Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glucokinase gene regions that are important for liver specific expression of the enzyme have been functionally identified using transient transfection of rat hepatocytes. Maximal luciferase activity was elicited by a reporter plasmid with 3.4 kilobase pairs of genomic DNA flanking the liver glucokinase promoter. Deletion of a gene fragment between -1000 and -600 with respect to the start of transcription resulted in a 60% decrease in luciferase activity. Further reduction, close to background level, occurred upon deletion of a 90-base pair sequence between -123 and -34. Reporter plasmids with the liver glucokinase promoter and any length of flanking sequence were minimally active in INS-1 insulinoma cells, and conversely reporters with the beta-cell-specific promoter were ineffective in primary hepatocytes. In FTO-2B hepatoma cells, a differentiated line expressing many liver-specific traits but not the endogenous glucokinase gene, the promoter proximal region between -123 and -34 markedly stimulated the expression of transfected plasmids above background. However, addition of the flanking region up to -1000 inhibited luciferase expression. The gene fragment from -1003 to -707 was shown to be a bona fide, hepatocyte-specific enhancer by the following criteria: 1) it stimulated reporter expression by more than 10- and 5-fold when inserted directly upstream of the glucokinase TATA box or complete promoter, respectively, regardless of orientation; 2) it stimulated gene expression from the heterologous SV 40 promoter 4-fold; 3) it was also effective from a downstream position; and 4) in contrast to the enhancer effect in primary hepatocytes, the sequence acted as a silencer in FTO-2B cells and was neutral in INS-1 cells. Both the promoter proximal and the enhancer regions were marked by DNase I hypersensitive sites in the chromatin of primary hepatocytes but not hepatoma or insulinoma cells. Seven footprinted elements termed A through G were mapped in the enhancer by the in vitro DNase I protection assay. Elements A-C may bind liver enriched factors, because they were not protected by spleen nuclear extract. In hepatocyte transfection, the downstream half of the enhancer containing elements A-C was about half as effective as the complete enhancer in stimulating glucokinase promoter activity. Site-directed mutagenesis of element A virtually abrogated the activity of the half-enhancer, whereas mutation of element C had a more moderate effect. The sequence between -732 and -578 upstream of the liver start of transcription in the human glucokinase gene displays 79% sequence identity with the downstream half of the rat enhancer. The human gene fragment ligated to the minimal rat liver glucokinase promoter was shown to work as an enhancer in the hepatocyte transfection system.
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PMID:Liver-specific enhancer of the glucokinase gene. 891 May 67

We have shown previously that rat hepatoma FTO-2B cells express two mRNAs, called F (fetal) and L (liver), from distinct promoters of the same gene coding for 6-phosphofructo-2-kinase (PFK-2). This enzyme catalyzes the synthesis of fructose 2,6-bisphosphate, an allosteric stimulator of glycolysis. We have now found that glucose, as well as lactate and pyruvate, increases the concentration of the F and L mRNAs. The effect of glucose was mimicked by xylitol, a precursor of xylulose 5-phosphate, and hence of intermediates of the pentose phosphate and glycolytic pathways, and was inhibited by okadaic acid, an inhibitor of protein phosphatases. Transfection experiments showed that the F promoter region is a target of the glucose effect, with glucose stimulating F promoter activity in a way probably similar to mitogens. Another region of the gene, located between the F and L promoters, also behaved as a glucose-sensitive element. This region corresponds to a cluster of DNase I-hypersensitive sites that were induced in chromatin following glucose treatment. The sequence organization of this region is very similar to the functional architecture of the glucose-sensitive insulin gene promoter. We propose a model for the concerted regulation by glucose metabolites of three pathways that lead to increased PFK-2 activity.
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PMID:Glucose response elements in a gene that codes for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. 932 10

