Interindividual Differences In Expression Research Articles

Global, cell non-autonomous gene regulation drives individual lifespan among isogenic C. elegans.

Across species, lifespan is highly variable among individuals within a population. Even genetically identical Caenorhabditis elegans reared in homogeneous environments are as variable in lifespan as outbred human populations. We hypothesized that persistent inter-individual differences in expression of key regulatory genes drives this lifespan variability. As a test, we examined the relationship between future lifespan and the expression of 22 microRNA promoter::GFP constructs. Surprisingly, expression of nearly half of these reporters, well before death, could effectively predict lifespan. This indicates that prospectively long- vs. short-lived individuals have highly divergent patterns of transgene expression and transcriptional regulation. The gene-regulatory processes reported on by two of the most lifespan-predictive transgenes do not require DAF-16, the FOXO transcription factor that is a principal effector of insulin/insulin-like growth factor (IGF-1) signaling. Last, we demonstrate a hierarchy of redundancy in lifespan-predictive ability among three transgenes expressed in distinct tissues, suggesting that they collectively report on an organism-wide, cell non-autonomous process that acts to set each individual's lifespan.

Interindividual Differences in the Expression of ATP-Binding Cassette and Solute Carrier Family Transporters in Human Skin: DNA Methylation Regulates Transcriptional Activity of the Human ABCC3 Gene.

The identification of drug transporters expressed in human skin and interindividual differences in gene expression is important for understanding the role of drug transporters in human skin. In the present study, we evaluated the expression of ATP-binding cassette (ABC) and solute carrier (SLC) transporters using human skin tissues. In skin samples, ABCC3 was expressed at the highest levels, followed by SLCO3A1, SLC22A3, SLC16A7, ABCA2, ABCC1, and SLCO2B1. Among the quantitated transporters, ABCC3 accounted for 20.0% of the total mean transporter mRNA content. The expression of ABCC3 mRNA showed large interindividual variability (9.5-fold). None of the single nucleotide polymorphisms tested (-1767G>A, -1328G>A, -1213C>G, -897delC, -260T>A, and -211C>T) in the promoter region of the ABCC3 gene showed a significant change in ABCC3 mRNA levels. ABCC3 expression levels negatively correlated with the methylation status of the CpG island (CGI) located approximately 10 kilobase pairs upstream of ABCC3 (Rs: -0.323, P < 0.05). The reporter gene assay revealed a significant increase in transcriptional activity in the presence of CGI. ABCC3 mRNA was upregulated in HaCaT cells by the demethylating agent 5-aza-2'-deoxycytidine. Furthermore, the deletion of the region surrounding CGI using the clustered regularly interspaced short palindromic repeat/Cas9 system resulted in significantly lower ABCC3 mRNA levels than those in control clones in HaCaT cells. Herein, we demonstrated large interindividual differences in the expression of drug transporters in human skin. CGI may function as an enhancer of the transcription of ABCC3, and methylation levels in CGI contribute to the variability of ABCC3 expression in human skin.

Basic Mechanisms of Action of the Antiepileptic Drugs

Abstract Available antiepileptic drugs interact with a variety of different molecular targets. The mechanism of action of most anticonvulsants is most often complex with a number of affected regions. The combination of mechanisms of action of drugs in particular proportions can possibly determine the showcase of its antiepileptic activity. The common factor between the different supposed mechanisms for a number of drugs includes the possibility for modulating the excitatory and inhibitory neurotransmission through effects upon the voltage-gated ion channels, synaptic plasticity, heterogeneous receptors, and metabolism of neurotransmitters. There are controversial data on the extent to which a specific action can be the reason for the wholesome anticonvulsive characteristics of various medications, as well as the relation with the presence of undesired drug effects. The complexity of the action of some antiepileptic drugs creates conditions for optimal choice during therapy. In many cases, the insufficient familiarity with individual genetic differences and the disease related receptor damages can hinder defining a particular drug action. Characterizing the mechanisms of action of the present antiepileptic medications would increase the understanding for the pathophysiological mechanisms of epileptic seizures, as well as the development of new therapeutic strategies. The development of novel antiepileptic drugs and the ongoing research regarding the mechanism of action of established antiepileptic drugs, are continuously increasing the level of complexity in the spectrum of molecular targets relevant for epilepsy therapy. The current state of knowledge as well as the limitations in our understanding should guide future research aiming for a more detailed elucidation of the impact of genetics and pathophysiological mechanisms on interindividual differences in expression and function of antiepileptic drug targets.

