Biomedical subjects

S Chevalier

Publications and source records attributed to S Chevalier.

At least 19 recordsLinked to original sources

PubMed2000

PPARalpha-dependent alteration of GRP94 expression in mouse hepatocytes.

The adverse effects of the peroxisome proliferators (PPs), a class of rodent nongenotoxic hepatocarcinogens, include suppression of apoptosis, induction of hepatocyte proliferation, and liver enlargement which eventually leads to tumours. The response to PPs is mediated by the peroxisome proliferator activated receptor alpha (PPARalpha). We carried out proteomic analyses of PP-treated hepatocytes from wild-type and PPARalpha-null mice to identify the molecular pathways underlying the adverse effects of PPs. We have identified eighteen protein spots exhibiting differential expression in PP-treated wild-type mouse hepatocytes. Several proteins involved in lipid metabolism pathways, but also ATP synthase beta subunit, which are regulated by PPs were identified. In addition, both 2D silver-stained gels and Western blotting analysis indicated that the anti-apoptotic glucose-regulated protein 94 (GRP94) is consistently overexpressed upon stimulation with PPs, providing us with novel insights into the anti-apoptotic mechanism activated by PPs.

Animals↗

PubMed2000

Identification of a gp130 cytokine receptor critical site involved in oncostatin M response.

Gp130 cytokine receptor is involved in the formation of multimeric functional receptors for interleukin-6 (IL-6), IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor, and cardiotrophin-1. Cloning of the epitope recognized by an OSM-neutralizing anti-gp130 monoclonal antibody identified a portion of gp130 receptor localized in the EF loop of the cytokine binding domain. Site-directed mutagenesis of the corresponding region was carried out by alanine substitution of residues 186-198. To generate type 1 or type 2 OSM receptors, gp130 mutants were expressed together with either LIF receptor beta or OSM receptor beta. When positions Val-189/Tyr-190 and Phe-191/Val-192 were alanine-substituted, Scatchard analyses indicated a complete abrogation of OSM binding to both type receptors. Interestingly, binding of LIF to type 1 receptor was not affected, corroborating the notion that in this case gp130 mostly behaves as a converter protein rather than a binding receptor. The present study demonstrates that positions 189-192 of gp130 cytokine binding domain are essential for OSM binding to both gp130/LIF receptor beta and gp130/OSM receptor beta heterocomplexes.

Alanine↗

PubMed2000

Links between Fer tyrosine kinase expression levels and prostate cell proliferation.

In our cloning strategy to identify tyrosine kinases implicated in the regulation of prostate growth, the dog fer cDNA was obtained and shown to be highly homologous to known fer cDNAs. Using a polyclonal Fer antibody directed against a C-terminal peptide, we studied its associations with cortactin, beta-catenin and p120Cas in human prostate carcinoma PC-3 cells. In contrast to previous reports, no interactions were observed. To assess its functional role, fer cDNA constructs were transfected in PC-3 cells. Antisense clones exhibiting a marked diminution of Fer expression had a reduced growth rate (doubling time of 29 vs. 42 h) and were unable to form colonies in soft agar. In agreement with these results, Fer protein expression was linked to human prostatic proliferative diseases, with enhanced levels in extracts from cancer tissues as compared to those from normal and hyperplastic ones, and was also expressed in the human prostate carcinoma cell lines DU145 and LNCaP. In the dog model, Fer expression was up-regulated in dividing versus resting prostate epithelial cells in vitro, and also in vivo when basal cell hyperplasia and metaplasia was induced by estrogen after castration. Minimal effects were observed when renewing the luminal epithelium with androgens. Taken together, these results show that Fer expression is associated with prostate cell proliferation and enhanced in prostate cancer.

Amino Acid Sequence↗

PubMed2000

Purification of the acyl-CoA elongase complex from developing rapeseed and characterization of the 3-ketoacyl-CoA synthase and the 3-hydroxyacyl-CoA dehydratase.

