Nutrient-deprived Conditions Research Articles

Integrated omics-analysis reveals Wnt-mediated NAD+ metabolic reprogramming in cancer stem-like cells.

Abnormal tumor cell metabolism is a consequence of alterations in signaling pathways that provide critical selective advantage to cancer cells. However, a systematic characterization of the metabolic and signaling pathways altered in cancer stem-like cells (CSCs) is currently lacking. Using nuclear magnetic resonance and mass spectrometry, we profiled the whole-cell metabolites of a pair of parental (P-231) and stem-like cancer cells (S-231), and then integrated with whole transcriptome profiles. We identified elevated NAAD+ in S-231 along with a coordinated increased expression of genes in Wnt/calcium signaling pathway, reflecting the correlation between metabolic reprogramming and altered signaling pathways. The expression of CD38 and ALP, upstream NAAD+ regulatory enzymes, was oppositely regulated between P- and S-231; high CD38 strongly correlated with NAADP in P-231 while high ALP with NAAD+ levels in S-231. Antagonizing Wnt activity by dnTCF4 transfection reversed the levels of NAAD+ and ALP expression in S-231. Of note, elevated NAAD+ caused a decrease of cytosolic Ca2+ levels preventing calcium-induced apoptosis in nutrient-deprived conditions. Reprograming of NAD+ metabolic pathway instigated by Wnt signaling prevented cytosolic Ca2+ overload thereby inhibiting calcium-induced apoptosis in S-231. These results suggest that “oncometabolites” resulting from cross talk between the deranged core cancer signaling pathway and metabolic network provide a selective advantage to CSCs.

480 - BRAF inhibition promotes BRAF mutant human melanoma cell survival under nutrient-deprived conditions through activation of mitochondrial metabolism

480 - BRAF inhibition promotes BRAF mutant human melanoma cell survival under nutrient-deprived conditions through activation of mitochondrial metabolism

Therapeutic inhibition of mitochondrial function induces cell death in starvation-resistant renal cell carcinomas.

Renal cell carcinomas (RCC) have two types of cells for carbon metabolism and for cell signaling under nutrient-deprivation conditions, namely starvation-resistant and starvation-sensitive cells. Here, we evaluated the mitochondrial characteristics of these cell types and found that the resistant type possessed higher activities for both mitochondrial oxidative phosphorylation and glycolysis than the sensitive types. These higher activities were supported by the stored carbon, lipid and carbohydrate sources, and by a low level of mitochondrial reactive oxygen species (ROS) due to sustained SOD2 expression in the resistant RCC cells. In metastatic RCC cases, higher SOD2 expression was associated with a significantly shorter survival period. We found that treatment with the drugs etomoxir and buformin significantly reduced mitochondrial oxidative phosphorylation and induced cell death under glucose-deprivation conditions in starvation-resistant RCC cells. Our data suggest that inhibitory targeting of mitochondria might offer an effective therapeutic option for metastatic RCC that is resistant to current treatments.

Autophagy inhibition in endogenous and nutrient-deprived conditions reduces dorsal root ganglia neuron survival and neurite growth in vitro.

Peripheral neuropathies can result in cytoskeletal changes in axons, ultimately leading to Wallerian degeneration and cell death. Recently, autophagy has been studied as a potential target for improving axonal survival and growth during peripheral nerve damage. This study investigates the influence of autophagy on adult dorsal root ganglia (DRG) neuron survival and axonal growth under control and nutrient deprivation conditions. Constitutive autophagy was modulated with pharmacological activators (rapamycin; Rapa) and inhibitors (3-methyladenine, bafilomycin A1) in conjunction with either a nutrient-stable environment (standard culture medium) or a nutrient-deprived environment (Hank's balanced salt solution + Ca(2+) /Mg(2+) ). The results demonstrated that autophagy inhibition decreased cell viability and reduced neurite growth and branching complexity. Although autophagy was upregulated with nutrient deprivation compared with the control, it was not further activated by rapamycin, suggesting a threshold level of autophagy. Overall, both cellular and biochemical approaches combined to show the influence of autophagy on adult DRG neuron survival and growth. © 2016 Wiley Periodicals, Inc.

Recurrent neomorphic mutations of MTOR in central nervous system and testicular germ cell tumors may be targeted for therapy.