Rat hepatoma cells were engineered to express, in a regulated manner, mature human insulin as an approach to the development of artificial beta-cells for insulin-dependent diabetes mellitus (IDDM) gene therapy. A chimeric gene obtained by linking a 2.4-kb fragment of the P-enolpyruvate carboxykinase (PEPCK) gene promoter to a human proinsulin gene (PEPCK/Insm), containing genetically engineered furin endoprotease cleavage sites, was stably transfected into FTO-2B rat hepatoma cells. The FTOInsm cells expressed high levels of insulin mRNA and protein after Northern blot or immunocytochemical analysis. High-performance liquid chromatography (HPLC) fractionation of culture medium and cell extracts revealed that about 90% of the proinsulin was processed to mature insulin. Insulin secretion was very fast, and 15 min after induction with dibutyryl cyclic AMP (Bt2cAMP) plus dexamethasone significant amounts of the hormone were released. Moreover, during the first hour, the rise in insulin concentration in the medium was 10-fold that detected in nontreated FTOInsm cells. Insulin produced by FTOInsm cells was biologically active because it blocked endogenous PEPCK gene expression and induced glucose uptake and lactate production. Thus, our results showed that genetically engineered FTOInsm hepatoma cells synthesized, processed, and secreted active insulin. The implantation of encapsulated engineered FTOInsm cells might provide a safe and practical therapeutic approach for IDDM treatment.
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PMID:Regulated production of mature insulin by non-beta-cells. 944 78

Hepatoma cell lines can be characterized by their expression of hepatocyte- and biliary-specific genes and by their response to differentiating agents in a lineage-dependent manner. These characteristics can be used to map the maturational lineage position of the cell lines. Tissue-specific gene expression and regulation by heparin, dimethylsulfoxide (DMSO), and sodium butyrate (SB) were examined in three rat hepatoma cell lines and two rat liver epithelial cell lines. Based on antigenic profiles and gene expression in serum-supplemented medium, the hepatoma cell lines could be organized in distinct categories of hepatic differentiation. All three hepatomas expressed the following five genes: gamma-glutamyl transpeptidase (GGT), glutathione-S-transferase pi (Yp), glutamine synthetase, and alpha 5 and beta 1 integrin. Cell line H4AzC2 also expressed alpha-fetoprotein (AFP), albumin. IGF II receptor, and the biliary/oval cell antigens OC.2 and OC.3, a phenotype characteristic of fetal hepatocytes. FTO-2B cells lacked AFP, OC.2, and OC.3 but expressed albumin and IGF II receptor in addition to the five commonly expressed genes, consistent with a more hepatocyte-like phenotype. Cell line H5D-7 expressed neither albumin nor the IGF II receptor, but did express OC.2, OC.3, and alpha 3 integrin in addition to the five commonly expressed genes, characteristic of biliary epithelial cells. Regulation of gene expression by heparin, DMSO, and SB was examined in cells cultured in hormonally defined medium. The patterns of regulation of AFP, albumin, GGT, and Yp were dependent upon the state of differentiation of the cell. FTO-2B cells regulated genes in a manner similar to that of E16 fetal hepatocytes, H4AzC2 regulated genes characteristic of both hepatocytic and biliary lineages, and H5D.7 regulated only biliary genes. Suppression of GGT by DMSO was uniformly observed. The three cell lines expressed equal amounts of HNF-4, but FTO-2B cells expressed more HNF-3 beta and less HNF-3 alpha, while the reverse was true of H4AzC2 and H5D.7 cells.
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PMID:Phenotypic characterization of rat hepatoma cell lines and lineage-specific regulation of gene expression by differentiation agents. 974 12

Studies on the pathobiology of chronic (long-term) hepatitis B virus (HBV) infection and in vitro drug testing have been hampered by the lack of appropriate systems for culturing susceptible cells chronically infected with HBV. Most of the in vitro studies of HBV replication have been performed with HBV genome-transduced cell lines. In this system, viral production is mainly the result of chromosomal replication. In an in vitro infection system, owing to medium refreshment (which leads to the removal of infectious particles necessary for the perpetuation of infection) and to trypsinization for cell passages, it is difficult, if not impossible, to maintain chronic HBV infection, despite the use of susceptible cells. To circumvent these unfavourable factors for chronic HBV infection in vitro, we cultured microcarrier-attached immortalized human hepatocytes, infected with HBV, in molecularporous (MW 12,000-14,000) membrane (dialysis) bags for a duration of 2 months. HBV covalently-closed-circular (ccc) DNA, HBV precore/core and X mRNAs were detected in the cells cultured in this system following infection until the end of the experiment (day 58), while in classical culture conditions (monolayer), markers of HBV replication were also detected. Production of hepatitis B surface antigen (HBsAg) and HBV DNA was detected and their levels in culture medium (collected at the end of experiments from the molecularporous membrane bags) were increased 2.86- and 3.28-fold respectively. Using Southern blot analysis, HBV replicative intermediates could also be demonstrated throughout the experiments. However, integrated HBV DNA was not present. In contrast, HBV ccc DNA, HBV precore/core and X mRNAs, and replicative intermediates were not demonstrable in FTO 2B rat hepatoma cells infected in the same manner in parallel experiments. This in vitro infection system, using susceptible, immortalized human hepatocytes, therefore provides a new tool for studying the long-term effect of HBV infection, mainly involving episomal replication in hepatocytes, and for drug testing.
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PMID:Hepatitis B virus infection in microcarrier-attached immortalized human hepatocytes cultured in molecularporous membrane bags: a model for long-term episomal replication of HBV. 985 47

