Cloning and functional expression of a first inducible avian cytochrome P450 of the CYP3A subfamily (CYP3A37).

CYP3As represent a family of cytochromes P450 involved in the metabolism of both endogenous and exogenous natural and synthetic compounds. Well described in mammals, none have yet been cloned and characterized in avian species. In this paper, we report the cloning and analysis of an avian CYP3A (CYP3A37). Using an RNA differential display approach, an 80-bp phenobarbital-inducible cDNA fragment was amplified from chicken embryo liver. Based on its homology with mammalian CYP3As, this fragment was used to clone a full-length cDNA consisting of 1638 bp encoding a putative protein of 509 amino acids. The sequence shares between 57.4 and 62% identity at the amino acid level with CYP3As of other species. This cDNA was designated CYP3A37 according to the current cytochrome P450 nomenclature. When expressed in COS1 cells, the CYP3A37 cDNA produced a protein of congruent with55 kDa, which was recognized by polyclonal anti-rat CYP3A1 antiserum. In a bacterial expression system, the CYP3A37 cDNA produced a protein capable of steroid 6beta-hydroxylation. At a substrate concentration of 100 microM, progesterone, testosterone, and androstenedione were found to be 6beta-hydroxylated at a rate of 15.4, 11.7, 12.2 nmol/min/nmol P450, respectively. Used as control, the human CYP3A4 gave similar hydroxylation rates. Finally, in both chicken embryo liver and chicken hepatoma cells (LMH), CYP3A37 mRNA was increased after treatment with typical CYP3A inducers, such as metyrapone, phenobarbital, dexamethasone, and pregnenolone 16alpha-carbonitrile, but not rifampicin. CYP2H1, a well-characterized inducible chicken cytochrome P450, also was induced by the same compounds, suggesting similar regulation of CYP3 and CYP2 genes in this species.

In vitro and in vivo secretion of cloned antibodies by genetically modified myogenic cells.

In vivo production of recombinant antibodies by engineered cells may have applications for gene therapy of certain cancers and of certain severe viral diseases. It would also permit the development of new animal models of autoimmune diseases and new approaches for in vivo ablation of specific cell types for fundamental purposes. Using gene transfer of an anti-human thyroglobulin monoclonal antibody, we show here that several cell types permitting autologous grafting of genetically engineered cells are efficiently able to secrete antibodies in vitro. Those cells include skin fibroblasts, hepatocytes, and myogenic cells. We also show that the secreted antibodies display an affinity for the antigen close to that of the parental antibody, with, however, slight differences varying according to the cell type. This indicates that the foldings of antigen combining sites of antibodies produced in B cell- and non-B cell contexts are very similar. Finally, we report that, when implanted in the forelimb of a mouse, genetically modified myogenic cells are able to secrete antibodies for at least 4 months. Taken together, our observations point to the notion that genetic modification of patient cells may be used for long-term antibody-based gene therapies.

Effect of cell density and epidermal growth factor on the inducible expression of CYP3A and CYP1A genes in human hepatocytes in primary culture.

The influence of cell density and epidermal growth factor (EGF) on the expression and inducibility of cytochrome P450 (CYP) genes of the CYP3A and CYP1A families in adult human hepatocytes in primary culture has been evaluated. Only when cultured at subconfluence and in the presence of EGF did hepatocytes exhibit a proliferative response, assessed by measuring DNA synthesis and cyclin A accumulation. In the absence of EGF, the accumulation of CYP3A4 and CYP1A2 messenger RNAs (mRNAs) in response to their respective inducers (rifampicin and dioxin) was dramatically decreased in subconfluent culture with respect to confluent cultures. The presence of EGF only slightly decreased the accumulation of these mRNAs in both confluent and subconfluent cultures. The accumulation of CYP2D6 and CYP2E1 proteins, which are constitutively expressed in confluent cultures, and the production of fibrinogen and apolipoprotein (Apo) B100 exhibited similar behavior, while nicotinamide adenine dinucleotide phosphate cytochrome c reductase activity was affected neither by cell density nor by EGF. In contrast, the accumulation of CYP1A1 mRNA in response to dioxin was similar in confluent and subconfluent cultures, irrespective of the presence of EGF. Interestingly, CYP3A7, a gene that is preferentially expressed in the fetal liver, was expressed constitutively neither in confluent nor in subconfluent cultures, irrespective of the presence of EGF. It is concluded that the loss of cell-cell contacts rather than the proliferative status of cells per se is responsible for the dramatic decrease in the expression of CYP genes, normally expressed in the adult human liver.

Lipid-mediated transfection of normal adult human hepatocytes in primary culture.

The aim of this work was to develop a procedure for the lipid-mediated transfection of DNA into normal adult human hepatocytes in culture. Cells were plated in a serum-free culture medium at various cell densities, on plastic or collagen-coated dishes, both in the absence and in the presence of epidermal growth factor (EGF). The cells were incubated for various periods of time with mixtures of DNA-lipofectin or DNA-3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) liposomes, and the efficiency of transfection was assessed by measuring the activity of reporter genes, beta-galactosidase or chloramphenicol acetyl-transferase (CAT). For comparison, similar experiments were carried out with human cell lines including HepG2, Caco-2, and WRL68. The efficiency of transfection (in percentage of cells) was not significantly different after transfection with lipofectin or DC-chol and comprised between 0.04 and 1.7% (extreme values) for different cultures. The efficiency of transfection decreased as the age or density of the culture increased and increased in cultures treated with EGF. Direct measurement of the rate of DNA synthesis suggested that the efficiency of transfection was related to the number of cells entering the S phase. Under the same conditions, the efficiency of transfection was one to two orders of magnitude greater in the three cell lines. A plasmid harboring 660 bp of the 5'-flanking region of CYP1A1 (containing two xenobiotic enhancer elements) fused upstream of the promoter of thymidine kinase and the CAT reporter gene was constructed. When this plasmid was transfected in human hepatocytes, CAT activity was induced as expected. We conclude that normal adult human hepatocytes can be transfected with exogenous DNA and that the transfected construct is regulated in the manner expected from in vivo studies.