Germ cell tumors constitute a heterogeneous group that displays a broad spectrum of morphology. They often arise in testes; however, extragonadal occurrence, in particular brain, is not uncommon, and whether they share a common pathogenesis is unknown. We performed whole exome sequencing in 41 pairs of central nervous system germ cell tumors (CNS GCTs) of various histology and their matched normal tissues. We then performed targeted sequencing of 41 selected genes in a total of 124 CNS GCTs, 65 testicular germ cell tumors (tGCTs) and 8 metastatic GCTs to the CNS. The results showed that mutually exclusive mutations of genes involved in the MAPK pathway were most common (48.4%), typically in KIT (27.4%), followed by those in the PI3K pathway (12.9%), particularly in MTOR (6.5%), among the 124 CNS GCTs. Pure germinomas and non-germinomatous germ cell tumors (NGGCTs), as well as CNS and testicular GCTs, showed similar mutational profiles, suggesting that GCTs share a common molecular pathogenesis. Mutated MTOR identified in CNS GCTs upregulated phosphorylation of the AKT pathway proteins including AKT and 4EBP1 in nutrient-deprived conditions and enhanced soft-agar colony formation; both events were suppressed in a dose-dependent manner by addition of the MTOR inhibitor pp242. Our findings indicate that the dominant genetic drivers of GCTs regardless of the site of origin are activation of the MAPK and/or PI3K pathways by somatic point mutations. Mutated MTOR represents a potential target for novel targeted therapies for refractory GCTs.

MicroRNA-495 regulates starvation-induced autophagy by targeting ATG3.

The functions of some essential autophagy genes are regulated by microRNAs. However, an ATG3-modulating microRNA has never been reported. Here we show that the transcription of miR-495 negatively correlates with the translation of ATG3 under nutrient-deprived or rapamycin-treated conditions. miR-495 targets ATG3 and regulates its protein levels under starvation conditions. miR-495 also inhibits starvation-induced autophagy by decreasing the number of autophagosomes and by preventing LC3-I-to-LC3-II transition and P62 degradation. These processes are reversed by the overexpression of an endogenous miR-495 inhibitor. Re-expression of Atg3 without miR-495 response elements restores miR-495-inhibited autophagy. miR-495 sustains cell viability under starvation conditions but has no effect under hypoxia. Moreover, miR-495 inhibits etoposide-induced cell death. In conclusion, miR-495 is involved in starvation-induced autophagy by regulating Atg3.

Tumor microenvironment derived exosomes pleiotropically modulate cancer cell metabolism

Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions.

A simplified process of swine slurry treatment by primary filtration and Haematococcus pluvialis culture to produce low cost astaxanthin

A simplified process for treating swine slurry through primary filtration and subsequent depuration of the filtrate with the astaxanthin-rich microalga Haematococcus pluvialis is proposed. The first step comprises a low-cost filtration system capable of reducing 66% of ammonia, 7% of phosphorus and 19% of chemical oxygen demand, and increasing the concentration of nitrate, being this useful for subsequent growth of the algae. The second step comprises the discontinuous cultivation of H. pluvialis in diluted filtered slurry. The optimal dilution was researched by testing undiluted and 2, 4 and 8-fold diluted filtrate. This step led to a drastic reduction in macro and micronutrients concentration (up to 99% for NO3-N and NH4-N, 98% for TP and 26% for chemical oxygen demand). After H. pluvialis growth the accumulation of astaxanthin took place for 14d in nutrient-deprived conditions: an astaxanthin accumulation of 1.27% on a dry weight basis was measured. These results indicate the possibility to couple low-cost filtration and microalgae production to recover nutrients from swine wastewaters and to add value by producing valuable astaxanthin for the feed market or for an on-farm utilization as feed addictive.

The long-term survival of Acinetobacter baumannii ATCC 19606(T) under nutrient-deprived conditions does not require the entry into the viable but non-culturable state.

Acinetobacter baumannii possesses a tremendous potential to thrive under hostile conditions. To learn more about its survival strategy and capacity to persist in the environment, we studied the effect of temperature, nutrient deprivation and dryness on the long-term survival of two A. baumannii strains (ATCC 19606(T) and a clinical isolate). Our results revealed that both strains show a great persistence under stress that appears to involve a bust-and-boom strategy. Bacterial survival was differentially affected by temperature and physical environment: Desiccation favored cell resistance to stress at 20 and 37°C, while survival in aqueous environments was temperature dependent and led to changes in several cellular characteristics. In addition, we tested the ability of the A. baumannii ATCC 19606(T) strain to form biofilms by monitoring the expression of adhesion-/biofilm-related genes (ompA, bfmR and csuAB). The observed downregulation of these genes suggests that the potential difficulties to adhere to solid surfaces and form biofilms likely limit the capacity of starved cells to spread and colonize abiotic surfaces.

