Showing papers on "Liver cell published in 1997"

Comprehensive allelotyping of human hepatocellular carcinoma.

Abstract: Hepatocellular carcinoma (HCC) is one of the most common cancers in many parts of the world, however the molecular mechanisms underlying liver cell transformation remain obscure. A genome-wide scan of loss of heterozygosity (LOH) in tumors provides a powerful tool to search for genes involved in neoplastic processes. To identify recurrent genetic alterations in liver tumors, we examined DNAs isolated from 120 HCCs and their adjacent non tumorous parts for LOH using a collection of 195 microsatellite markers located roughly every 20 cM throughout 39 autosomal arms. The mean heterozygosity was 73%. Our findings provide additional support that LOH for loci on chromosomal arms 1p, 4q, 6q, 8p, 13q and 16p is significantly elevated in HCC. The highest percentage of LOH is found for a locus in 8p23 (42% of informative csaes). This corresponds to one of the most common genetic abnormalities reported to date in these tumors. In addition, high ratio of LOH (⩾35%) is observed on chromosome arms which had not been implicated in previous studies, notably on 1q, 2q and 9q. No correlation was found between LOH of specific chromosomal regions and etiologic factors such as chronic infections with hepatitis B or C viruses. This first report of an extensive allelotypic analysis of HCC should help in identifying new genes whose loss of function contributes to the development of liver cancer.

CREB controls LAP/C/EBP beta transcription.

Abstract: LAP/C/EBP beta is a member of the C/EBP family of transcription factors and is involved in hepatocyte-specific gene expression. Recently we showed that, besides its posttranscriptional regulation, LAP/C/EBP beta mRNA is modulated during liver regeneration. Therefore, in this study we investigated mechanisms which control LAP/C/EBP beta gene transcription. Deletion analysis of the 59-flanking region, located upstream of the start site of transcription in the LAP/C/EBP beta gene, demonstrated that a small region in close proximity to the TATA box is important in maintaining a high level of transcription of the luciferase reporter gene constructs. In gel shift experiments two sites were identified which are important for specific complex formation within this region. Further analysis by cross-linking, super shift, and competition experiments was performed with liver cell nuclear extracts, hepatoma cell nuclear extracts, or recombinant CREB protein. These experiments conclusively demonstrated that CREB binds to both sites in the LAP/C/EBP beta promoter with an affinity similar to that with the CREB consensus sequence. Transfection experiments with promoter constructs where the CREB sites were mutated showed that these sites are important to maintain both basal promoter activity and LAP/C/EBP beta inducibility through CREB. Northern blot analysis and runoff transcription assays demonstrated that the protein kinase A pathway not only stimulated the activity of the luciferase reporter construct but also the transcription of the endogenous LAP/C/EBP beta gene in different cell types. Western blot analysis of rat liver cell nuclear extracts and runoff transcription assays of rat liver cell nuclei after two-thirds hepatectomy showed a functional link between the induction of CREB phosphorylation and LAP/C/EBP beta mRNA transcription during liver regeneration. These results demonstrate that the two CREB sites are important to control LAP/C/EBP beta transcription in vivo. As several pathways control CREB phosphorylation, our results provide evidence for the transcriptional regulation of LAP/C/EBP beta via CREB under different physiological conditions.

CD95/CD95L-mediated apoptosis of the hepatic stellate cell. A mechanism terminating uncontrolled hepatic stellate cell proliferation during hepatic tissue repair.

