In vitro and in vivo effects of glucocorticoids on gene transfer to skeletal muscle.

As a pharmacological approach to potentially improve gene transfer efficiency into skeletal muscle cells, glucocorticoids were shown here to allow efficient transfection of cultured and mouse human myoblasts, human pulmonary A549 cells, but not dog myoblasts, independently of the transfection protocol, the reporter gene and the transcription promoter employed. Transduction with adenovirus was also increased by dexamethasone. Pretreatment of cells 48 h prior to transfection was most effective and was shown to be concentration-dependent. This effect is mediated by binding to the glucocorticoid receptor, but not by glucocorticoid responsive elements present in the vectors. The acute dexamethasone effect could be due to increased plasmid entry into the cells as suggested by Southern blot, whereas the sustained increase of luciferase activity in dexamethasone-treated cultures may be related to intracellular mechanisms following cell entry. In mice in vivo, a similar increase of luciferase activity upon glucocorticoid treatment was found.

Establishment of a V79 transfected cell line highly producing recombinant human gamma-glutamyltransferase.

gamma-Glutamyltransferase (GGT) is a glutathione-metabolizing enzyme whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse and induction by drugs. To elucidate the implication of GGT in various metabolic pathways, we established a stable transfected V79 cell line highly producing the human GGT. A full length cDNA, encoding the human hepatoma Hep G2 GGT, was subcloned into an expression vector under the control of the SV40 early promoter and was used to transfect V79 cells. A cell line was selected, exhibiting a GGT activity of 2 units per mg of protein, one of the highest levels reported to date. The recombinant GGT purified from this cell line showed the expected heterodimeric structure, with two subunits existing as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. Catalytic features were found to be identical to those of human serum and Hep G2 GGTs. Thus, the newly engineered cell line should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.

Stable expression of enzymatically active human pancreatic lipase in V79 cells: purification and characterization of the recombinant enzyme.

The serum activity of human pancreatic lipase (HPL; EC 3.1.1.3), the main lipolytic enzyme secreted by the pancreas, is a valuable marker of pancreatic disorders. However, determining lipase activity in human serum is difficult because the substrates used vary in their lipase specificity. A lipase reference material is therefore needed. We describe the production of recombinant HPL (rHPL) in V79 Chinese hamster lung cells. A cDNA encoding the sequence of HPL was subcloned into the pcDNAI eukaryotic expression vector, which was then used to transfect V79 cells. The 50-kDa purified recombinant enzyme is fully active, is glycosylated, and has a pI of 7.0. The catalytic properties of rHPL, determined by using triolein as the substrate, were similar to those of the native enzyme. The V79rHPL cell line we developed might be useful for the production of rHPL suitable for the preparation of a reference material.

Molecular and functional characterization of recombinant human gamma-glutamyltransferase. Coupling of its activity to glutathione levels in V79 cells.

We previously described the establishment of a transfected cell line (V79HGGT) that stably produces the highest recombinant human gamma-glutamyltransferase (GGT) activity. We now report the utilization of V79HGGT as a model system for studying human GGT. The papain-solubilized recombinant enzyme has been highly purified from cultured cells by a new procedure. Studies on the purified enzyme, either by N-terminal sequencing or by characterization of its enzymic activities, confirmed that recombinant GGT shares structural and catalytic identity with native human enzymes. The circular dichroism analysis indicated an alpha-helical content of 19%. Based on these data, we have undertaken a study on the functional consequences of elevated GGT activity on the reduced glutathione (GSH) content. GSH status was followed in V79 and V79HGGT cells throughout growth. A particular pattern was observed for each cell line, depending on, but differentially affected by, alteration of the culture medium. Elevated GGT activity was associated with a 2.5-fold reduced GSH content, clearly suggesting a negative influence of the highly expressed enzyme on the GSH level under normal growth conditions. Possible mechanisms involved are proposed. Our findings pointed out that, among the GSH-related enzymes, GGT could constitute an important factor determining the steady-state content of GSH.

High-level expression of enzymatically active mature human gamma-glutamyltransferase in transgenic V79 Chinese hamster cells.

gamma-Glutamyltransferase [GGT; (5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2] is a glutathione-metabolizing enzyme, whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse, and induction by xenobiotics. To elucidate the implication of GGT in various metabolic pathways, we established a stable transgenic V79 cell line, highly producing the human GGT. A full-length cDNA, encoding the human hepatoma HepG2 GGT, was subcloned in an expression vector under the control of the simian virus 40 early promoter and was used to transfect V79 cells. We selected a cell line exhibiting a GGT activity of 2 units per mg of protein, the highest GGT expression level reported to date. As described for the human kidney and liver enzymes, the recombinant GGT purified from this cell line showed a heterodimeric structure. Its two subunits existed as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. However, catalytic features were found to be identical to those of human serum and HepG2 GGTs. The newly engineered cell line thus should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.