Genetic architecture of gene expression underlying variation in host response to porcine reproductive and respiratory syndrome virus infection

It has been shown that inter-individual variation in host response to porcine reproductive and respiratory syndrome (PRRS) has a heritable component, yet little is known about the underlying genetic architecture of gene expression in response to PRRS virus (PRRSV) infection. Here, we integrated genome-wide genotype, gene expression, viremia level, and weight gain data to identify genetic polymorphisms that are associated with variation in inter-individual gene expression and response to PRRSV infection in pigs. RNA-seq analysis of peripheral blood samples collected just prior to experimental challenge (day 0) and at 4, 7, 11 and 14 days post infection from 44 pigs revealed 6,430 differentially expressed genes at one or more time points post infection compared to the day 0 baseline. We mapped genetic polymorphisms that were associated with inter-individual differences in expression at each day and found evidence of cis-acting expression quantitative trait loci (cis-eQTL) for 869 expressed genes (qval < 0.05). Associations between cis-eQTL markers and host response phenotypes using 383 pigs suggest that host genotype-dependent differences in expression of GBP5, GBP6, CCHCR1 and CMPK2 affect viremia levels or weight gain in response to PRRSV infection.

Pharmacogenomic or -epigenomic biomarkers in drug treatment: Two sides of the same medal?

Interindividual differences in expression of ADME genes are controlled by both genetic and epigenetic factors. Much emphasis has been made to describe the genetic influence, whereas the epigenetic part is not fully understood. Currently, we utilize mainly genetic biomarkers for optimization of drug therapy, although many rare genetic variants are not taken into consideration. Now, also epigenomic biomarkers are at hand and together genetic and epigenetic biomarkers can indeed improve the predictability of drug treatment.

Diversified expression of aryl hydrocarbon receptor dependent genes in human laryngeal squamous cell carcinoma cell lines treated with β-naphthoflavone

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study the effect of administration of β-naphthoflavone (BNF), potent AhR ligand, on the expression of AhR, AhRR, CYP1A1, CYP1A2, CYP1B1, NQO1, GSTA1, ALDH3A1 and UGT1A genes encoding the enzymes controlled by AhR were examined in thirteen laryngeal tumor cell lines and in HepaRG cell line. The analyzed cell lines were derived from patients with squamous laryngeal cancer, with history of cigarette smoking and without signs of human papillomavirus types 16 and 18 infection in investigated cells. Quantitative real-time RT-PCR analysis revealed huge interindividual differences in expression of genes from AhR regulatory network. Our results strongly suggest predominant effect of DNA methylation on induction of CYP1A1 expression by AhR ligands as well. Our results indicate that differentiated HepaRG cell line appeared to be very good substitute for human liver in studies on xenobiotic metabolism by AhR regulated enzymes.

Regulation of UDP-Glucuronosyltransferase 1A1 Expression and Activity by MicroRNA 491-3p