Oleoyl-CoA elongase catalyzes four successive reactions: condensation of malonyl-CoA to oleoyl-CoA, reduction, dehydration, and another reduction. Evidence supporting this mechanism and the multienzymatic nature of the elongation complex are reported. A particulate membrane fraction from rapeseed is able to elongate intermediates (R,S) 3-hydroxy-20:0-CoA and (E) 2,3-20:1-CoA to very long chain fatty acids in the presence of malonyl-CoA. Studies of the 3-ketoacyl-CoA synthase activities showed that maximal activity could be measured by using 15 to 30 microM 18:1-CoA and 30 microM malonyl-CoA, and that 18:0-CoA and 18:1-CoA were the best substrates. Comparison of the condensation and the overall elongation activities indicated that condensation is the rate-limiting step of the elongation process. The 3-hydroxyacyl-CoA dehydratase activity was maximal in the presence of 75 microM Triton X-100 and 25 microg of proteins. Finally, the acyl-CoA elongase complex was solubilized and purified. During the purification process, the 3-hydroxyacyl-CoA dehydratase copurified with the elongase complex, strongly suggesting that this enzyme belongs to the elongase complex. The apparent molecular mass of 700 kDa determined for the elongase complex, and the fact that four different polypeptide bands were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified fraction, further suggest that the acyl-CoA elongase is a multienzymatic complex.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed2000

Evidence for association of lipopolysaccharide with Pseudomonas fluorescens strain MF0 porin OprF.

Lipopolysaccharide (LPS) was found to be associated with the major outer membrane protein OprF of the psychrotrophic bacterium Pseudomonas fluorescens MF0, using two OprF purification procedures. OprF, purified under mild conditions, presented two types of association with LPS: tight (tLPS) and slight (sLPS), both of type R. LPS protected OprF from heat modification and trypsin degradation and facilitated the reincorporation of purified OprF into an artificial lipid bilayer without affecting its pore-forming activity. The size of the OprF channel depended on cell growth temperature, as did the extent of LPS phosphorylation: we suggest that LPS may be involved in modifications of OprF pore formation.

Electrophoresis, Polyacrylamide Gel↗

PubMed2000

Characterization of an OprF-deficient mutant suggests that OprF is an essential protein for Pseudomonas fluorescens strain MF0.

A stable OprF-deficient mutant for Pseudomonas fluorescens strain MF0 was constructed using reverse genetics. This mutant, called MF372, showed a rounded morphology and grew more slowly in minimal medium, but not in rich medium. Contrary to other Pseudomonas strains, the loss of OprF for strain MF0 was accompanied by an altered outer membrane composition. At least three outer membrane proteins were overexpressed, apparently as a consequence of adaptive mutations. The N-terminal sequence of two of them revealed strong similarities with porins of the OprD family from P. aeruginosa. The data presented here shows that OprF may be an essential protein for this P. fluorescens strain.

Molecular Weight↗

PubMed2000

CLF associates with CLC to form a functional heteromeric ligand for the CNTF receptor complex.

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of various cell types in the peripheral and central nervous systems. Its receptor complex consists of a non-signaling alpha chain, CNTFR, and two signaling beta chains, gp130 and the leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second, developmentally important ligand. We have identified this factor as a stable secreted complex of cardiotrophin-like cytokine (CLC) and the soluble receptor cytokine-like factor-1 (CLF). CLF expression was required for CLC secretion, and the complex acted only on cells expressing functional CNTF receptors. The CLF/CLC complex activated gp130, LIFR and signal transducer and activator of transcription 3 (STAT3) and supported motor neuron survival. Our results indicate that the CLF/CLC complex is a second ligand for CNTFR with potentially important implications in nervous system development.

Animals↗

PubMed2000

Proteomic analysis of differential protein expression in primary hepatocytes induced by EGF, tumour necrosis factor alpha or the peroxisome proliferator nafenopin.