Influence of sample preparation on cellular glutathione recovery from adherent cells in culture.

During the last decade, the unbound glutathione content of cultured adherent cells has become a very important biological marker for many pharmacological and toxicological in vitro studies with regard to the protective role of the tripeptide in its reduced form (GSH). However, the literature does not provide extensive information on the influence of sample preparation on cellular GSH and thiol analyses. Using the fibroblast-like V79 cell line as model, we undertook a comparative study of the efficiency of different procedures reported in the literature with respect to GSH recovery. Depending on the preanalytical step, up to 10-fold discrepancies could be observed in the recovery of intracellular GSH. Different parameters that must be controlled in order to maximize GSH recovery are discussed. The optimal strategy consisted in rapid perchloric acid deproteinization performed directly in the dish, which was extremely valuable for preparing GSH samples from adherent cells, and especially from cells expressing elevated gamma-glutamyl transferase activity.

Transfection of myoblasts in primary culture with isomeric cationic cholesterol derivatives.

Transfection of satellite cells from dog muscle (myoblasts) in primary culture has been optimized with respect to the position of the cholesteryl moiety along the polyamine chain of spermidine or spermine. Spermidine or spermine were derivatized with cholesterylchloroformate giving rise to three isomers in the case of spermidine and two isomers for spermine that were separated by reversed-phase high-performance liquid chromatography (rp-HPLC). The position of the cholesteryl moiety was assigned by 13C-NMR and coelution with synthetic isomers of defined structure. The isomeric cationic lipids were evaluated for their transfection activity in myoblasts from dog muscle and a human lung epithelial cell line (A549) using plasmid DNA expressing the luciferase reporter gene. The results showed that the position of the cholesteryl moiety is of critical importance for efficient transfection of myoblasts in primary culture with isomers having a derivatized secondary amine being significantly more effective than those with a derivatized primary amine. On the contrary, differences in the A549 cell line were less pronounced and did not follow the same pattern. The results show that slight structural differences between cationic lipids lead to significantly different transfection efficiencies for myoblasts in primary culture. This may also represent an advantage in view of cell or organ targeting.

Expression of rat renal gamma-glutamyltransferase cDNA in Escherichia coli.

To obtain the expression of rat kidney gamma-glutamyltransferase (GGT) cDNA in E. coli, plasmids containing the cDNA sequences coding for various parts of GGT were constructed. Transformation of E. coli cells by these hybrid vectors results in a production of unglycosylated recombinant proteins, immunologically recognized by specific antirat kidney GGT antibodies. Plasmid, expressing the complete coding sequence of GGT cDNA, allows the production of enzymatically active proteins localized in the periplasmic space, while the same sequence without the N-terminal hydrophobic region results in a production of cytoplasmic proteins. These recombinant proteins present a very basic isoelectric point (pI greater than 9). These results suggest that the presence of the N-terminal region seems to be necessary to direct the expressed proteins enzymatically active in the periplasmic space.

Rapid liquid chromatographic assay of glutathione in cultured cells.

A rapid, sensitive and selective method for the assay of glutathione in cultured cells has been developed using ion-pair reversed phase rapid high performance liquid chromatography. The use of a 4 micron particle, 5 cm long column (Superspher 100 RP 18 end-capped) allowed complete analysis of glutathione within 3 min. A postcolumn derivatization reaction with o-phthalaldehyde and fluorometric detection made the assay fully selective with regard to other endogenous thiols and sensitive (the detection limit was 0.5 ng of glutathione injected). The linearity range was between 0.1 and 2.0 microgram/mL with good repeatability (relative standard deviation less than 5% for the lowest concentration quantitated). Recoveries of GSH from cultured cell samples were above 98%. The rapid analysis enabled the processing of a large number of samples in a short time (up to 20 per hour). The method was applied to the measurement of the intracellular glutathione amount in V79 fibroblasts along cell growth in culture.

Application of gene transfer technologies to the production of enzyme reference materials: example of gamma-glutamyltransferase.