The UDP-glucuronosyltransferase (UGT) 1A enzymes are involved in the phase II metabolism of many important endogenous and exogenous compounds. The nine UGT1A isoforms exhibit high interindividual differences in expression, but their epigenetic regulation is not well understood. The purpose of the present study was to examine microRNA (miRNA) regulation of hepatic UGT1A enzymes and determine whether or not that regulation impacts enzymatic activity. In silico analysis identified miRNA 491-3p (miR-491-3p) as a potential regulator of the UGT1A gene family via binding to the shared UGT1A 3'-untranslated region common to all UGT1A enzymes. Transfection of miR-491-3p mimic into HuH-7 cells significantly repressed UGT1A1 (P < 0.001), UGT1A3 (P < 0.05), and UGT1A6 (P < 0.05) mRNA levels. For UGT1A1, this repression correlated with significantly reduced metabolism of raloxifene into raloxifene-6-glucuronide (ral-6-gluc; P < 0.01) and raloxifene-4'-glucuronide (ral-4'-gluc; P < 0.01). In HuH-7 cells with repressed miR-491-3p expression, there was a significant increase (~80%; P < 0.01) in UGT1A1 mRNA and a corresponding increase in glucuronidation of raloxifene into ral-6-gluc (50%; P < 0.05) and ral-4'-gluc (22%; P < 0.01). Knockdown of endogenous miR-491-3p in HepG2 cells did not significantly alter UGT1A1 mRNA levels but did increase the formation of ral-6-gluc (50%; P < 0.05) and ral-4'-gluc (34%; P < 0.001). A significant inverse correlation between miR-491-3p expression and both UGT1A3 (P < 0.05) and UGT1A6 (P < 0.01) mRNA levels was observed in a panel of normal human liver specimens, with a significant (P < 0.05) increase in UGT1A3 and UGT1A6 mRNA levels observed in miR-491-3p nonexpressing versus expressing liver specimens. These results suggest that miR-491-3p is an important factor in regulating the expression of UGT1A enzymes in vivo.

Abstract 3089: miR-491-3p regulation of the UDP-glucuronosyltransferase (UGT) 1A gene family.

Abstract Background: The UDP-Glucuronosyltransferases (UGTs) are phase II metabolic enzymes that prime endogenous and exogenous compounds for excretion from the body. The UGTs are often found to have high inter-individual differences in expression, and this impacts patients’ response to drug therapy. Studies examining the epigenetic regulation of the UGT enzymes have not previously been performed. Recently, it has been shown that the phase I metabolic cytochrome P-450s (CYPs) are negatively regulated by microRNA (miRNA). miRNA are short, endogenous strands of RNA roughly 22-24 nucleotides in length that regulate gene expression. MiRNA specifically bind to the 3’ untranslated region (UTR) of mature messenger RNA (mRNA) and post-transcriptionally repress protein expression. Purpose: The goal of this study is to identify and evaluate miRNA regulation of UGT1A expression and whether this regulation contributes to differences in UGT1A enzymatic activity. Results: miR-491-3p was identified using the miRanda prediction program. Luciferase assays showed a significant reduction in luciferase activity with an attached UGT1A 3’UTR in the presence of miR-491-3p mimic (P&amp;lt;0.03). This reduction was lost when the miR-491-3p binding site in the UGT1A 3’ UTR was mutated. Mature miR-491-3p expression levels were identified and quantified in numerous cancer cell lines and normal human tissues. There was a significant increase in expression of miR-491-3p in four human liver cancer cell lines (HepG2, Hep3B, HuH-7, and SK-HEP-1) compared to normal liver (P&amp;lt;0.001) and a significant decrease in expression between normal human colon compared to Caco-2 colorectal cancer cells (P=0.0041), and between normal lung compared to A-549 lung cancer cells (P=0.0002). In HuH-7 liver cancer cells, we determined that transient transfection of miR-491-3p significantly reduced mRNA levels of UGT1A1 (P&amp;lt;0.0001), UGT1A3 (P&amp;lt;0.05), and UGT1A6 (P&amp;lt;0.001). Inversely, inhibitor knockdown of endogenous miR-491-3p levels in HuH-7 cells showed a significant increase in the expression of UGT1A1 (P&amp;lt;0.003) and UGT1A10 (P&amp;lt;0.05) mRNA. No alteration in expression was observed for UGT2B7. miR-491-3p over-expression affected the enzymatic activity of the UGT1As against the chemotherapeutic compound raloxifene (P&amp;lt;0.01), a drug metabolized by several members of the UGT1A family. Conclusion: miR-491-3p regulates the expression of several members of the UGT1A gene family. This regulation affects UGT1A enzymatic activity against the drug raloxifene in vitro. This is the first evidence implicating the epigenetic regulation of the UGT enzymes by miRNA. Citation Format: Douglas F. Dluzen, Anna Salzberg, Dongxiao Sun, Nathan Jones, Ryan Bushey`, Philip Lazarus. miR-491-3p regulation of the UDP-glucuronosyltransferase (UGT) 1A gene family. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3089. doi:10.1158/1538-7445.AM2013-3089

