Subtle metabolic and liver gene transcriptional changes underlie diet-induced fatty liver susceptibility in insulin-resistant mice.

AIMS/HYPOTHESIS: Complex changes in gene expression are associated with insulin resistance and non-alcoholic fatty liver disease (NAFLD) promoted by feeding a high-fat diet (HFD). We used functional genomic technologies to document molecular mechanisms associated with diet-induced NAFLD. MATERIALS AND METHODS: Male 129S6 mice were fed a diet containing 40% fat (high-fat diet, HFD) for 15 weeks. Glucose tolerance, in vivo insulin secretion, plasma lipid profile and adiposity were determined. Plasma metabonomics and liver transcriptomics were used to identify changes in gene expression associated with HFD-induced NAFLD. RESULTS: In HFD-fed mice, NAFLD and impaired glucose and lipid homeostasis were associated with increased hepatic transcription of genes involved in fatty acid uptake, intracellular transport, modification and elongation, whilst genes involved in beta-oxidation and lipoprotein secretion were, paradoxically, also upregulated. NAFLD developed despite strong and sustained downregulation of transcription of the gene encoding stearoyl-coenzyme A desaturase 1 (Scd1) and uncoordinated regulation of transcription of Scd1 and the gene encoding sterol regulatory element binding factor 1c (Srebf1c) transcription. Inflammatory mechanisms appeared to be stimulated by HFD. CONCLUSIONS/INTERPRETATION: Our results provide an accurate representation of subtle changes in metabolic and gene expression regulation underlying disease-promoting and compensatory mechanisms, collectively contributing to diet-induced insulin resistance and NAFLD. They suggest that proposed models of NAFLD pathogenesis can be enriched with novel diet-reactive genes and disease mechanisms.

ST7-mediated suppression of tumorigenicity of prostate cancer cells is characterized by remodeling of the extracellular matrix.

Multiple lines of evidence have provided compelling evidence for the existence of a tumor suppressor gene (TSG) on chromosome 7q31.1. ST7 may be the target of this genetic instability but its designation as a TSG is controversial. In this study, we show that, functionally, ST7 behaves as a tumor suppressor in human cancer. ST7 suppressed growth of PC-3 prostate cancer cells inoculated subcutaneously into severe combined immunodeficient mice, and increased the latency of tumor detection from 13 days in control tumors to 23 days. Re-expression of ST7 was also associated with suppression of colony formation under anchorage-independent conditions in MDA-MB-231 breast cancer cells and ST7 mRNA expression was downregulated in 44% of primary breast cancers. Expression profiling of PC-3 cells revealed that ST7 predominantly induces changes in genes involved in re-modeling the extracellular matrix such as SPARC, IGFBP5 and several matrix metalloproteinases. These data indicate that ST7 may mediate tumor suppression through modification of the tumor microenvironment.

Effects of ras and von Hippel-Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3'-kinase/Akt signaling pathway.

Many oncogenes induce expression of vascular endothelial growth factor (VEGF), a key factor in tumor angiogenesis. Phosphatidylinositol 3'-kinase (PI3K)/Akt is a common signaling pathway for oncogenes and tumor suppressor genes and is involved in VEGF regulation. Because hypoxia is a major stimulus for VEGF production, we examined the effects of LY294002, a selective PI3K inhibitor, on hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression and on endogenous VEGF responses to hypoxia. A panel of breast cancer cell lines reflecting the different genetic changes occurring in human breast cancer was analyzed. LY294002 inhibited HIF-1alpha induction and phosphorylation under hypoxia. However, HIF-2alpha expression was not affected. Basal and hypoxia-inducible VEGF expression was reduced at both mRNA and protein levels by 50%. V12-ras overexpression resulted in an increase in hypoxia-induced HIF-1alpha and HIF-2alpha expression. This effect was blocked by PI3K inhibitor, demonstrating one mechanism for ras synergy with hypoxia-mediated induction of genes. The decreased HIF-1alpha expression was not dependent on VHL interaction because a renal carcinoma cell line with VHL mutation and constitutive high HIF-1alpha expression also showed down-regulation of HIF-1alpha after treatment with LY294002. These results have implications for the use of PI3K inhibitors to inhibit synergistic effects of hypoxia with a wide range of common oncogenes.

Cloning and characterization of a novel transcription factor involved in cellular proliferation arrest: PATF.

Cell cycle withdrawal involves several transcription factors such as E2Fs members that play a key role in cell growth control. Here we describe a novel putative bZIP transcription factor isolated from the retina and involved in neuronal proliferation arrest at the terminal differentiation: PATF (Proliferation Arrest Transcription Factor). We show that PATF associates with E2F4 protein and interacts with the E2F consensus site. PATF expression increases with establishment of quiescent state. Furthermore, the nuclear PATF localization like E2F4, depends on cell growth arrest. The decrease of PATF amount, using a retroviral antisense strategy, results in pursued neuroretina cell mitosis. Our results indicate that PATF could be a new molecular signal implicated in the final neuronal cell cycle withdrawal.

