Letter from the editor
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Journal Article↗
Biomedical subjects
E Bresnick
Publications and source records attributed to E Bresnick.
The existence of the 4S polycyclic aromatic hydrocarbon-protein binding in 14-day-old chick embryo liver.
Cytochrome P-450IA1, the isozyme most closely associated with aryl hydrocarbon hydroxylase (AHH), is regulated by two high-affinity binding proteins, the 4S polycyclic aromatic hydrocarbon (PAH)-binding protein which primarily binds PAHs and the 8S Ah (dioxin) receptor which binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and like congeners. The present study was conducted to determine whether the 4S protein existed in 14-day-old chick embryo liver when AHH activity is maximal to determine if they are linked as is the 8S Ah receptor and to confirm the existence of the dioxin receptor by investigating their ligand binding characteristics in the presence and absence of sodium molybdate, an agent that stabilizes steroid hormone receptors and partially stabilizes the dioxin receptor. Competitive ligand binding studies were performed with liver cytosol from livers of male 14-day-old chick embryos using [3H]-benzo[a]pyrene (B[a]P) or [3H]-TCDD in the presence and absence of a 200-fold excess of B[a]P, benzo[e]pyrene (B[e]P), 3-methylcholanthrene (3-MC), and tetrachlorodibenzofuran (TCDBF). Specific PAH-binding activity was assayed using sucrose gradient analysis. In the absence of molybdate, the 4S PAH-binding protein had high affinity for B[a]P, B[e]P, 3-MC, but very low affinity for TCDBF; the Ah receptor exhibited high affinity for TCDBF. In the presence of sodium molybdate, the Ah receptor was stabilized while the 4S PAH-binding protein was relatively unaffected. These results affirm the existence of two distinct PAH-binding proteins in 14-day-old chick embryo liver cytosol and suggest a linkage of the 4S protein to AHH.
Animals↗
Innovative approaches to the prevention, diagnosis, and therapy of cancer: fourth joint conference of the American Association for Cancer Research (AACR) and the Japan Cancer Association (JCA).
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Animals↗
Polycyclic aromatic hydrocarbon (PAH)-binding protein and glycine N-methyltransferase (GNMT)
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Animals↗
Glycine N-methyltransferase is an example of functional diversity. Role as a polycyclic aromatic hydrocarbon-binding receptor.
The cytochrome P-4501A1 (CYP1A1) gene is regulated by several trans-acting factors including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein, which has recently been identified as glycine N-methyltransferase (GNMT) (Raha, A., Wagner, C., Macdonald, R. G., and Bresnick, E. (1994) J. Biol. Chem. 269, 5750-5756). The role of GNMT as a 4 S PAH-binding protein in mediating the induction of cytochrome P-4501A1 has been investigated further. GNMT cDNA, which was cloned into a pMAMneo vector containing the Rous sarcoma virus promoter and the neomycin resistance gene, was stably transfected into D422 Chinese hamster ovary (CHO) cells. Several positive clones were selected by reverse transcription-polymerase chain reaction and assayed for the expression of recombinant protein. Western blot analysis indicated the expression of significant levels of the 4 S protein in the stably transfected CHO cells (CHO-GNMT). Cytosolic preparations from the CHO-GNMT showed high benzo[a]pyrene (B[a]P) binding but no 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding activity when compared with clones transfected with the pMAMneo vector alone (CHO-neo) or the parental CHO cells. Challanging the CHO-GNMT cells with 4 microM B[a]P resulted in elevated levels of CYP1A1 mRNA. Equally effective in inducing CYP1A1 mRNA were benzo[e]pyrene and 3-methylcholanthrene. On the other hand, TCDD did not induce CYP1A1 gene expression in these cells. B[a]P-treated CHO-GNMT, expressing the 4 S protein, also showed CYP1A1 protein by Western blotting and exhibited ethoxyresorufin-O-deethylase activity; neither the CHO-neo or parental CHO cells were positive for any of these measures. No Ah receptor message or protein was detectable in the parental CHO, CHO-neo, or CHO-GNMT cells. Furthermore, no XRE binding activity was observed in TCDD-treated cytosolic preparations or nuclear extracts from CHO-GNMT cells that were treated with TCDD. These studies unequivocally establish that GNMT is a PAH-binding protein that can mediate the induction of CYP1A1 by PAHs such as B[a]P through an Ah receptor-independent pathway.
