We identified genetic polymorphisms in the 5'-flanking region of the human cytochrome P450IIE1 gene and investigated the effect of these polymorphisms on the transcriptional regulation of the gene. PCR direct sequencing of the two homozygous alleles [types A (c1/c1) and C (c2/c2)] revealed the existence of several point mutations in the distal 5'-flanking region of the gene, but no differences in the proximal promoter region. The DNA segment (-1372 to -960) placed upstream of SV40 promoter and the chloramphenicol acetyltransferase (CAT) gene enhanced the expression of the gene, and the enhancement of expression by type C DNA was about 10 times that by its type A counterpart. DNase I footprinting analysis showed at least one protected region in which one of the polymorphic loci (RsaI polymorphism) was located. The DNase I sensitivities and protection profiles of the two genotypes were different. The protected region had high homology to the consensus sequence of the binding region of liver specific transcription factor HNF1 (LF-B1), and this was confirmed by gel retardation assay. These results indicate that genetic polymorphisms in the 5'-flanking region of the human P450IIE1 gene affect its binding of trans-acting factor and change its transcriptional regulation. This may lead to inter-individual differences of microsomal drug oxidation activity.

A good correlation was observed between enhanced lung cancer risk and restriction fragment length polymorphisms (RFLPs) of the P450IAl gene with the restriction enzyme. MspI. Genotype frequencies of 0.49 for the predominant homozygote, 0.40 for the heterozygote, and 0.11 for the homozygous rare allele were observed in a healthy population. Among lung cancer patients, the frequency of homozygous rare allele of P450IAl gene was found to be about 3-fold higher than that among healthy population, and this difference was statistically significant. This is the first report to identify the genetically high risk individuals to lung cancer at the gene level.

Individuals with high genetic risk of lung cancer had previously been identified by MspI polymorphisms of the cytochrome P450IA1 gene. In the present study we analyzed the structures of individual P450IA1 genes by PCR direct sequencing of genomic DNA of each genotype raised by the MspI polymorphisms, which were ascribed to a single point mutation in the 3'-flanking region. We then found a novel point mutation in the coding region of the gene which results in the substitution of Ile for Val at residue 462 in the heme binding region. We further analyzed the genetic association between this amino acid replacement and MspI polymorphisms in the general population, using a new method to detect polymorphisms not recognized by restriction enzymes. The results showed that there are at least two forms of human P450IA1 protein with different primary structures and that one of the forms is closely linked with the lung cancer-susceptible genotype of MspI polymorphisms. Thus MspI polymorphisms, which are associated with increased risk of lung cancer, are linked to at least one amino acid substitution, which gives an important clue, at the molecular level, toward elucidation of increased susceptibility to lung cancer.

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that binds DNA in the form of a heterodimer with the Ahr nuclear translocator (hypoxia-inducible factor 1␤). We found in this study that Ahr contains both nuclear localization and export signals in the NH 2 -terminal region. A fusion protein composed of ␤-galactosidase and full-length Ahr translocates from the cytoplasm to the nucleus in a liganddependent manner. However, a fusion protein lacking the PAS (Per-Ahr nuclear translocator-Sim homology) domain of the Ahr showed strong nuclear localization activity irrespective of the presence or absence of ligand. A minimum bipartite Ahr nuclear localization signal (NLS) consisting of amino acid residues 13-39 was identified by microinjection of fused proteins with glutathione S-transferase-green fluorescent protein. A NLS having mutations in bipartite basic amino acids lost nuclear translocation activity completely, which may explain the reduced binding activity to the NLS receptor, PTAC58. A 21-amino acid peptide (residues 55-75) containing the Ahr nuclear export signal is sufficient to direct nuclear export of a microinjected complex of glutathione S-transferase-Ahr-green fluorescent protein. These findings strongly suggest that Ahr act as a ligandand signal-dependent nucleocytoplasmic shuttling protein.The aryl hydrocarbon receptor (Ahr) 1 binds a variety of environmentally important carcinogens, including polycyclic aromatic hydrocarbons and certain halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Before binding ligands, Ahr is located in the cytoplasm as one component of a complex that has a molecular mass of about 280 kDa (1). This complex is composed of Ahr, two molecules of the 90-kDa heat shock protein, and possibly a 43-kDa protein (2). After ligand binding, Ahr dissociates from the complex and translocates to the nucleus (3). The heterodimer of Ahr and Ahr nuclear translocator (ARNT) constitutes a transcription factor and binds specific DNA sequences called XREs (xenobioticresponsive elements) in the enhancer regions of the CYP1A1 and several other proteins involved in xenobiotic metabolism (4). Because these enzymes are involved in the metabolism of polycyclic aromatic hydrocarbons to active genotoxic metabolites, Ahr plays an important role in carcinogenesis caused by these compounds (5-7).Because ARNT was first cloned as a factor required for ligand-dependent nuclear translocation of Ahr from the cytoplasm to the nucleus (8), the subcellular localization of ARNT was believed to be cytoplasmic. In fact, most ARNT was recovered in the cytosolic fraction by cell fractionation. However, immunohistochemical analysis has shown that ARNT is localized predominantly in the nucleus, regardless of the presence or absence of ligands (9, 10). This controversial subject was clarified by our recent study in which a nuclear localization signal (NLS) of the amino acid residues between 39 and 61 of human ARNT was found to be a novel bipartite type recognized by th...

