Representational difference analysis was used to isolate cDNAs corresponding to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible genes from mouse Hepa-1 cells. One cDNA encoded a novel cytochrome P450. The human homolog was also isolated and later proved to be human CYP2S1. The induction of mouse CYP2S1 mRNA by dioxin represents a primary response and required the aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins. The induction of CYP2S1 also occurred in mouse liver and lung, with the highest expression found in lung. CYP2S1 was also inducible in a human lung epithelial cell line. The dioxin-inducibility of CY2S1 is exceptional, because all previously well-characterized cases of the induction of cytochromes P450 by dioxin involve members of the CYP1 family.The cytochrome P450 (P450) superfamily of proteins is involved in the metabolism of a vast array of carcinogens, drugs, and endogenous metabolites. The three members of the CYP1 cytochrome P450 family are inducible by ligands for the aryl hydrocarbon receptor, which include important classes of chemical carcinogens such as PAHs, aromatic amines, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin). All three CYP1 family members also play important roles in the metabolic activation of a number of these same compounds to carcinogenic derivatives. CYP1A1 and CYP1B1 both activate PAHs to carcinogenic intermediates, whereas CYP1A2 activates aromatic amines. Dioxin is the most carcinogenic substance evaluated (Hankinson, 1995). In contrast to PAHs and aromatic amines, dioxin is refractory to biotransformation, and the parent compound itself acts as a carcinogen. The induction of CYP1A1 has been well-studied. After binding the ligand, the AHR translocates to the nucleus, where it dimerizes with the ARNT protein. The AHR/ARNT dimer then binds to xenobiotic-responsive elements in the 5Ј flanking region of the CYP1A1 gene, leading to stimulation of transcription of the gene (Whitlock, 1999). Transcriptional activation of the CYP1A2 and CYP1B1 genes seems to occur in a similar fashion. Induction of CYP1A2 is restricted to the liver, whereas CYP1A1 and CYP1B1 are both inducible in many tissues (Savas et al., 1994;Dey et al., 1999).The aim of this study was to identify novel dioxin-inducible genes, with the notion that such genes might be involved in carcinogenesis by PAHs, dioxin, and related compounds. Using representational difference analysis (RDA) on the mouse hepatoma cell line Hepa-1, we identified several genes not known previously to be inducible by dioxin. This report focuses on one of these genes, a novel cytochrome P450 that belongs to the CYP2 family rather than to the CYP1 family, which includes all other previously well-characterized, dioxin-inducible cytochromes P450.
Adaptation to hypoxia is essential for tumor progression. Transcriptional activation of hypoxia-regulated genes is mediated by hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1K K and ARNT (Ah receptor nuclear translocator; HIF-1L L). Using representational di¡erence analysis, we identi¢ed three novel hypoxia-inducible genes: MIG-6 (gene 33), adipophilin and tuftelin. The mRNAs for these genes were inducible by 1% O 2 in the human HepG2 and MCF-7 cell lines. Hypoxic induction of the MIG-6 and tuftelin proteins was also observed. Induction was ARNT-dependent. Induction also occurred in livers of mice treated with CoCl 2 , which mimics hypoxia. The potential roles of these genes in adaptation to hypoxia and in tumorigenesis will be of considerable interest. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
The objective of this study was to design and validate a next-generation sequencing assay (NGS) to detect BRCA1 and BRCA2 mutations. We developed an assay using random shearing of genomic DNA followed by RNA bait tile hybridization and NGS sequencing on both the Illumina MiSeq and Ion Personal Gene Machine (PGM). We determined that the MiSeq Reporter software supplied with the instrument could not detect deletions greater than 9 base pairs. Therefore, we developed an alternative alignment and variant calling software, Quest Sequencing Analysis Pipeline (QSAP), that was capable of detecting large deletions and insertions. In validation studies, we used DNA from 27 stem cell lines, all with known deleterious BRCA1 or BRCA2 mutations, and DNA from 67 consented control individuals who had a total of 352 benign variants. Both the MiSeq/QSAP combination and PGM/Torrent Suite combination had 100% sensitivity for the 379 known variants in the validation series. However, the PGM/Torrent Suite combination had a lower intra- and inter-assay precision of 96.2% and 96.7%, respectively when compared to the MiSeq/QSAP combination of 100% and 99.4%, respectively. All PGM/Torrent Suite inconsistencies were false-positive variant assignments. We began commercial testing using both platforms and in the first 521 clinical samples MiSeq/QSAP had 100% sensitivity for BRCA1/2 variants, including a 64-bp deletion and a 10-bp insertion not identified by PGM/Torrent Suite, which also suffered from a high false-positive rate. Neither the MiSeq nor PGM platform with their supplied alignment and variant calling software are appropriate for a clinical laboratory BRCA sequencing test. We have developed an NGS BRCA1/2 sequencing assay, MiSeq/QSAP, with 100% analytic sensitivity and specificity in the validation set consisting of 379 variants. The MiSeq/QSAP combination has sufficient performance for use in a clinical laboratory.