Peroxisome proliferators are nongenotoxic rodent-liver carcinogens that have been shown to cause both an induction of hepatocyte proliferation and a suppression of apoptosis. Both epidermal growth factor (EGF) and the peroxisome proliferator nafenopin induce DNA replication in primary rat hepatocyte cultures, but apparently through different signalling pathways. However, both EGF and nafenopin require tumour necrosis factor alpha (TNFalpha) signalling to induce DNA replication. By examining proteins isolated from rat primary hepatocyte cultures using two-dimensional gel electrophoresis and mass spectrometry, we found that proteins showing an altered expression pattern in response to nafenopin differed from those showing altered expression in response to EGF. However, many proteins showing altered expression upon stimulation with TNFalpha were common to both the EGF and nafenopin responses. These proteome profiling experiments contribute to a better understanding of the molecular mechanisms involved in the response to peroxisome proliferators. We found 32 proteins with altered expression upon stimulation with nafenopin, including muscarinic acetylcholine receptor 3, intermediate filament vimentin and the beta subunit of the ATP synthase. These nonperoxisomal protein targets offer insights into the mechanisms of peroxisome proliferator-induced carcinogenesis in rodents and provide opportunities to identify toxicological markers to facilitate early identification of nongenotoxic carcinogens.

Actins↗

PubMed2000

Endoglin expression on human microvascular endothelial cells association with betaglycan and formation of higher order complexes with TGF-beta signalling receptors.

Transforming growth factor-beta (TGF-beta) plays an important role in angiogenesis and vascular function. Endoglin, a transmembrane TGF-beta binding protein, is highly expressed on vascular endothelial cells and is the target gene for the hereditary haemorrhagic telangiectasia type I (HHT1), a dominantly inherited vascular disorder. The specific function of endoglin responsible for HHT1 is believed to involve alterations in TGF-beta responses. The initial interactions on the cell surface between endoglin and TGF-beta receptors may be an important mechanism by which endoglin modulates TGF-beta signalling, and thereby responses. Here it is shown that on human microvascular endothelial cells, endoglin is co-expressed and is associated with betaglycan, a TGF-beta accessory receptor with which endoglin shares limited amino acid homology. This complex formation may occur in either a ligand-dependent or a ligand-independent manner. In addition, the occurrence of three higher order complexes containing endoglin, type II and/or type I TGF-beta receptors, on these cells is demonstrated. Our findings suggest that endoglin may modify TGF-beta signalling by interacting with both betaglycan and the TGF-beta signalling receptors at physiological receptor concentrations and ratios.

Affinity Labels↗

PubMed1999

Species differences in sequence and activity of the peroxisome proliferator response element (PPRE) within the acyl CoA oxidase gene promoter.

In rats and mice, peroxisome proliferators (PP) cause liver enlargement, hepatocarcinogenesis and peroxisome proliferation associated with induction of enzymes such as acyl CoA oxidase (ACO). However, humans appear to be non-responsive to the adverse effects of PPs such as ACO induction. PPs activate the peroxisome proliferator activated receptor alpha (PPARalpha) that binds to DNA at peroxisome proliferator response elements (PPREs) within the promoters of PP-responsive genes. When the human ACO promoter was cloned previously (Varanasi et al., 1996. Journal of Biological Chemistry, 271, 2147-2155), it was reported to contain a PPRE (5' AGGTCA C TGGTCA 3') that bound PPARalpha and could be activated in vitro by Wyeth-14,643 (at >1 mM) or DEHP (at > 1.5 mM). In contrast, when we cloned the ACO gene promoter from a human liver biopsy, it was non-responsive to PPs and differed at three positions (5' AGGTCA G CTGTCA 3') from that reported previously (Woodyatt et al., 1999. Carcinogenesis, 20, 369-375). Subsequent to this, Varanasi et al. re-sequenced their constructs and obtained the same sequence as we have described (Varanasi et al., 1998. Journal of Biological Chemistry, 273, 30832). However, the observation that the errant sequence (5' AGGTCA C TGGTCA 3') was able to bind PPARalpha still remained since it appears that this sequence was used by Varanasi et al. (1996) to design oligonucleotides for their DNA binding analyses. Thus, if the 5' AGGTCA C TGGTCA 3' sequence did exist in some individuals, it could be active. To address this, we used site-directed mutagenesis to create a promoter fragment that contained the errant sequence. This reporter gene was transfected into NIH3T3 cells together with a plasmid expressing mPPARalpha, and assessed for its ability to drive PP-mediated gene transcription using a non-toxic concentration of Wyeth-14,643 (100 microM). This human ACO promoter was also inactive, unlike the equivalent rat ACO promoter fragment used as a positive control. Next, we used site directed mutagenesis to convert the PPRE found in the active rat ACO promoter (3' AGGACA A AGGTCA 5') to our inactive human sequence (AGGTCA G CTGTCA). This human PPRE was unable to drive PP-induced gene transcription even in the context of the rat ACO promoter suggesting that the activity of the rat promoter is conferred principally by the PPRE sequence, even though it may be enhanced by flanking sequences. These data confirm that neither the native nor the errant human ACO gene PPRE can respond to PPs. The absence of a responsive PPRE contributes to our understanding of the lack of response of humans to some of the adverse effects of the PP class of non-genotoxic hepatocarcinogens.