Previously, we have found that human liver annexin V (hA-V; in earlier reports referred as Endonexin II) is a specific hepatitis B surface antigen (HBsAg) binding protein. In this study, we demonstrate that transfection of rat hepatoma FTO 2B cells, a cell line that is not infectable by hepatitis B virus (HBV) and does not express hA-V, with a construct containing the hA-V gene, resulted in hA-V expressing cells susceptible to HBV infection. After in vitro infection, transfected FTO cells (assigned as FTO 9.1 cells) expressing hA-V in cultures were shown to contain HBV-precore/core, X mRNAs, and covalently closed circular (ccc) DNA as detected by polymerase chain reaction (PCR). The presence of HBV ccc and replicative intermediate DNA was also demonstrated by Southern blot hybridization assay. HBV DNA secreted in the culture medium was also evident as determined by quantitative branched DNA (bDNA) assay. HBsAg and hepatitis B core antigen (HBcAg) could also be detected by an immunocytochemical method in 10% to 15% of the cells at day 3 and day 5 after infection. Infectivity of in vitro-propagated HBV was demonstrated by infection of the naive FTO 9.1 cells with the culture supernatant from HBV-carrier cultures. In contrast to primary cultures of human hepatocytes and FTO 9.1 cells, primary rat and mouse hepatocytes, as well as rat hepatoma cell lines that do not express hA-V, are not susceptible to HBV infection. These findings suggest that hA-V plays a key role in the initial step of HBV infection and that the species-specific susceptibility to HBV infection and replication in hepatocytes is associated with the expression of hA-V.
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PMID:Transfection of a rat hepatoma cell line with a construct expressing human liver annexin V confers susceptibility to hepatitis B virus infection. 991 38

In a previous study [Nachaliel et al., 1993], we identified an RNA-binding protein (RBP) in FTO-2B rat hepatoma cells whose activity was stimulated upon the dissociation of a protein factor. We report in this article that the RBP is a complex protein of about 400 kDa, composed of RNA-binding subunit(s) (RBS), and regulatory subunit(s) (RS). We purified the RS to near-homogeneity (Mr approximately 25,000) and determined the amino acid sequence of a peptide derived from RS. On the basis of this sequence information, the cDNA for RS was obtained. Recombinant RS protein expressed in Escherichia coli had the capacity to bind RBS and inhibit its RNA-binding activity. The cDNA contains the complete coding sequence because the recombinant protein has the same electrophoretic mobility as that of the native RS in SDS-polyacrylamide gels. Sequence comparison showed that RS is almost identical to DJ-1, a recently discovered protein with an oncogenic potential, and CAP1, a rat sperm protein. However, the protein does not contain any known motifs that can provide a clue as to its exact function. Indirect immunofluorescence analyses showed that in addition to the cytoplasm, where RS is associated with microtubular filaments, the polypeptide is localized to the cell nucleus. The possible role of RS is discussed.
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PMID:Identification and characterization of a novel protein that regulates RNA-protein interaction. 1002 24