Notch stimulates growth by direct regulation of genes involved in the control of glycolysis and the tricarboxylic acid cycle.

Glycolytic shift is a characteristic feature of rapidly proliferating cells, such as cells during development and during immune response or cancer cells, as well as of stem cells. It results in increased glycolysis uncoupled from mitochondrial respiration, also known as the Warburg effect. Notch signalling is active in contexts where cells undergo glycolytic shift. We decided to test whether metabolic genes are direct transcriptional targets of Notch signalling and whether upregulation of metabolic genes can help Notch to induce tissue growth under physiological conditions and in conditions of Notch-induced hyperplasia. We show that genes mediating cellular metabolic changes towards the Warburg effect are direct transcriptional targets of Notch signalling. They include genes encoding proteins involved in glucose uptake, glycolysis, lactate to pyruvate conversion and repression of the tricarboxylic acid cycle. The direct transcriptional upregulation of metabolic genes is PI3K/Akt independent and occurs not only in cells with overactivated Notch but also in cells with endogenous levels of Notch signalling and in vivo. Even a short pulse of Notch activity is able to elicit long-lasting metabolic changes resembling the Warburg effect. Loss of Notch signalling in Drosophila wing discs as well as in human microvascular cells leads to downregulation of glycolytic genes. Notch-driven tissue overgrowth can be rescued by downregulation of genes for glucose metabolism. Notch activity is able to support growth of wing during nutrient-deprivation conditions, independent of the growth of the rest of the body. Notch is active in situations that involve metabolic reprogramming, and the direct regulation of metabolic genes may be a common mechanism that helps Notch to exert its effects in target tissues.

Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa.

14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1–5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses.

Reconfiguration of Transcriptional Control of Lysine Biosynthesis in Candidaalbicans Involves a Central Role for the Gcn4 Transcriptional Activator.

Evolution of transcriptional control is essential for organisms to cope with diversification into a spectrum of environments, including environments with limited nutrients. Lysine biosynthesis in fungi occurs in eight enzymatic steps. In Saccharomycescerevisiae, amino acid starvation elicits the induction of LYS gene expression, mediated by the master regulator Gcn4 and the pathway-specific transcriptional regulator Lys14. Here, we have shown that the activation of LYS gene expression in the human fungal pathogen Candidaalbicans is predominantly controlled by Gcn4 under amino acid starvation conditions. Multiple lines of study showed that the four C.albicans LYS14-like genes have no role in the regulation of lysine biosynthesis. Whereas Gcn4 is dispensable for the growth of S.cerevisiae under lysine deprivation conditions, it is an essential regulator required for the growth of C.albicans under these conditions, as gcn4 deletion caused lysine auxotrophy. Gcn4 is required for the induction of increased LYS2 and LYS9 mRNA but not for the induction of increased LYS4 mRNA. Under lysine or isoleucine-valine deprivation conditions, Gcn4 recruitment to LYS2 and LYS9 promoters was induced in C.albicans. Indeed, in contrast to the S.cerevisiae LYS gene promoters, all LYS gene promoters in C.albicans harbored a Gcn4 binding site but not all harbored the S.cerevisiae Lys14 binding site, indicating the evolutionary divergence of cis-regulatory motifs. Thus, the transcriptional rewiring of the lysine biosynthetic pathway in C.albicans involves not only neofunctionalization of the four LYS14-like genes but the attendant strengthening of control by Gcn4, indicating a coordinated response with a much broader scope for control of amino acid biosynthesis in this human pathogen. IMPORTANCE Microbes evolve rapidly so as to reconfigure their gene expression to adapt to the metabolic demands in diverse environmental niches. Here, we explored how conditions of nutrient deprivation regulate lysine biosynthesis in the human fungal pathogen Candidaalbicans. We show that although both Saccharomyces cerevisiae and C.albicans respond to lysine deprivation by transcriptional upregulation of lysine biosynthesis, the regulatory factors required for this control have been reconfigured in these species. We found that Gcn4 is an essential and direct transcriptional regulator of the expression of lysine biosynthetic genes under lysine starvation conditions in C.albicans. Our results therefore suggest that the regulation of the lysine biosynthetic pathway in Candida clade genomes involves gain of function by the master transcriptional regulator Gcn4, coincident with the neofunctionalization of the S.cerevisiae pathway-specific regulator Lys14.