Animals↗
The homodimeric form of glycine N-methyltransferase acts as a polycyclic aromatic hydrocarbon-binding receptor.
In the rat, cytochrome P4501A1 gene expression is thought to be regulated by several trans-acting factors including the 4S polycyclic aromatic hydrocarbon (PAH)-binding protein, which was recently identified as glycine N-methyltransferase [Raha, A., Wagner, C., MacDonald, R. G., and Bresnick, E. (1994) J. Biol. Chem. 268, 5750-5756]. Glycine N-methyltransferase (GNMT) is one of those unique proteins which exhibit diversity in function. Different subunit configurations are involved in its enzymatic role as a methyltransferase and as PAH-binding receptor. Here we report a systematic study of the oligomeric state of GNMT in the presence of benzo[a]pyrene (B[a]P) in vivo and in vitro. We have used chemical cross-linking and denaturing polyacrylamide gel electrophoresis to show that the B[a]P-binding unit of GNMT is a homodimer. We recently reported that phosphorylation is involved in the interaction of B[a]P with the 4S PAH-binding protein [Bhat, R., Weaver, J. A., Wagner, C., Bodwell, J. E., and Bresnick, E. (1996) J. Biol. Chem. 271, 32551-32556]. In the present study, this observation has been amplified by using bacterially expressed GNMT, which was post-translationally modified by a reticulocyte lysate and ATP-generating system. This modification was also accompanied by the formation of homodimers in the presence of B[a]P. These results indicate that post-translational modification is involved in determining the final configuration, i.e., dimeric form, of GNMT which then acts as a PAH-binding receptor.
Animals↗
Identification of positive and negative regulatory elements of the human cytochrome P4501A2 (CYP1A2) gene.
We previously demonstrated an enhancer-like positive regulatory element within a 259-bp sequence (-2352 to -2094 bp) of the human CYP1A2 gene in HepG2 cells. Three protein binding sites were identified by DNase I footprinting analyses within the 259-bp sequence: protected region A PRA; -2283 to -2243 bp), PRB (-2218 to -2187 bp), and PRC (-2124 to -2098 bp) (I. Chung and E. Bresnick, Mol. Pharmacol. 47, 677-685, 1995). In the present study, the functional significance of those protected regions was examined. Transfection experiments with deletion and substitution mutants defined the PRB and PRC as containing positive and negative regulatory elements, respectively. Human breast carcinoma MCF-7 cells were cotransfected with a hepatocyte nuclear factor-1 (HNF-1) expression vector and CYP1A2 promoter- or thymidine kinase promoter-luciferase reporter gene constructs. HNF-1, which contributes to the liver specificity of genes, enhanced reporter gene activity in a PRC sequence-dependent manner. These results suggested that PRC could exist bound to a repressor which was displaceable by other transcription factors such as HNF-1. Results obtained by transfection of HepG2 hepatoma cells with various PRB substitution mutant-luciferase gene fusion constructs indicated that the entire sequence of PRB was necessary for promoter activity. Consequently, the regulation of CYP1A2 expression is very complex, requiring a number of both positive and negative regulatory factors.
Base Sequence↗
ATP depletion affects the phosphorylation state, ligand binding, and nuclear transport of the 4 S polycyclic aromatic hydrocarbon-binding protein in rat hepatoma cells.