The effects of the liver tumor promoters phenobarbital, clofibrate, dieldrin, and DDT on transforming growth factor-beta1 (TGFbeta)-induced apoptosis were studied in FTO-2B hepatoma cells. Inhibition of apoptosis by these compounds was strongly correlated with a decrease in CPP32-like caspase activity. Similar effects were obtained with insulin and dexamethasone. CPP32-like activity may thus provide a useful tool for quantiation of apoptosis under various treatment conditions. Diverse effects on apoptosis-associated cellular signaling proteins were observed: insulin led to an activation of the MAP kinases ERK1/2, of PKB/Akt and of NF-kappaB, phenobarbital and clofibrate enhanced NF-kappaB activity solely, while dexamethasone slightly enhanced NF-kappaB activity and increased the expression of Bcl-xL. Since inhibition of apoptosis was still detectable if the anti-apoptotic compounds were administered more than 10 h after TGFbeta, the diverse primary signals appear to converge at a presumably late stage of apoptosis, but upstream of activation of CPP32 or related caspases.
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PMID:Inhibition of transforming growth factor beta1-induced hepatoma cell apoptosis by liver tumor promoters: characterization of primary signaling events and effects on CPP32-like caspase activity. 1020 May 66

The mammalian mitochondrial branched-chain 2-oxoacid dehydrogenase (BCOD) complex is regulated by a reversible phosphorylation (inactivation)/dephosphorylation (activation) cycle. In the present study, the effects of glucocorticoids on the level of BCOD kinase mRNA were investigated in rat hepatoma cell lines (H4IIE and FTO-2B), as well as in the rat. In H4IIE cells, dexamethasone was found to significantly reduce steady-state concentrations of BCOD kinase mRNA after a 48 h culture, and this was correlated with a 2-fold increase in the dephosphorylated form of the BCOD complex. The half-life of the kinase mRNA in H4IIE cells was not affected by dexamethasone treatment. Therefore, the decrease in the steady-state kinase mRNA level resulting from dexamethasone treatment was not caused by changes in mRNA stability, which raised the possibility of regulation at the level of gene transcription. To identify the negative glucocorticoid-responsive element in the kinase promoter, nested deletion constucts in the 3.0 kb promoter region were examined in H4IIE cells cultured in the presence or absence of dexamethasone. No significant differences in promoter activity were observed on either transient or stable transfection. The data showed that the glucocorticoid-responsive element was located outside the 3. 0 kb promoter region. At the physiological level, hepatic BCOD kinase mRNA levels were reduced in rats injected intraperitoneally with dexamethasone. This effect was liver-specific, and was not detected in other tissues. These results suggest that the down-regulation of kinase gene expression by glucocorticoids is mediated through a liver-specific or -enriched transcription factor(s).
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PMID:Down-regulation of rat mitochondrial branched-chain 2-oxoacid dehydrogenase kinase gene expression by glucocorticoids. 1021 86

Type 1 diabetic patients depend dramatically on insulin replacement therapy, which involves the administration of intermediate- or long-acting insulin, together with short-acting insulin to mimic physiological insulin profiles. However, the delayed-action preparations available are not generally able to produce smooth background levels of insulin. Muscle cells were tested for long-term delivery of active human insulin as an approach to achieve a constant basal level of insulin. Thus, C2C12 mouse myoblast cells were stably transfected with a chimeric gene obtained by linking the myosin-light chain 1 (MLC1) promoter to the human proinsulin gene, containing genetically engineered furin endoprotease cleavage sites (MLC1/Insm). When differentiated, C2C12Insm myotube cells expressed high levels of insulin mRNA and protein, whereas no insulin was detected in myoblast cells. HPLC fractionation of culture medium and cell extracts from differentiated C2C12Insm cells revealed that about 90% of the proinsulin was processed to mature insulin. In addition, these cells released significant levels (about 100 microU/10(6) cells/hr) of mature insulin to the medium. The hormone was biologically active since it increased glucose consumption and utilization by the differentiated C2C12Insm cells and was able to block the expression of the endogenous phosphoenolpyruvate carboxykinase (PEPCK) gene in FTO-2B rat hepatoma cells. Furthermore, when C2C12Insm myoblast cells were transplanted into diabetic mice an increase in insulinemia and a decrease in hyperglycemia were observed. Thus, our results suggest that the use of engineered myotube cells continuously secreting a defined level of insulin might be a useful approach to improve the efficacy of insulin injection treatment.
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PMID:Insulin production by engineered muscle cells. 1034 May 52


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