Two RFLPs associated with the human P1450 gene linked to the MPI locus on chromosome 15 [HGM8 D15S8].
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Chromosome Mapping↗
Biomedical subjects
D W Nebert
Publications and source records attributed to D W Nebert.
An example of P-450 catalytic activities not correlated with corresponding P-450 mRNA concentrations.
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Animals↗
The murine aromatic hydrocarbon responsiveness locus: a comparison of receptor levels and several inducible enzyme activities among recombinant inbred lines.
The aromatic hydrocarbon responsiveness (Ah) locus has been correlated with genetic differences in the risk of drug toxicity, teratogenesis, chemical carcinogenesis, and mutagenesis. Hepatic cytosolic Ah receptor levels, 2-amino-5-chlorobenzoxazole (zoxazolamine) paralysis time following beta-naphthoflavone treatment and aryl hydrocarbon hydroxylase (AHH3, acetanilide 4-hydroxylase (Ac4H), and NAD(P)H:menadione oxidoreductase (NMOR)4, induction by 3-methylcholanthrene were studied in (a) the progenitors C57BL/6J (Ahb/Ahb) and DBA/2J (Ahd/Ahd) and 25 BXD recombinant inbred lines, (b) the progenitors C57BL/6N and C3H/HeN and 14 B6NXC3N recombinant inbred lines, and (c) the progenitors C57BL/6J and C3H/HeJ and 12 BXH recombinant inbred lines. The Ahb phenotype exhibits greater than 5 femtomole receptor/mg of cytosolic protein, less than or equal to 15 minutes zoxazolamine paralysis time, and twofold to 15-fold induction of these three hepatic enzyme activities; the Ahd phenotype exhibits less than or equal to 2 fmol receptor/mg protein, greater than 15 minutes zoxazolamine paralysis time, and less than 30% induction of these three activities. Among the BXD lines but especially among the B6NXC3N and BXH lines, high frequencies of recombination were found; the phenotype of each of the five parameters did not segregate with the phenotype of each of the other parameters in four or more recombinant lines. This report shows for the first time that AHH induction by 3-methylcholanthrene can occur in the Ahd phenotype mouse. These data underline the complexity of this genetic system when genes from C57BL/6 and DBA/2 are combined and particularly when genes from C57BL/6 and C3H/He inbred mouse strains are combined.
Animals↗
Dioxin-inducible enhancer region upstream from the mouse P(1)450 gene and interaction with a heterologous SV40 promoter.
In mouse hepatoma Hepa-1 cells, polycyclic aromatic compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activate transcription of the mouse P(1)450 gene via trans-acting regulatory factors that include the TCDD X receptor complex. The positive control element in the P(1)450 5'-flanking region was examined in control and TCDD-treated Hepa-1 stable transformants that had been transfected with either of two expression vectors containing the chloramphenicol acetyltransferase (CAT) gene: pA10-cat, which has the simian virus 40 (SV40) early core promoter (without enhancers) immediately upstream from the CAT gene; and pSV0-cat, which has no promoter or enhancer. When the 1-kb DNA fragment from -1,647 to -611 upstream from the P(1)450 gene is inserted in either orientation--immediately upstream or almost 2 kb further upstream--from the SV40 promoter in pA10-cat, there is enhancement of CAT activity that can be further induced three- to fourfold by TCDD. When the same experiment is carried out with the -1,247 to -823 fragment or the -1,051 to -823 fragment, but not the -1,247 to -1,052 fragment, TCDD responsiveness is lost, or at least masked, because of a large increase in constitutive CAT activity. pSV0-cat mutants containing internal deletions in the upstream flanking sequences of P(1)450 were constructed. A region of 300 bases (-1,218 to -918) is shown to be required for TCDD responsiveness, and one TCDD-inducible element can be dissociated from an enhancer of constitutive gene expression, whereas one or more other TCDD-inducible elements cannot.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetyltransferases↗
P450 genes: evolution, regulation, and relationship to human cancer and pharmacogenetics.