Association of ARNT splice variants with estrogen receptor-negative breast cancer, poor induction of vascular endothelial growth factor under hypoxia, and poor prognosis.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a basic helix-loop-helix transcription factor that forms heterodimers with the aryl hydrocarbon receptor (AhR) or hypoxia inducible factor-1alpha to activate transcription via xenobiotic response element or hypoxia response element, respectively. Thus, it plays a major role in two key biochemical pathways involved in tumor growth. We previously showed that estrogen receptor (ER)-negative breast cancer cell lines expressed a splice variant of ARNT that was associated with Ah nonresponsiveness. We have now used a sensitive PCR method to analyze the expression of the variant in a series of 92 breast cancers to assess interactions with the ER and prognosis. The splice variant could be detected in all of the cases examined, with high ratios of variant:full-length ARNT (> or =10) characterized in 10 cases. When the patient group was split into quartiles by increasing splice variant ratios, there was an inverse relationship of ER status to ARNT splice-variant ratios (P = 0.01, chi(2)). Univariate analysis showed that cases with high ARNT splice-variant ratios > or =10 had a worse relapse-free and overall survival (P > or = 0.03; hazard ratio, 2.7; and P = 0.006; hazard ratio, 3.9, respectively). In multivariate analysis for relapse-free and overall survival, ARNT splice-variant ratio was the strongest independent factor and, although inversely related to ER, remained a separate risk factor. At least two potential mechanisms could explain this phenomenon: the loss of aryl hydrocarbon receptor-mediated antiestrogenic activity or the blockade of a proapoptotic pathway induced by hypoxia. Because several enzymes involved in drug resistance are induced through a xenobiotic response element, the tumors presenting high ARNT splice-variant ratios may be specifically targeted by drugs normally degraded or inactivated. This study shows the biological importance of ARNT splice variants in the behavior of human breast cancer and suggests that the breast cell lines in which the splice variant was discovered may be useful models for further investigation.

SDS -PAGE and Western Blotting Techniques.

The goal of Western blotting, or more correctly, immunoblotting, is to identify with a specific antibody a particular antigen within a complex mixture of proteins that has been fractionated in a polyacrylamide gel and immobilized onto a membrane. Immunoblotting can be used to determine a number of important characteristics of protein antigens-the presence and quantity of an antigen, the relative molecular weight of the polypeptide chain, and the efficiency of extraction of the antigen.Immunoblotting occurs in six stages: (1) extraction and quantification of protein samples; (2) resolution of the protein sample in sodium dodecyl sulfatepolyacrylamide denaturing gel electrophoresis (SDS-PAGE); (3) transfer of the separated polypeptides to a membrane support; (4) blocking nonspecific binding sites on the membrane; (5) addition of antibodies; and (6) detection.Sample preparation is important for obtaining accurate separation of the proteins on the basis of molecular weight. Depending on whether an antigen is primarily extracellular, cytoplasmic, or membrane-associated different procedures might be required to prepare the sample initially. Although there are exceptions, many soluble nuclear and cytoplasmic proteins can be solubilized by lysis buffers that contain the nonionic detergent Nonidet P-40 (NP-40) and either no salt at all or relatively high concentrations of salt (e.g., 0.5 M NaCl). However, the efficiency of extraction is often greatly affected by pH of the buffer and the presence or absence of chelating agents such EDTA.

Relationship of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression to vascular endothelial growth factor induction and hypoxia survival in human breast cancer cell lines.

Hypoxia-inducible factors (HIF-1 and HIF-2) are two closely related protein complexes that activate transcription of target genes in response to hypoxia. Expression of HIF-1alpha and HIF-2alpha and their effects on survival under hypoxia were studied in six human breast cancer cell lines. We also evaluated the basal and inducible expression of two hypoxically regulated genes, vascular endothelial growth factor (VEGF) and lactate dehydrogenase-A (LDH-A). All of the cell lines studied expressed HIF-1alpha at various levels, but HIF-2alpha was low or absent from the more aggressive cell lines. There was an inverse correlation between HIF-1alpha and HIF-2alpha induction and clonogenic survival under hypoxia. Thus, cell lines with reduced induction of HIF-1alpha or HIF-2alpha showed high basal levels of VEGF and improved survival under hypoxia. A reduction in HIF expression was also associated with a more aggressive phenotype in vivo. To confirm these results, we carried out stable transfection of the MDA 435 cell line with human HIF-2alpha cDNA. There was no change in the growth rate in monolayer culture. However, in vitro growth as colonies and in vivo tumor growth of the HIF-2alpha overexpressing cells were significantly impaired compared with the control transfectants. Thus, despite the fact that HIF proteins are necessary for optimal tumor growth and angiogenesis in vivo, overexpression of these molecules seems detrimental to tumor growth. A balance between the angiogenic and tumor-inhibiting levels of HIF proteins may, therefore, be necessary for optimal tumor growth.

