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.

Evidence for a new human CYP1A1 regulation pathway involving PPAR-alpha and 2 PPRE sites.

BACKGROUND AND AIMS: Cytochrome P450 1A1 catalyzes the degradation of endobiotics (estradiol, fatty acids, and so on) and the bioactivation of numerous environmental procarcinogens, such as arylamines and polycyclic aromatic hydrocarbons, that are found in food. Several peroxisome proliferators and arachidonic acid derivatives enhance cytochrome P450 1A1 activity, but the mechanisms involved remain unknown. The aim of this work was to study the role of peroxisome proliferator-activated receptors in cytochrome P450 1A1 gene induction. METHODS: The role of peroxisome proliferator-activated receptor transcription factors in cytochrome P450 1A1 induction was assessed by means of enzymatic activities, quantitative real-time polymerase chain reaction, gene reporter assays, mutagenesis, and electrophoretic mobility shift assay. RESULTS: We show that peroxisome proliferator-activated receptor-alpha agonists (WY-14643, bezafibrate, clofibrate, and phthalate) induce human cytochrome P450 1A1 gene expression, whereas 2,4-thiazolidinedione, a specific peroxisome proliferator-activated receptor-gamma agonist, represses it. The induction of cytochrome P450 1A1 transcripts by WY-14643 was associated with a marked increase of ethoxyresorufin O -deethylase activity (10-fold at 200 mumol/L). Transfection of peroxisome proliferator-activated receptor-alpha complementary DNA enhanced cytochrome P450 1A1 messenger RNA induction by WY-14643, although WY-14643 failed to activate xenobiotic responsive element sequences. Two peroxisome proliferator response element sites were located at positions -931/-919 and -531/-519 of the cytochrome P450 1A1 promoter. Their inactivation by directed mutagenesis suppressed the inductive effect of WY-14643 on cytochrome P450 1A1 promoter activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay experiments showed that the 2 cytochrome P450 1A1 peroxisome proliferator response element sites bind the peroxisome proliferator-activated receptor-alpha/retinoid X receptor-alpha heterodimer. CONCLUSIONS: We describe here a new cytochrome P450 1A1 induction pathway involving peroxisome proliferator-activated receptor-alpha and 2 peroxisome proliferator response element sites, indicating that peroxisome proliferator-activated receptor-alpha ligands, which are common environmental compounds, may be involved in carcinogenesis.

Speculations on the role of the microtubule network in glucocorticoid receptor signaling.

The glucocorticoid receptor (GR) is an important player in the life of a cell. This is underlined by a cohort of protein and nucleic acid structures interacting with the GR. Among many issues surrounding GR activity that are under active investigation, the role of microtubules (MTs) is still unclear. This article aims to evaluate the ayes and noes in favor of microtubule importance and then form a hypothesis on their function in GR activity.

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.

Comparative effect of colchicine and colchiceine on cytotoxicity and CYP gene expression in primary human hepatocytes.

The aims of the present study were (1) to determine the cytotoxicity of colchiceine (EIN) in comparison with that of colchicine (COL); (2) to evaluate the effect of EIN on cytochrome P450 (CYP) expression and activity. Primary human hepatocytes were the model of choice for cytotoxicity and CYP expression experiments. LDH leakage and albumin secretion served as cytotoxicity parameters. EIN was less toxic than COL based on both parameters within the concentration range 1-100 microM. 10 microM concentration of EIN did not induce the expression of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 isoforms, which were evaluated at the levels of mRNAs, proteins and specific activities in culture. EIN in concentrations up to 200 microM had no effect on marker activities of CYP1A2, 2C9, 2E1 and 3A4 in human liver microsomes. It was concluded that EIN in concentrations up to 10 microM is not cytotoxic in primary human hepatocytes as revealed by albumin secretion and LDH leakage. Possible drug-drug interactions of EIN due to effects on cytochromes P4501A2, 2C9, 2E1 and 3A4 isoforms are unlikely because inhibition/induction studies show any lack of such effects. As EIN was shown to have better antifibrotic properties than COL (European Journal of Clinical Investigation 1997, 2, 77), it can be used as a COL substitute with anticipated fewer side-effects.