Abstract: During liver tissue repair, hepatic stellate cells (HSC), a pericyte-like mesenchymal liver cell population, transform from a "quiescent" status ("resting" HSC) into myofibroblast-like cells ("activated" HSC) with the latter representing the principle matrix synthesizing cell of the liver. Presently, the mechanisms that terminate HSC cell proliferation when tissue repair is concluded are poorly understood. Controlled cell death known as apoptosis could be a mechanism underlying this phenomenon. Therefore, apoptosis and its regulation were studied in HSC using an in vitro and in vivo approach. Spontaneous apoptosis became detectable in parallel with HSC activation because resting cells (2 days after isolation) displayed no sign of apoptosis, whereas apoptosis was present in 8% (+/- 5%) of "transitional" cells (day 4) and in 18% (+/- 8%) of fully activated cells (day 7). Both CD95 (APO-1/Fas) and CD95L (APO-1-/Fas-ligand) became increasingly expressed during the course of activation. Apoptosis could be fully blocked by CD95-blocking antibodies in normal cells and HSC already entering the apoptotic cycle. Using CD95-activating antibodies, transition of more than 95% cells into apoptosis was evident at each activation step. The apoptosis-regulating proteins Bcl-2 and p53 could not be detected in resting cells but were found in increasing amounts at days 4 and 7 of cultivation. Whereas p53 expression was induced by the CD95-activating antibody, no change was inducible in Bcl-2 expression. The Bcl-2-related protein bax could be found at days 2 and 4 in similar expression, was considerably up-regulated at day 7, but was not regulated by CD95-agonistic antibodies. In vivo, acute tissue damage was first accompanied by activation and proliferation of HSC displaying no sign of apoptosis. In the recovery phase, apoptotic HSC were detectable in parallel to a reduction in the total number of HSC present in the liver tissue. The data demonstrate that apoptosis becomes detectable in parallel with HSC activation, which suggests that apoptosis might represent an important mechanism terminating proliferation of activated HSC.

Cellular adhesion molecules: regulation and functional significance in the pathogenesis of liver diseases

Abstract: Adhesion molecules are cell surface glycoproteins that are critical for the localization of leukocytes at sites of inflammation. This review discusses the current knowledges of adhesion molecule expression in normal liver and its upregulation on individual liver cell types during liver inflammation. Cytokines, chemokines, complement factors, and lipid-derived mediators are critical for increased gene transcription and activation of constitutively expressed adhesion molecules. The specific role of selectins, integrins, and members of the immunoglobulin gene superfamily in sinusoidal and venular leukocyte sequestration, transendothelial migration, and adherence to target cells in the liver is described. Increased understanding of these basic mechanisms of communication between resident liver cells and infiltrating leukocytes (neutrophils, lymphocytes, macrophages) not only advances our insight into the pathophysiology of hepatic inflammation but also identifies promising new targets for therapeutic interventions and expands the spectrum of diagnosis and treatment of liver diseases, including alcoholic hepatitis and cirrhosis, viral hepatitis, ischemia-reperfusion injury (transplantation, tumor resection, shock), sepsis- or endotoxin-induced liver injury, acute and chronic rejection, primary biliary cirrhosis, and primary sclerosing cholangitis.

Glucokinase Regulatory Protein May Interact With Glucokinase in the Hepatocyte Nucleus

Abstract: Glucokinase (GK) plays a central role in the sensing of glucose in pancreatic β-cells and parenchymal cells of the liver. Glucokinase regulatory protein is a physiological inhibitor of GK in the liver. To understand the role of the interaction of these two proteins in glucose sensing, we carried out a series of experiments to localize the protein in the liver cell. The regulatory protein was found to be present mainly in the nucleus of the cell under a variety of conditions that mimicked the glucose status of the fed and fasted state. GK was localized in the nucleus when the cells were exposed to low glucose concentrations. At higher glucose concentrations or in the presence of low concentrations of fructose, GK translocated to the cytoplasm. The effect of fructose was more robust and rapid than the effect of high glucose concentrations. Furthermore, the effect of fructose and high glucose on the translocation of GK from the nucleus could be partially reversed by glucagon. This unusual localization and behavior suggests a role for GK and its regulatory protein in hepatic energy metabolism that may be broader than glucose phosphorylation.

α -Tocopherol Transfer Protein Stimulates the Secretion of α -Tocopherol from a Cultured Liver Cell Line through a Brefeldin A-Insensitive Pathway

Abstract: Vitamin E (α-tocopherol) is a fat-soluble antioxidant that is transported by plasma lipoproteins in the body. α-Tocopherol taken up by the liver with lipoprotein is thought to be resecreted into the plasma in very low density lipoprotein (VLDL). α-Tocopherol transfer protein (αTTP), which was recently identified as a product of the causative gene for familial isolated vitamin E deficiency, is a cytosolic liver protein and plays an important role in the efficient recycling of plasma vitamin E. To throw light on the mechanism of αTTP-mediated α-tocopherol transfer in the liver cell, we devised an assay system using the hepatoma cell line McARH7777. Using this system, we found that the secretion of α-tocopherol was more efficient in cells expressing αTTP than in matched cells lacking αTTP. Brefeldin A, which effectively inhibits VLDL secretion by disrupting the Golgi apparatus, had no effect on α-tocopherol secretion, indicating that αTTP-mediated α-tocopherol secretion is not coupled to VLDL secretion. Among other agents tested, only 25-hydroxycholesterol, a modulator of cholesterol metabolism, inhibited α-tocopherol secretion. This inhibition is most likely mediated by oxysterol-binding protein. These results suggest that αTTP present in the liver cytosol functions to stimulate secretion of cellular α-tocopherol into the extracellular medium and that the reaction utilizes a novel non-Golgi-mediated pathway that may be linked to cellular cholesterol metabolism and/or transport.