3T3 Cells↗

PubMed1999

Hepatocyte differentiation of WIF-B cells includes a high capacity of interleukin-6-mediated induction of alpha 1-acid glycoprotein and alpha 2-macroglobulin.

Responsiveness to cytokine-mediated acute inflammatory stimuli of the highly differentiated and polarized WIF-B hybrid cell line was studied by measuring the induction of alpha 1-acid glycoprotein and alpha 2-macroglobulin mRNAs after interleukin-1, interleukin-6 and tumor necrosis factor-alpha treatments in the presence of dexamethasone. Compared with their Fao parent, WIF-B cells were 10 times more responsive to 24-h interleukin-6 induction regarding alpha 2-macroglobulin induction. At variance from the response measured in Fao cells, the late effects of interleukin-6 treatment confirmed the higher sensitivity of WIF-B cells to this cytokine as a 72-h treatment as 10 times more effective than a 24-h treatment at inducting alpha 1-acid glycoprotein mRNA. These findings highlight the hepatocyte differentiation of WIF-B cells compared with other hepatoma cell lines, with respect to the regulation of acute-phase protein gene expression. They also make WIF-B cells a convenient model to study the molecular effects of interleukin-6 in terms of transduction and/or transcription, and the many cross-talks that occur during the regulation of acute-phase protein gene expression.

Animals↗

PubMed1999

Suppression of apoptosis and induction of DNA synthesis in vitro by the phthalate plasticizers monoethylhexylphthalate (MEHP) and diisononylphthalate (DINP): a comparison of rat and human hepatocytes in vitro.

Diethylhexylphthalate (DEHP) and diisononylphthalate (DINP) are plasticizers with many important commercial, industrial and medical applications. However, both DEHP and DINP are rodent peroxisome proliferators (PPs), a class of compounds that cause rodent liver tumours associated with peroxisome proliferation, induction of hepatic DNA synthesis and the suppression of apoptosis. Despite these effects in the rodent, humans appear to be nonresponsive to the adverse effects of PPs. Previously, we have shown that the fibrate hypolipidaemic peroxisome proliferator, nafenopin, induced DNA synthesis and suppressed apoptosis in rat but not in human hepatocytes. In this work, we have examined species differences in the response of rat and human hepatocytes to DEHP and DINP in vitro. In rat hepatocytes in vitro, both DINP and MEHP (a principle metabolite of DEHP and the proximal peroxisome proliferator) caused a concentration-dependent induction of DNA synthesis and suppression of both spontaneous and transforming growth factor beta1 (TGFbeta1)-induced apoptosis. Similarly, both MEHP and DINP caused a concentration-dependent induction of peroxisomal beta-oxidation although the response to DINP was less robust. In contrast to the pleiotropic response noted in rat hepatocytes, neither DINP nor MEHP caused an induction of beta-oxidation, stimulation of DNA synthesis and suppression of apoptosis in human hepatocytes cultured from three separate donors. These data provide evidence for species differences in the hepatic response to the phthalates DEHP and DINP, confirming that human hepatocytes appear to be refractory to the hepatocarcinogenic effects of PPs first noted in rodents.

Animals↗

PubMed1999

Searching for an intrinsic neuroendocrine cell in the kidney. An immunohistochemical study of the fetal, infantile and adult kidney.