Functional characterization of NAD dependent de-acetylases SIRT1 and SIRT2 in B-Cell Chronic Lymphocytic Leukemia (CLL)

ABSTRACTSirtuins (SIRT) are nicotinamide adenine dinucleotide (NAD+) dependent deacetylases or ADP- ribosyl transferases (ARTs) that deacetylate lysine residues on various proteins regulating a variety of cellular and metabolic processes. These enzymes regulate metabolism, cell survival, differentiation and DNA repair. SIRT proteins play an important role in the survival and drug resistance of cancer cells. The purpose of the present study was to investigate the expression and role of SIRT in chronic lymphocytic leukemia (CLL). We analyzed the expression of SIRT1 and SIRT2 in CLL and normal B cells using the Oncomine database as well as by Western blotting of fresh CLL cells from patients and pro-lymphocytic leukemia (PLL) cell lines, JVM-3 and MEC-2. We showed that both primary CLL cells and JVM-3 and MEC-2 cell lines overexpress high levels of functional SIRT1 and SIRT2. SIRT inhibitors EX-527 and sirtinol impair cell growth, induce ROS production, loss of mitochondrial membrane potential and apoptosis in primary CLL cells and cell lines. Using shRNA knock down of SIRT1 and SIRT2 in JVM-3 and MEC-2 cell lines, we showed that expression of both proteins is crucial for the survival of these cells. Furthermore, studies in nutrient deprived conditions suggest a role of SIRT in metabolism in CLL. These results demonstrate that the inhibition of SIRT1 and SIRT2 activity may be a new therapeutic approach for CLL.

Identification of the Factors Responsible for the Selective in vitro Cytotoxic Activity of Isoprenylated Coumarin Derivatives under Nutrient-deprived Conditions.

Pancreatic cancer is one of the most devastating forms of human cancer. The lack of effective clinical treatments for pancreatic cancer has led to one of the lowest five-year survival rates among all cancers. Recently, our laboratory has developed a novel series of isoprenylated coumarin derivatives that have exhibited anti-pancreatic cancer activity exclusively under nutrient-deprived conditions. In this study, we report the effect of the various cell culture medium components on the preferential cytotoxicity of our lead isoprenylated coumarin compound against the pancreatic adenocarcinoma cell line PANC-1. In particular, our findings show a clear link between observed cytotoxicity and glucose deprivation, suggesting that our compound targets a salvage pathway when glycolysis is no longer an option for cancer cell survival. The cytotoxicity of our lead compound was also examined in vitro against two other pancreatic cancer cell lines, BxPC-3 and Capan-2 under both nutrient-rich and nutrient-deprived conditions.

Novel mechanisms of neuroprotective effects of Quercetin on human striatal neuroblasts

Human striatal precursor (HSP) primary cell cultures were isolated from ganglionic eminence of 9-12 week old human fetuses and extensively characterized in vitro (1). Our studies demonstrated that these cultures consists of a mixed population of neural stem cells, neuronal-restricted progenitors and striatal neurons that express and are responsive to many trophic factors, as BDNF and FGF2, and possess an adaptive response to stress conditions as nutrient deprivation and hypoxia through mechanisms involving different factors and neurotrophins (1,2). In the last decades, several in vitro and in vivo studies have provided evidence for neuroprotective effects by Quercetin, a polyphenol widely present in nature, passively absorbed in the small intestine and able to traverse the blood brain barrier (3). However, the mechanisms through which Quercetin exerts its neuroprotective effects are not fully delucidated. Our study was aimed at investigating the effects of Quercetin on HSP cells and its contribution to cell survival in nutrient deprivation condition, obtained replacing culture medium with Phosphate Buffer Saline (PBS). Quercetin treatment significantly promoted cell survival and strongly decreased apoptosis induced by nutrient deprivation condition, as evaluated by MTT assay, Trypan Blue staining and western blot analysis of cell death and proliferation markers. Moreover, since the adhesive capacities of cells are essential for cell survival, we next analysed the expression of some adhesion molecules such as Pancadherin and Focal Adhesion Kinase; our results interestingly showed that PBS exposure determined a strong decrease in all the analysed adhesion molecules, while in presence of Quercetin the expression was significantly increased. Our results add new mechanicistic insights into the comprehension of neuroprotective action of Quercetin treatment, thus suggesting possible implications in sustaining striatal neuron survival during neurodegenerative disorders, such as Huntington Disease.

