Kaname Kawajiri

Identification of genetically high risk individuals to lung cancer by DNA polymorphisms of the cytochrome P45 0IA1 gene

A good correlation was observed between enhanced lung cancer risk and restriction fragment length polymorphisms (RFLPs) of the P450IA1 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 P450IA1 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.

High Susceptibility to Lung Cancer Analyzed in Terms of Combined Genotypes of P450IA1 and Mu-class Glutathione S-Transferase Genes

Lung cancer is closely associated with cigarette smoking. Aromatic hydrocarbons in smoke, including benzo[n]pyrene, first require metabolic activation by Phase I enzymes, cytochrome P450, to their ultimate forms, and these activated forms are then subjected to detoxification by Phase II enzymes, especially glutathione S‐transferases. Thus, genetically determined susceptibility to lung cancer may depend on the metabolic balance between Phase I and Phase II enzymes. In this study, we identified individuals genetically at high risk of lung cancer in terms of polymorphisms of the P450IA1 gene and GST1 gene. The relative risk of individuals with a combination of the genotypes of both a homozygous rare allele of the P450IA1 gene and the nulled GST1 gene was remarkably high at 5.8 for lung cancer and 9.1 for squamous cell carcinoma compared with other combinations of genotypes.

Association of CYP1B1 genetic polymorphism with incidence to breast and lung cancer

Cytochrome P450 1B1 (CYP1B1) participates in the metabolic activation of a number of procarcinogens including benzo[a]pyrene and the hydroxylation of 17beta-estradiol at the C-4 position. In this study, we investigated the association between CYP1B1 genetic polymorphism and breast or lung cancer incidence. The Ala-Ser polymorphism at codon 119 in presumed substrate recognition site 1 was significantly associated with the incidence of breast or squamous cell carcinoma of the lung. On the other hand, Leu-Val polymorphism at codon 432 did not show any association to the cancers. An allele containing both Ala and Leu simultaneously, comprised 75% of alleles among 315 Japanese healthy controls, was significantly inversely associated with breast cancer incidence. When expressed in a recombinant system, this CYP1B1 cDNA showed the lowest 17beta-estradiol 4-hydroxylase activity among four different variant forms of CYP1B1. Thus, inter-individual differences in activation of procarcinogens or metabolism of oestrogen originating from genetic polymorphisms of the human CYP1B1 gene may contribute to the susceptibility of human cancers.

A Nuclear Localization Signal of Human Aryl Hydrocarbon Receptor Nuclear Translocator/Hypoxia-inducible Factor 1β Is a Novel Bipartite Type Recognized by the Two Components of Nuclear Pore-targeting Complex

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a component of the transcription factors, aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1, which transactivate their target genes, such as CYP1A1 and erythropoietin, in response to xenobiotic aromatic hydrocarbons and to low O2concentration, respectively. Since ARNT was isolated as a factor required for the nuclear translocation of AhR from the cytoplasm in response to xenobiotics, the subcellular localization of ARNT has been of great interest. In this investigation, we analyzed the subcellular distribution of ARNT using transient expression of a fusion gene with β-galactosidase and microinjection of recombinant proteins containing various fragments of ARNT in the linker region of glutathioneS-transferase/green fluorescent protein. We found a clear nuclear localization of ARNT in the absence of exogenous ligands to AhR, and identified the nuclear localization signal (NLS) of amino acid residues 39–61. The characterized NLS consists of 23 amino acids, and can be classified as a novel variant of the bipartite type on the basis of having two separate regions responsible for efficient nuclear translocation activity, but considerable deviation of the sequence from the consensus of the classical bipartite type NLSs. Like the well characterized NLS of the SV40 T-antigen, this variant bipartite type of ARNT NLS was also mediated by the two components of nuclear pore targeting complex, PTAC58 and PTAC97, to target to the nuclear rim in an in vitro nuclear transport assay.

P450 and human cancer

Most of the chemical carcinogens in our environment are activated mainly by a restricted number of cytochrome P450 species, P450 1A1,1A2, 2E1, and 3A. This metabolic activation of procarcinogens is a crucial part of the initial host response to the environmental exposure, since most chemical carcinogens do not show any carcinogenicity by themselves. Inter‐individual variability in the metabolic activity may thus be a key host factor to explain the differences in susceptibility to chemical carcinogenesis among individuals. Recent studies on P450s in cancer etiology have provided some valuable insights into this problem.

Polymorphisms of human Ah receptor gene are not involved in lung cancer.

The Ah receptor (Ahr) is a ligand-dependent transcription factor that positively regulates inducible expression of the CYP1A1 gene. Based on the sequence information of the human Ahr and the intron-exon junctions of the mouse counterpart, an analysis of single-strand conformational polymorphism (SSCP) was carried out to detect subtle base differences in the coding region of the gene among individuals. We found that the Ahr protein has at least two forms of variants in a Japanese gene pool, and that these variants can be ascribed to one amino acid replacement of Arg by Lys at codon 554. The frequencies of Arg-coded and Lys-coded alleles were 0.57 and 0.43, respectively. We found, however, that this germ line polymorphism of the Ahr gene did not show a significant association with aryl hydrocarbon hydroxylase (AHH) inducibility nor with lung cancer incidence.

