Networking of differentially expressed genes in human cancer cells resistant to methotrexate

BACKGROUND: The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). METHODS: Seven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software. RESULTS: Dikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. CONCLUSIONS: Biological association networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX.

Networking of differentially expressed genes in human cancer cells resistant to methotrexate

BACKGROUND: The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). METHODS: Seven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software. RESULTS: Dikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. CONCLUSIONS: Biological association networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX.

Increased alpha-defensins 1-3 production by dendritic cells in HIV-infected individuals is associated with slower disease progression.

Defensins are natural endogenous antimicrobial peptides with potent anti-HIV activity and immuno-modulatory effects. We recently demonstrated that immature dendritic cells (DC) produce α-defensins1-3 and that α-defensins1-3 modulate DC generation and maturation. Since DC-HIV interaction plays a critical role during the first steps of HIV infection, we investigated the possible impact of α-defensins1-3 production by DC on disease progression.

Increased alpha-defensins 1-3 production by dendritic cells in HIV-infected individuals is associated with slower disease progression.

Defensins are natural endogenous antimicrobial peptides with potent anti-HIV activity and immuno-modulatory effects. We recently demonstrated that immature dendritic cells (DC) produce α-defensins1-3 and that α-defensins1-3 modulate DC generation and maturation. Since DC-HIV interaction plays a critical role during the first steps of HIV infection, we investigated the possible impact of α-defensins1-3 production by DC on disease progression.

Hepatocyte nuclear factor-4 alpha regulates the human apolipoprotein AV gene: Identification of a novel response element and involvement in the control by peroxisome proliferator-activated receptor-gamma coactivator-1 alpha, AMP-activated protein kinase, and mitogen-activated protein kinase pathway

The recently discovered apolipoprotein AV (apoAV) gene has been reported to be a key player in modulating plasma triglyceride levels. Here we identify the hepatocyte nuclear factor-4 (HNF-4 ) as a novel regulator of human apoAV gene. Inhibition of HNF-4 expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4 directly regulates human apoAV promoter through DR1 [a direct repeat separated by one nucleotide (nt)], and via a novel element for HNF-4 consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor- coactivator-1 was capable of stimulating the HNF-4 -dependent transactivation of apoAV promoter. Furthermore, analyses in human hepatic cells demonstrated that AMP-activated protein kinase (AMPK) and the MAPK signaling pathway regulate human apoAV expression and suggested that this regulation may be mediated, at least in part, by changes in HNF-4 . Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4 gene revealed a species-distinct regulation of apoAV by HNF-4 , which resembles that of a subset of HNF-4 target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4 and underscore the role of HNF-4 in regulating triglyceride metabolism.

Heparin attenuates TNF-alpha induced inflammatory response through a CD11b dependent mechanism

Background In addition to its anticoagulant properties, heparin has anti-inflammatory effects, the molecular and mechanistic bases of which are incompletely defined. AIMS The current studies were designed to test the hypothesis that heparin abrogates the expression or function of leucocyte-endothelial adherence molecules which are fundamental to the acute inflammatory response. Methods The effects of heparin on tumour necrosis factor alpha (TNF-¿) induced leucocyte rolling, adhesion, and migration as well as vascular permeability were assessed in rat mesenteric venules using intravital microscopy. Expression of adhesion molecules was quantitated using a double radiolabelled monoclonal antibody (mAb) binding technique in vivo (P-selectin, intercellular cell adhesion molecule type 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1)) or flow cytometry (CD11a, CD11b, and L-selectin). Ex vivo binding of heparin to neutrophils was assessed by flow cytometry. RESULTS TNF-alpha induced a significant increase in leucocyte rolling, adhesion, and migration, and vascular permeability, coincident with a significant increase in expression of P-selectin, ICAM-1, and VCAM-1. Ex vivo assessment of blood neutrophils showed significant upregulation of CD11a and CD11b and significant downregulation of L-selectin within five hours of TNF-¿ administration. Heparin pretreatment significantly attenuated leucocyte rolling, adhesion, and migration but did not affect expression of cell adhesion molecules or vascular permeability elicited by TNF-¿ administration. Binding of heparin was significantly increased on blood neutrophils obtained five hours after TNF-¿ administration. Preincubation with an anti-CD11b mAb but not with an anti-CD11a or anti-L-selectin antibody significantly diminished heparin binding ex vivo.

PGC-1α induces mitochondrial and myokine transcriptional programs and lipid droplet and glycogen accumulation in cultured human skeletal muscle cells

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a chief activator of mitochondrial and metabolic programs and protects against atrophy in skeletal muscle (skm). Here we tested whether PGC-1α overexpression could restructure the transcriptome and metabolism of primary cultured human skm cells, which display a phenotype that resembles the atrophic phenotype. An oligonucleotide microarray analysis was used to reveal the effects of PGC-1α on the whole transcriptome. Fifty-three different genes showed altered expression in response to PGC-1α: 42 upregulated and 11 downregulated. The main gene ontologies (GO) associated with the upregulated genes were mitochondrial components and processes and this was linked with an increase in COX activity, an indicator of mitochondrial content. Furthermore, PGC-1α enhanced mitochondrial oxidation of palmitate and lactate to CO2, but not glucose oxidation. The other most significantly associated GOs for the upregulated genes were chemotaxis and cytokine activity, and several cytokines, including IL-8/CXCL8, CXCL6, CCL5 and CCL8, were within the most highly induced genes. Indeed, PGC-1α highly increased IL-8 cell protein content. The most upregulated gene was PVALB, which is related to calcium signaling. Potential metabolic regulators of fatty acid and glucose storage were among mainly regulated genes. The mRNA and protein level of FITM1/FIT1, which enhances the formation of lipid droplets, was raised by PGC-1α, while in oleate-incubated cells PGC-1α increased the number of smaller lipid droplets and modestly triglyceride levels, compared to controls. CALM1, the calcium-modulated δ subunit of phosphorylase kinase, was downregulated by PGC-1α, while glycogen phosphorylase was inactivated and glycogen storage was increased by PGC-1α. In conclusion, of the metabolic transcriptome deficiencies of cultured skm cells, PGC-1α rescued the expression of genes encoding mitochondrial proteins and FITM1. Several myokine genes, including IL-8 and CCL5, which are known to be constitutively expressed in human skm cells, were induced by PGC-1α.