In the rat, cytochrome P4501A1 gene expression is thought to be regulated by several trans-acting factors including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein. Phosphorylation and dephosphorylation have been suggested to influence the function of many cytosolic receptors and transcription factors. The ATP level within H4IIE rat hepatoma cells could be depleted by treatment with sodium azide or 2,4-dinitrophenol; restoration of the original ATP levels occurred with addition of glucose to the cell culture. ATP depletion reduced the phosphate content of the 4 S protein by approximately 25-30%, which lowered the binding of benzo[a]pyrene (B[a]P) to the 4 S protein by >60%. This effect could not be reversed by the addition of ATP to the binding reaction mixtures. Alkaline phosphatase treatment of the purified 4 S protein in a cell-free system also reduced the B[a]P binding to the protein. Cells treated with a protein phosphatase inhibitor, okadaic acid, and a protein kinase inhibitor, staurosporin, affected the B[a]P binding of the 4 S protein positively and negatively, respectively. These data suggested that phosphorylation is involved in the interaction of the 4 S protein with the PAH. The nuclear translocation of the predominantly cytosolic binding protein has been investigated after ligand binding. Western blots with the immunopurified 4 S PAH-binding protein from cytosolic and nuclear lysates showed significant differences in the distribution of the 4 S receptor between cytosolic and nuclear compartments in control and ATP-depleted cells. Ligand binding stimulated the movement of the receptor into the nucleus, which was completely blocked by reducing the intracellular ATP concentration. These findings provide new information on the role of ATP and phosphorylation on the interaction of B[a]P with 4 S PAH-binding protein and its nuclear translocation.
2,4-Dinitrophenol↗
Retrovirus-mediated transfer of the human O6-methylguanine-DNA methyltransferase gene into a murine hematopoietic stem cell line and resistance to the toxic effects of certain alkylating agents.
O6-Methylguanine-DNA methyltransferase (MGMT) is an important DNA repair protein that plays a key role in cancer chemotherapy by alkylating agents such as carmustine (BCNU) and Dacarbazine (DTIC). Therapy by BCNU and DTIC is reduced by dose-limiting hematological toxicity as a result of low MGMT repair activity in bone marrow cells. In this study, we have constructed a Moloney murine leukemia virus retroviral vector containing the human mgmt gene. High-titer retrovirus producer cells lines have been generated. Retroviral-mediated transfer of the human mgmt gene into murine multi-potent hematopoietic stem cells, FDCP-1, resulted in the expression of a high level of MGMT activity. In comparison with the control cells that were transduced with the parent vector, the MGMT-expressing clones were considerably more resistant to the cytotoxicity of the methylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine, N-nitroso-N-methyl-urea, and temozolomide, as well as the chloroethylating agents 1-(2-chloroethyl)-1-nitrosourea and BCNU. The protection provided by MGMT could be eliminated by the MGMT inactivator O6-benzylguanine. Thus, the principal lethal lesions produced by these alkylating agents in the murine hematopoietic stem cells and the MGMT deficiency in these cells can be complemented by retroviral-mediated gene transduction.
3T3 Cells↗
Nuclear transcription factor Oct-1 binds to the 5'-upstream region of CYP1A1 and negatively regulates its expression.
The cytochrome P450-dependent monooxygenases, which represent an extended superfamily, catalyze the biotransformation of many endogenous and exogenous substances. One of these hemoproteins, cytochrome P4501A1, is most closely associated with the bioactivation of polycyclic aromatic hydrocarbons such as benzo[a]pyrene, which may play a role in environmental carcinogenesis. A negative regulatory element (NRE) has been localized in the 5'-upstream region of the cytochrome P4501A1 gene (CYP1A1) at -843 to -746 base pairs from the site of transcription. The purpose of this research was to define any interactions of trans-acting proteins with this cis element. Rat liver nuclei were used as the source of trans-acting proteins and a biotinylated NRE-bearing fragment (-782 to -843 bp) from a plasmid which contained the CYP1A1 was prepared by the polymerase chain reaction technique. Gel mobility shift assays were used to demonstrate interactions between this NRE fragment and nuclear proteins. The specific binding to an octamer-containing motif in the 5'-upstream region of CYP1A1 was demonstrated; this was used as a step in the partial purification from rat liver of the transcription factor, Oct-1. Conventional chromatographic procedures and DNA recognition site affinity chromatography were also used. HepG2 human hepatoma cells were transfected with both pMCoLUC+ which contains the luciferase gene as a reporter gene driven by the CYP1A1 promoter (including the NRE), and an Oct-1 expression vector. Luciferase activity/mg protein in the doubly-transfected cells was significantly lower than in cells containing only pMCoLUC+. A nuclear transcription factor Oct-1 interacts with a portion of the NRE of the rat CYP1A1, suppressing the expression of this gene. These findings may help to explain the low level of basal expression of CYP1A1 in mammalian systems.