Cytochrome P4502S1 (CYP2S1) is expressed at high levels in epithelial tissues and is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) via the aryl hydrocarbon receptor (AHR). Transcriptional initiation of mouse Cyp2s1 was found to occur at three regions, ϳ198, 102, and 22 nucleotides from the translational initiation codon. Approximately 400 nucleotides upstream of its translational initiation codon, mouse Cyp2s1 contains three overlapping xenobiotic-responsive element (XRE) sequences, which make a major contribution toward dioxin inducibility. Each XRE sequence in this trimeric XRE can bind the AHR/aryl hydrocarbon receptor nuclear translocator (ARNT) dimer in a dioxin-dependent fashion in vitro and can mediate dioxin-dependent transcription. Cyp2s1 is also markedly inducible by hypoxia. Induction is dependent on hypoxiainducible factor-1 (HIF-1) and is mediated in large part by three overlapping hypoxia response elements (HREs) embedded within the trimeric XRE segment. Although each HRE within this segment can bind HIF-1␣/ARNT in vitro, the most 3 HRE contributes the most toward hypoxia inducibility. AHR/ARNT and HIF-1␣/ARNT dimers bind to the region containing the trimeric XRE segment of the endogenous Cyp2s1 gene in vivo in a dioxin-dependent fashion and hypoxia-dependent fashion, respectively. These observations identify a novel regulatory cassette that mediates changes in Cyp2s1 expression.The cytochrome P450 superfamily consists of at least 57 genes in humans and 102 genes in mice (1). In general, mammalian cytochrome P450s in families 1-4 metabolize foreign compounds (xenobiotics), although they also frequently metabolize endogenous molecules, such as steroids and fatty acids. Families 1-4 are often also inducible by xenobiotics. Members of family 1 are inducible by the potent tumor promoter, 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), 6 and carcinogenic polycyclic aromatic hydrocarbons via the aryl hydrocarbon receptor (AHR) (2). Rylander et al. (3) identified human CYP2S1 by performing a homology search in a sequence data base, whereas we cloned mouse Cyp2s1 as a dioxin-inducible transcript in the mouse hepatoma cell line, Hepa-1, in which cells it is maximally induced about 10-fold (4). CYP2S1 is unusual for a non-CYP1 family member in being inducible by dioxin (although another Cyp2 family member, Cyp2a5, has recently been shown also to be dioxin-inducible (5)). Human CYP2S1 has also been shown to be inducible in skin by coal tar, which contains high concentrations of polycyclic aromatic hydrocarbon ligands for AHR. Human CYP2S1 can convert alltrans-retinoic acid to the catabolic products, 4-hydroxyretinoic acid and 5,6-epoxyretinoic acid (6), and it has been reported that the enzyme can metabolize naphthalene to two products (7). Both mouse and human CYP2S1 are expressed robustly in most epithelial surfaces and tissues, including the lung and intestinal tract and at all stages of embryogenesis (3, 8 -10).The facts that all other previously well characterized dioxininducible cytochrome P45...
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