Metabolism and CYP-inducer properties of astaxanthin in man and primary human hepatocytes.

Previous investigations in the rat have shown that the non-provitamin A carotenoid astaxanthin is metabolized into 3-hydroxy-4-oxo-beta-ionone and 3-hydroxy-4-oxo-7,8-dihydro-beta-ionone and, in addition, is a potent CYP1A gene inducer. Here we investigated the metabolism of this compound as well as its capacity to induce CYP genes in primary cultures of human hepatocytes. Free metabolites of 14C-astaxanthin produced in this cellular model were purified by high pressure liquid chromatography (HPLC) and identified by gas chromatography-mass spectrometry (GC-MS) analyses as 3-hydroxy-4-oxo-beta-ionol and 3-hydroxy-4-oxo-beta-ionone. In addition, deconjugation of polar compounds by glusulase and further analyses with HPLC and GC-MS revealed four radiolabeled metabolites including: 3-hydroxy-4-oxo-beta-ionol, 3-hydroxy-4-oxo-beta-ionone, and their reduced forms, 3-hydroxy-4-oxo-7, 8-dihydro-beta-ionol and 3-hydroxy-4-oxo-7,8-dihydro-beta-ionone. The same four metabolites were identified in human plasma from two volunteers who had orally taken 100 mg astaxanthin 24 h before blood collection. In cultured hepatocytes, astaxanthin was a significant inducer of the major cytochrome P450 enzyme, CYP3A4 as well as of CYP2B6, but not of other CYPs, including those from CYP1A and CYP2C families. The lack of autoinduction of astaxanthin metabolism in human hepatocytes suggests that neither CYP3A4 nor CYP2B6 contribute to the formation of metabolites. We conclude that metabolism of astaxanthin and its CYP-inducing capacity are different in humans and in rats. The novel methodology used in our studies could be extended to evaluating the role of metabolites of more important carotenoids such as beta-carotene in differentiation and carcinogenicity.

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).

The human pregnane X receptor: genomic structure and identification and functional characterization of natural allelic variants.

The pregnane X receptor (PXR)/steroid and xenobiotic receptor (SXR) transcriptionally activates cytochrome P4503A4 (CYP3A4) when ligand activated by endobiotics and xenobiotics. We cloned the human PXR gene and analysed the sequence in DNAs of individuals whose CYP3A phenotype was known. The PXR gene spans 35 kb, contains nine exons, and mapped to chromosome 13q11-13. Thirty-eight single nucleotide polymorphisms (SNPs) were identified including six SNPs in the coding region. Three of the coding SNPs are non-synonymous creating new PXR alleles [PXR*2, P27S (79C to T); PXR*3, G36R (106G to A); and PXR*4, R122Q (4321G to A)]. The frequency of PXR*2 was 0.20 in African Americans and was never found in Caucasians. Hepatic expression of CYP3A4 protein was not significantly different between African Americans homozygous for PXR*1 compared to those with one PXR*2 allele. PXR*4 was a rare variant found in only one Caucasian person. Homology modelling suggested that R122Q, (PXR*4) is a direct DNA contact site variation in the third alpha-helix in the DNA binding domain. Compared with PXR*1, and variants PXR*2 and PXR*3, only the variant PXR*4 protein had significantly decreased affinity for the PXR binding sequence in electromobility shift assays and attenuated ligand activation of the CYP3A4 reporter plasmids in transient transfection assays. However, the person heterozygous for PXR*4 is normal for CYP3A4 metabolism phenotype. The relevance of each of the 38 PXR SNPs identified in DNA of individuals whose CYP3A basal and rifampin-inducible CYP3A4 expression was determined in vivo and/or in vitro was demonstrated by univariate statistical analysis. Because ligand activation of PXR and upregulation of a system of drug detoxification genes are major determinants of drug interactions, it will now be useful to extend this work to determine the association of these common PXR SNPs to human variation in induction of other drug detoxification gene targets.

