Expression and transcriptional activities of nuclear receptors involved in regulation of drug-metabolizing enzymes are not altered by colchicine: focus on PXR, CAR, and GR in primary human hepatocytes.

Recent findings show that colchicine (COL) in submicromolar concentrations downregulates the expression of major drug-metabolizing P450 enzymes in human hepatocytes. Concomitantly, the expression of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) was diminished by COL, whereas expression of glucocorticoid receptor (GR) remained unaltered. A tentative mechanism is perturbation of the GR-PXR/CAR-CYP2/3 signaling cascade, resulting in restricted transcriptional activity of GR receptor by colchicine. In this work we focused on 10-demethylcolchicine (colchiceine; EIN), a structural analogue and a putative metabolite of COL that lacks tubulin-binding activity. We investigated the effects of EIN on the expression of PXR, CAR, and GR receptors in primary cultures of human hepatocytes. In contrast with the effects of COL, EIN does not alter the expression of PXR, CAR, and/or GR receptors mRNAs. In addition, EIN had no effects on transcriptional activities of PXR, CAR, and GR receptors in reporter gene assays using transfected cell lines. Considering that COL and EIN are structurally very close and differ only in their tubulin-binding activity, the data presented imply that the deleterious effects of COL on the GR-PXR/CAR-CYP2/3 cascade are primarily due to perturbation of the microtubule network. Our data support the idea of replacing COL by EIN, which is less toxic and does not interact with xenoreceptors.

Cross-talk between xenobiotic detoxication and other signalling pathways: clinical and toxicological consequences.

1. Recent investigations on nuclear receptors and other transcription factors involved in the regulation of genes encoding xenobiotic metabolizing and transport systems reveal that xenobiotic-dependent signalling pathways are embedded in, and establish functional interactions with, a tangle of regulatory networks involving the glucocorticoid and oestrogen receptors, the hypoxia-inducible factor, the vitamin D receptor and other transcription factors/nuclear receptors controlling cholesterol/bile salt homeostasis and liver differentiation. 2. Such functional interferences provide new insight, first for understanding how xenobiotics might exert adverse effects, and second how physiopathological stimuli affect xenobiotic metabolism.

The expression of CYP2B6, CYP2C9 and CYP3A4 genes: a tangle of networks of nuclear and steroid receptors.

Numerous chemicals increase the metabolic capability of organisms by their ability to activate genes encoding various xenochemical-metabolizing enzymes, such as cytochromes P450 (CYPs), transferases and transporters. For example, natural and synthetic glucocorticoids (agonists and antagonists) as well as other clinically important drugs induce the hepatic CYP2B, CYP2C and CYP3A subfamilies in man, and these inductions might lead to clinically important drug-drug interactions. Only recently, the key cellular receptors that mediate such inductions have been identified. They include nuclear receptors, such as the constitutive androstane receptor (CAR, NR1I3), the retinoid X receptor (RXR, NR2B1), the pregnane X receptor (PXR, NR1I2), and the vitamin D receptor (VDR, NR1I1) and steroid receptors such as the glucocorticoid receptor (GR, NR3C1). There is a wide promiscuity of these receptors in the induction of CYPs in response to xenobiotics. Indeed, this adaptive system appears now as a tangle of networks, where receptors share partners, ligands, DNA response elements and target genes. Moreover, they influence mutually their relative expression. This review is focused on these different pathways controlling human CYP2B6, CYP2C9 and CYP3A4 gene expression, and the cross-talk between these pathways.

Dual effect of dexamethasone on CYP3A4 gene expression in human hepatocytes. Sequential role of glucocorticoid receptor and pregnane X receptor.