Three pharmacogenetic differences (the acetylation, debrisoquine 4-hydroxylase, and Ah locus polymorphisms) appear to be associated with increased risk of environmentally caused human cancer. The latter two polymorphisms represent differences in P450 gene expression. It is predicted that molecular biological studies will soon provide a means (RFLP patterns or expression vector assays) of predicting individual cancer risk related to these polymorphisms. At the present time, only the Ah locus polymorphism has been explored at the molecular biological level. More than 30 P450 genes, including eight from the human, have been isolated and sequenced to date. The P450 gene superfamily comprises at least eight families, including one gene family that has diverged more recently into at least five subfamilies. The P450 gene superfamily can be regarded as ancient, with Pseudomonas and human amino acid sequences displaying a small but significant resemblance in the region of the enzyme active-site. P450 enzymic activity can be depressed 30-60% by immunosuppressive agents. The mechanism of this effect is unknown. This phenomenon can become clinically important if a cancer patient who is taking any chemotherapeutic drug that is detoxified by P450 then receives one of these agents. The Ah receptor-controlled gene expression, positive and negative control elements, and negative autoregulatory loop--best characterized in the mouse P(1)450 gene and upstream sequences, plus the receptor-defective and the P(1)450 enzymic activity-negative mutants in mouse cell cultures--provide the molecular geneticist with an exciting model system for studying the regulation of a clinically relevant gene family. The high degree of homology in a 220-bp segment between mouse and human P(1)450 sequences (about 1140 to 920 bases upstream from the mRNA cap site) suggests that this positive regulatory element (dioxin-inducible enhancer which also controls constitutive gene expression) is conserved between Mus domesticus and Homo sapiens.
Amino Acid Sequence↗
Tissue-specific expression of the mouse dioxin-inducible P(1)450 and P(3)450 genes: differential transcriptional activation and mRNA stability in liver and extrahepatic tissues.
Expression of the P(1)450 and P(3)450 genes was examined in liver and five extrahepatic tissues of mice after they were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylcholanthrene. All six tissues were shown to have increased P(1)450 and P(3)450 mRNA concentrations after treatment with these inducers. P(3)450 mRNA induction was more sensitive than P(1)450 mRNA induction to small doses of TCDD in liver, kidney, and lung. When transcription run-on assays were compared with mRNA prevalence, control P(3)450 mRNA in liver, kidney, and lung was shown to be 20 to 30 times more stable than control P(1)450 mRNA. After TCDD treatment the increases in mRNA concentrations did not necessarily parallel the increases in transcriptional rate. Thus, the inducer appeared to enhance mRNA stability in some instances. This was evident for liver P(1)450 mRNA, in which an 8-fold rise in transcription was associated with a 27-fold increase in mRNA content, and for kidney P(3)450 mRNA, in which a 2-fold rise in transcription was accompanied by a 12-fold increase in mRNA content. In the kidney and lung of control and TCDD-treated mice, transcriptional rates of the P(3)450 gene were at least 10-fold less than those of the P(1)450 gene. These data indicate that even though both genes are controlled by the same receptor, striking tissue-specific differences in transcription and mRNA stabilization affect the final mRNA concentrations.
Animals↗
Current cellular assays for measuring clinical drug metabolizing capacity--impact of new molecular biologic techniques.
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Antibodies, Monoclonal↗
Autoregulation plus upstream positive and negative control regions associated with transcriptional activation of the mouse P1(450) gene.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to interact with a cytosolic receptor and, in turn, activate transcription of the mouse P1(450) gene. Various lengths of DNA upstream of the P1(450) gene were inserted into the pSV0-cat expression vector, with and without addition of the Harvey murine sarcoma virus (Ha-MSV) 72-bp repeat enhancer element. The constructs were cotransfected with pSV2-neo into mouse hepatoma wild-type cells and two variant cell lines. One variant is believed to result from a mutation in the P1(450) structural gene and expresses high levels of P1(450) mRNA constitutively; the other variant has a defect in nuclear translocation of the inducer-receptor complex. After selection in G418, the cells were treated with control medium, TCDD, cycloheximide, or TCDD plus cycloheximide and then assayed for chloramphenicol acetyltransferase (CAT) activity. The data are consistent with the presence of several functional regions within the upstream sequence: a promoter region, a region that is negatively autoregulated, possible repressor-binding and inducer-receptor complex-binding sites, and an upstream activation element that is required for transcriptional activation by TCDD. The Ha-MSV enhancer can substitute for this upstream activation element.