Pharmacological treatment strategies: Mechanisms of antiepileptic drugs

Both, the development of novel antiepileptic drugs as well as ongoing research regarding the mechanism of action of established antiepileptic drugs, are continuously increasing the level of complexity in the spectrum of molecular targets relevant for epilepsy therapy. Although we are far from a reliable prediction of the optimal choice for polytherapy in individual patients, evidence exists that the mechanisms of action of the combination partners can matter regarding tolerability and efficacy. Moreover, the current state of knowledge as well as the limitations in our understanding should guide future research aiming for a more detailed elucidation of the exact mechanisms of target modulation as well as a more comprehensive investigation of the impact of genetics and pathophysiological mechanisms on interindividual differences in expression and function of antiepileptic drug targets.Therefore, this review gives a short overview of the mechanism of action of currently marketed antiepileptic drugs.

Effect of chronic variable stress on corticosterone levels and hippocampal extracellular 5-HT in rats with persistent differences in positive affectivity.

Objective:The trait of experiencing positive affect could make a unique contribution to the pathogenesis of affective disorders. Animal models of positive emotionality are scarce but 50-kHz ultrasonic vocalizations (USVs) in rats have been associated with rewarding experience. We have previously reported that persistent inter-individual differences in expression of 50-kHz USVs (chirps) exist, and that male rats producing fewer 50-kHz USVs are more sensitive to chronic variable stress (CVS). In this study we examined the effect of CVS on extracellular serotonin (5-HT) levels in hippocampus, comparing high-chirping (HC) and low-chirping (LC) rats.Methods:Male rats were classified as HC- and LC-rats on the basis of stable levels of USV response using sessions of tickling-like stimulation. CVS procedure lasted 4 weeks. The administration of citalopram (1 μM) and measurements of levels of 5-HT were done by microdialysis. Corticosterone levels were also measured from trunk blood.Results:Male LC-rats were more sensitive to CVS: the effect of stress on body weight gain was larger and corticosterone levels from full blood were higher in the stressed LC animals as compared to both the unstressed groups and the stressed HC animals. While no baseline differences in extracellular 5-HT levels in hippocampus were found between groups, the increase in extracellular 5-HT levels induced by citalopram was much higher in LC-rats.Conclusion:Chronic stress appears to modify hippocampal 5-HT overflow in rats with low positive affectivity. This finding supports the notion of greater vulnerability to CVS in male rats with low positive affectivity.

Systematic Functional Characterization of Cytochrome P450 2E1 Promoter Variants in the Chinese Han Population

CYP2E1 promoter polymorphisms can lead to significant interindividual differences in expression of CYP2E1. Using a database of CYP2E1 gene polymorphisms established in 2010, our study aimed to functionally characterize the single nucleotide polymorphisms (SNPs) of the promoter region and corresponding haplotypes in the Chinese Han population. Six novel SNPs and seven haplotypes with a frequency equal to or greater than 0.01 were constructed on a luciferase reporter system on the basis of site-directed mutagenesis. Dual luciferase reporter systems were used to analyze regulatory activity. The constructs including single novel SNP mutations exhibited insignificant change in luciferase activity, whereas, the activity produced by Haplo1(GTTGCTATAT), Haplo2 (CTTGCTATAT) and Haplo7 (GAGCTCACAT), containing a −333T>A polymorphism was significantly greater than for the wild type in Hep G2 cells (p<0.05), being 1.5−, 2.0− and 1.4− times greater respectively. These findings suggest the possibility of significant clinical prediction of adverse drug reaction and the facilitation of personalized medicine.