Enhancement by indole-3-carbinol of liver and thyroid gland neoplastic development in a rat medium-term multiorgan carcinogenesis model

Abstract: The modifying effects of indole-3-carbinol (I3C), a naturally occurring compound in cruciferous vegetables, were assessed using a rat multiorgan carcinogenesis model. One hundred male Sprague-Dawley (SD) rats were divided randomly into three groups. Groups 1 and 2 were sequentially treated with diethylnitrosamine (DEN), N-methyl-N-nitrosourea (MNU), and dihydroxy-di-N-propylnitrosamine (DHPN) for four weeks (DMD treatment) and Group 3 was given treatment without mutagen. Animals of groups 1 and 3 were given a diet containing 0.25% I3C for 20 weeks after the initiation period, followed by a return to the basal diet for 28 weeks, and subgroups were killed at weeks 24 and 52. I3C caused significant increases in both density and area (mm2/ cm2) of liver cell foci positive for glutathione S-transferase placental form, assessed at week 24 of the experiment (P < 0.01, 0.001). The incidence of hepatocellular adenomas in the DMD → I3C group at week 52 similarly tended to be elevated as compared to the initiation only (DMD-alone) group. The incidence of thyroid gland tumors in the DMD → I3C group was significantly increased compared with the DMD-alone group values at week 52 of the experiment (P < 0.01). In conclusion, I3C was shown to enhance liver and thyroid gland neoplastic development when given during the promotion stage in a rat medium-term multiorgan carcinogenesis model.

Class I-deficient hemopoietic cells and nonhemopoietic cells dominantly induce unresponsiveness of natural killer cells to class I-deficient bone marrow cell grafts.

Abstract: NK cells in normal mice reject bone marrow transplants from class I-deficient mice. In contrast, class I-deficient mice do not reject autologous cells, suggesting that NK cell tolerance is acquired. We employed fetal liver irradiation chimeras to assess two potential mechanisms that could account for the tolerance of NK cells in class I-deficient mice to class I-deficient cells: 1) a positive model, in which recognition of class I+ cells molecules by NK cells is necessary to induce functional NK cell maturation; and 2) a negative model, in which interactions of NK cells with class I-deficient cells induce tolerance. In class I+ chimeras reconstituted with mixtures of class I+ and class I-deficient fetal liver cells, the rejection of class I-deficient bone marrow cell grafts was significantly impaired, supporting the negative model. We further addressed whether nonhemopoietic cells are also able to induce NK cell tolerance. Class I- mice reconstituted with class I+ fetal liver cells were tolerant of class I-deficient cells, favoring this idea. Furthermore, class I-deficient mice reconstituted with a mixture of class I-deficient and class I+ fetal liver cells were more tolerant to class I-deficient cells than were class I+ mice reconstituted with the same fetal liver cell mixture. These results suggest that maximal tolerance induction requires the presence of class I-deficient nonhemopoietic cells.

Development of hepatic sinusoidal structure with special reference to the Ito cells.

Abstract: To elucidate sinusoidal cell structure and function under normal conditions and their behavior in diseased settings, an understanding of their developmental aspects is needed. At day 10 of gestation in mice and rats or at 5 weeks of gestation in humans, the hepatic cords grow into the mesenchymal tissue of the septum transversum, and the primitive sinusoidlike structure is simultaneously observed between the liver cell cords. In the margin of the growing liver primordium, mesenchymal cells in the septum transversum are trapped in the subendothelial space. These subendothelial cells are at the early stages of organogenesis and become progenitors of the Ito cells. By days 12-14 of gestation in mice and rats or 8 weeks of gestation in humans, the basic structure of the sinusoids has developed. Embryonic hepatic sinusoids are usually lined by a continuous endothelium without basement membranes, and an incompletely fenestrated sinusoid appears at the middle gestational stage. In the late gestational stages, the Ito cells exhibit myofibroblastlike features in humans, mice, and rats. In association with this event, perisinusoidal reticular networks are gradually intensified. After birth until days 4-5 in mice and rats, the sinusoidal and perisinusoidal structures are almost completely formed, although slight morphological differences from those in adult livers still exist. What happens to sinusoidal endothelial cells and Ito cells in hepatic fibrosis-cirrhosis of the adult may be a deviated or uncontrolled occurrence of what goes on during the fetal period, i.e., a continuous nonfenestrated sinusoidal lining in the early embryonic stage and a myofibroblastlike transformation of Ito cells in late fetal life.