Although CYP3A induction by dexamethasone has been extensively documented, its mechanism is still unclear because both the role of the glucocorticoid receptor and the ability of dexamethasone to activate the human pregnane X receptor have been questioned. In an attempt to resolve this problem, we investigated the response of CYP3A4 to dexamethasone (10 nm-100 microm) in primary human hepatocytes and HepG2 cells, using a variety of methods: kinetic analysis of CYP3A4 and tyrosine aminotransferase expression, effects of RU486 and cycloheximide, ligand binding assay, cotransfection of HepG2 cells with CYP3A4 reporter gene constructs and vectors expressing the glucocorticoid receptor, pregnane X receptor or constitutively activated receptor. In contrast to rifampicin (monophasic induction), dexamethasone produces a biphasic induction of CYP3A4 mRNA consisting of a low-dexamethasone component (nmol concentrations) of low amplitude (factor of 3-4) followed by a high-dexamethasone component (supramicromolar concentrations) of high amplitude (factor of 15-30). We show that the low-dexamethasone component results from the glucocorticoid receptor-mediated expression of pregnane X receptor and/or constitutively activated receptor which, in turn, are able to transactivate CYP3A4 in a xenobiotic-independent manner. At supramicromolar concentrations (>10 microm), dexamethasone binds to and activates pregnane X receptor thus producing the high-dexamethasone component of CYP3A4 induction. We conclude that, in contrast to the other xenobiotic inducers of CYP3A4, glucocorticoids play a dual role in CYP3A4 expression, first by controlling the expression of PXR and CAR under physiological conditions (submicromolar concentrations) through the classical glucocorticoid receptor pathway, and second by activating the pregnane X receptor under bolus or stress conditions (supramicromolar concentrations).

Calcium channel modulators of the dihydropyridine family are human pregnane X receptor activators and inducers of CYP3A, CYP2B, and CYP2C in human hepatocytes.

The expression of three cytochromes P450 (CYP3A4, CYP2C9, and CYP2B6) was investigated in primary human hepatocyte cultures following treatment with four calcium channel modulators (CCM) of the dihydropyridine family, three antagonists (nifedipine, nicardipine, and isradipine), and one agonist (BK8644). Induction of CYP3A4 was studied by Northern blot, Western blot, and enzymatic activity. Induction began between 1 and 10 microM CCM and was dependent on the presence of dexamethasone (100 nM) in the medium. CYP3A4 mRNA accumulation started only after 16 h of treatment because pregnane X receptor (hPXR) synthesis was needed. Cotransfection experiments showed that the proximal and the distal PXR response elements of the CYP3A4 promoter and hPXR (HepG2 cells) or dexamethasone-induced hPXR (primary hepatocytes) were necessary to obtain full induction. Furthermore, glutathione S-transferase pull-down assays demonstrated that the CCM tested can act as hPXR ligands. In addition, cotransfection experiments in CV1 cells showed that these compounds failed to reverse CAR (constitutively activated receptor) inactivation by androstenol. Finally, 10 microM CCM induced both CYP2C9 and CYP2B6, strengthening the evidence that hPXR is involved in the regulation of these genes. All together, these results widen the field of hPXR activators to a new class of ligand, namely the CCM of the dihydropyridine family.

Dexamethasone enhances constitutive androstane receptor expression in human hepatocytes: consequences on cytochrome P450 gene regulation.

