A selective system for hepatoma cells producing gluconeogenic enzymes

Bertolotti, Roger

doi:10.1007/bf01542966

Cited by 58 publications

(29 citation statements)

References 44 publications

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“…To prepare glucosefree (G Ϫ ) medium, glucose is omitted and serum is extensively dialyzed. G ϩ medium consists of G Ϫ medium supplemented with glucose (2). For reversion analysis, cells were first seeded in a series of dishes in medium supplemented with undialyzed serum and then adapted for 2 to 3 days in G ϩ medium.…”

Section: Methodsmentioning

confidence: 99%

Liver-Enriched Transcription Factors Uncoupled from Expression of Hepatic Functions in Hepatoma Cell Lines

Chaya

Fougère-Deschatrette

Weiss

1997

Molecular and Cellular Biology

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Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver.Genetic analysis of hepatoma-derived cell lines revealed, many years ago, that expression of differentiated functions is regulated in trans by mechanisms whose final effects are negative (extinction) or positive (activation) (78). More recent studies of these hepatoma lines further showed that maintenance of the hepatic phenotype is an active process operating at the level of transcription (21,42). By analysis of DNA sequences implicated in liver-specific transcription, the identification and cloning of members of four major families of liverenriched transcription factors (LETF) have been achieved. These families, each characterized by structurally related DNA binding domains, include the hepatocyte nuclear factor families HNF1, HNF3, and HNF4 and the CCAAT enhancer binding protein (C/EBP) family (13,82). Determination of the tissue distribution of these factors and analysis of their hierarchical relations led to the hypothesis that the combinatorial action of LETF together with the ubiquitous transcriptional machinery of the cell is necessary and maybe even sufficient for the maintenance of liver-specific gene transcription (30, 81). Indeed, the H4IIEC3 differentiated rat hepatoma cells and their derivatives that stably express an extensive set of adult hepatic functions express all the LETF identified to date, while in the rare dedifferentiated rat hepatoma cells that have lost expression of all these hepatic functions, HNF4 and HNF1 are systematically absent. In addition,...

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Section: Methodsmentioning

confidence: 99%

Liver-Enriched Transcription Factors Uncoupled from Expression of Hepatic Functions in Hepatoma Cell Lines

Chaya

Fougère-Deschatrette

Weiss

1997

Molecular and Cellular Biology

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“…Gluconeogenesis and Glycolysis in FAO Cells-FAO cells contain all of the gluconeogenic enzymes required for growth and survival in the absence of glucose (11,12), and they can produce glucose from gluconeogenic precursors (13). However, quantitative studies on glucose metabolism in these cells have not been reported.…”

Section: Consequences Of Overexpression Of the Wild-type Andmentioning

confidence: 99%

“…Mutation of Ser-32 to Ala prevents cAMP-dependent phosphorylation of the enzyme (8), while mutation of His-258 to alanine abolishes bisphosphatase activity, since this residue is known to mediate catalysis via a phosphohistidine intermediate (9,10). Since they are capable of producing glucose from 3-carbon precursors (11)(12)(13)(14), cultured rat hepatoma FAO cells were chosen to overexpress the enzyme forms. Thus, the switching mechanism between glycolysis and gluconeogenesis, hypothesized for the bifunctional enzyme and Fru-2,6-P 2 , can be studied.…”

mentioning

confidence: 99%

Adenovirus-mediated Overexpression of Liver 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase in Gluconeogenic Rat Hepatoma Cells

Argaud

Lange

Becker

et al. 1995

Journal of Biological Chemistry

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6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been postulated to be a metabolic signaling enzyme, which acts as a switch between glycolysis and gluconeogenesis in mammalian liver by regulating the level of fructose 2,6-bisphosphate. The effect of overexpressing the bifunctional enzyme was studied in FAO cells transduced with recombinant adenoviral constructs of either the wild-type enzyme or a double mutant that has no bisphosphatase activity or protein kinase phosphorylation site. With both constructs, the mRNA and protein were overexpressed by 150-and 40-fold, respectively. Addition of cAMP to cells overexpressing the wild-type enzyme increased the S 0.5 for fructose 6-phosphate of the kinase by 1.5-fold but had no effect on the overexpressed double mutant. When the wild-type enzyme was overexpressed, there was a decrease in fructose 2,6-bisphosphate levels, even though 6-phosphofructo-2-kinase maximal activity increased more than 22-fold and was in excess of fructose-2,6-bisphosphatase maximal activity. The kinase:bisphosphatase maximal activity ratio was decreased, indicating that the overexpressed enzyme was phosphorylated by cAMP-dependent protein kinase. Overexpression of the double mutant resulted in a 28-fold increase in kinase maximal activity and a 3-4-fold increase in fructose 2,6-bisphosphate levels. Overexpression of this form inhibited the rate of glucose production from dihydroxyacetone by 90% and stimulated the rate of lactate plus pyruvate production by 200%. In contrast, overexpression of the wild-type enzyme enhanced glucose production and inhibited lactate plus pyruvate production. These results provide direct support for fructose 2,6-bisphosphate as a regulator of gluconeogenic/glycolytic pathway flux and suggest that regulation of bifunctional enzyme activities by covalent modification is more important than the amount of the protein.