An insight into the cells’ glycans and lectin-glycosensing sites

Glycosylation produces a diverse and abundant repertoire of glycans that help protein folding and cellular secretion [1]. Glycoproteins often sprout sialic acid (alias N-acetyl neuraminic acid, abbreviated as Neu5Ac) at the termini of their Nand O-glycans in a process called sialylation [2]. Alterations to the normal function of the glycosylation machinery, including increased branching of N-linked glycans and sialylation, are major hallmarks of cancer progression. Particularly, increased sialylation on the cell surface induces tumor proliferation by promoting cell detachment from primary tumors through charge repulsion [3]. Despite the potential of increased or altered sialylation as diagnostic and prognostic biomarkers, there is still no early glycosensing platform that can be ubiquitously applied to detect glycans sialylation in diverse bio-samples with high selectivity and sensitivity. Further analyses have been recently found that the metabolic control of glycan’s sialylation is regulated by two factors: (1) the rate of flux through the sialic acid pathway and (2) the expression of an appropriate complement of sialyltransferase. However, none of these factors have been exploited with sufficient precision to achieve specific endpoints. Metabolic glycoengineering has been established as a very useful technique for the modulation of glycan sialylation structures on cell surfaces, and various constituents of the target sialoglycans are studied by lectins, a famous class of glycan-binding proteins [4]. Structurally, lectins have small or shallow binding sites, which results in relatively weak and non-covalent glycan interactions, often with binding constants in the millimolar range. However, the weak binding affinity and non-covalent characteristics of the lectin glyco-interactive sites can be enhanced dramatically by multivalent binding approaches [5]. Among these, the most powerful one is sensing the structural motifs and intensity of the accessible cell surface sialylation constellations [6, 7]. Djansugurova group from Kazakh Institute of General Genetics and Cytology, in collaboration with Ahmed group in the University of Maryland, reported in a preliminary communication the restoration of surface sialylation sites in a T47D breast cancer line compared to HB4A normal cells after sialic acid supplementation under conditions of nutrient deprivation [8]. Looking forward, Ahmed’s group [9] focused further efforts on modulating cell surface glycan sialylation and found increases in cell surface binding for wheat germ agglutinin (WGA), a lectin that binds to both 2,3and 2,6-linked sialic acid with slightly greater enhancement in the cancer cells (T47D, MCF7 and MDA MB231) when compared to the normal lines (MCF10A and HB4A). However, upon using plant lectins that discriminate between 2,3and 2,6-linkages, the Sambucus nigra lectin (SNA) binding to 2,6-sialic acids revealed roughly proportional increases in all lines, whereas Maackia amurensis lectin I (MAL-I) binding to 2,3-linked sialic acid showed greater enhancement in the malignant lines, compared to the normal cells. These research results highlight, for the first time, a detailed differential lectin-glycosensing site approach for the application of cell surface sialylation detection, specifically the highly dense clustered patches of sialo-epitopes NeuAc(2–3)bGal(1–4)bGlcNAc/Glc, which are displayed on the mammal cell surface envelope, enabling the detection of targeted sialoglycans in a highly sensitive and cost-effective manner [10]. More work along this line will potentially be useful for both cytochemical C.-Z. Li (&) Nanobioengineering and Bioelectronics Lab, Department of Biomedical Engineering, Florida International University, Miami, FL 33174, USA e-mail: licz@fiu.edu