Animals↗
Oct-1 transcription factor is a negative regulator of rat CYP1A1 expression via an octamer sequence in its negative regulatory element.
The rat CYP1A1 negative regulatory element (NRE) contains AP-1 and Oct-1 motifs at -808 to -788 bp. The CYP1A1 sequence from -813 to -779 bp and an identical sequence bearing a point mutation in the octamer motif were synthesized. Gel mobility shift assays showed the formation of two complexes with the wild-type CYP1A1 sequence and nuclear extracts from H4IIE and HepG2 hepatoma cells and from rat liver. The formation of the major complex was significantly reduced with the mutant octamer-containing oligomer and was specifically competed by an Oct-1 oligodeoxyribonucleotide. The addition of Oct-1 antibody caused a supershift of the major complex. The presence of the wild-type sequence, but not the mutant octamer sequence, caused a 3-fold decrease in SV40 enhancerless promoter activity in transfected HepG2 cells. Co-transfection of an Oct-1 expression vector with rat CYP1A1 NRE octamer-containing, promoter/reporter gene constructs specifically further decreased promoter activity of the wild-type octamer-containing constructs in HepG2 cells. The results indicate that Oct-1 binds to the rat CYP1A1 promoter NRE and is a negative regulator of rat CYP1A1 expression.
Animals↗
Glycine N-methyltransferase is a mediator of cytochrome P4501A1 gene expression.
Cytochrome P4501A1, the isozyme most closely approximating aryl hydrocarbon hydroxylase activity under conditions of induction, is thought to be regulated by several trans-acting factors, including the 4S polycyclic aromatic hydrocarbon-binding protein; this protein has recently been identified as glycine N-methyltransferase (Raha et al. (1994) J. Biol. Chem. 269, 5750-5756). Previous studies had shown that partially purified liver preparations containing the 4S binding protein interacted with 5'-flanking regions of the cytochrome P4501A1 gene. Consequently, the ability of the 4S binding protein to serve as a mediator in the regulation of the cytochrome P4501A1 gene was investigated further. Introduction of an antisense 24-mer oligonucleotide to glycine N-methyltransferase cDNA into rat hepatoma H4IIE cells by lipofectin resulted in a 60% reduction in the benzo(a)pyrene-mediated induction of ethoxyresorufin-O-deethylase activity and protein over the sense and scrambled antisense oligonucleotide controls. In addition, the antisense oligonucleotide caused a marked reduction in the steady-state level of cytochrome P4501A1 mRNA; no such effect was observed with the sense oligonucleotide. Introduction of GNMT polyclonal antibodies into H4IIE cells by a streptolysin-O permeabilization technique markedly reduced both benzo(a)pyrene-binding and benzo(a)-pyrene-induced ethoxyresorufin-O-deethylase activities, but had no effect on 2,3,7,8-tetrachlorodibenzo-p-dioxin induction. Collectively, these findings suggest that, in addition to the Ah (dioxin) receptor, glycine N-methyltransferase appears to be both a polycyclic aromatic hydrocarbon-binding protein and a mediator of the induction of the cytochrome P4501A1 gene by polycyclic hydrocarbons such as benzo(a)pyrene.
Animals↗
Regulation of the constitutive expression of the human CYP1A2 gene: cis elements and their interactions with proteins.