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“…Studies using these clones and the tools of somatic cell genetics (12,23,34) and molecular genetics (4,24,25) have provided evidence that many liver functions are regulated in these cells in a concerted manner. For example, cell lines can be selected which no longer produce the two liver-specific enzymes central to the ability of normal hepatocytes to synthesize glucose (1,2). Loss of the ability of variant cells grow without glucose is accompanied by the loss of other liver functions (e.g., serum albumin production) (23).…”

mentioning

confidence: 99%

Liver-specific RNA metabolism in hepatoma cells: variations in transcription rates and mRNA levels.

Clayton¹,

Weiss²,

Darnell³

1985

Mol. Cell. Biol.

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The transcription rate and abundance of several liver-specific mRNAs as well as mRNAs common to many cel types were compared in a series of rodent hepatoma cell lines, normal liver cells, and primary hepatocyte cultures. The rat hepatoma cell line, Fao, which displays a liver-specific phenotype, contained eight of eight liver-specific mRNAs examined. However, the transcription rates of most liver-specific mRNAs were found to be low (1 to 30%) compared with normal liver in this and other differentiated cell lines. This low rate is similar to the transcription rates of liver-specific mRNA sequences measured in primary cultures of hepatocytes. Several variant ceU lines that had lost differentiated traits contained few or none of the liver-specific mRNAs; clonal descendents which had regained differentiated function regained the tissue-specific mRNAs as a group, but at various concentrations. Because all of the changes observed in mRNA levels were not accompanied by parallel changes in transcription of the same sequences, differential posttranscriptional stabilization of the liver-specific mRNAs must also occur in the different cell lines. These results qualify the utility of cultured cell lines in the study of tissue-specific transcriptional control, but raise the possibility that posttranscriptional mechanisms act in cooperation with transcriptional controls to bring the level of tissue-specific mRNAs closer to those found in liver cells.Cell lines derived from liver tumors have been widely used in attempts to study various processes that normally occur in hepatocytes, including the mechanisms of tissue-specific gene control. Particular hepatoma clones secrete or contain numerous liver-specific plasma proteins and enzymes and have been considered well differentiated (13). Studies using these clones and the tools of somatic cell genetics (12,23,34) and molecular genetics (4, 24, 25) have provided evidence that many liver functions are regulated in these cells in a concerted manner. For example, cell lines can be selected which no longer produce the two liver-specific enzymes central to the ability of normal hepatocytes to synthesize glucose (1, 2). Loss of the ability of variant cells grow without glucose is accompanied by the loss of other liver functions (e.g., serum albumin production) (23). When revertant cell lines are then selected for their ability to grow in glucose-free medium, the expression of many or all of these other liver functions is regained (12).However, two important and general questions about these experiments, and cultured differentiated cell lines in general, remain unanswered. First, do immortalized, aneuploid, and culture-adapted cells exhibit patterns of gene control that resemble normal differentiated cells, or do they diverge in some potentially instructive way? For example, is the amount of a given liver-specific gene product in a well-differentiated hepatoma cell line quantitatively similar to that seen in the normal liver? How many separate liver functions are retained?Second, when he...

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A selective system for hepatoma cells producing gluconeogenic enzymes

Cited by 58 publications

References 44 publications

Liver-Enriched Transcription Factors Uncoupled from Expression of Hepatic Functions in Hepatoma Cell Lines

Liver-Enriched Transcription Factors Uncoupled from Expression of Hepatic Functions in Hepatoma Cell Lines

Adenovirus-mediated Overexpression of Liver 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase in Gluconeogenic Rat Hepatoma Cells

Liver-specific RNA metabolism in hepatoma cells: variations in transcription rates and mRNA levels.

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