Claude Forest

Adenovirus enhancement of polyethylenimine-mediated transfer of regulated genes in differentiated cells

Efficient gene transfer is a prerequisite for analysing regulation of transfected promoters. We combined the DNA binding property of the cationic polymer polyethylenimine (PEI) and the potent endocytic activity of adenovirus in a PEI–DNA–adenovirus complex which provided efficient plasmid delivery in differentiated cultured cells. We transfected 3T3-F442A adipocytes, C2.7 myocytes and FAO hepatoma cells with a construct containing the simian virus 40 promoter fused to the chloramphenicol acetyltransferase (CAT) gene, using a combination of PEI and 200 p.f.u. per cell of replication-deficient type 5 adenovirus. Resulting CAT activities varied according to the cell type reaching about 0.6, 8 and 38 units/mg protein for respectively 3T3-F442A, FAO and C2.7 cells. Increases in transfection efficiencies were 140- to 300-fold when compared with those obtained with PEI alone. Then we tested physiologically regulated promoters: the phosphoenolpyruvate carboxykinase gene promoter in 3T3-F442A or FAO cells and the hexokinase II gene promoter in C2.7 myocytes. Gene expression was appropriately increased by clofibrate, dexamethasone and insulin for 3T3-F442A, FAO and C2.7 cells, respectively. Thus, the combination of PEI and adenovirus is a simple, efficient, inexpensive and versatile method of gene transfer which is applicable to several differentiated cells and provides a physiologically coherent transgene regulation. We name this method PEI- adenofection.

Peroxisome proliferator activated receptor-γ, leptin and tumor necrosis factor-α mRNA expression during very low calorie diet in subcutaneous adipose tissue in obese women

PPARγ, leptin and TNFα are three major factors that play a key role in influencing adipocyte differentiation and both adipose tissue function and metabolism. However, the regulation of these three genes during a dynamic period of weight loss is unknown. We therefore investigated the concomitant regulation of the mRNA expression of PPARγ, leptin and TNFα in adipose tissue during a 21‐day very low calorie diet (VLCD) in 12 non‐diabetic obese women.

MECHANISM OF ADENOVIRUS IMPROVEMENT OF CATIONIC LIPOSOME-MEDIATED GENE TRANSFER

Substantial effort has been focused on the development of highly efficient gene transfer strategies. Although viral and non-viral methods have been elaborated, mechanisms of gene delivery are still poorly understood. We exploited our recent observation that replication-deficient type 5 adenovirus dramatically enhances lipofectAMINE-mediated gene transfer (lipoadenofection) in differentiated cells to elucidate the mechanism of adenovirus action in this process. Heat-induced denaturation of viral capsid abolishes adenovirus action whereas inactivation of viral genome by short treatment with UV has no effect. Electron microscopic observations reveal the formation of a complex containing adenovirus and lipofectAMINE which probably carries DNA into cells via endocytosis. Anti-adenovirus antiserum or monoclonal anti-alpha(v)beta3 integrin antibody inhibits lipoadenofection, at least partially. Neutralization of endosomal compartments with chloroquine, ammonium chloride or monensin does not prevent adenovirus improvement of gene transfer. Hence, adenovirus-lipofectAMINE-DNA complexes in which viral particles are each encompassed by three lipid layers, penetrate cells via an endocytic pathway involving probably the adenovirus receptor and alpha(v)beta3 integrin. The resulting efficient transfer and expression of plasmid DNA proceeds from a mechanism in which adenoviral endosomolytic activity appears to be required while viral genome is not essential.

Complex Hormone Response Unit Regulating Transcription of the Phosphoenolpyruvate Carboxykinase Gene: From Metabolic Pathways to Molecular Biology

Publisher Summary This chapter discusses the history of metabolic regulation and the relevant concepts of molecular biology before showing how these historical antecedents allow for an analysis of a gene critically involved in metabolic regulation. The chapter describes the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter in an attempt to define the hormonal regulation of the rate-limiting step of gluconeogenesis at the molecular level. Cyclic adenosine monophosphate and glucocorticoids increase gluconeogenesis by increasing the rate of transcription of the PEPCK gene, and this results in an enhanced rate of synthesis of the protein. Insulin exerts an opposite, dominant negative effect. The chapter defines the way in which hormone response elements in the PEPCK promoter interact to provide coordinate regulation of this gene.

