Hormonal regulation of human apolipoprotein E gene expression in HepG2 cells

• 1.1. Hormonal regulation of apolipoprotein E (apoE) gene expression by insulin and thyroid hormone was studied in a human hepatoma cell line, HepG2.
• 2.2. Changes at the mRNA level, mRNA translation, in vivo synthesis and secretion were monitored.
• 3.3. Both insulin and triiodothyronine were found to have no significant effect on apoE mRNA levels.
• 4.4. Insulin treatment caused an inhibition of: (a) the in vitro translation of endogenous apoE mRNA in a HepG2 cell-free system (25%), and (b) the incorporation of radioactivity into newly-synthesized apoE in an in vivo pulse-chase labeling experiment (32%).
• 5.5. Interestingly, apoE secretion rate was found to be significantly reduced with insulin (84%) suggesting that a major portion of newly-synthesized apoE may be shunted into a degradative pathway.
• 6.6. Using a similar experimental approach, triiodothyronine showed no significant effect on the rate of apoE synthesis or translation (6–15% decrease), however a slight reduction (20%) in secretion rate was shown.
• 7.7. Overall, apoE gene expression does not appear to be influenced by triiodothyronine significantly but is modulated by insulin at the translational and post-translational level.

     

Expression of apolipoprotein E by cultured vascular smooth muscle cells is controlled by growth state

Rat vascular smooth muscle cells (SMC) in culture synthesize and secrete a approximately 38,000-Mr protein doublet or triplet that, as previously described (Majack and Bornstein. 1984. J. Cell Biol. 99:1688-1695), rapidly and reversibly accumulates in the SMC culture medium upon addition of heparin. In the present study, we show that this approximately 38,000-Mr heparin-regulated protein is electrophoretically and immunologically identical to apolipoprotein E (apo-E), a major plasma apolipoprotein involved in cholesterol transport. In addition, we show that expression of apo-E by cultured SMC varies according to growth state: while proliferating SMC produced little apo-E and expressed low levels of apo-E mRNA, quiescent SMC produced significantly more apo-E (relative to other proteins) and expressed markedly increased levels of apo-E mRNA. Northern analysis of RNA extracted from aortic tissue revealed that fully differentiated, quiescent SMC contain significant quantities of apo-E mRNA. These data establish aortic SMC as a vascular source for apo-E and suggest new functional roles for this apolipoprotein, possibly unrelated to traditional concepts of lipid metabolism.     

Expression of fungal desaturase genes in cultured mammalian cells

Long-chain polyunsaturated fatty acids (LC-PUFA) are important components of cellular structure and function. Most of LC-PUFA are derived from linoleic acid and a-linolenic acid. In plants and fungi, these two acids can be synthesized from oleic acid via the action of two enzymes, 12 and 15-desaturases. Due to lack of these enzymatic activities and the ability to synthesize these two essential fatty acids, animals must obtain them from the diet. In this report, we demonstrated the expression of a fungal 12-desaturase gene in mouse L cells incubated in serum-free medium. The results showed a significant increase in the amount of linoleic acid with a concomitant decrease of oleic acid in cellular lipids. Most of the newly formed linoleic acid was incorporated into cellular phospholipids, particularly phosphatidylcholine. The increase of linoleic acid provided the substrate for the endogenous synthesis of (n-6) LC-PUFA, such as eicosadienoic acid (EDA), dihomo--linoleic acid (DGLA) and arachidonic acid (AA). Prolonged incubation further increased the levels of linoleic acid derived from oleic acid by the action of 12-desaturase, and the levels of 20:2n-6 produced from linoleic acid by the action of the endogenous elongase. However, prolonged incubation suppressed significantly the formation of DGLA and AA. In a separate study, a fungal 6-desaturase gene has also been expressed in the mouse L cells incubated in serum-containing medium. The result shows a significant increase in levels of 20:3n-6 and 20:4n-6. These findings demonstrate that through genetic modification, it is possible to (1) generate cell lines which no longer require dietary 'essential' fatty acids and (2) alter the endogenous fatty acid metabolism to enhance the production of LC-PUFA and their derivatives.     

Two copies of the human apolipoprotein C-I gene are linked closely to the apolipoprotein E gene

The gene for human apolipoprotein (apo) C-I was selected from human genomic cosmid and lambda libraries. Restriction endonuclease analysis showed that the gene for apoC-I is located 5.5 kilobases downstream of the gene for apoE. A copy of the apoC-I gene, apoC-I', is located 7.5 kilobases downstream of the apoC-I gene. Both genes contain four exons and three introns; the apoC-I gene is 4653 base pairs long, the apoC-I' gene 4387 base pairs. In each gene, the first intron is located 20 nucleotides upstream from the translation start signal; the second intron, within the codon of Gly-7 of the signal peptide region; and the third intron, within the codon for Arg39 of the mature plasma protein coding region. The upstream apoC-I gene encodes the known apoC-I plasma protein and differs from the downstream apoC-I' gene in about 9% of the exon nucleotide positions. The most important difference between the exons results in a change in the codon for Gln-2 of the signal peptide region, which introduces a translation stop signal in the downstream gene. Major sequence differences are found in the second and third introns of the apoC-I and apoC-I' genes, which contain 9 and 7.5 copies, respectively, of Alu family sequences. The apoC-I gene is expressed primarily in the liver, and it is activated when monocytes differentiate into macrophages. In contrast, no mRNA product of the apoC-I' gene can be detected in any tissue, suggesting that it may be a pseudogene. The similar structures and the proximity of the apoE and apoC-I genes suggest that they are derived from a common ancestor. Furthermore, they may be considered to be constituents of a family of seven apolipoprotein genes (apoE, -C-I, -C-II, -C-III, -A-I, -A-II, and -A-IV) that have a common evolutionary origin.     