The Effects of Glutamine Supplementation on Markers of Autophagy and Apoptosis in Peripheral Blood Mononuclear Cells from Patients with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is a hemoglobinopathy associated with an increased risk of pulmonary hypertension (PH) due to a number of mechanisms that includes iron overload, hemolysis, erythrocyte-derived arginase (which limits both nitric oxide and arginine bioavailability), functional splenectomy, and a hypercoagulable state among others. Glutathione (GSH, and its oxidized pair glutathione disulfide GSSG) is the principal thiol redox buffer in erythrocytes, which has been linked to hemolysis when depleted. Glutamine is not only a precursor to GSH, but also plays an anti-oxidant role through preservation of the intracellular nicotinamide adenine dinucleotide (NAD) levels, required for reducing GSSG back to GSH, thus decreasing the risk for hemolysis. Low erythrocyte glutamine levels are associated with risk of PH as defined by a tricuspid regurgitant jet velocity of (TRV) ≥2.5 m/s measured by Doppler echocardiography. SCD also exhibits an elevated level of circulating leukocytes, known as leukocytosis, which may contribute to vascular occlusion. The mechanism by which leukocytosis occurs is currently unknown. However, leukocyte cell death can be informative on the regulation of leukocyte cell numbers and measurement of mitochondrial BAX and caspase 9, are classic indicators of an active intrinsic cell death pathway. Autophagy is responsible for the turnover of macromolecules and organelles via the lysosomal degradative pathway. In this pathway, LC3 is important for the maturation and transport of autophagosomes and therefore, a reflection of autophagic activity. Autophagy ensures cell survival under certain conditions of nutrient deprivation or growth factor withdrawal and has also been implicated in innate and adaptive immune responses. In this study, the mitochondrial apoptotic marker BAX and the autophagy marker LC3 were examined in a SCD trial of glutamine therapy in patients at risk for PH.Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples taken from SCD (n=13) and control patients (n=7) and BAX and LC3 were measured via western blot analysis. Western blot results were evaluated via densitometry. SCD patients with PH-risk were treated with oral L-glutamine supplementation (10 mg TID) with the objective of estimating the level of cell death and autophagy proteins in circulating PBMCs from SCD patients at baseline and after glutamine supplementation. SCD patients were sampled at baseline (BL),and then at two weeks (W2), four weeks (W4), six weeks (W6), and eight weeks (W8) during the glutamine therapy.Results: Mean age for patients with SCD was 46±14; 39% were male with 54% having Hb-SS, while 46% had Hb-SC. Mean TRV was 3.0±0.6 m/s. The mean age for controls was 32± 12 and 57% were male; all controls were Hb-AA with a mean TRV of 1.8±0.6. At baseline there was no statistical difference in BAX expression between control and SCD patients. In comparison to baseline, however, supplementation with glutamine in SCD patients resulted in significantly increased expression of BAX in PMBCs by 15% over the 8 weeks of therapy; potentially indicating a restorative effect of glutamine on the intrinsic mitochondrial apoptotic pathway, which may ultimately reduce leukocytosis. In contrast, glutamine supplementation over 8 weeks, significantly reduced LC3 expression by 42% in PBMCs, suggesting a decrease in cellular autophagy, thus reducing the ability for PBMCs to remain in circulation.Conclusions: At baseline there was no difference in BAX expression between control and SCD patients, however, after 8 weeks of glutamine supplementation, PBMCs had an increased BAX expression and a decreased LC3 expression. This suggests that PBMCs from glutamine supplemented SCD patients may lose their ability to remain in circulation via apoptosis upregulation and autophagy downregulation DisclosuresWalter:Novartis: Research Funding.

Functional Characterization of NAD Dependent De-Acetylases SIRT1 and SIRT2 in B-Cell Chronic Lymphocytic Leukemia (CLL)