Protein reference materials are traditionally prepared by purification from mammalian or human tissues. The supply of these tissues is limited; consequently, there is a growing need for applied molecular and cellular biology technologies for the production of human recombinant proteins. This is especially true when only small amounts of the proteins are available in the tissues. We review the current knowledge necessary for high-level production of such proteins in different heterologous expression systems, using our data on gamma-glutamyltransferase (EC 2.3.2.2) as an example. We describe the steps required to achieve the expression of enzymes and other proteins in Escherichia coli, yeast, or mammalian cells. We list many of the problems investigators may face in preparing recombinant proteins, and provide information on selecting the most appropriate system as well as the most favorable experimental conditions. Depending on the expression system, recombinant proteins can potentially be obtained for most, if not all, enzymes of interest in clinical chemistry, and such proteins should possess characteristics very similar to those of the corresponding human native proteins. Studies suggest that these products can be used as reference materials in clinical chemistry laboratories.

Induction of recombinant human gamma-glutamyl transferase by sodium butyrate in transfected V79 and CHO Chinese hamster cells.

Sodium butyrate was used to enhance biosynthesis rates of recombinant human gamma-glutamyl transferase (GGT) expressed under the control of the SV40 or the cytomegalovirus immediate early promoter, respectively, in transfected V79 and CHO Chinese hamster cell lines. Maximal induction of GGT specific activity in butyrate-treated cells ranged from 3 to 5-fold and resulted from a strong increase in the GGT mRNA ratio. We also observed that maximal transcription level in V79 cells occurred within 12 hr of treatment, whilst the cell proliferation was transiently arrested. Despite its processing requirements, induced GGT exhibited unchanged catalytic and physico-chemical features relative to human serum or hepatoma enzyme, thus appearing as an excellent model for further studies on human GGT.

Electrophoretic mobility of gamma-glutamyltransferase in rat liver subcellular fractions. Evidence for structure difference from the kidney enzyme.

Adult rat liver gamma-glutamyltransferase (GGT) has been poorly characterized because of its very low concentration in the tissue. In contrast with the kidney, the liver enzyme is inducible by some xenobiotics, and its relationship to hepatic ontogeny and carcinogenesis seems to be important. Liver GGT polypeptides were identified by immunoblot analysis in subcellular fractions (rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi membranes and plasma membranes). Rat liver GGT appeared as a series of polypeptides corresponding to different maturation steps. Polypeptides related to the heavy subunit of GGT were detected in rough endoplasmic reticulum at 49, 53 and 55 kDa, and in Golgi membranes at 55, 60 and 66 kDa. Two polypeptides related to the light subunit of GGT were also observed in Golgi membranes. In plasma membranes GGT was composed of 100 kDa, 66 kDa and 31 kDa polypeptides. The 66 kDa component could correspond to the heavy subunit of the rat liver enzyme, and if so has a molecular mass higher than that of the purified rat kidney form of GGT (papain-treated). These data suggest different peptide backbones for the heavy subunits of liver GGT and kidney GGT.

Immune rejection of human dystrophin following intramuscular injections of naked DNA in mdx mice.

Intramuscular administration of plasmid expressing full-length human dystrophin in dystrophin-deficient adult mdx mice resulted in humoral and weak specific T cell responses against the human dystrophin protein. Following plasmid injection, human dystrophin was detected in the injected muscles at 7 days, but decreased thereafter. Anti-dystrophin antibodies were found 21 days following plasmid injection, which coincided with transient myositis. This immune rejection prevented the mice from expressing human dystrophin after a second plasmid injection. No anti-DNA antibodies were found. Anti-dystrophin antibodies were seen in a smaller proportion of plasmid-injected dystrophin-competent C57BL/10 mice, suggesting that the immune rejection of dystrophin may be explained partially by species differences in the dystrophin protein.

Gene transfer into canine myoblasts.

We have developed and characterized cultures of healthy and dystrophic canine myoblasts for the evaluation of various gene transfer protocols. The number of desmin-positive myoblasts was elevated (>>80%) in cultures of myoblasts obtained from different muscle territories, the diaphragm muscle giving rise to the purest cultures. Myoblasts from dogs turned out to be a very convenient source of well transfectable and transducible cells. Transfection with plasmid DNA allowed efficient transgene expression (50% of beta-galactosidase positive cells and about 375 ng luciferase/mg protein after transfection with a calcium phosphate-precipitated plasmid). Infection with high concentrations of adenoviral and retroviral vectors allowed transgene (beta-galactosidase or mini-dystrophin) detection in about 75 to 90% of the canine cells. Therefore, primary dog myoblast cultures represent a useful in vitro model for viral and non-viral gene delivery, as well as for functional evaluation and cell grafting with applications in genetic diseases, vaccination or production of circulating therapeutic proteins.