The regulation of gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
J P Whitlock
Publications and source records attributed to J P Whitlock.
Control of gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Multiple dioxin-responsive domains 5'-ward of the cytochrome P1-450 gene.
In mouse hepatoma cells, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) stimulates cytochrome P1-450 gene transcription via the formation of TCDD-receptor complexes and the accumulation of the complexes in the nucleus. Here, we show that the DNA that flanks the 5'-end of the cytochrome P1-450 gene contains at least two discrete TCDD-responsive domains. Each domain has properties analogous to those of transcriptional enhancers. Each domain requires TCDD-receptor complexes for its function. There is no significant nucleotide sequence homology between the two TCDD-responsive domains.
Animals↗
Control of cytochrome P1-450 gene expression: analysis of a dioxin-responsive enhancer system.
We analyzed the function and sequence of a dioxin-responsive genomic element flanking the 5' end of the cytochrome P1-450 gene in high-activity variant mouse hepatoma cells. The element can regulate the mouse mammary tumor virus promoter. The element retains responsiveness to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) when the distance, the 5' or 3' position, and/or the 5' or 3' orientation with respect to the promoter are varied. The function of the element requires TCDD-receptor complexes. The element remains responsive to TCDD when transfected into cells from either a heterologous mouse tissue or a heterologous species (human). The DNA element and TCDD receptors together constitute a dioxin-responsive enhancer system.
Acetyltransferases↗
The regulation of cytochrome P-450 gene expression.
Explore the source record for details and available documents.
Animals↗
Superinduction of cytochrome P1-450 gene transcription by inhibition of protein synthesis in wild type and variant mouse hepatoma cells.
Inhibition of protein synthesis superinduces transcription of the cytochrome P1-450 gene in Hepa 1c1c7 mouse hepatoma cells. The superinduced transcription rate is 10-15-fold higher than the maximal rate of transcription induced by the known inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alone. Superinduction is maximal within 30-40 min and remains maximal for at least 90 min. Cytochrome P1-450 mRNA is the same length in TCDD-induced and superinduced cells. Superinduction does not occur in variant cells in which TCDD-receptor complexes bind weakly to nuclei and which do not transcribe the cytochrome P1-450 gene in response to TCDD. Inhibition of protein synthesis does not alter several properties of TCDD-receptor complexes. The results imply that a second control mechanism modulates the action of the TCDD-receptor complex in regulating cytochrome P1-450 gene transcription.
Animals↗
Control of cytochrome P1-450 gene expression by dioxin.
The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may produce its effects by altering gene expression in susceptible cells. In mouse hepatoma cells, TCDD induces the transcription of the cytochrome P1-450 gene, whose product, aryl hydrocarbon hydroxylase, contributes both to the detoxification and to the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons. A DNA fragment containing sequences flanking the 5' end of the cytochrome P1-450 gene was isolated and analyzed. This DNA fragment contains a cis-acting control element with at least three functional domains: a putative promoter, an inhibitory domain upstream from the promoter that blocks its function, and a TCDD-responsive domain still farther (1265 to 1535 base pairs) upstream of the promoter. These findings, together with results from earlier studies, imply that transcription of the cytochrome P1-450 gene is under both positive and negative control by at least two trans-acting regulatory factors.
Acetyltransferases↗
Biochemical and genetic analysis of variant mouse hepatoma cells which overtranscribe the cytochrome P1-450 gene in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Using the fluorescence-activated cell sorter, we have isolated a population of variant mouse hepatoma cells which have a markedly increased ability to metabolize benzo(a)pyrene. Compared with wild-type (Hepa 1c1c7) cells, the variant cells exhibit increased aryl hydrocarbon hydroxylase activity and increased responsiveness of the aryl hydrocarbon hydroxylase induction mechanism to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cell fusion experiments indicate that the variant phenotype is co-dominant with respect to wild-type. Filter hybridization analyses indicate that increased accumulation of cytochrome P1-450-specific mRNA accounts for the overproduction of aryl hydrocarbon hydroxylase activity. Measurements of RNA synthesis in isolated nuclei reveal that the variants exhibit an increased rate of transcription of the cytochrome P1-450 gene in response to TCDD. The variant cells contain no detectable alteration in their TCDD receptors, nor is the cytochrome P1-450 gene amplified in the variants. Filter hybridization analyses of restriction endonuclease-digested DNA indicate that the variant cytochrome P1-450 gene is relatively undermethylated, compared with the wild-type gene. We conclude that the variant cells contain an altered cis-acting genomic element(s) which regulates the expression of the cytochrome P1-450 gene.