Fas system and apoptosis in viral hepatitis

Abstract: Hepatitis C virus (HCV) and hepatitis B virus (HBV) are the major causative agents of chronic liver disease. However, the mechanisms responsible for liver cell injury remain to be clarified. Playing crucial roles in the clearance of viral infection are cytotoxic T lymphocytes. Recently, it has been demonstrated that perforin- and Fas-based mechanisms account for all T cell-mediated cytotoxicity. Therefore, we examined the correlation between liver cell damage and the Fas system in the liver of patients with chronic hepatitis C. Fas is a cell surface protein that mediates apoptosis with treatment of the Fas ligand or the anti-Fas antibody. To investigate the role of Fas in type C hepatitis, we examined the correlation between liver cell damage and Fas expression. Fas expression was found mainly in the cytoplasm of hepatocytes and these positive cells were found particularly among infiltrating lymphocytes. A high prevalence of Fas expression was shown in liver tissue with more severe inflammation. The Fas system-mediated death signal requires the interaction of Fas ligand with Fas on target cells. We isolated a 1.9 kb cDNA clone for the human Fas ligand and examined the expression of the Fas ligand in liver-infiltrating mononuclear cells obtained from patients with chronic hepatitis C. The open reading frame encodes 281 amino acids. Next, we examined the expression of the Fas ligand in liver-infiltrating mononuclear cells obtained from patients with chronic hepatitis C. The amplified products (231 bp) derived from Fas ligand transcripts were detected in liver-infiltrating mononuclear cells, whereas no signal was observed in liver tissues. In HCV infection, Fas expression in hepatocytes is up-regulated in accordance with the severity of liver inflammation. When HCV-specific T cells migrate into hepatocytes and recognize the viral antigen via the T cell receptor, they become activated and express Fas ligand that can transduce the apoptotic death signal to Fas-bearing hepatocytes. Thus, the Fas system plays an important role in liver cell injury by HCV infection.

Human hepatic preneoplasia: phenotypes and proliferation kinetics of foci and nodules of altered hepatocytes and their relationship to liver cell dysplasia

Abstract: Foci of altered hepatocytes (FAH) represent preneoplastic lesions, as shown in various animal models of hepatocarcinogenesis, but their significance in the human liver has not been established. The cellular composition, size distribution and proliferation kinetics of FAH in 163 explanted and resected human livers with or without hepatocellular carcinoma (HCC) and their possible association with small-cell change of hepatocytes (SCC) were therefore studied. FAH, including glycogen-storing foci, were found in 84 of 111 cirrhotic livers, demonstrating higher incidences in cases with (29/32) than in those without HCC (55/79). FAH were observed more frequently in HCC-free cirrhosis associated with hepatitis B or C virus or chronic alcoholic abuse (high-risk group) (37/47) than in that due to other causes (low-risk group) (12/21). MCF, predominant in cirrhotic livers of the high-risk group, were more proliferative, larger and more often involved in formation of nodules of altered hepatocytes (39.3%) than were GSF (8.5%). The results suggest that the FAH are preneoplastic lesions, MCF being more advanced than GSF. Oncocytic and amphophilic cell foci were also observed, but their significance remains to be clarified. Two types of SCC, namely diffuse and intrafocal SCC, were identified, but only intrafocal SCC was found to be related to increased proliferative activity and more frequent nodular transformation of the FAH involved, suggesting a close association with progression from FAH to HCC.

Nuclear factor-kappaB/Rel blocks transforming growth factor beta1-induced apoptosis of murine hepatocyte cell lines.