Abstract 1530: Modulation of drug transporter expression in pancreatic cancer cells by indole-3-carbinol and gemcitabine: Potential transporter-targeted therapy

Abstract Increased expression of drug transporters that induces multidrug resistance is a critical event in poor prognosis and treatment for numerous cancers, particularly pancreatic cancer. Due to its aggressive and resistance characteristics, prognosis is often fatal. Recently, investigators have focused on the use of inhibitors of proteins transcribed by various drug transporters as a potential target for improved therapy of various chemotherapeutics. Gemcitabine is the current treatment for pancreatic cancer, although some patients respond poorly to the treatment. Interindividual differences in expression of drug transporters relating to drug resistance has been a major factor in different drug outcomes. In the present study, pancreatic cancer cells (Panc-1 and SU86. 86) showed expression of the following drug transporters, ABCB1, ABCA5, MRP5, SLCO1B1, and SLC5A6. Gemcitabine treatment alone down-regulated expression of ABCA5, SLCO1B1, and SLC5A6 (P&amp;lt;0.05) transporters. Gemcitabine (0.1 µM) or I3C (100 µM) alone had no effect on ABCB1 expression. However, combination of gemcitabine (0.1µM) and indole-3-carbinol(100 µM) significantly down-regulated ABCB1. ABCB1 codes for the multidrug protein, pgp. This combination significantly inhibited growth of pancreatic cancer cells by 70%. Gemcitabine and indole-3 carbinol also decreased expression of MRP5 expression. These data suggest that dietary agents in combination with chemotherapeutics are potential inhibitors of multidrug resistance by down-regulating these proteins thus allowing for increased efficacy of the drug for treatment of the cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1530.

Quantitative Evaluation of the Expression and Activity of Five Major Sulfotransferases (SULTs) in Human Tissues: The SULT “Pie”

Expression levels of the major human sulfotransferases (SULTs) involved in xenobiotic detoxification in a range of human tissues (i.e., SULT "pies") are not available in a form allowing comparison between tissues and individuals. Here we have determined, by quantitative immunoblotting, expression levels for the five principal human SULTs-SULT1A1, SULT1A3/4, SULT1B1, SULT1E1, and SULT2A1-and determined the kinetic properties toward probe substrates, where available, for these enzymes in cytosol samples from a bank of adult human liver, small intestine, kidney, and lung. We produced new isoform-selective antibodies against SULT1B1 and SULT2A1, which were used alongside antibodies against SULT1A3 and SULT1A1 previously produced in our laboratory or available commercially (SULT1E1). Expression levels were derived using purified recombinant enzymes to construct standard curves for each individual isoform and immunoblot. Substantial intertissue and interindividual differences in expression were observed. SULT1A1 was the major enzyme (>50% of total, range 420-4900 ng/mg cytosol protein) in the liver, followed by SULT2A1, SULT1B1, and SULT1E1. SULT1A3 was completely absent from this tissue. In contrast, the small intestine contained the largest overall amount of SULT of any of the tissues, with SULT1B1 the major enzyme (36%), closely followed by SULT1A3 (31%), and SULT1A1, SULT1E1, and SULT2A1 more minor forms (19, 8, and 6% of total, respectively). The kidney and lung contained low levels of SULT. We provide a unique data set that will add value to the study of the role and contribution of sulfation to drug and xenobiotic metabolism in humans.

Functional annotation of the human retinal pigment epithelium transcriptome

BackgroundTo determine level, variability and functional annotation of gene expression of the human retinal pigment epithelium (RPE), the key tissue involved in retinal diseases like age-related macular degeneration and retinitis pigmentosa. Macular RPE cells from six selected healthy human donor eyes (aged 63–78 years) were laser dissected and used for 22k microarray studies (Agilent technologies). Data were analyzed with Rosetta Resolver, the web tool DAVID and Ingenuity software.ResultsIn total, we identified 19,746 array entries with significant expression in the RPE. Gene expression was analyzed according to expression levels, interindividual variability and functionality. A group of highly (n = 2,194) expressed RPE genes showed an overrepresentation of genes of the oxidative phosphorylation, ATP synthesis and ribosome pathways. In the group of moderately expressed genes (n = 8,776) genes of the phosphatidylinositol signaling system and aminosugars metabolism were overrepresented. As expected, the top 10 percent (n = 2,194) of genes with the highest interindividual differences in expression showed functional overrepresentation of the complement cascade, essential in inflammation in age-related macular degeneration, and other signaling pathways. Surprisingly, this same category also includes the genes involved in Bruch's membrane (BM) composition. Among the top 10 percent of genes with low interindividual differences, there was an overrepresentation of genes involved in local glycosaminoglycan turnover.ConclusionOur study expands current knowledge of the RPE transcriptome by assigning new genes, and adding data about expression level and interindividual variation. Functional annotation suggests that the RPE has high levels of protein synthesis, strong energy demands, and is exposed to high levels of oxidative stress and a variable degree of inflammation. Our data sheds new light on the molecular composition of BM, adjacent to the RPE, and is useful for candidate retinal disease gene identification or gene dose-dependent therapeutic studies.