Animals↗
Regulation of cytochrome P1-450 gene transcription by 2,3,7, 8-tetrachlorodibenzo-p-dioxin in wild type and variant mouse hepatoma cells.
Using RNA synthesized in isolated nuclei, we have measured the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the rate of synthesis of cytochrome P1-450 mRNA in wild type mouse hepatoma (Hepa 1c1c7) cells and in variant (BPrc1) cells, which fail to accumulate the TCDD-receptor complex within the nucleus. In wild type cells, TCDD induces a 20-fold increase in the rate of synthesis of cytochrome P1-450 mRNA within 30 min. This effect persists for at least 18 h. In contrast, TCDD has no effect on cytochrome P1-450 mRNA synthesis in the variant cells. The results demonstrate that TCDD increases the rate of transcription of the cytochrome P1-450 gene and suggest that transcription requires nuclear localization of the TCDD-receptor complex.
Animals↗
2,3,7,8-Tetrachlorodibenzo-p-dioxin receptors in wild type and variant mouse hepatoma cells. Nuclear location and strength of nuclear binding.
The volume of aqueous solvent present during subcellular fractionation of mouse hepatoma (Hepa 1c1c7) cells influences the distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptors between the nuclear and cytosolic fractions. When the effects of dilution are minimized, at least 80% of the receptors associate with nuclei. The receptors bind relatively strongly to nuclei, as measured by their release by KCl. TCDD-receptor complexes bind more strongly to nuclei than do unoccupied receptors. A temperature-dependent event further enhances the binding of TCDD-receptor complexes to nuclei. A class of variant cells contains receptors which bind relatively weakly to nuclei; this defect accounts for the variant phenotype. We conclude that, in the intact cell, TCDD receptors are located within the nucleus and that the temperature-dependent event in the induction of cytochrome P1-450 gene expression is one which strengthens the binding of the TCDD-receptor complex to chromatin.
Animals↗
Induction of mRNA specific for cytochrome P1-450 in wild type and variant mouse hepatoma cells.
We have used a cDNA probe specific for an aromatic hydrocarbon-inducible form of cytochrome P-450 to analyze the accumulation of enzyme-specific mRNA in wild type Hepa 1c1c7 cells and in variant cells defective in aryl hydrocarbon hydroxylase induction. In wild type cells, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces a 25-50-fold increase in the steady state concentration of cytochrome P1-450-specific RNA. This RNA binds to oligo(dT)-cellulose and migrates in agarose gels as a single species of about 2900 nucleotides. Dot hybridization analyses of total RNA indicate that half-maximal induction of P1-450 mRNA occurs at about 10 pM TCDD; maximal induction occurs at about 100 pM TCDD. Parallel increases occur in aryl hydrocarbon hydroxylase activity. RNA induction precedes aryl hydrocarbon hydroxylase induction by 2-4 h. Kinetic analyses of mRNA induction in response to submaximal concentrations of TCDD reveal no change in the degradation of P1-450 mRNA during induction. We detect an increase in P1-450 mRNA 30 min after TCDD administration. Full accumulation of mRNA occurs in cells in which protein synthesis is inhibited by 95-97%. Variant cells with decreased TCDD receptors exhibit decreased basal and inducible levels of P1-450 mRNA. Variant cells with a defect in nuclear localization of the inducer-receptor complex exhibit virtually no basal or inducible levels of P1-450 mRNA. Hybrid cells formed by fusing these different variants contain wild type basal and inducible levels of P1-450 mRNA. We conclude that expression of the cytochrome P1-450 gene is under transcriptional control and requires nuclear localization of the TCDD receptor.