The pathogenesis of primary renal carcinoid tumor is unknown. One hypothesis has implied derivation from a yet unrecognized intrinsic neuroendocrine cell in the renal parenchyma/hilum either as a minute endocrineparacrine constituent or resulting from entrapped/misplaced progenitor cells of the so-called dispersed neuroendocrine system during organogenesis. Immunohistochemical staining for chromogranin and serotonin was systematically performed on a whole-mount and geographically mapped normal adult kidney, kidneys from 15 fetuses (age range: 15 to 38 weeks), and renal specimens from 18 infants/children (age range: 7 days to 123 months). Minute paraganglion nests (composed of chromogranin positive/serotonin negative chief cells and S-100 protein positive dendritic cells) were incidentally detected within the renal hilum primitive stroma (unilaterally) of two fetuses at 22 and 26 weeks. Sequestration and persistence of such paraganglion nests during renal growth and maturation would offer a basis for the rare occurrence of extra-adrenal paraganglioma involving the renal hilum/pedicle. Otherwise, no neuroendocrine cell was detected within the renal parenchyma or hilum, therefore not validating/sustaining the aforementioned hypothesis in the pathogenesis of renal carcinoid tumor.

Carcinoid Tumor↗

PubMed1999

Evidence that oleoyl-CoA and ATP-dependent elongations coexist in rapeseed (Brassica napus L.).

The elongation of different substrates was studied using several subcellular fractions from Brassica napus rapeseed. In the presence of malonyl-CoA, NADH and NADPH, very-long-chain fatty acid (VLCFA) synthesis was observed from either oleoyl-CoA (acyl-CoA elongation) or endogenous primers (ATP-dependent elongation). No activity was detected using oleic acid as precursor. Acyl-CoA and ATP-dependent elongation activities were mainly associated with the 15 000 g/25 min membrane fraction. Reverse-phase TLC analysis showed that the proportions of fatty acids synthesized by these activities were different. Acyl-CoA elongation increased up to 60 microM oleoyl-CoA, and ATP-dependent elongation was maximum at 1 mM ATP. Both activities increased with malonyl-CoA concentration (up to 200 microM). Under all conditions tested, acyl-CoA elongation was higher than ATP-dependent elongation, and, in the presence of both ATP and oleoyl-CoA, the elongation activity was always lower. ATP strongly inhibited acyl-CoA elongation, whereas ATP-dependent elongation was slightly stimulated by low oleoyl-CoA concentrations (up to 15 microM) and decreased in the presence of higher concentrations. CoA (up to 150 microM) had no effect on the ATP-dependent elongation, whereas it inhibited the acyl-CoA elongation. These marked differences strongly support the presence in maturing rapeseed of two different elongating activities differently modulated by ATP and oleoyl-CoA.

Acyl Coenzyme A↗

PubMed1999

G1-arrested FaO cells re-enter the cell cycle upon stimulation with the rodent non-genotoxic hepatocarcinogen nafenopin.

The peroxisome proliferators are rodent non-genotoxic hepatocarcinogens that suppress apoptosis and induce DNA replication, cell proliferation and liver tumours. In order to investigate the effect of peroxisome proliferators on cell cycle progression, we arrested the well-differentiated rat hepatoma cell line FaO in the G1 phase of the cell cycle. Under these conditions, CDK2 and CDK4 protein expression remained unchanged compared with proliferating cells, but expression of cyclin D1 and p27(KIP1) was down-regulated and cyclin E accumulated in the inactive form. G1-arrested cells were able to enter the cell cycle on addition of exogenous growth factors such as epidermal growth factor (EGF) or hepatocyte growth factor (HGF) and replicate their DNA within 12 to 24 h of re-stimulation. Upon release from G1 arrest, CDK2 protein expression was down-regulated and, surprisingly, p27(KIP1) expression was restored. Cyclin D1 and phosphorylated cyclin E accumulated at 12 h but were degraded by 24 h after addition of EGF. Importantly, the peroxisome proliferator nafenopin and tumour necrosis factor alpha were able to induce DNA replication. Thus, the profile of expression of cell cycle regulatory proteins upon stimulation with nafenopin is comparable with that induced by growth factors such as EGF.