Multiple sclerosis: chemokine receptor expression on circulating lymphocytes in correlation with radiographic measures of tissue injury

Background Leukocytes expressing inflammatory chemokine receptors (CKRs), most consistently CCR2, CCR5, and CXCR3, have been identified in multiple sclerosis (MS) tissue lesions and provide attractive therapeutic targets. Our previous studies found large inter-individual differences in expression of these CKRs but stable levels over time within subjects. This observation suggests a CKR “set-point” within individuals, which might relate to inflammatory injury in MS. We evaluated the correlation between CKR levels and magnetic resonance imaging (MRI) measures of disease activity. Methods Fifty-five relapsing remitting MS (RRMS) and secondary progressive MS (SPMS) patients were prospectively followed with annual CKR and MRI studies. Multiparameter flow cytometry was used to determine CCR2, CCR5, and CXCR3 expression on CD4 and CD8 cells. Simultaneous cranial MRIs were performed, and quantitative measures of T2, T1, and gadolinium lesions, brain parenchymal fraction (BPF), and whole brain and fractionated magnetization transfer ratio (MTR) were performed using automated software. Spearman’s rank correlations evaluated the relationship between CKR levels and MRI measures. Results Significant correlations were observed between CXCR3 expression on CD8 cells and measures of new (T1) and total (T1, T2) lesion volumes, lesion MTR, and BPF; higher levels of CXCR3 expression were correlated with greater injury on MRI (|r| = 0.27–0.42). In contrast, CD4 cell CKR expression was only minimally correlated with MRI measures. Conclusions Over 2 years, we observed significant correlations between the percent of CD8 cells expressing CXCR3 and MRI measures of MS inflammatory activity and tissue destruction. These observations are consistent with a pathogenic role for cytotoxic T cells in MS brain and have significant implications regarding T-cell targeted therapeutic strategies.

Tissue distribution and interindividual variation in human UDP-glucuronosyltransferase activity: relationship between UGT1A1 promoter genotype and variability in a liver bank

The variability in a liver bank and tissue distribution of three probe UDP-glucuronosyltransferase (UGT) activities were determined as a means to predict interindividual differences in expression and the contribution of extrahepatic metabolism to presystemic and systemic clearance. Formation rates of acetaminophen-O-glucuronide (APAPG), morphine-3-glucuronide (M3G), and oestradiol-3-glucuronide (E3G) as probes for UGT1A6, 2B7, and 1A1, respectively, were determined in human kidney, liver, and lung microsomes, and in microsomes from intestinal mucosa corresponding to duodenum, jejunum and ileum. While formation of E3G and APAPG were detectable in human kidney microsomes, M3G formation rates from kidney microsomes approached the levels seen in liver, indicating significant expression of UGT2B7. Interestingly, rates of E3G formation in human intestine exceeded the hepatic rates by several fold, while APAPG and M3G formation rates were low. The intestinal apparent Km value for E3G formation was essentially identical to that seen in liver, consistent with intestinal UGT1A1 expression. No UGT activities were observed in lung. Variability in APAPG and M3G activity across a bank of 20 human livers was modest (< or = 7-fold), compared to E3G formation, which varied approximately 30-fold. The E3G formation rates were found to segregate by UGT1A1 promoter genotype, with wild-type (TA)6 rates significantly greater than homozygous mutant (TA)7 individuals. Kinetic analyses were performed to demonstrate that the promoter mutation altered apparent Vmax without significantly affecting apparent Km. These results suggest that glucuronidation, and specifically UGT1A1 activity, can profoundly contribute to intestinal first pass metabolism and interindividual variability due to the expression of common allelic variants.