Abstract: Treatment of hepatocytes with transforming growth factor beta1 (TGF-beta1) induces growth arrest, which is followed by extensive cell death by apoptosis. Previously, we found that TGF-beta1 down-modulates nuclear factor (NF)-kappaB/Rel activity in murine B cell lymphomas, inducing apoptosis. Furthermore, p65 (RelA)-deficient mice died during gestation due to apoptosis of liver cells. Here we have explored the effects of TGF-beta1 on hepatocytes, using two untransformed murine hepatocyte cell lines, AML-12 and NMH, which constitutively express classical NF-kappaB. TGF-beta1 treatment caused increased NF-kappaB binding that was followed by a dramatic decrease in NF-kappaB levels that preceded apoptosis. Ectopic c-Rel expression ablated apoptosis induced by TGF-beta1. The down-regulation in NF-kappaB activity correlated with elevated IkappaB-alpha expression due to hypophosphorylation and increased IkappaB-alpha protein stability. Thus, NF-kappaB factor expression acts directly to promote liver cell survival. Furthermore, these findings characterize a novel signaling pathway for TGF-beta1 in epithelial cells involving down-regulation of NF-kappaB/Rel factors activity through posttranslational modification of IkappaB-alpha protein.

Two mechanisms by which ATP depletion potentiates induction of the mitochondrial permeability transition

Abstract: The present and a previous study [J. W. Snyder, J. G. Pastorino, A. M. Attie, and J. L. Farber, Am. J. Physiol. 264 (Cell Physiol. 33): C709-C714, 1993] define two mechanisms whereby ATP depletion promotes liver cell death. ATP depletion and cell death are linked by the mitochondrial permeability transition (MPT). Mitochondrial deenergization promotes the MPT, and ATP maintains a membrane potential by reversal of ATP synthase. With an increased influx of Ca2+ induced by the ionophore A-23187, oligomycin depleted the cells of ATP without loss of the mitochondrial membrane potential and further elevated the intracellular Ca2+ concentration. Cyclosporin A (CyA) prevented the accompanying cell killing. Fructose also preserved the viability of the cells. With the increased cytosolic Ca2+ imposed by A-23187, viability is maintained by ATP-dependent processes. Upon depletion of ATP, Ca2+ homeostasis cannot be maintained, and the MPT is induced. Rotenone also depleted the cells of ATP, and A-23187 accelerated the loss of the mitochondrial membrane potential occurring with rotenone alone. CyA and fructose prevented the cell killing with rotenone and A-23187. Oligomycin did not prevent this action of fructose. We conclude that ATP is needed to maintain Ca2+ homeostasis to prevent the MPT and the resultant liver cell death. ATP is also needed to maintain mitochondrial energization when electron transport is inhibited.

HRS/SRp40-Mediated Inclusion of the Fibronectin EIIIB Exon, a Possible Cause of Increased EIIIB Expression in Proliferating Liver

Abstract: Serine-arginine (SR)-rich proteins are believed to be important in mediating alternative pre-mRNA splicing. HRS/SRp40 expression is elevated in liver cell proliferation during development, regeneration, and oncogenesis. We tested whether HRS expression correlates with the appearance of alternatively spliced fibronectin transcripts during liver growth. HRS was highly expressed during the proliferative phase of liver development, correlating with expression of the fibronectin EIIIB alternative exon. In regenerating liver, HRS protein was induced in a time course consistent with the observed increase in fibronectin transcripts containing the EIIIB exon, particularly in nonparenchymal liver cells. Furthermore, in an in vivo assay, HRS, and not other SR proteins, directly mediated EIIIB exon inclusion in the fibronectin transcript. This alternative splicing was dependent on a purine-rich region within the EIIIB exon to which HRS specifically bound. We have established that HRS has the potential to contribute to the regulation of fibronectin pre-mRNA splicing during liver growth. Changes in fibronectin forms may be important in modifying liver architecture during the proliferative response, thus providing a potential mechanism by which SR proteins may participate in cellular growth control.

Long-term liver cell cultures in bioreactors and possible application for liver support.