Introduction: CLL is characterized by abnormal proliferation and accumulation of mature CD5 positive B-lymphocytes in blood, bone marrow, spleen and lymph nodes. Outcome and survival is determined in part by the presence of 11q deletions and17p deletion/TP53 mutation with complex karyotype. Sirtuins are NAD+ dependent ADP-ribosyl transferases with evolutionary conserved function in cellular metabolism and chromatin regulation. Seven sirtuins (SIRT1-SIRT7) have been identified in mammals at distinct subcellular locations and targeting different substrates. SIRT1, 2, 6, and 7 are primarily found in the nucleus, SIRT2 in the cytoplasm and SIRT3, 4, 5 in the mitochondria. Sirtuins are associated with cancer as they deacetylate cancer associated transcription factors, and SIRT1 is overexpressed in acute myeloid leukemia, colon and prostate cancers. Several studies reported SIRT2 as a tumor suppressor as it is down regulated in human gliomas. SIRT1 and SIRT6 are reported to be significantly increased in CLL. We hypothesized that sirtuins play an important role in the development and maintenance of CLL and might therefore be a target. Methods: We measured SIRT 1 and 2 expression in fresh primary CLL cells, in the B-cell pro-lymphocytic cell lines JVM-3 and MEC-2 and by data-mining of the Oncomine microarray gene expression datasets. Oncomine is a bioinformatics initiative that collects, standardizes, analyzes, and delivers cancer transcriptome data to the biomedical research community. We then inhibited SIRT activity in primary CLL cells and cell lines by pharmacologic inhibitors EX-529 and sirtinol, and by knock down using shRNA in cell lines and then measured cell viability, apoptosis, reactive oxygen species formation and mitochondrial membrane potential. To determine the metabolic contribution to SIRT activity, we studied the effect of SIRT inhibition under conditions of nutrient deprivation. Results: We observed an increase in SIRT1mRNA expression in CLL by data mining of the independent microarray dataset in the Oncomine database, with a total of 2022 leukemia samples and 74 normal controls (Figure1). SIRT1was significantly up regulated in CLL compared with normal PBMC as well as other leukemia types. We found that SIRT inhibitors EX-527 and sirtinol impair cell growth (IC50 50-100 microM for EX-527 and 10-20 microM for sirtinol), cause apoptosis (&gt;2-fold increase in apoptosis in cell lines JVM-3 and MEC-2), induce ROS production (up to 90% increase in mean fluorescence intensity (MFI) with EX-527 and sirtinol), loss of mitochondrial membrane potential, (MFI from 4 to &lt;1 after treatment with SIRT inhibitors) and increase alpha-tubulin acetylation in primary CLL cells and cell lines. Using shRNA knock down of SIRT1 and SIRT2 in JVM-3 and MEC-2 cell lines, we showed that expression of both proteins is crucial for the survival of these cells. Furthermore, studies in nutrient deprived conditions suggest a role of SIRT in metabolism in CLL. Conclusion: These findings suggest that CLL cells are characterized by increased expression and function of SIRT1 and SIRT2, both directly inhibited by SIRT inhibitors. SIRT1 and SIRT2 inhibition using specific inhibitors could be a novel therapeutic approach for the treatment of CLL and other SIRT expressing hematologic malignancies. Figure 1. Figure 1. Disclosures Gordon: Northwestern University: Employment; Dr Leo I. Gordon: Patents &amp; Royalties: Patent for gold nanoparticles pending.

Catabolic pathways regulated by mTORC1 are pivotal for survival and growth of cancer cells expressing mutant Ras.

Oncogenic Ras stimulates macropinocytosis, a clathrin-independent endocytosis that increases the uptake of extracellular fluid. However, the functional significance of and regulatory mechanisms driving macropinocytosis in cancer cells remain largely unknown. Here, we show that extracellular macromolecules, such as albumin, internalized by Ras-expressing cells can support growth and survival under the nutrient-deprived conditions like those found in tumors. Moreover, we demonstrate that autophagy, a lysosome-mediated catabolic pathway, is required for the uptake and degradation of macropinocytic vesicles. Intracellular metabolites derived from macropinocytosis and autophagy directly influence the activity and localization of mTOR, which is ultimately responsible for the restoration of cell growth. Surprisingly, suppression of mTORC1, which typically triggers anabolic processes, facilitates macropinocytosis and thus supports cell growth and survival under the nutrient-deprived conditions. In a mouse xenograft model of pancreatic ductal adenocarcinoma, concomitant inhibition of macropinocytosis/autophagy and mTOR activity resulted in antitumor effects. These data suggest that novel anti-cancer strategies interrupting these metabolic processes and related signaling molecules may represent promising therapeutic avenues.

Analysis of Yeast Sporulation Efficiency, Spore Viability, and Meiotic Recombination on Solid Medium.

Under conditions of nutrient deprivation, yeast cells initiate a differentiation program in which meiosis is induced and spores are formed. During meiosis, one round of genome duplication is followed by two rounds of chromosome segregation (meiosis I and meiosis II) to generate four haploid nuclei. Meiotic recombination occurs during prophase I. During sporogenesis, each nucleus becomes surrounded by an individual spore wall, and all four haploid spores become contained as a tetrad within an ascus. Important insights into the meiotic function(s) of a gene of interest can be gained by observing the effects of gene mutations on spore viability and viability patterns among tetrads. Moreover, recombination frequencies among viable spores can reveal potential involvement of the gene during meiotic exchange between homologous chromosomes. Here, we describe methods for inducing spore formation on solid medium, determining spore viability, and measuring, via tetrad analysis, frequencies of crossing over and gene conversion as indicators of meiotic chromosome exchange.