not known. Therefore, we here analyzed by microarray technology the expression of a set of genes related to angiogenesis and endothelial cell biology in order to identify factors that could explain our previous data related to vascular biology in the context of celiac disease. To this end, in vitro models using human umbilical vein endothelial cells (HUVECs) or in vivo models of angiogenesis were used. A total of 116 genes were analyzed after treatment with celiac patient autoantibodies against TG2. Compared to treatment with control IgA celiac patient, total IgA induced a consistent expression change of 10 genes, the up-regulation of four and down-regulation of six. Of these genes the up-regulated RhoB was selected for further studies. RhoB expression was found to be up-regulated at both messenger RNA and protein level in response to celiac patient total IgA as well as anti-TG2-specific antibody derived from a celiac patient. Interestingly, down-regulation of RhoB by specific small interfering RNA treatment in endothelial cells could rescue the deranged endothelial length and tubule formation caused by celiac disease autoantibodies. RhoB function is controlled by its post-translational modification by farnesylation. This modification of RhoB required for its correct function can be prevented by the cholesterol lowering drug simvastatin, which was also able to abolish the anti-angiogenic effects of celiac anti-TG2 autoantibodies. Taken together, our results would suggest that RhoB plays a key role in the response of endothelial cells to celiac disease-specific anti-TG2 autoantibodies.

Sensitization of Cervical Cancer Cells to Cisplatin by Genistein: The Role of NFB and Akt/mTOR Signaling Pathways

Cervical cancer is among the top causes of death from cancer in women. Cisplatin-based chemotherapy has been shown to improve survival; however, cisplatin treatment is associated with toxicity to healthy cells. Genistein has been used as an adjunct to chemotherapy to enhance the activity of chemotherapeutic agents without causing increased toxicity. The present study was designed to investigate the effect of genistein (25 μM) on antitumor activity of cisplatin (250 nM) on HeLa cervical cancer cells. We have examined the alterations in expression of NF-κB, p-mTOR, p-p70S6K1, p-4E-BP1, and p-Akt protein levels in response to treatment. The combination of 25 μM genistein with 250 nM cisplatin resulted in significantly greater growth inhibition (P < 0.01). Genistein enhanced the antitumor activity of cisplatin and reduced the expression of NF-κB, p-mTOR, p-p70S6K1, p-4E-BP1, and p-Akt. The results in the present study suggest that genistein could enhance the activity of cisplatin via inhibition of NF-κB and Akt/mTOR pathways. Genistein is a promising nontoxic nutritional agent that may enhance treatment outcome in cervical cancer patients when given concomitantly with cisplatin. Clinical trials of genistein and cisplatin combination are warranted to test this hypothesis.

Abstract P048: The Role of p62 in Cardiac Protein Quality Control

Rationale: Two recent studies suggest an important role of autophagy in protection against αB-crystallin-based (CryAB R120G ) desmin-related cardiomyopathies (DRC) but this has not been demonstrated in a different model of cardiac proteinopathy. Moreover, mechanisms underlying the response of cardiomyocytes to increased expression of misfolded proteins remain incompletely understood. Objective: First,to determine whether and how the autophagic activity is changed in a mouse model of desminopathy; second, to investigate the role of p62 in the protein quality control of cardiomyocytes. Methods and Results: Using an autophagosome reporter and determining changes in LC3-II protein levels in response to lysosomal inhibition, we found significantly increased autophagic flux in mouse hearts with transgenic overexpression of a DRC-linked mutant desmin. Similarly, autophagic flux was increased in cultured neonatal rat ventricular myocytes (NRVMs) expressing a mutant desmin. Suppression of autophagy by 3-methyladenine increased, whereas enhancement of autophagy by rapamycin reduced, the ability of a comparable level of mutant desmin overexpression to accumulate ubiquitinated proteins in NRVMs. Furthermore, p62 mRNA and protein expression was significantly upregulated in cardiomyocytes by transgenic overexpression of the mutant desmin or CryAB R120G both in intact mice and in vitro. p62 depletion impaired aggresome and autophagosome formation, exacerbated cell injury, and decreased cell viability in cultured NRVMs expressing the misfolded proteins. Conclusions: Autophagic flux is increased in desminopathic hearts, serving as an adaptive response to overexpression of misfolded proteins. p62 is upregulated in mouse proteinopathic hearts. p62 protects cardiomyocytes against proteotoxic stress by promoting aggresome formation and autophagy activation.