Expression and DNA-binding activity of C/EBPalpha and C/EBPbeta in human liver and differentiated primary hepatocytes.

BACKGROUND/AIMS: Limited information is available on the expression and role of C/EBP factors in human liver and hepatocytes. We investigated the expression and DNA-binding activity of C/EBPalpha and C/EBPbeta in human liver needle biopsies, surgical lobectomies and differentiated cultured hepatocytes derived from lobectomies. METHODS: RNA and protein extracts were analyzed by RNAse protection, immunoblot and gel shift assays. RESULTS: C/EBP mRNAs, isoforms and DNA-binding activities were low/undetectable in lobectomies. In contrast, several C/EBPalpha (47, 45, 35 and 33 kDa) and C/EBPbeta isoforms (47, 43, 40, 35 and 21 kDa) were observed in needle biopsies. In cultured hepatocytes, the C/EBP expression pattern dramatically changed with time. C/EBPalpha mRNA and the 45 kDa isoform increased in parallel, reaching a maximum after 3-4 weeks coincident with weak DNA-binding activity. C/EBPbeta mRNA and isoform expression increased rapidly reaching a plateau within 1-2 weeks; all C/EBPbeta isoforms were phosphorylated. C/EBPbeta exhibited greater DNA-binding activity than C/EBPalpha, and this activity paralleled C/EBPbeta isoform expression. CONCLUSIONS: C/EBP isoforms exhibit markedly different expression patterns in lobectomies, needle biopsies and cultured hepatocytes. Stress stimuli during and/or after surgery for lobectomy resections may account for this difference. The pattern of C/EBP isoform expression in long-term highly differentiated cultured hepatocytes is close to that observed in needle biopsies.

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.

Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression.

Variation in the CYP3A enzymes, which act in drug metabolism, influences circulating steroid levels and responses to half of all oxidatively metabolized drugs. CYP3A activity is the sum activity of the family of CYP3A genes, including CYP3A5, which is polymorphically expressed at high levels in a minority of Americans of European descent and Europeans (hereafter collectively referred to as 'Caucasians'). Only people with at least one CYP3A5*1 allele express large amounts of CYP3A5. Our findings show that single-nucleotide polymorphisms (SNPs) in CYP3A5*3 and CYP3A5*6 that cause alternative splicing and protein truncation result in the absence of CYP3A5 from tissues of some people. CYP3A5 was more frequently expressed in livers of African Americans (60%) than in those of Caucasians (33%). Because CYP3A5 represents at least 50% of the total hepatic CYP3A content in people polymorphically expressing CYP3A5, CYP3A5 may be the most important genetic contributor to interindividual and interracial differences in CYP3A-dependent drug clearance and in responses to many medicines.

Induction of CYP2C genes in human hepatocytes in primary culture.

The expression and inducibility of four CYP2C genes, including CYP2C8, -2C9, -2C18, and -2C19, was investigated in primary cultures of human hepatocytes. By the use of RNase protection assay and specific antibodies, each CYP2C mRNA and protein were quantified unequivocally. The four CYP2C mRNAs were expressed in human livers and cultured primary hepatocytes, but only the CYP2C18 protein was not detected. Compounds known to activate the pregnane X receptor (PXR) such as rifampicin, or the constitutively activated receptor (CAR) such as phenobarbital, induced CYP2C8, CYP2C9, and to a lesser extent CYP2C19 mRNAs and proteins. CYP2C18 mRNA was expressed but not inducible. The concentration dependence of CYP2C8 and CYP2C9 mRNAs in response to rifampicin and phenobarbital paralleled that of CYP3A4 and CYP2B6, the maximum accumulation being reached with 10 microM rifampicin and 100 microM phenobarbital. In contrast, dexamethasone produced maximum induction of CYP2C8 and CYP2C9 mRNAs at 0.1 microM while in these conditions neither CYP3A4 nor CYP2B6 was significantly induced. Moreover, the concentration dependence of CYP2C8 and CYP2C9 mRNAs in response to dexamethasone paralleled that of tyrosine aminotransferase. Furthermore, dexamethasone, which has been recently shown to up-regulate PXR and CAR expression through the glucocorticoid receptor, potentiated CYP2C8 and CYP2C9 mRNA induction in response to rifampicin and phenobarbital. Collectively, these results suggest the possible implication of at least three receptors in the regulation of CYP2C8 and CYP2C9 expression, i.e., glucocorticoid receptor, PXR, and/or CAR.