Cytochrome P4501A2 (CYP1A2) is a member of the cytochrome P450 family that is involved in phase I drug metabolism in vertebrates. To understand how the constitutive expression of the human CYP1A2 gene is regulated, its 5' flanking region was analyzed. The promoter activity of a human CYP1A2 gene sequence [base pairs (bp) -3203 to +58 bp] was measured in transiently transfected HepG2 cells using fusion constructs containing the luciferase reporter gene. Using 5'-end deletion analysis, two functionally important cis elements, i.e., a proximal 42-bp DNA from bp -72 to bp -31 and a distal 259-bp DNA from bp -2352 to bp -2094, were identified. The proximal sequence (bp -72 to -31) contained CCAAT and GC boxes, with which well characterized transcription factors such as nuclear factor-1/CCAT transcription factor and simian virus 40 promoter factor-1 could interact. With regard to the 259-bp fragment (bp -2352 to bp -2094), gel mobility shift analyses with HepG2 nuclear lysates indicated high affinity, specific interactions of several trans-acting factors. Three protein binding sites within the 259-bp fragment were identified by DNase 1 footprinting analysis; these sites contained activator protein-1, nuclear factor-E1.7, and one-half hepatic nuclear factor-1 (HNF-1) binding consensus sequences. Only the region from bp -2124 to bp -2098, in which the HNF-1 binding site was located, was markedly protected by a HepG2 nuclear extract, compared with a MCF7 human breast cancer nuclear extract. These results suggested that the 259-bp DNA fragment contained positive regulator binding sites and HNF-1 could contribute to the liver-specific expression of human CYP1A2.
Base Sequence↗
Analysis of within-subject variation of caffeine metabolism when used to determine cytochrome P4501A2 and N-acetyltransferase-2 activities.
Cytochrome P4501A2 (CYP1A2) and N-acetyltransferase-2 (NAT2) are hepatic enzymes that may activate some procarcinogens. Previous reports have determined CYP1A2 and NAT2 phenotypes by quantitating relative amounts of urinary caffeine and metabolites. However, a number of experimental issues with this approach remain. To address these, we measured caffeine and 4 metabolites in urine samples from 20 healthy volunteers on 3 separate occasions at 7-day intervals. Two additional volunteers were studied to measure the pattern of excretion of these analytes in urine over time. The molar ratio of two compounds (1,7-dimethylxanthine/1,3,7-trimethylxanthine) was used to phenotype CYP1A2, while the molar ratio of two other compounds (5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine) served to phenotype NAT2. Within-subject variation was less than 25% for most participants. In instances when within-subject variation of the metabolic ratio was > 25%, metabolite peaks were usually present in one or more control urine samples. Some caffeine metabolites were observed in urine samples at detectable levels up to 48 h after caffeine ingestion. We conclude that: (a) this assay for determining CYP1A2 and NAT2 activities (phenotyping) has an acceptably low within-subject variation over 3 consecutive weeks for most subjects who were caffeine-free for 36 h prior to study; (b) collecting and analyzing urine samples prior to testing can indicate if subjects are excreting caffeine metabolites and will aid in locating metabolite peaks on chromatograms; (c) refraining from caffeine for 48 h before testing is the best compromise between convenience for the subject and obtaining reproducible results; (d) determining metabolite molar ratios in urine collected 4-5 h after ingesting caffeine provides an acceptable time for the measurement; and (e) different ratios of metabolites for determining CYP1A2 phenotype have different advantages.
Adult↗
Rat liver cytosolic 4 S polycyclic aromatic hydrocarbon-binding protein is glycine N-methyltransferase.
In the rat, cytochrome P-450IA1 gene expression, which is most closely associated with aryl hydrocarbon hydroxylase activity, is thought to be regulated by several trans-acting factors, including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein. This protein has been purified to homogeneity from rat liver using ion exchange, gel permeation, hydrophobic interaction, and affinity chromatographies. Partial sequencing of the 33-kDa band indicated its identity as glycine N-methyltransferase (GNMT). Polyclonal antibodies to GNMT immunoprecipitated PAH-binding activity from rat liver cytosol. Methyltransferase and PAH-binding activities copurified during the course of protein purification. GNMT protein and PAH binding activity were colocalized in various cytosolic fractions. These data all indicate that the 4 S PAH-binding protein and GNMT are one and the same protein or very similar proteins. Western blot analyses yielded a positive signal under denaturing (33 kDa) and nondenaturing (150 kDa, tetramer) conditions; the PAH-binding protein also was an oligomer. GNMT was detected by immunohistochemistry in nuclei from H4IIE rat hepatoma cells and rat liver. The localization of GNMT in liver nuclei is in accordance with a role in modulating cytochrome P-450IA1 gene expression.