CD28-Mediated Pro-Survival Signaling in Multiple Myeloma

Abstract 2921Multiple myeloma (MM) is a plasma cell neoplasm which is resident in the bone marrow. It has been observed that MM cells are critically dependent upon the bone marrow (BM) microenvironment for survival, which likely plays a role in therapeutic resistance. Thus, a better understanding of the critical interactions of MM with the BM microenvironment may yield new therapeutic targets. One approach to elucidate these survival mechanisms is to characterize cell surface receptors and their stromal ligands. In particular, it has been observed that MM cells express CD28, a molecule best understood as the prototypic T-cell costimulatory receptor. It has since been demonstrated that expression of CD28 correlates with increasing disease burden and that expression at the time of diagnosis correlated with worse prognosis, which is consistent with CD28's function as a potential pro-survival molecule in MM. CD86, one of the ligands for CD28, has also been identified on MM cell lines and in patient biopsies and correlates with worse prognosis than patient biopsies which are CD86 negative. Together, these data suggest that CD28 is a pro-survival molecule in MM. However, the molecular mechanisms by which CD28 may be mediating a pro-survival response in MM remain largely uncharacterized.The ligands for CD28, CD80 and CD86, are expressed on APCs, particularly dendritic cells (DC), which are found in the BM microenvironment. We and others have observed that DCs infiltrate myelomatous portions of patient BM biopsies and murine plasmacytomas. Therefore, we hypothesize that DC in the microenvironment are providing the pro-survival signal to the MM via a CD28 :CD80/86 interaction. To test this in vitro, we have co-cultured MM cells and DC + 2 μM melphalan. CD28 signaling was then blocked either on the CD28 side with an anti-CD28 blocking antibody (CD28.6) or the CD80/CD86 side with the fusion protein CTLA4-Ig. In melphalan alone, MM cells have markedly reduced survival. With the addition of DC, MM cell survival significantly increases. However, this pro-survival signal is abrogated by blockade of CD28 signaling via CD28.6 or CTLA4-Ig.To better understand the mechanisms by which CD28 signaling may be increasing MM cell survival, we first examined expression of apoptotic factors. In particular, it has been shown that diminished levels of the pro-apoptotic molecule Bim plays a critical role in the survival of MM cells. Consistent with these observations, we have data which demonstrate that activation of CD28 decreases Bim protein expression (correlated with increased survival of the MM cell), and blockade of CD28 signaling increase Bim protein expression (correlated with decreasing survival). This mechanism is at least in part transcriptionally regulated; we have demonstrated via RT-PCR that chemotherapy alone increases Bim expression levels, and CD28 activation decreases Bim expression levels without chemotherapy and maintains these lower levels in the presence of chemotherapy.Additionally, MM cells must be highly metabolically efficient due to their forced production of high levels of immunoglobulin (explaining one mechanism by which proteasome inhibitors may be functioning). In the T cell field, it has been shown that CD28 activation increases expression of the glucose transporter Glut1. Similarly, CD28 activation in MM cells also increases Glut1 protein expression, especially in the absence of glucose. These data demonstrate that CD28 may increase metabolic efficiency in low nutrient conditions. Moreover, we have data which demonstrate that MM cells increase Glut1 protein levels in response to CD28 activation, particularly in the presence of chemotherapies.Taken together, these data demonstrate that cells in the microenvironment which provide the CD28 ligands (such as DC) are critical for MM cell survival. Moreover, our data show that this CD28-mediated protection of MM cells occurs via at least two separate pathways and affects both apoptosis and metabolism. These data demonstrate that the CD28 receptor and its downstream signaling component represent novel therapeutic targets for the treatment of multiple myeloma. Disclosures:No relevant conflicts of interest to declare.

Abstract C92: Single cell network profiling (SCNP) assay application to bladder cancer (BC) samples: Identifying rare epidermal growth factor (EGF) responsive epithelial cancer cells sensitive to PI3K inhibition from bladder washings.