The barbiturate phenobarbital induces the transcription of cytochromes P450 (CYPs) 2B through the constitutive androstane receptor (CAR; NR1I3). CAR is a member of the nuclear receptor family (NR1) mostly expressed in the liver, which heterodimerizes with retinoid X receptor (RXR) and was shown to transactivate both the phenobarbital responsive element module of the human CYP2B6 gene and the CYP3A4 xenobiotic response element. Because previous studies in rodent hepatocyte cultures have shown that the phenobarbital-mediated induction of CYP2B genes is potentiated by glucocorticoids, we examined the role of activated glucocorticoid receptor in this process. We show that submicromolar concentrations of dexamethasone enhance phenobarbital-mediated induction of CYP3A4, CYP2B6, and CYP2C8 mRNA in cultured human hepatocytes. In parallel, we observed that glucocorticoid agonists, such as dexamethasone, prednisolone, or hydrocortisone, specifically increase human car (hCAR) mRNA expression. Accumulation of hCAR mRNA parallels that of tyrosine aminotransferase: both mRNAs reach a maximum at a concentration of 100 nM dexamethasone and are down-regulated by concomitant treatment with the glucocorticoid antagonist RU486. Moreover, the effect of dexamethasone on hCAR mRNA accumulation appears to be of transcriptional origin because the addition of protein synthesis inhibitor cycloheximide has no effect, and dexamethasone does not affect the degradation of hCAR mRNA. Furthermore, dexamethasone increases both basal and phenobarbital-mediated nuclear translocation of CAR immunoreactive protein in human hepatocytes. The up-regulation of CAR mRNA and protein in response to dexamethasone explains the synergistic effect of this glucocorticoid on phenobarbital-mediated induction of CYP2B genes and the controversial role of the glucocorticoid receptor on phenobarbital-mediated CYP gene inductions.

Interleukin-6 negatively regulates the expression of pregnane X receptor and constitutively activated receptor in primary human hepatocytes.

The marked impairment of hepatic drug metabolism during inflammation and infections has been known for many years and shown to result from down-regulation of cytochrome P450s (CYP) by cytokines. However, the mechanism of this repression is unknown. Using primary cultures of human hepatocytes, we show here that interleukin-6 (IL-6) rapidly and markedly decreases the expression of PXR (pregnane X receptor) and CAR (constitutively activated receptor) mRNAs, but does not affect the levels of dioxin receptor and glucocorticoid receptor mRNA. In parallel, IL-6 decreases both rifampicin- and phenobarbital-mediated induction of CYP2B6, CYP2C8, CYP2C9, and CYP3A4. As the transcriptional activity of PXR and CAR is not affected by IL-6 in cell-based reporter assays, our data suggest that the loss of CYP2 and CYP3 inducibility results from the negative regulation of PXR and CAR gene expression by this cytokine.

Dexamethasone induces pregnane X receptor and retinoid X receptor-alpha expression in human hepatocytes: synergistic increase of CYP3A4 induction by pregnane X receptor activators.

In this report we show that submicromolar concentrations of dexamethasone enhance pregnane X receptor (PXR) activator-mediated CYP3A4 gene expression in cultured human hepatocytes. Because this result is only observed after 24 h of cotreatment and is inhibited by pretreatment with cycloheximide, we further investigated which factor(s), induced by dexamethasone, might be responsible for this effect. We report that dexamethasone increases both retinoid X receptor-alpha (RXRalpha) and PXR mRNA expression in cultured human hepatocytes, whereas PXR activators such as rifampicin and clotrimazole do not. Accumulation of RXRalpha and PXR mRNA reaches a maximum at a concentration of 100 nM dexamethasone after treatment for 6 to 12 h and is greatly diminished by RU486. A similar pattern of expression is observed with tyrosine aminotransferase mRNA. Moreover, the effect of dexamethasone on PXR mRNA accumulation seems to be through direct action on the glucocorticoid receptor (GR) because the addition of cycloheximide has no effect, and dexamethasone does not affect the degradation of PXR mRNA. Furthermore, dexamethasone induces the accumulation of a RXRalpha-immunoreactive protein and increases the nuclear level of RXRalpha:PXR heterodimer as shown by gel shift assays with a CYP3A4 ER6 PXRE probe. This accumulation of latent PXR and RXRalpha in the nucleus of hepatocytes explains the synergistic effect observed with dexamethasone and PXR activators together on CYP3A4 induction. These results reveal the existence of functional cross talk between the GR and PXR, and may explain some controversial aspects of the role of the GR in CYP3A4 induction.

Evidence for the presence of a functional pregnane X receptor response element in the CYP3A7 promoter gene.