Acetyltransferases↗
Comparison of aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase induction by polycyclic aromatic compounds in human and mouse cell lines.
The human MCF-7 and the mouse Hepa-1 cell culture lines were compared for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]anthracene (BA) and TCDD- and BA-specific binding in the cytosol and nucleus. The effective concentration of BA in the growth medium required to induce either enzyme to 50% of its maximally inducible activity (EC50) was the same (5-11 microM) in both MCF-7 and Hepa-1 cells. On the other hand, the EC50 for TCDD in MCF-7 cells (5-25 nM) was more than 40-fold greater than that in Hepa-1 cells (0.4 to 0.6 nM). P1-450- and P3-450-specific mouse cDNA probes were used to quantitate mRNA induction in the Hepa-1 cell line. P1-450 mRNA was induced markedly by TCDD and benzo[a] anthracene, whereas P3-450 mRNA was induced negligibly. A P1-450-specific human cDNA probe was used to quantitate P1-450 mRNA induction in the MCF-7 cell line. Aryl hydrocarbon hydroxylase inducibility by TCDD or BA always paralleled P1-450 mRNA inducibility in either the mouse or human line. Although the cytosolic Ah receptor in Hepa-1 cells was easily detected by sucrose density gradient centrifugation, gel permeation chromatography, and anion-exchange high-performance liquid chromatography, the cytosolic receptor cannot be detected in MCF-7 cells. Following in vivo exposure of cultures to radiolabeled TCDD, the intranuclear concentration of inducer-receptor complex was at least fifty times greater in Hepa-1 than MCF-7 cultures. The complete lack of measurable cytosolic receptor and almost totally absent inducer-receptor complex in the nucleus of MCF-7 cells was, therefore, out of proportion to its capacity for aryl hydrocarbon hydroxylase and acetanilide 4-hydroxylase inducibility. This MCF-7 line should provide an interesting model for a better understanding of the mechanisms of drug-metabolizing enzyme induction by polycyclic aromatic compounds, including the Ah receptor-mediated mechanism.
Animals↗
Regional linkage analysis of the dioxin-inducible P-450 gene family on mouse chromosome 9.
The dioxin-inducible P-450 gene family in the C57BL/6N mouse comprises two genes, P1-450 and P3-450. Restriction endonuclease-digested genomic DNA was probed with P1-450 and P3-450 full-length cDNA clones in an attempt to find species-specific fragment length differences between mouse and hamster cell lines and any restriction fragment length polymorphism among four inbred mouse strains. With this Southern blot hybridization technique, PstI fragments were used to distinguish between the mouse and hamster P1-450/P3-450 genes, and PvuII fragments were used to distinguish P3-450 differences between the AKR/J and C57L/J inbred strains. Analysis of nineteen mouse X hamster somatic cell hybrid lines and sixteen AKXL (AKR/J X C57L/J) recombinant inbred lines showed that the P1-450/P3-450 genes are located near the Mpi-1 locus, between the Thy-1 and Pk-3 loci, in the middle portion of mouse chromosome 9.
Animals↗
Human P1-450 gene sequence and correlation of mRNA with genetic differences in benzo[a]pyrene metabolism.
The human P1-450 gene (6,311 base pairs), as well as the 5' (1,604 bases) and 3' (113 bases) flanking regions, have been completely sequenced. Four highly homologous boxes (61, 82, 56 and 97 base pairs) between the human and mouse P1-450 genes are found in the "TATA" box promoter region, -226, -338, and -450 upstream from the cap site, respectively. Nine genomic-DNA samples were digested with each of 23 restriction endonucleases and probed with human P1-450 cDNA fragments; restriction fragment length polymorphisms are detected, although it remains to be seen whether such a recombinant DNA test will be useful in determining individuals at increased risk for cigarette smoking-induced cancer and toxicity. We show in this report, however, that human inducible P1-450 mRNA concentrations are very highly correlated (r = 0.98; N = 6) with genetic differences in benzo[a]pyrene metabolism in mitogen-activated lymphocyte cultures.
Base Composition↗
Complete cDNA and protein sequence of a pregnenolone 16 alpha-carbonitrile-induced cytochrome P-450. A representative of a new gene family.