Animals↗

PubMed1999

Dietary restriction alters retinol and retinol-binding protein metabolism in aging rats.

Recent studies reported that retinoid metabolism was influenced by long-term dietary restriction (DR) in rats. Because plasma retinol was decreased in rats subjected to DR, it was thought that this dietary manipulation may have an effect on retinol-binding protein (RBP) metabolism. Thus, the aim of this study was to assess retinoids, RBP, and transthyretin (TTR) levels in plasma and liver of young (3 months), adult (12 months), and old (22 months) female Sprague-Dawley rats fed ad libitum (AL) or subjected to a 40% DR, enriched (DR+), or not (DR), with vitamins and minerals. Results indicate that hepatic total retinoid concentrations and content increased with age in all the groups. DR+ rats showed higher hepatic retinoid concentrations than age-matched AL and DR rats. Adult and old DR and DR+ rats exhibited significantly lower plasma RBP-retinol and higher total retinoic acid levels than corresponding controls, although these parameters were not influenced by aging. Liver RBP levels were also decreased in DR and DR+ rats when compared to respective AL controls. There was a slight age-related decline in plasma TTR levels in DR and DR+ rats which was not associated with modifications in liver TTR levels. Hepatic gene expression of RBP and TTR, as evaluated by Northern blot hybridization, did not change with age or diet, suggesting that the lower levels of plasma RBP-retinol and liver RBP in vitamin A-sufficient rats subjected to DR may reflect post-transcriptional alterations and/or accelerated degradation of hepatic RBP. The elevated plasma levels of retinoic acid may represent an adaptive mechanism developed by DR rats to maintain retinoid-dependent functions.

Actins↗

PubMed1999

Study of the 3-hydroxy eicosanoyl-coenzyme A dehydratase and (E)-2,3 enoyl-coenzyme A reductase involved in acyl-coenzyme A elongation in etiolated leek seedlings

(R,S)-[1-14C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L. ) seedling microsomes. 3-Hydroxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of (E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had a marked preference for (R)-3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-14C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-14C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-14C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and NADPH or NADH, [1-14C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-14C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-14C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg-1 h-1, which is the highest ever reported for a plant system.

Journal Article↗

PubMed1999

Induction of DNA replication by peroxisome proliferators is independent of both tumour necrosis factor (alpha) priming and EGF-receptor tyrosine kinase activity.

Peroxisome proliferators (PPs) cause hepatocyte proliferation and tumorigenesis in rodent liver. PPs induce hepatocyte DNA synthesis although the mechanism is unclear. Tumour necrosis factor (alpha) (TNF(alpha)) and epidermal growth factor (EGF) have been implicated in mediating this growth response since these factors induce a threefold and 17.2-fold increase, respectively, in DNA synthesis in rat primary hepatocyte cultures. Previously, others have suggested that TNF(alpha) acts as a primer to sensitise hepatocytes to the proliferative effects of growth factors. Indeed, here we show that costimulation with TNF(alpha) and a suboptimal (4-20% of optimal) concentration of EGF permits an 11.7-fold increase in DNA synthesis in rat primary hepatocyte cultures. The PP nafenopin induced a 2. 3-fold increase in DNA synthesis but there was no further increase upon co-administration of either TNF(alpha) or a suboptimal concentration of EGF. Furthermore, there was no gross dysregulation of the CDK and cyclin protein expression profile upon stimulation with nafenopin. Using a specific epidermal growth factor receptor tyrosine kinase inhibitor (4-(3-chloro-4-fluorophenylamino)-7-methoxy-6- (3-¿1-pyrolidino)-propoxyquinazoline, EGFR-TKI), we show that signalling through EGF-R is not required for nafenopin-induced DNA synthesis. The EGFR-TKI also prevented progression into S phase upon stimulation with TNF(alpha), but DNA synthesis was not reduced to control levels, indicating that TNF(alpha) has a mitogenic activity in the absence of EGF signalling. Therefore, although TNF(alpha) can act as a priming factor for growth factors such as EGF, nafenopin does not appear to act via this mechanism.

Animals↗