Functional interaction between peroxisome proliferator-activated receptors-alpha and Mef-2C on human carnitine palmitoyltransferase 1beta (CPT1beta) gene activation

Muscle-type carnitine palmitoyltransferase 1 (CPT1β) is considered to be the gene that controls fatty acid mitochondrial β-oxidation. A functional peroxisome proliferator-activated receptor (PPAR) responsive element (PPRE) and a myocite-specific (MEF2) site that binds MEF2A and MEF2C in the promoter of this gene had been previously identified. We investigated the roles of the PPRE and the MEF2 binding sites and the potential interaction between PPARα and MEF2C regulating the CPT1β gene promoter. Mutation analysis indicated that the MEF2 site contributed to the activation of the CPT1β promoter by PPAR in C2C12 cells. The reporter construct containing the PPRE and the MEF2C site was synergistically activated by co-expression of PPAR, retinoid X receptor (RXR) and MEF2C in non-muscle cells. Moreover, protein-binding assays demonstrated that MEF2C and PPAR specifically bound to one another in vitro. Also for the synergistic activation of the CPT1β gene promoter by MEF2C and PPARα-RXRα, a precise arrangement of its binding sites was essential.

Simultaneous determination of gross alpha, gross beta and Ra-226 in natural water by liquid scintillation counting.

The determination of gross alpha, gross beta and 226Ra activity in natural waters is useful in a wide range of environmental studies. Furthermore, gross alpha and gross beta parameters are included in international legislation on the quality of drinking water [Council Directive 98/83/EC].1 In this work, a low-background liquid scintillation counter (Wallac, Quantulus 1220) was used to simultaneously determine gross alpha, gross beta and 226Ra activity in natural water samples. Sample preparation involved evaporation to remove 222Rn and its short-lived decay daughters. The evaporation process concentrated the sample ten-fold. Afterwards, a sample aliquot of 8 mL was mixed with 12 mL of Ultima Gold AB scintillation cocktail in low-diffusion vials. In this study, a theoretical mathematical model based on secular equilibrium conditions between 226Ra and its short-lived decay daughters is presented. The proposed model makes it possible to determine 226Ra activity from two measurements. These measurements also allow determining gross alpha and gross beta simultaneously. To validate the proposed model, spiked samples with different activity levels for each parameter were analysed. Additionally, to evaluate the model's applicability in natural water, eight natural water samples from different parts of Spain were analysed. The eight natural water samples were also characterised by alpha spectrometry for the naturally occurring isotopes of uranium (234U, 235U and 238U), radium (224Ra and 226Ra), 210Po and 232Th. The results for gross alpha and 226Ra activity were compared with alpha spectrometry characterization, and an acceptable concordance was obtained.

Preparation of alpha-labeled aldehydes by base-catalyzed exchange reactions

A simple, efficient protocol for the preparation of α-labeled aldehydes based on H/D exchange catalyzed by 4-(N,N-dimethylamino)pyridine or Et3N is described. High chemical yields and ratios of isotope incorporation were obtained even when small amounts (1 mmol) of aldehyde were used.

Preparation of alpha-labeled aldehydes by base-catalyzed exchange reactions

A simple, efficient protocol for the preparation of α-labeled aldehydes based on H/D exchange catalyzed by 4-(N,N-dimethylamino)pyridine or Et3N is described. High chemical yields and ratios of isotope incorporation were obtained even when small amounts (1 mmol) of aldehyde were used.

Simultaneous determination of gross alpha, gross beta and Ra-226 in natural water by liquid scintillation counting.

The determination of gross alpha, gross beta and 226Ra activity in natural waters is useful in a wide range of environmental studies. Furthermore, gross alpha and gross beta parameters are included in international legislation on the quality of drinking water [Council Directive 98/83/EC].1 In this work, a low-background liquid scintillation counter (Wallac, Quantulus 1220) was used to simultaneously determine gross alpha, gross beta and 226Ra activity in natural water samples. Sample preparation involved evaporation to remove 222Rn and its short-lived decay daughters. The evaporation process concentrated the sample ten-fold. Afterwards, a sample aliquot of 8 mL was mixed with 12 mL of Ultima Gold AB scintillation cocktail in low-diffusion vials. In this study, a theoretical mathematical model based on secular equilibrium conditions between 226Ra and its short-lived decay daughters is presented. The proposed model makes it possible to determine 226Ra activity from two measurements. These measurements also allow determining gross alpha and gross beta simultaneously. To validate the proposed model, spiked samples with different activity levels for each parameter were analysed. Additionally, to evaluate the model's applicability in natural water, eight natural water samples from different parts of Spain were analysed. The eight natural water samples were also characterised by alpha spectrometry for the naturally occurring isotopes of uranium (234U, 235U and 238U), radium (224Ra and 226Ra), 210Po and 232Th. The results for gross alpha and 226Ra activity were compared with alpha spectrometry characterization, and an acceptable concordance was obtained.