Cell suspensions, cell cultures, and tissue slices--important metabolic in vitro systems.

In vitro subcellular and cellular systems have important and irreplaceable roles in the metabolic investigations that precede the development of new potential drugs. Of these model systems, tissue slices are probably the nearest to in vivo conditions. From the experimental and complexity points of view, perfused organs lie midway between tissue slices and whole organism. Preparation and working with liver slices is quick and easy, and, excess material can be cryopreserved and stored untill the next experiment. Slices can be prepared from a wide variety of organs and it is possible to co-incubate them. Another important feature is the possibility of interspecies comparison of slices. Different experiments can be run both in the short-term as well as long-term incubations. Each in vitro system has an important place for example, in the development of new medicaments. It is therefore important to compare and supplement experimental results from different in vitro systems when extrapolating to in vivo situations is done.

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.

Axonal regulation of Schwann cell proliferation and survival and the initial events of myelination requires PI 3-kinase activity.

In this report, we have investigated the signaling pathways that are activated by, and mediate the effects of, the neuregulins and axonal contact in Schwann cells. Phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase kinase (MAPK kinase) are strongly activated in Schwann cells by glial growth factor (GGF), a soluble neuregulin, and by contact with neurite membranes; both kinase activities are also detected in Schwann cell-DRG neuron cocultures. Inhibition of the PI 3-kinase, but not the MAP kinase, pathway reversibly inhibited Schwann cell proliferation induced by GGF and neurites. Cultured Schwann cells undergo apoptosis after serum deprivation and can be rescued by GGF or contact with neurites; these survival effects were also blocked by inhibition of PI 3-kinase. Finally, we have examined the role of these signaling pathways in Schwann cell differentiation in cocultures. At early stages of coculture, inhibition of PI 3-kinase, but not MAPK kinase, blocked Schwann cell elongation and subsequent myelination but did not affect laminin deposition. Later, after Schwann cells established a one-to-one relationship with axons, inhibition of PI 3-kinase did not block myelin formation, but the myelin sheaths that formed were shorter, and the rate of myelin protein accumulation was markedly decreased. PI 3-kinase inhibition had no observable effect on the maintenance of myelin sheaths in mature myelinated cocultures. These results indicate that activation of PI 3-kinase by axonal factors, including the neuregulins, promotes Schwann cell proliferation and survival and implicate PI 3-kinase in the early events of myelination.

Effect of colchicine and its derivatives on the expression of selected isoforms of cytochrome P450 in primary cultures of human hepatocytes.

The study addressed the effect of colchicine and its derivatives on the protein levels of cytochrome P450 (CYP) 1A2, 2A6, 3A4, 2C9/19, and 2E1 isoforms. Primary human hepatocyte culture was the model of choice. Levels of individual CYP isoforms were detected using immunoblotting. Colchicine caused an increase of CYP2E1 protein content, colchiceine and N-deacetylcolchiceine induced isoforms CYP2C9, 2E1 and 3A4 whereas colchicoside induced CYP2C9 and 2E1. The levels of CYP1A2 and 2A6 were unaffected by any of tested compounds. Demecolcine and 3-demethylcolchicine had no effect on any studied P450 isoform. Since colchicine is an exclusive substrate of CYP3A4 whereas it induces CYP2E1, there is a suspicion rather at protein stabilization than at gene induction concerning induction origin.