Abstract Background: SCNP is a multiparametric flow cytometry-based assay that simultaneously provides measurements, at the single cell level, of extracellular surface markers and quantitative changes in the activation levels of intracellular signaling proteins in response to extracellular modulators (Kornblau et al. Clin Cancer Res 2010). Studies in hematologic malignancies have shown the value of quantitatively measuring single cell signaling networks under modulated conditions as a basis for the development of prognostic and predictive tests. The aim of this study was to assess the feasibility of applying SCNP to the examination of signaling networks in an epithelial cancer, using bladder washings as a BC sample source and EGF +/− the PI3K inhibitor GDC-0941 as modulators. Method: Bladder washes from non-cancer (NC) patients (pts) (n=8) and confirmed/suspected BC pts (n=20) were collected using standard practice and shipped overnight on ice for processing within 24 hours. Antibodies against CD45, CytoKeratin (CK), EpCam, and cleaved-PARP (cPARP) were used to differentiate non-apoptotic epithelial cells from leukocytes, while measurements of DNA aneuploidy (DAPI stain) allowed for distinction between tumor and normal epithelial cells. Signaling activity in the PI3K and MAPK pathways was assessed by measuring intracellular levels of p-AKT and p-ERK both at baseline and in response to pathway modulation. Upon delivery cells were pelleted, counted and incubated for 1 hr at 37°C. GDC-0941 (200nM) or vehicle control was added for 1 hr prior to stimulation with EGF (5 min) or vehicle control. After cell fixation and permeabilization, samples were stained with fluorophore-conjugated antibodies/DAPI cocktail, and data acquired using multi-parametric flow cytometry. The epithelial bladder carcinoma cell line HT-1376 was used as a control for the epithelial phenotype and EGF signaling. DNA index was determined using the lymphoid population in the pt samples as a diploid G0/G1 reference. Results: 50% (N=10) of BC samples and 25% (N=2) of NC samples met the “evaluable” criteria i.e., at least 400,000 total cells upon sample receipt and &amp;gt;2% of cells acquired containing an epithelial phenotype (DAPI+, CD45Low, CK+, and EpCam+). The majority of epithelial cells detected in BC samples were non-apoptotic (i.e. &amp;gt;77% cPARPneg) and therefore suitable for functional pathway analysis. In 3/10 BC samples a quantifiable increase in EGF-induced p-AKT and p-ERK signaling was identified (2–3 fold over baseline) and was preventable by GDC-0941 pre-incubation, whereas no EGF-induced signaling was observed in the NC specimens. Conclusion: This study demonstrates the feasibility of applying SCNP, using multi-parametric flow cytometry, to the functional characterization of BC. Ongoing studies are currently focused on correlation of the individual patient in vitro network signaling profile with clinical outcome to develop prognostic and predictive tools for improved BC management to inform treatment options. The feasibility of extending such SCNP analyses to other solid tumor indications, using alternative tissue sources is also under investigation, e.g., using circulating tumor cells or cells obtained from body fluids such as pleural effusions, ascites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C92.

CCAAT/ enhancer‐binding protein β activation by capsaicin contributes to the regulation of CYP1A1 expression, mediated by the aryl hydrocarbon receptor

Capsaicin, a constituent of peppers, has been linked to the suppression of tumorigenesis and carcinogenesis. The influence of capsaicin on cytochrome P450 (CYP) 1A1, which is involved in metabolism of carcinogens, and the underlying mechanisms remain unclear. Here, we examined the effect of capsaicin on CYP1A1 expression in mouse hepatoma cells. Murine hepatoma Hepa-1c1c7 cells were incubated with capsaicin and/or 3-methylcholanthrene (3-MC). Effects of capsaicin on CYP1A1 levels were determined by analysing mRNA expression, transcription activity and protein expression. Regulation of CYP1A1 was investigated by determining transcriptional factor expression, activation and binding activity with cotreatment with target signal antagonists. Capsaicin alone slightly induced CYP1A1 activity, mRNA expression, protein level and promoter activity. Treatment with transient receptor potential vanilloid type-1 receptor (TRPV1) or aryl hydrocarbon receptor (AhR) antagonist decreased induction of CYP1A1 expression by capsaicin. Additionally, capsaicin significantly inhibited 3-MC-induced CYP1A1 mRNA and protein level and xenobiotic response element-luciferase activity. Capsaicin also inhibited 3-MC-induced AhR transactivation and nuclear localization of AhRs. Moreover, capsaicin increased Ca(2+) /calmodulin (CaM)-dependent protein kinase (CaMK) and CCAAT/ enhancer-binding protein β (C/EBPβ) activation, downstream of TRPV1 receptors. Capsaicin-induced C/EBPβ activation inhibited induction of CYP1A1 mRNA and protein by 3-MC. Capsaicin alone weakly induced CYP1A1 expression, and 3-MC-induced CYP1A1 levels were suppressed by capsaicin. Activation of C/EBPβ and inhibition of 3-MC-induced AhR transactivation by capsaicin contributed to the suppression of CYP1A1 expression. Capsaicin has a potential chemopreventive effect through inhibiting induction of CYP1A1 by poly aryl hydrocarbons.