A full-length cDNA complementary to rat liver mRNA coding for pregnenolone 16 alpha-carbonitrile-induced cytochrome P-450 (P-450PCN) was isolated and completely sequenced. P-450PCN mRNA is 2038 nucleotides in length and has a continuous reading frame (82-1596) that encodes a protein of 504 amino acids (Mr = 57,917). The amino-terminal sequence of 18 residues of the purified P-450PCN protein agrees with the open reading frame of the cDNA sequence. The P-450PCN mRNA nucleotide and amino acid sequences clearly establish that this cytochrome is a member of a separate P-450 family different from the phenobarbital-induced (e.g. P-450e) and 3-methyl-cholanthrene-induced (e.g. P-450c) P-450 gene families. P-450PCN shares 38 and 37% nucleotide similarity and 33 and 33% amino acid similarity with P-450e and P-450c, respectively. P-450PCN, P-450e, and P-450c exhibit greater homology in the C-terminal half than in the N-terminal half of the proteins. Included in this region is the cysteinyl fragment (surrounding residue 443 in P-450PCN), which appears to be the most conserved among all fragments of other P-450 proteins. Of interest, the N-terminal region of P-450PCN does not contain the cysteine residue previously thought to contribute the thiolate ligand to the heme iron in P-450 proteins; these data establish more firmly the cysteine residue located in the carboxylterminal region as serving this function. These sequence studies further support the conclusion derived from chromosomal localization studies and Southern blot analyses that P-450PCN represents a member of a distinct third family of P-450 genes, which diverged from a common ancestor more than 200 million years ago.
Amino Acid Sequence↗
Benzo[a]pyrene metabolism in mouse liver. Association of both 7,8-epoxidation and covalent binding of a metabolite of the 7,8-diol with the Ah locus.
The 7,8-epoxidation of benzo[a]pyrene, and the 9,10-epoxidation of benzo[a]-pyrene trans-7,8-dihydrodiol coupled with covalent binding of the highly reactive diol-epoxide, are two key P-450-mediated reactions believed to be important in cancer initiation, mutagenesis and teratogenesis. New assays for these two reactions were developed with mouse liver microsomes. These two activities have apparent Km values (approximately 6 microM) similar to that of aryl hydrocarbon hydroxylase activity. Twenty-six individual 3-methylcholanthrene-treated Ahb/Ahd and Ahd/Ahd progeny of the (C57BL/6N)(DBA/2N) F1 X DBA/2N backcross were studied. Both of the newly described activities appear to represent P-450 protein(s) that are responsible for aryl hydrocarbon hydroxylase activity and that are coordinately controlled by the Ahb allele.
Animals↗
Comparison of the flanking regions and introns of the mouse 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome P1-450 and P3-450 genes.
The C57BL/6N inbred mouse cytochrome P1-450 and P3-450 genes, two genes in the same family and under control by the Ah receptor, have been completely sequenced. The transcription initiation sites were confirmed by primer extension studies. An additional 823 and 893 bp of the 5' upstream flanking regions of P1-450 and P3-450, respectively, and 1771 and 1251 bp of the 3' downstream flanking regions of P1-450 and P3-450, respectively, were sequenced and studied. P1-450 exons total 2619 nucleotides, and the gene spans 6215 bp. P3-450 exons total 1892 nucleotides, and the gene spans 6716 bp. Three interesting highly homologous regions of 11 or 12 bp, upstream between -280 and -530 from the cap site of both genes, are noted as possible candidates for binding by the inducer-Ah receptor complex (and/or other DNA-binding regulatory proteins). Several stretches of DNA upstream from the cap site, in several introns, and in the 3' flanking region of both genes have a high degree of homology with known core enhancer sequences. Other interesting stretches (DNA with Z-DNA-forming properties, DNA with recombinational potential, highly repetitive and middle repetitive sequences between 50 and 360 bp in length, and "simple" sequences presumably having no function in gene expression) exist throughout many of the introns and flanking regions in both the positive and negative strands of both genes. The mouse 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible and rat phenobarbital-inducible P-450 genes were compared for the amino acid residue number at each exon-intron junction, the location in the coding triplet at which the exons are split, and homologies among introns and exons. It can be shown that these two gene families probably diverged from a common ancestor more than 200 million years ago and that P1-450 and P3-450 split from each other about 65 million years ago.