Pregnane X Receptor (PXR) has been recently shown to regulate the inducible expression of CYP3A genes in response to xenobiotics and steroids. PXR forms a heterodimer with the retinoic acid receptor (RXR) and this complex binds to and transactivates an 18bp region containing two everted repeats TGA(A/C)CT separated by 6 nucleotides (ER6) and located at approximately -150 in the CYP3A4 promoter. In this work we have isolated and sequenced the proximal 5'-flanking region of CYP3A7 from two different human genomic libraries. In contrast to a previously reported sequence (Itoh et al., 1992), we did not observe any mutation in the 3'-half of the CYP3A7 ER6 element. Using electrophoretic mobility shift assays and cotransfection experiments we show that this element is able to bind the PXR:RXR complex and transactivates the expression of a down stream promoter in response to rifampicin, clotrimazole, and RU-486, three compounds known to specifically activate the human PXR. This is consistent with the fact that CYP3A7 mRNA is inducible in several primary cultures of human hepatocytes from different patients, as well as in two hepatocarcinoma cell lines HuH7 and HepG2, in response to these compounds. In contrast to a previous report (Blumberg et al., 1998), based on the sequence published by Itoh et al., we conclude that CYP3A7, like CYP3A4, is inducible in response to xenobiotics and presumably in a large proportion of the population.

The antibiotic rifampicin is a nonsteroidal ligand and activator of the human glucocorticoid receptor.

The glucocorticoid receptor (GR) belongs to a superfamily of ligand-regulated nuclear steroid hormone receptors. The steps in the signal transduction pathway leading to the biological effects of glucocorticoids (GCs) include sequentially binding of the steroid to the GR ligand binding domain (LBD), receptor transformation, nuclear translocation and either positive or negative gene transactivation. Rifampicin (RIF) is a macrocyclic antibiotic used as an antituberculosis agents. As the incidence of tuberculosis has been increasing, in part because of the AIDS epidemic, a growing number of patients are being exposed to the adverse effects of this antibiotic. Indeed, this compound, as are the GCs, is often implicated in noxious drug interactions, because of its strong ability to induce drug-metabolizing enzymes. Moreover, in humans, RIF, as are the GCs, has been described as a potential immunodepressor, associated notably with the reduction of mitogenic responsiveness of human peripheral blood lymphocytes. Here, we report that RIF activates the human glucocorticoid receptor (hGR). Transient expression of wild-type, deleted or mutated GRs; sucrose density gradient sedimentation; and the BIAcore technique strongly suggest that RIF binds to the receptor with the physiological consequence that this antibiotic acts as an immunodepressor. Given the wide use of RIF in the treatment of coinfection of tuberculosis and HIV, this report is highly relevant to current medical practice.

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.

Sequence of the 5'-flanking region of CYP3A5: comparative analysis with CYP3A4 and CYP3A7.

Two genomic clones containing the 3'-end of CYP3A7, the whole 5'-flanking region plus the first exon of CYP3A5 were isolated from two different human genomic banks and sequenced. The 5'-flanking region of CYP3A5, from nucleotide -1 to -1434, was 60% and 59% similar with the corresponding regions of CYP3A4 and CYP3A7, respectively. However, the similarity increased to 74% when regions from nucleotide -1 to -700 were compared and dropped to less than 42% for regions upstream from -700. The CYP3A5 promoter contains a CATA box instead of the typical TATA box and a basic transcription element (BTE). Other consensus sequences previously found in CYP3A4 and CYP3A7 were also identified in the 5'-flanking of CYP3A5 from -1 to -700. Both CYP3A5 and CYP3A7 genes appear to be tandemly associated and transcribed in the same direction on chromosome 7.

Regulation of two rat serine-protease inhibitor gene promoters by somatotropin and glucocorticoids. Study with intact hepatocytes and cell-free systems.