Expression of glutathione S-transferase theta class isoenzymes in human colorectal and gastric cancers.

Glutathione S-transferases (GSTs) are a superfamily of detoxification enzymes, which play an important role in the protection of tissues against potentially harmful compounds. In humans, two theta class isoenzymes, GSTT1-1 and GSTT2-2, have been described so far. Both enzymes were claimed to have an important role in human carcinogenesis. In colorectal and gastric tissues, the expression of the other isoenzymes changes after malignant transformation. No data on the expression levels of the theta isoenzymes in these tissues are available. The aim of this study was to determine the protein levels of the two theta class isoenzymes in human colorectal and gastric cancers and paired normal tissue. Cytosolic fractions of normal and matched tumor tissue samples from 20 patients with colorectal or gastric adenocarcinomas were analyzed on immunoblots using specific antibodies against GSTT1-1 and GSTT2-2, respectively. In addition paraffin-embedded sections of these tissues were examined immunohistochemically for GSTT1-1 expression. In both types of tissue, theta class isoenzymes were highly expressed. Expression of GSTT1-1 was higher in gastric than in colorectal tissues. The GSTT2-2 levels were comparable in both tissues. A great interindividual difference in expression was demonstrated. In colon, there was no change in the theta class isoenzyme levels after malignant transformation. Gastric tumors had significantly lower expression of both theta class isoenzymes compared with the normal mucosa. In colon, GSTT1-1 was expressed in the enterocytes and goblet cells. In gastric tissues, staining was seen in upper and deeper mucous cells, chief cells and, to a lesser extent, in parietal cells. In both types of tumors, staining was seen in adenomatous cells. In conclusion, in both normal human colonic and gastric mucosa, GSTT1-1 and GSTT2-2 are present at high levels, whereas after malignant degeneration, expression is not influenced or is even downregulated.

Mutations in the exons and exon-intron junction regions of human cytochrome P-4502E1 gene and alcoholism.

Cytochrome P-4502E1 (CYP2E1) is a major component of the microsomal ethanol-oxidizing system (MEOS) and is also involved in the metabolism of a variety of foreign compounds, including carcinogens. It has been shown that there are interindividual variations in the expression of human CYP2E1. Gene-environmental interactions have been suggested to account for the difference. In this study, we screened nine exons and exon-intron junctions of the human CYP2E1 gene for detecting allelic variants in genomic DNA samples obtained from 115 Japanese controls, 96 alcoholics, and 44 patients with alcoholic liver diseases. A novel missense mutation in exon 2 (V72L) was found in Japanese controls, but the frequency was low (2.6%). In addition, two novel silent mutations (T303T and F420F), together with one mutation in intron 2, were found. However, no association of these mutations with alcoholism and alcoholic liver diseases was found. Our data indicate that nucleotide replacement in the open reading frame of CYP2E1 gene is not a major factor for interindividual differences in expression of CYP2E1 and susceptibility to alcohol-related disorders.

Interindividual difference in expression of human Ah receptor and related P450 genes.

The genomic clones of human aryl hydrocarbon receptor (Ahr) and Ahr nuclear translocator (Arnt) were isolated, and the structures of exon-intron junctions of these genes were partially determined. Based on the sequence information, a quantitative RT-PCR analysis was developed, and the expression of these genes was studied in various human tissues. mRNAs for Ahr and Arnt were widely expressed in human tissues and abundantly in lung. Individual difference in expression levels of Ahr and Arnt mRNA was observed in liver, lung and blood. In order to examine whether expression levels of Ahr and Arnt were associated with those of CYP1A1, we studied the expression of these mRNAs in blood among 20 healthy subjects, taking account of individuals' cigarette smoking habits. We found that the expression levels of CYP1A1 appeared to associate with those of Ahr and Arnt mRNAs (P < 0.06), and also that the expression of Ahr and Arnt was influenced by cigarette smoking. The expression of human Ahr and Arnt is reported here for the first time, providing a quantitative RT-PCR analysis as a useful tool for further studies.