Role of the liver-enriched transcription factors C/EBP alpha and DBP in the expression of human CYP3A4 and CYP3A7.

In the human fetal liver, CYP3A7 is expressed as early as the 13th week of gestation. This continues to the perinatal period when it is sharply repressed prior to birth. Concomitantly, the expression of CYP3A4, not detectable in the fetus, sharply increases in the perinatal period to remain elevated throughout adulthood. The mechanisms controlling these developmental patterns of expression have not yet been elucidated at the molecular level. The aim of the present work was to make a functional analysis of the 5'-flanking regions of CYP3A4 and CYP3A7 in different cell lines, including CHO, HepG2, WRL68 and Caco-2 TC7, after cotransfection with two hepato-specific transcription factors, C/EBP alpha and DBP. Six deletions of different length of the 5'-flanking region of each gene, spanning from -1240 to +11 for CYP3A4 and from -1157 to +13 for CYP3A7, were analysed by reporter gene assay. With the CYP3A4 constructs, C/EBP alpha stimulated the transcriptional activity in CHO cells in a way that suggested the presence of at least two C/EBP alpha-responsive elements, one downstream of -55 and one upstream of this position. In CYP3A7, the proximal element exhibited comparable stimulation to the corresponding one in CYP3A4, although the more distal one appeared to respond to a much smaller extent. CYP3A4 and CYP3A7 constructs also responded to C/EBP alpha in HepG2 and WRL68. However, only CYP3A4 and not CYP3A7 was transactivated by this factor in the Caco-2-TC7 cell line. In CHO cells, only the shortest proximal promoter deletion of CYP3A4 (downstream of -57) responded to DBP, while neither the longer constructs nor the CYP3A7 deletions were transactivated. Although preliminary, our results suggest that C/EBP alpha, and possibly other members of the C/EBP family, play a prominent part in the expression of the CYP3A family in man, and that the two genes respond differently to C/EBP alpha and DBP, two factors that exhibit a strict proliferation-dependent pattern of expression in the liver.

Differential effects of cytokines on the inducible expression of CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture.

We have investigated the effect of cytokines, including interleukin-6 (Il-6), interleukin-1 alpha (Il-1 alpha), and tumor necrosis factor-alpha (TNF-alpha), on the inducible expression of cytochrome P450s (CYP) CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture. The ability of these cultures to mimic the acute phase response when stimulated with cytokines was evaluated using immunoblotting to measure the production of albumin, ferritin, fibrinogen, and ceruloplasmin. The cytokines exhibited specific patterns of action on the production of these proteins. Albumin was depressed by all the cytokines. In contrast to Il-6 and Il-1 alpha, TNF-alpha reduced the production of fibrinogen and ceruloplasmin but stimulated the production of ferritin. When cells were treated with the CYP inducer alone, large increases in the expression of CYP1A1 and CYP1A2 by beta-naphthoflavone and of CYP3A4 by rifampicin were observed at messenger RNA (mRNA) and protein levels, by ribonuclease protection and immunoblotting, respectively. When the cells were treated with the inducer plus cytokines, the induction of mRNA was greatly reduced. Again, specific patterns of action were revealed: Il-6 had the most potent effect on CYP3A4, whereas TNF-alpha was the most potent with CYP1A genes. In all cases, changes at the protein levels paralleled changes at the mRNA levels. In cells preinduced with beta-naphthoflavone or rifampicin, the decay with time of the levels of the CYP1A2 or CYP3A4 proteins, after the removal of the inducer, was not affected by cytokines. We conclude that cytokines strongly repress the inducibility of CYP1As and CYP3A4 genes at a transcriptional or a posttranscriptional level, but affect neither the rate of translation of CYP mRNAs nor the rate of degradation of the CYP proteins in these cultures.

Omeprazole and lansoprazole are mixed inducers of CYP1A and CYP3A in human hepatocytes in primary culture.

The ability of several gastric antiulcer drugs including lansoprazole, cimetidine and ranitidine to affect the expression of human liver microsomal cytochromes P450 comparatively to omeprazole, reported previously to be a CYP1A inducer, was evaluated in primary cultures of human hepatocytes. Poly (A)+ RNA and microsomes extracted from the cells were analyzed in Northern and Western blots with specific cDNA probes and antibodies, and assayed for form-specific monoxygenase activities. Lansoprazole induced both CYP1A1 and CYP1A2 as omeprazole and did not apparently bind to the aryl hydrocarbon receptor with high affinity. Omeprazole sulfone was not an inducer of CYP1A. Omeprazole, omeprazole sulfone and lansoprazole induced CYP3A in approximately 50% of tested cultures, whereas 100% of tested cultures responded to omeprazole and to rifampicin in terms of CYP1A and CYP3A induction, respectively. Finally, cimetidine and ranitidine were not inducers. We conclude that omeprazole and lansoprazole constitute a new class of mixed inducers of CYP1A and CYP3A in human hepatocytes in primary culture and that the induction of CYP3A in response to these molecules could be polymorphic in humans.