Only two out of the three serine-protease inhibitor genes (SPI 2.1, 2.2 and 2.3) expressed in rat liver are tightly controlled by somatotropin acting mainly at the transcriptional level, thus making this gene system particularly suitable to study its molecular mechanism of action. In these studies, we analyzed SPI promoter activities in cultured hepatocytes transfected by electroporation or in cell-free extracts. The proximal SPI 2.1 promoter region contains two somatotropin-responsive sites which are functional in intact cells. The more distal element that maps at positions -175 to -114, and is analogous to the one originally described by Yoon et al. (1990) [Yoon, J. B., Berry, S. A., Seelig, S. & Towle, H. C. (1990) J. Biol. Chem. 265, 19947-19954], behaves as a weak enhancer whose activity is strongly potentiated by proximal 5' downstream sequences that contains potential CCAAT-enhancer binding protein (C/EBP) sites. An additional proximal hormone-sensitive site is located in the close vicinity of the transcription-start site between positions -41 and +8, and also requires the first C/EBP-binding element to be active. The distal element appears to contribute more importantly (60%) than the proximal one (40%) to the overall somatotropin stimulation of chimeric gene expression. Nonetheless, both displayed similar dose-dependence, with half-maximal and maximal effects occurring at 0.5-1 nM and 5-10 nM, respectively. The somatotropin refractoriness of the SPI 2.3 gene appears to be due to the presence of distal (-2300 to -200) inhibitory element(s) in the promoter. Glucocorticoids exert both positive and negative effects on SPI promoter activity. Their stimulatory action appears to involve sequences located between positions -114 and -82, together with a more distal half glucocorticoid-responsive element, whereas their inhibitory effect is more likely mediated by sequences located between positions -41 and +8. In vitro transcription assays, performed with promoter-deletion mutants and competitor oligonucleotides, revealed the presence of a major functional C/EBP site located immediately upstream from the transcription-start point. Unfortunately, the regulatory features of SPI gene expression observed in intact cells were completely obliterated by breaking down the cell structure, and could not therefore be studied using cell-free systems.

Functional characterization of the promoter of pp63, a gene encoding a natural inhibitor of the insulin receptor tyrosine kinase.

PP63 is a liver specific phosphorylated glycoprotein encoded by a single copy gene, which has the property of inhibiting both autophosphorylation and tyrosine kinase activity of the insulin receptor. In this study, we have analyzed the structure activity relationship of the pp63 gene promoter. Five protein binding sites were found in the proximal 5' flanking region of the gene (-223 to +4). Using oligonucleotides as competitors and purified recombinant C/EBP in footprinting and gel retardation assays, we identified two typical C/EBP sites (X1 and X3) plus a heterogenous, C/EBP-NF1 like site (X5), separated by two classical NF1 binding sites (X2 and X4). C/EBP or the related proteins were predominantly involved in supporting cell-free transcription. Occupancy of the first high affinity C/EBP site conferred almost maximal promoter efficiency, in vitro. However, this pp63 promoter activity remained very low as compared to that in intact hepatocytes. In these cells, occupancy of the first C/EBP (X1) and NF1 (X2) sites was already required for achieving a weak transcriptional activity. The use of the second C/EBP site (X3) strongly enhanced transcription, up to 60-70% of the maximum, whereas occupancy of the two more distal sites (X4 and X5) was necessary to fully activate the promoter. Thus, the strength of the promoter as well as the liver specific expression of pp63 gene appear to result from the interplay of several DNA-protein complexes involving mainly C/EBP and/or related proteins as well as the ubiquitous NF1 factor(s), rather than from the interaction of a more liver specific trans-acting factor with the promoter.

Analysis of proteins binding to the proximal promoter region of two rat serine protease inhibitor genes.