Intestinal cancer is one of the most common human cancers. Aberrant activation of the canonical Wnt signaling cascade, for example, caused by adenomatous polyposis coli (APC) gene mutations, leads to increased stabilization and accumulation of ␤-catenin, resulting in initiation of intestinal carcinogenesis. The aryl hydrocarbon receptor (AhR) has dual roles in regulating intracellular protein levels both as a ligand-activated transcription factor and as a ligand-dependent E3 ubiquitin ligase. Here, we show that the AhR E3 ubiquitin ligase has a role in suppression of intestinal carcinogenesis by a previously undescribed ligand-dependent ␤-catenin degradation pathway that is independent of and parallel to the APC system. This function of AhR is activated by both xenobiotics and natural AhR ligands, such as indole derivatives that are converted from dietary tryptophan and glucosinolates by intestinal microbes, and suppresses intestinal tumor development in Apc Min/؉ mice. These findings suggest that chemoprevention with naturally-occurring and chemically-designed AhR ligands can be used to successfully prevent intestinal cancers.cecal cancer ͉ ubiquitin ligase ͉ ␤-catenin ͉ tumor chemoprevention

Four polymorphic human cytochrome P450 (CYP) 1B1 allelic variants, namely Arg48,Ala119,Leu432,Asn453, Arg48,Ser119,Leu432,Asn453, Arg48, Ala119,Val432,Asn-453 and Arg48,Ser119,Val432,Asn453, were expressed in Escherichia coli together with human NADPH-P450 reductase and the recombinant proteins (in bacterial membranes) were used to assess whether CYP1B1 polymorphisms affect catalytic activities towards a variety of P450 substrates, including diverse procarcinogens and steroid hormones. Activities for activation of 19 procarcinogens to DNA-damaging products by these four CYP1B1 variants in a Salmonella typhimurium NM2009 umu response system were found to be essentially similar, except that a Arg48, Ser119,Leu432,Asn453 variant was slightly more active (1.2- to 1.5-fold) than the other three CYP1B1 enzymes in catalyzing activation of (+)- and (-)-benzo[a]pyrene-7, 8-diols, 7,12-dimethylbenz[a]anthracene-3,4-diol, benzo[g]chrysene-11,12-diol, benzo[b]fluoranthene-9,10-diol, 2-amino-3,5-dimethylimidazo[4,5-f]quinoline, 2-amino-3-methylimidazo[4,5-f]quinoline and 2-aminofluorene. Kinetic analysis of 17beta-estradiol hydroxylation showed that V(max) values for 4-hydroxylation ranged between 0.9 and 1.5 nmol/min/nmol P450 for 4-hydroxylation and 0.3 and 0.6 nmol/min/nmol P450 for 2-hydroxylation in these CYP1B1 variants, with K(m) values ranging from 1 to 9 microM. Interestingly, the ratio of product formation of 4-hydroxyestradiol to 2-hydroxyestradiol was higher for the Val432 variants of CYP1B1 variants than the Leu432 variants of the enzyme. The same trend was noted in the ratio of estrone 4-hydroxylation to estrone 2-hydroxylation catalyzed by CYP1B1 variants. Mutation in the CYP1B1 genes also affected the K(m) and V(max) values in the 6beta-hydroxylation of testosterone and 6beta- and 16alpha-hydroxylation of progesterone. These results indicate that the polymorphisms in the human CYP1B1 gene cause some alterations in catalytic function towards procarcinogens and steroid hormones and thus may make some contribution to susceptibilities of individuals towards mammary and lung cancers in humans.

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The p53 tumor suppressor gene is mutated in diverse types of human cancer, and the normal allele encodes a nuclear protein that regulates expression of cell cycle-related genes as a transcription factor. The wild-type of p53 protein exists as at least two forms of variants among human populations, ascribed to amino acid replacement at codon 72 of Arg by Pro. In this study, we show that this germ line Arg-Pro polymorphism at codon 72 of the p53 gene is associated with genetically determined susceptibility to smoking-induced lung cancer; a susceptible genotype Pro/Pro has a 1.7-fold higher risk of this cancer compared with other genotypes. This p53 polymorphism modulates risk to smoking-induced lung cancer independently of other genetic risk factors such as germ line polymorphism of CYP1A1 or GST1 genes.