The molecular basis of the hypoxia response pathway: tumour hypoxia as a therapy target.

Hypoxia induces a cascade of physiological responses that includes glycolysis, erythropoiesis, angiogenesis, changes in adrenergic signal transduction and vascular cellular proliferation. Hypoxia-inducible genes are relevant to growth and behaviour of cancer as well as the adaptation and survival of normal tissues. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric DNA binding complex composed of two basic-helix-loop-helix PAS-proteins: HIF-1 beta/ARNT (aryl hydrocarbon receptor nuclear translocator), which is constitutively expressed, and HIF-1 alpha, which is not present in normoxic cells but induced under hypoxic conditions. Recently another member of the bHLH-PAS family, EPAS-1 has been reported and shares similar properties with HIF-1 alpha, although it is considered endothelial specific. In addition, the presence of other DNA-binding motifs in the promoter of hypoxia-inducible genes highlight the occurrence of cross-talk between transcription factors in the modulation of hypoxic gene expression. In this review, we present a survey of the hypoxia response pathway and we discuss attempts to use gene therapy activated by the low oxygen environment or by necrotic regions of tumours.

Retinal dysplasia in mice lacking p56lck.

The product of the proto-oncogene p56lck is a non-receptor tyrosine kinase member of the Src family. It is found in T cells (Marth et al., 1985, 1988) and in the mouse brain (Omri et al., 1996; Van Tan et al., 1996). In this report, we describe experiments showing that Lck is present in the mouse retina neurons. Lck gene expression was identified after isolating and sequencing the specific 5' and 3' part of the cDNA obtained by RT-PCR. In adult retina Lck immunoreactivity was most abundant in photoreceptor cells and within the outer plexiform layers. Staining was also observed in the inner nuclear and plexiform layers. In transgenic mice, the disruption of the Lck gene had serious consequences on the organization of the retina causing retinal dysplasia. These mice have partial retinal detachment with infolding and rosette formation in the photoreceptor sheet. These retinal abnormalities observed in Lck deficient mice lead to the loss of normal architecture of the photoreceptor and the inner nuclear layers, and provide an important role of Lck protein in the retina development. The lack of the Lck protein produces a spectrum of retinal pathology that resembles human retinopathy of prematurity (ROP).

Nectinepsin: a new extracellular matrix protein of the pexin family. Characterization of a novel cDNA encoding a protein with an RGD cell binding motif.

We report the isolation and characterization of a novel cDNA from quail neuroretina encoding a putative protein named nectinepsin. The nectinepsin cDNA identifies a major 2.2-kilobase mRNA that is detected from ED 5 in neuroretina and is increasingly abundant during embryonic development. A nectinepsin mRNA is also found in quail liver, brain, and intestine and in mouse retina. The deduced nectinepsin amino acid sequence contains the RGD cell binding motif of integrin ligands. Furthermore, nectinepsin shares substantial homologies with vitronectin and structural protein similarities with most of the matricial metalloproteases. However, the presence of a specific sequence and the lack of heparin and collagen binding domains of the vitronectin indicate that nectinepsin is a new extracellular matrix protein. Furthermore, genomic Southern blot studies suggest that nectinepsin and vitronectin are encoded by different genes. Western blot analysis with an anti-human vitronectin antiserum revealed, in addition to the 65- and 70-kDa vitronectin bands, an immunoreactive protein of about 54 kDa in all tissues containing nectinepsin mRNA. It seems likely that the form of vitronectin found in chick egg yolk plasma by Nagano et al. ((1992) J. Biol. Chem. 267, 24863-24870) is the protein that corresponds to the nectinepsin cDNA. This new protein could be an important molecule involved in the early steps of the development.

A novel cDNA corresponding to transcripts expressed in retina post-mitotic neurons.

The long term objective of this study is to isolate genes specifically expressed at the onset of neuronal cell cycle withdrawal. As an experimental paradigm we have used a quail neuroretinal cell clone (clone K2) immortalized by a thermosensitive mutant of Rous Sarcoma Virus. K2 cells proliferate at 36 degrees C but stop synthesizing DNA after a shift to 41.5 degrees C. We have constructed a cDNA library from K2 cells transferred to 41.5 degrees C and autosubtracted with RNAs from K2 cells maintained at 36 degrees C. This strategy has led to the isolation of cDNAs which recognize mRNAs expressed in quail neuroretina (NR) during development. We report here one of these cDNAs, cDNA QN1, that hybridizes with transcripts expressed in retina neurons, in parallel with their withdrawal from the cell cycle. QN1 ORF codes for a 138 kDa polypeptide corresponding to the protein observed in Western blot analysis. A role of QN1 product(s) on neuronal quiescence is suggested by the positive effect of an antisense oligonucleotide on DNA synthesis of K2 cells.