Isolation and characterization of the mouse cytosolic phosphoenolpyruvate carboxykinase (GTP) gene: evidence for tissue-specific hypersensitive sites

A 72 kilobase pair DNA fragment that contains the mouse phosphoenolpyruvate carboxykinase (PEPCK) gene locus, pck1, was isolated from a genomic bacterial artificial chromosome library. The region from approximately -5.5 to +6.6 kilobase pairs relative to the pck1 transcription start site was sequenced and exhibits a high degree of homology to the rat and human genes. Additionally, the chromatin structure of the PEPCK gene in mouse liver resembles that seen in rat. Backcross panel analysis of a microsatellite sequence confirms that the gene is located on chromosome 2. Hypersensitive site analysis was performed on nuclei isolated from the adipocyte cell line 3T3-F442A in the preadipose and adipose states. Several hypersensitive sites are present in the undifferentiated 3T3-F442A cells, before PEPCK mRNA is detected. The same sites are present after differentiation, however, the sensitivity of mHS 3 increases relative to the others. We conclude that the chromatin is open in 3T3-F442A cells and that factors are able to bind in the undifferentiated state but that something else is required for transcription.

Establishment of a human cell line after transformation by a plasmid containing the early region of the SV40 genome

A human clonal cell line, designated TAH9, has been established from cells of adipose tissue by fusion with E. coli protoplasts containing a recombinant plasmid carrying the early genes of Simian virus 40. The establishment of the cell line is preceeded by a delayed growth crisis. The results indicate that, after cell transformation, another event(s) of low frequency is required for cell immortalization. Some biological properties of TAH9 cells are also described.

Glucocorticoids use a positive liver element to repress fibrate-induced adipose transcription of the phosphoenolpyruvate carboxykinase gene.

Glucocorticoids inhibit basal and hormone-induced phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in adipocytes whereas beta-adrenergic agonists and fibrates are stimulatory. Here we show that dexamethasone inhibits the induction of PEPCK mRNA by isoprenaline or clofibrate in 3T3-F442A adipocytes. RU 38486 antagonizes dexamethasone effect, suggesting the involvement of the glucocorticoid receptor. In H4IIE hepatoma cells, glucocorticoids enhance PEPCK gene transcription through a complex region which encompasses an element, AF1, with a direct repeat 1-type sequence. Mutations in the AF1 sequence abolish binding of nuclear factors from liver and from 3T3-F442A adipocytes. We transiently transfected 3T3-F442A cells with a wild type or an AF1-mutated PEPCK-CAT construct comprising -2100 to +69 base pairs of the promoter fused to the chloramphenicol acetyltransferase (CAT) gene. With both constructs, CAT activity is decreased by dexamethasone and is increased by isoprenaline or by clofibrate. However, dexamethasone is unable to inhibit clofibrate induction of CAT activity in cells transfected with the AF1-mutated construct whereas it prevents isoprenaline action on both constructs. Hence, although a single hormone can repress stimulations originating from different intracellular routes, sites in the promoter which mediate inhibition of a specific stimulation are distinct.

Transcriptional and posttranscriptional mechanisms of glucocorticoid-mediated repression of phosphoenolpyruvate carboxykinase gene expression in adipocytes

Glucocorticoids exert pleiotropic effects, among which negative regulation of transcription has been recognized as of crucial importance. While glucocorticoids induce phosphoenolpyruvate carboxykinase (PEPCK) gene expression in liver cells, it represses gene activity in adipose cells. We used the 3T3‐F442A adipocytes to analyze the underlying mechanisms. In these cells, the synthetic glucocorticoid dexamethasone exerts a dominant repression either on basal or on β‐agonist stimulation of PEPCK gene expression. To determine whether glucocorticoid action required protein synthesis, we employed cycloheximide, anisomycin, and puromycin, three different translation inhibitors. None of these affected induction by isoprenaline or repression by dexamethasone of isoprenaline stimulation. In contrast, dexamethasone inhibitory action on basal PEPCK mRNA was totally prevented by the three translation inhibitors. Time courses of glucocorticoid action on basal and on induction by β‐agonist were similar. Half‐maximal effect of dexamethasone on isoprenaline‐induced PEPCK mRNA was obtained at about 10 nM, a tenfold higher concentration than that observed for the reduction of basal mRNA. Using the transcription inhibitor DRB, we showed that dexamethasone did not alter mRNA half‐life, while isoprenaline strongly stabilized mRNA. In a 3T3‐F442A stable transfectant bearing −2,100 base pairs of the PEPCK promoter fused to the chloramphenicol acetyltransferase (CAT) gene, isoprenaline stimulated CAT activity, whereas dexamethasone reduced basal and isoprenaline‐induced CAT expression. Hence, β‐agonists exert both transcriptional and posttranscriptional regulation, while glucocorticoid action is purely transcriptional. However, mechanisms of glucocorticoid repression of basal and of β‐agonist stimulation appear different. J. Cell. Biochem. 66:386–393, 1997.

Regulation of the aspartate and alanine aminotransferases in humans and rodents

We have studied the regulation of transaminase gene expression by hormones and drugs in humans and rodents. In rodents, most of the regulation is hormonal and is consistent with the role of these enzymes in gluconeogenesis.In humans, these genes are up-regulated by drugs such as fibrates, which may partially account for the increase in the activities in serum observed in some treated patients. Interestingly, fibrates down-regulate these genes in the rat, suggestingthat there ae important differences in the regulation of gene expression between rodents and humans.