The three serine protease inhibitor (SPI) rat genes expressed preferentially in liver share considerable structural features and, nonetheless, are transcriptionally regulated in completely different manners, more particularly after hypophysectomy or upon acute inflammation. DNase I footprinting and gel mobility shift analyses of the SPI 2.1 and 2.3 proximal promoter regions reveal the presence of three common protein binding sites (1 to 3, 3' to 5') located immediately upstream from the transcription start site. C/EBP, the liver-enriched factor, specifically interacts with site 1 whereas its related proteins (e.g.; DBP, LAP/NFIL6) most likely recognize sites 2 and 3. Another ubiquitous unidentified factor also binds to site 2. A liver-specific protein dependent on growth hormone, whose binding is competed out by an oligonucleotide reproducing an HNF3 motif, interacts exclusively with site 3. The 42 bp sequence which is found only within the SPI 2.3 promoter interacts with two ubiquitous factors, one of which is related to NF kappa B. Acute inflammation does not significantly affect the protein binding patterns observed with the SPI 2.1 or 2.3 proximal promoter sequences. Our results show an apparent discrepancy between the large magnitude of in vivo changes in SPI gene transcription mediated by hormones and the small alterations detected in vitro, in the DNA-protein interactions on the promoters.

Lineage-specific patterns of p21ras proteins in immortalized cell lines derived from mouse teratocarcinoma.

The expression of proteins coded by the ras oncogene family was examined in mouse embryonal carcinoma (EC) cells and in immortalized cell lines derived from EC. These cell lines, which correspond to early stages of differentiation, express the simian virus 40 (SV40) T antigen and still proliferate. By 2-D gel electrophoresis of the immune complexes formed with monoclonal anti-ras antibodies, it was possible to distinguish the products of the Ha-, N- and Ki-ras genes and to correlate the observed patterns with the differentiation state of the cells. We show in this report (1) that the 2-D gel pattern of ras protein is identical for the various EC tested and is not influenced by SV40 transformation, (2) that p21Ki-ras is not detected in EC cells, although some EC cell lines are known to express a Ki-ras transcript, and (3) that the complex patterns of N- and Ha-ras observed in EC cells becomes simpler as differentiation proceeds, with a different, characteristic pattern for neuroectodermal, mesodermal and endodermal derivatives. Such patterns could prove useful as differentiation markers.

Activation of c-Ki-ras coexists with c-myc amplification in cells from a nude mouse tumor induced by the human breast carcinoma cell line SW 613-S.

In vitro transfection experiments have shown that cooperation between two different oncogenes can confer a fully malignant phenotype to primary rodent cells. We have previously reported that SW 613-Tul cells, derived from a tumor induced in a nude mouse by the human breast carcinoma cell line SW 613-S, showed a 30-fold amplification of the c-myc gene. In the present work, we show that these cells also harbor an activated c-Ki-ras gene capable of inducing the formation of foci upon transfection of NIH 3T3 cells with SW 613-Tul genomic DNA. Our results suggest that both the c-myc and c-Ki-ras oncogenes, activated by two different mechanisms, may cooperate in the full expression of the tumorigenic phenotype of SW 613-Tul cells.

[Lack of expression of the protein p53 gene in a human T-cell leukemia line]. / Absence d'expression du gène de la protéine p53 dans une lignée humaine issue d'une leucémie de type T.

Expression of the gene encoding the nuclear phosphoprotein p53 (a proto-oncogene classified in the same functional family as c-myc and E1a adenovirus gene) was examined in a human T-cell leukemia (KE-37R cell line). No p53 (or a modified product) could be detected by immunoprecipitation with monoclonal antibodies P Ab 421 and P Ab 122 in KE-37R cell extracts, and no p53-specific RNA was characterized by Northern blot analysis. Southern blot using a murine p53 cDNA clone as a probe, did not reveal any gross rearrangement in the structure of the gene. However, this molecular probe was not suited for investigating the 5' end of the gene which contains the promoter and the non coding exon 1. It is interesting to notice that in KE-37R cells, c-myc has been activated by a t(8; 14) (q24; q11) translocation, suggesting that the c-myc product might substitute to some functions normally requiring p53.