Identification of allelic variants of the human CYP1A1 gene.

Publisher Summary This chapter describes the identification of variant alleles of the human CYP1A1 gene. Individual differences in susceptibility to chemically induced cancers are ascribed partly to genetic differences in metabolic balance in the activation and detoxification of environmental procarcinogens. Human lung cancer requires exposure to the procarcinogens contained in cigarette smoke. CYP1A1 is expressed in the lung and metabolizes polycyclic aromatic hydrocarbons, such as benzo[ a ]pyrene, in cigarette smoke. If some of the genetic polymorphisms of the CYP1A1 gene correlate to the lung cancer incidence, this might be helpful in predicting the individual risk of cancer. The chapter proves that patients with susceptible genotypes of the CYP1A1 gene contracted carcinoma after smoking fewer cigarettes than those with other genotypes. These results were reconfirmed by a subsequent study in a different Japanese population. However, a lack of association between the CYP1A1 genotypes and lung cancer incidence has been reported in Caucasian populations. This discrepancy may be ascribed largely to the ethnic difference in the allelic frequency of the polymorphism.

Ablating the aryl hydrocarbon receptor (AhR) in CD11c+ cells perturbs intestinal epithelium development and intestinal immunity.

Diet and microbiome derived indole derivatives are known to activate the ligand induced transcription factor, the Aryl hydrocarbon Receptor (AhR). While the current understanding of AhR biology has confirmed its role in mucosal lymphocytes, its function in intestinal antigen presenting cells (APCs) is poorly understood. Here, we report that Cre-mediated deletion of AhR in CD11c-expressing cells in C57/BL6 mice is associated with altered intestinal epithelial morphogenesis in vivo. Moreover, when co-cultured with AhR-deficient DCs ex vivo, intestinal organoids showed reduced SRY (sex determining region Y)-box 9 and increased Mucin 2 expression, which correlates with reduced Paneth cells and increased goblet cell differentiation, similar to the data obtained in vivo. Further, characterization of intestinal APC subsets, devoid of AhR, revealed an expression pattern associated with aberrant intrinsic Wnt pathway regulation. At a functional level, the loss of AhR in APCs resulted in a dysfunctional epithelial barrier, associated with a more aggressive chemically induced colitis compared to wild type animals. Our results are consistent with a model whereby the AhR signalling pathway may participate in the regulation of innate immunity through intestinal epithelium development and mucosal immunity.

Biochemical basis for the resistance of guinea pigs to carcinogenesis by 2-acetylaminofluorene

Abstract Studies were made on why guinea pigs are resistant to carcinogenesis by 2-acetylaminofluorene. Cytochrome P-448 and arylhydrocarbon hydroxylase were not induced in either the microsomes and nuclei of guinea pigs by 3-methylcholanthrene treatment. 3-Methylcholanthrene treatment caused only 2-fold increase in the binding of 2-acetylaminofluorene to DNA in nuclei isolated from guinea pigs, while it caused 17-fold increase in the binding in rat nuclei. Microsomes from 3-methylcholanthrene treated rats had 5 times more effect than Microsomes from 3-methylcholanthrene treated guinea pigs on the binding of 2-acetylaminofluorene to DNA of nuclei from untreated guinea pigs. N-Hydroxy-2-acetylaminofluorene combined equally well with the DNA of rats and guinea pigs. In guinea pigs, there was a good correlation between the low inducibility of cytochrome P-448 and the low binding of 2-acetylaminofluorene to DNA. Our results clearly showed that guinea pigs are resistant to tumor induction by 2-acetylaminofluorene through inability to carry out the first step of activation of 2-acetylaminofluorene.

Aryl Hydrocarbon Receptor Suppresses Cecal Carcinogenesis

The aryl hydrocarbon receptor (AhR) has dual roles in regulating intracellular protein levels as both a ligand-activated transcription factor and a ligand-dependent E3 ubiquitin ligase. We showed that the AhR E3 ubiquitin ligase has a role in the suppression of intestinal carcinogenesis by a previously undescribed ligand-dependent β-catenin degradation pathway. 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 which suppresses intestinal tumor development in Apc Min/+ mice. To elucidate whether the tumors develop autonomously in AhR −/− mice as a result of impaired β-catenin degradation or in association with accelerated inflammation, we performed two kinds of experiments using germ-free (GF) AhR −/− mice or compound mutant mice lacking genes for AhR and adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which play an essential role in caspase-1 activation in inflammasomes. Both GF AhR −/− and AhR −/− •ASC −/− mice showed considerably reduced tumor development compared with that in AhR −/− mice, albeit in a “cancer-prone” state with aberrant β-catenin accumulation. These results revealed an important role of the bacteria-triggered or ASC-mediated inflammation signaling pathway in mouse intestinal tumorigenesis. Furthermore, they also suggest a possible chemical therapeutic intervention that involves AhR ligands and inhibitors of the inflammation pathway.