Phage particle-mediated gene transfer to cultured mammalian cells

Recombinant phage particles carrying the thymidine kinase (TK) gene of herpes simplex virus type 1, coprecipitated with calcium phosphate, efficiently transformed mouse Ltk- cells to the TK+ phenotype. The conditions necessary to achieve high efficiency of transfer of the TK gene by phage particle-mediated gene transfer were investigated. Of the parameters examined, the pH of the buffer used for coprecipitation of phage particles with calcium phosphate, the length of time of coprecipitation, and the length of the adsorption period were found to alter the transfer efficiency significantly. The optimal pH was 6.87 at 25 degrees C. The other optimal values for these parameters were as follows: coprecipitation time, 7 to 20 min; adsorption time, 18 to 30 h. Treatment with dimethyl sulfoxide, glycerol, or sucrose did not enhance gene transfer. The optimal conditions yielded about 1 transformant per 10(5) phage particles per 10(6) cells without carrier DNA. An increase in the dosage of phage particles, up to at least 5 x 10(7) phage particles per 100-mm dish, resulted in a linear increase in the number of transformants. Addition of carrier phage, up to 10(10) phage particles per dish, did not significantly affect the number of transformants.     

Expression of the E. coli fpg gene in mammalian cells reduces the mutagenicity of gamma-rays.

The E. coli fpg gene encodes the formamido-pyrimidine-DNA-glycosylase (FPG protein) which specifically removes the formamido-pyrimidine and C8-oxoGuanine residues from gamma-irradiated DNA. The fpg gene was ligated in the psV2 vector and transfected into the Chinese hamster CHO and V-79 cells. The transfected cells expressed a formamido-pyrimidine-DNA-glycosylase activity 30 to 40-fold over the constitutive level. The resistance of CHO and V-79 cells to the lethal effect of gamma-rays was similar in control and transfected cells. Furthermore CHO cells expressing the fpg gene had the same resistance to the lethal effect of hydrogen peroxide as control cells. However, the sensitivity to the mutagenic effect of gamma-rays, measured as 6-thioguanine resistance, decreased both in CHO and V-79 transfected cells. Since the lethal effect of gamma-rays was not modified in cells overproducing the FPG protein, the results suggest that this protein protects the cells against the mutagenic lesions formed by ionizing radiations, and among them C8-oxoguanine.     

Expression of the antibacterial peptide, cecropin, in cultured mammalian cells

We have stably transfected a Chinese hamster lung cell line V79 with a recombinant gene construct where the Drosophila cecropin A2 cDNA is under the control of Rous sarcoma virus, long terminal repeat (RSV LTR). We have not only been able to demonstrate expression at the RNA level by Northern analysis but also have detected an unprocessed peptide using an antiserum raised against Hyalophora cecropin.     

Expression of the plasmid-encoded type I dihydrofolate reductase gene in cultured mammalian cells: a novel selectable marker

A recombinant plasmid has been designed to express the gene encoding a type I methotrexate-resistant dihydrofolate reductase, derived from the bacterial plasmid R483, in DHFR- Chinese hamster ovary cells. Vectors containing the wild type gene, whose coding sequence initiates with a GTG codon, fail to direct the synthesis of detectable levels of protein. Substitution of the GTG codon by an AG codon using in vitro mutagenesis overcomes this block; cells transfected with the modified vector synthesize a functional prokaryotic protein that sustains the growth of these cells in the presence of dihydrofolic acid in the culture media. This property is consistent with the inability of the type I enzyme to reduce folate to dihydrofolate, and enabled the development of a selection strategy whereby prokaryotic and mammalian DHFRs genes could be used sequentially as independently selectable markers.     

Hypergravity signal transduction and gene expression in cultured mammalian cells

A number of studies have been conducted during space flight and with clinostats and centrifuges, suggesting that gravity effects the proliferation and differentiation of mammalian cells in vitro. However, little is known about the mechanisms by which mammalian cells respond to changes in gravitational stress. This paper summarizes studies designed to clarify the effects of hypergravity on the cultured human HeLa cells and to investigate the mechanism of hypergravity signal transduction in these cells.     

Expression of a bacterial repair gene in mammalian cells

The coding sequence of the uvrA gene from Escherichia coli has been fused to the early promoter, enhancer and origin of replication of the simian virus SV40, and was supplemented with splicing and polyadenylation sites arising from the same virus. Introduction of this hybrid gene into simian cos-1 cells results in the synthesis of a full length UvrA protein (114 kD) which has retained its ability to bind to single-stranded DNA.     

Identification of human apolipoprotein E variant gene: apolipoprotein E7 (Glu244,245----Lys244,245)

Apolipoprotein E (apoE) is one of the protein moieties of the human serum lipoproteins. Three major isoforms of apoE (apoE2, apoE3, and apoE4) and minor variant isoforms (apoE1, apoE5, and apoE7) have been detected by isoelectric focusing. In this study we have cloned the apoE7 gene from a patient with the apoE3/E7 phenotype associated with hypertriglyceridemia and diabetes mellitus. DNA sequencing revealed that the apoE7 gene has two base substitutions (G----A) changing Glu244,245----Lys244,245, compared with the apoE3 gene. The replacement of the two amino acids is consistent with the result of isoelectric focusing of the apoE7 isoprotein, which shifts to four positively charged units compared with the apoE3 isoprotein.     

The gene for human apolipoprotein CI is located 4.3 kilobases away from the apolipoprotein E gene on chromosome 19

Summary We have isolated a cDNA clone for apolipoprotein CI and a genomic clone for apolipoprotein E, and by hybridisation and mapping experiments found the gene for apoCI to be located on the genomic apoE clone. The distance between the loci was 4.3 kb.