Amino Acid Sequence↗
Inhibition of human O6-methylguanine-DNA methyltransferase by 5-methylcytosine.
The ability of cloned human O6-methylguanine-DNA methyltransferase to repair a methylated guanine in a CpG-containing sequence, i.e., island, was studied by using a synthetic double-stranded 20-mer oligonucleotide from codon 248 of the p53 gene and another designed sequence. The double-stranded oligonucleotides incorporating 5-methylcytosine (5mC) and O6-methylguanine (O6mG) in various combinations in a CpG site were 5' labeled with 32P and incubated with recombinant O6-methylguanine-DNA methyltransferase. The rate constant for O6-methylguanine-DNA methyltransferase repair of O6mG in this oligomer was always higher with the substrate which contained only the O6mG, as compared to the oligomer that included a 5mC adjacent in the 5'-position to the methylated guanine. The reduction in substrate activity ranged from 75% (modified p53 sequence) to 100% (in the designed oligomer). A 5mC opposite the O6mG reduced the rate slightly. These results suggest that O6-methylation of the guanine moiety at CpG islands may not be efficiently repaired when normal 5mC is present and this may contribute significantly to an increase in mutagenesis of p53 and like molecules.
5-Methylcytosine↗
3-Methylcholanthrene-mediated induction of cytochrome P4501A2 in human hepatoma HepG2 cells as quantified by the reverse transcription-polymerase chain reaction.
The feasibility of using HepG2 human hepatoma cells to study the regulation of the expression of the cytochrome P4501A2 gene (CYP1A2) was examined. The reverse transcription-polymerase chain reaction (RT-PCR) assay revealed that HepG2 cells constitutively express CYP1A2 and are able to respond to 3-methylcholanthrene (3MC) by an induction of CYP1A2 mRNA. In these studies, selected sequences from intron-exon junctions were used as mRNA-specific primers for both CYP1A2 and the beta-actin gene. The level of induction was quantitated based on two parameters within the exponential phase of the amplification: the difference in the number of cycles that yields the same level of amplification and the efficiency of the PCR reaction. Using this method, it was estimated that the CYP1A2 steady-state mRNA level increased to a maximum of 12-fold at 24 h after exposure of the cells to 3MC. Both a reduction in basal expression as well as an increased accumulation of CYP1A2 mRNA appeared responsible for the overall induction at 24 and 48 h. These results suggested that the HepG2 cell line would be appropriate for studying the regulation of CYP1A2 expression.
Base Sequence↗
Induction of CYP1A1 gene expression in mouse hepatoma cells by benzo[e]pyrene, a ligand of the 4S polycyclic hydrocarbon-binding protein.
Hepa 1c1c7 (WT), TAOc1BPrc1 (CI), and BPrc1 (CII) mouse hepatoma cells were exposed to benzo[e]pyrene (B[e]P) or benzo[a]pyrene (B[a]P). B[e]P induced activity of a rat CYP1A1 reporter gene construct (-3015 to +2545 bp) by 1.8- to 2-fold and 5-fold in WT and CI cells, respectively. B[e]P caused a 2-fold induction of a truncated CYP1A1 reporter gene construct (-658 to +2545 bp) in WT cells and induced ethoxyresorufin O-deethylase (EROD) activity by 24- and 13-fold in WT and CI cells. B[a]P also induced CYP1A1 reporter gene and EROD activity in these cells. WT and CII cells had both 8S (Ah) receptor and 4S polycyclic hydrocarbon (PAH)-binding activity, while CI cells exhibited a lower 4S binding activity; 8S binding activity was not detected in CI cells under two separate binding conditions. 8S binding activity in the presence of sodium molybdate was 60-fold greater in WT cells than in CII cells. The absence of sodium molybdate resulted in a dramatic decrease of 8S binding activity in WT cells. The ability of B[e]P to induce CYP1A1 promoter-reporter gene activity and EROD activity in WT and CI cells suggested a role for the 4S PAH-binding protein in the induction of CYP1A1. The lack of detectable 8S binding activity in CI cells was in concert with this role.
Animals↗