Abstract: Hybrid artificial liver systems are being developed as a temporary extracorporeal liver support therapy. A short overview is given which emphasizes the development of hepatocyte culture models for bioreactors, subsequent in vitro studies, animal studies and the clinical application of hybrid liver support systems. An own bioreactor construction has been designed for the utilization of hepatocytes and sinusoidal endothelial cells. The reactor is based on capillaries for hepatocyte aggregate immobilization, coated with biomatrix. Four separate capillary membrane systems, each permitting a different function, are woven in order to create a three-dimensional network. Cells are perfused via independent capillary membrane compartments. Decentralized oxygen supply and carbon dioxide removal with low gradients is possible. There is a decentralized co-culture compartment for nonparenchymal liver cells. The use of identical parallel units to supply a few hepatocytes facilitates scale-up.

Excessive liver oxidant stress causes mortality in response to burn injury combined with endotoxin and is prevented with antioxidants.

Abstract: We studied the effect of the oral administration of a water-soluble antioxidant solution containing ascorbic acid, glutathione, and a precursor for glutathione synthesis, N-Acetyl-L-cysteine, on liver antioxidant activity, liver cell energetics, and mortality in rats in response to a 20% third-degree burn injury challenged 5 days later with an intraperitoneal injection of 30 mg/kg endotoxin. Rats with burns were fluid-resuscitated with subcutaneous Ringer's lactate solution according to the Parkland formula (4 cc/kg/%burn). Rats challenged with endotoxin 5 days after burn were given an additional 100 ml/kg of subcutaneous Ringer's lactate solution immediately after the injection of endotoxin. A group of rats with burns challenged with endotoxin 5 days after burn were given an oral antioxidant solution beginning after burn injury. Liver cell energetics were measured as tissue energy charge potential (ECP), adenosine triphosphate (ATP) content, and total adenine nucleotides. The levels of endogenous liver glutathione, catalase, vitamin C, and vitamin E were measured to monitor antioxidant status. We found that burn injury alone did not produce any mortality over the 6-day period despite a 35% decrease in liver energy charge potential resulting from a decrease in ATP, a 34% decrease in liver catalase activity, and a 20% decrease in liver vitamin C. It was interesting that glutathione increased and vitamin E remained unchanged. We found that endotoxin injury combined with burn injury produced a 61% mortality rate with a 63% decrease in liver energy charge potential, again resulting from a decrease in ATP, a 74% decrease in liver catalase activity, a 16% decrease in vitamin C, and a 29% decrease in vitamin E. Glutathione was significantly decreased compared with burn alone. We compared the liver antioxidant status of survivors with that of nonsurvivors who were killed when appearing moribund and found that glutathione was decreased by 51% and vitamin C by 73% in nonsurvivors over that in survivors, whereas catalase and vitamin E levels were comparable between the two groups. The oral administration of the antioxidants prevented mortality and the decrease in antioxidant activity and attenuated the decrease in energy charge potential. We conclude that a 20% burn produces a modest decrease in liver energy charge potential and antioxidant defenses without producing mortality. The addition of endotoxin further decreases liver antioxidant defenses, liver energy charge potential, and markedly increases mortality. Antioxidants, given post-burn, restored antioxidant defenses, attenuated the altered cell energetics, and prevented mortality, indicating oxidants to be the cause of mortality. This data also suggests that a critical value of decreases in antioxidant defenses and ATP exists, resulting in mortality.

Extent and Character of Phenobarbital-Mediated Changes in Gene Expression in the Liver

Abstract: Phenobarbital elicits pleiotropic effects in the liver, including induction of enzymes involved in xenobiotic metabolism. The spectrum of this response was analyzed by differential display of a large population (approximately 7500) of mRNAs in chicken embryo liver treated in vivo with phenobarbital. We identified 29 cDNA fragments that reproducibly and significantly changed in intensity after a 48-hr in ovo treatment. Eighteen of these (62%) were increased, whereas 11 (38%) were decreased. Twenty strongly regulated cDNA fragments were subcloned and further analyzed. Nucleotide sequence analysis revealed three types of genes: (a) those previously described to be regulated by phenobarbital, including CYP2H1, glutathione S-transferase, and uridine diphosphate-glucuronosyltransferase; (b) genes reported herein for the first time to be regulated by phenobarbital, including fibrinogen beta-chain and gamma-chain, retinal glutamine synthetase, apolipoprotein B, two gene products with homologies to elongation factor 1delta and complement factor H, respectively, and (c) several novel genes with hitherto unknown functions. If these data are extrapolated to the entire population of mRNAs of a liver cell, phenobarbital seems to significantly modulate the expression of more than 50 different genes. Our results also demonstrate that a large fraction of genes is negatively regulated by drug treatment.