Animals↗
Human dioxin-inducible cytochrome P1-450: complementary DNA and amino acid sequence.
Induction of cytochrome P1-450 has been linked to susceptibility to certain chemically induced cancers in mouse and man. Treatment of the human cell line MCF-7 with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in high levels of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (P1-450) activity. This cell line was used to isolate a human P1-450 full-length complementary DNA (cDNA) clone. The cDNA is 2566 nucleotides in length, encodes a polyadenylated messenger RNA (2.8 kilobases in length), and has a continuous reading frame producing a protein with 512 residues (molecular weight, 58,151). The human P1-450 cDNA and protein are 63 percent and 80 percent similar to mouse P1-450 cDNA and protein, respectively. Whereas the mouse TCDD-inducible P-450 gene subfamily has two members (P1-450 and P3-450), the human TCDD-inducible gene subfamily appears to have only one gene (P1-450).
Amino Acid Sequence↗
Assignment of the human 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome P1-450 gene to chromosome 15.
The human 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome P1-450 full-length cDNA has been recently isolated and sequenced [Jaiswal, A.K., Gonzalez, F.J. and Nebert, D.W. (1985) Science, in press]. A 1521-bp 5' DNA fragment representing almost all of the translating region was used to probe DNA from human, mouse, hamster, 53 human X mouse somatic cell hybrids, and 36 human X hamster somatic cell hybrids. These data indicate that the P1-450 gene resides on human chromosome 15. Knowledge of the chromosomal assignment of this gene should help in our understanding of its regulation and role in development and disease.
Base Sequence↗
Mutations affecting the regulation of transcription of the cytochrome P1-450 gene in the mouse Hepa-1 cell line.
The cytochrome P1-450-dependent activity, aryl hydrocarbon hydroxylase, and cytochrome P1-450 messenger RNA levels were studied in wild-type Hepa1c1c7 cells and in aryl hydrocarbon hydroxylase-deficient mutants derived from this line. Tetrachlorodibenzo-p-dioxin (TCDD) induced both parameters approximately 50-fold in Hepa1c1c7 cells. Mutants in genes B and C that are affected in the functioning of the Ah receptor required for hydroxylase induction and dominant mutants had nondetectable or much reduced P1-450 mRNA levels and hydroxylase activities after TCDD treatment. Hybrids between wild-type cells and the dominant mutants were also deficient in these parameters. The dominant mutants therefore appear to express a trans-acting repressor of cytochrome P1-450 transcription. Mutants in gene A were heterogenous. Some lacked the mRNA completely; others were inducible for it; while still others (subgroup IV) had high levels even when they were grown without TCDD. These results suggest strongly that gene A is the structural gene for cytochrome P1-450. When subgroup IV mutants were cultured together with wild-type cells, they failed to express P1-450 mRNA in the absence of TCDD treatment. These cells probably accumulate an inducer that can be metabolized by aryl hydrocarbon hydroxylase. The inducer did not appear to be a component of the medium and therefore may be an endogenous ligand of the Ah receptor.
Animals↗
Measurements of the cytosolic Ah receptor among four strains of Drosophila melanogaster.
Four strains of Drosophila melanogaster exhibit differences in aryl hydrocarbon hydroxylase (AHH) inducibility by phenobarbital or Aroclor 1254, yet do not show the typical AHH induction response when exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[a]anthracene. Adult flies were nevertheless examined for the presence of cytosolic TCDD-specific binding (Ah receptor). Berlin-K and Haag 79 exhibit AHH induction by Aroclor 1254 and possess detectable amounts of Ah receptor. Hikone-R has negligible AHH inducibility by Aroclor 1254, yet possesses measurable amounts of the receptor. Oregon-K displays AHH induction by Aroclor 1254 but has no detectable levels of the cytosolic receptor. Specific (high-affinity, low-capacity and saturable) binding of [3H-1,6]TCDD to the Ah receptor in D. melanogaster was shown to be similar to that observed in C57BL/6 mouse liver. Similar specific binding of generally labeled [3H]benzo[a]anthracene in D. melanogaster cytosol was not found. These data suggest that the presence of the Ah receptor per se, or quantity of receptor, does not guarantee AHH inducibility by TCDD or benzo[a]anthracene in adults of these four fruit fly strains.
Animals↗