Animals↗
Biochemical and genetic analysis of variant mouse hepatoma cells defective in the induction of benzo(a)pyrene-metabolizing enzyme activity.
We have analyzed wild type mouse hepatoma (Hepa 1c1c7) cells and variant cells which are defective in the induction of benzo(a)pyrene-metabolizing enzyme activity. One type of variant has no detectable basal or inducible aryl hydrocarbon hydroxylase activity. This class contains apparently normal cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin, but is unable to translocate the inducer-receptor complex to the nucleus. The second type of variant has low levels of basal and inducible aryl hydrocarbon hydroxylase activity. This class contains cytosolic receptors which are decreased either in their number or in their ability to bind 2,3,7,8-tetrachlorodibenzo-p-dioxin; translocation of the inducer-receptor complex to the nucleus is apparently normal. Cell fusions indicate that both variant phenotypes are recessive with respect to wild type. Complementation analyses indicate that the defects are located on different genes.
Animals↗
Acetylation and calcium-dependent phosphorylation of histone H3 in nuclei from butyrate-treated HeLa cells.
In HeLa nuclei, 1 microM Ca2+ stimulates 3-fold the phosphorylation of histone H3. Prior treatment of cells with Na butyrate increases the degree of H3 phosphorylation and reveals a correlation between the extents of H3 acetylation and phosphorylation. Acetylation of H3 increases its accessibility to the calcium-dependent kinase. Phosphorylation of H3 occurs at a serine residue located in the trypsin-sensitive region of the protein. Brief digestion of nuclei with DNase I preferentially releases the phosphorylated form of H3 from chromatin.
Acetylation↗
Heterogeneity in the rate of benzo[a]pyrene metabolism in single cells: quantitation using flow cytometry.
We describe a method for quantitating heterogeneity in the rate of benzo[a]pyrene metabolism in single cells by using flow cytometry. We have used the technique to study the response of Hepa-1c1c7 mouse hepatoma cells to the microsomal enzyme inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cells responded in a relatively homogeneous fashion at different times of induction with a maximally inducing concentration of the inducer. However, the induction response could be heterogeneous at a submaximal inducer concentration. We found even higher heterogeneity of enzyme activity among low-activity variants derived from the Hepa-1c1c7 cell line. When cells of either high or low activity were isolated from such a clonal population, propagated, and reanalyzed, they displayed average enzyme activity and heterogeneity identical to the parental cells; therefore, the heterogeneity represents transient, nonheritable differences between cells within the population.
Animals↗
Efficient metabolism of benzo(a)pyrene at nanomolar concentrations by intact murine hepatoma cells.
We have studied the metabolism of benzo(a)pyrene (BP) by intact mouse hepatoma cells, at nM concentrations of the carcinogen, using an assay in which we directly measure the rate of BP fluorescence disappearance. The rate of BP metabolism is half-maximal, at limiting cell dilution, when the concentration of BP is about 4 nM. This apparent Km for BP metabolism is much lower than those reported previously for several reasons. (a) Partitioning of BP into cells markedly influences kinetic measurements, and we account for these effects. (b) Enzyme inducers can competitively inhibit BP metabolism and thus may introduce artifacts into kinetic measurements. (c) Under the conditions of this assay, phenolic BP metabolites are produced but do not accumulate, due to their further metabolism; therefore, assays of BP metabolism which measure the production of phenols, such as the commonly used aryl hydrocarbon hydroxylase assay, may markedly underestimate the rate of BP metabolism when intact cells and low substrate concentrations are used. Our results show that cells can efficiently metabolize BP when exposed to BP concentrations similar to those present in the environment.
Animals↗
Carcinogen-induced alteration of DNA structure.
We have prepared covalent complexes between defined length DNA fragments and a diol epoxide derivative of the carcinogenic polycyclic aromatic hydrocarbon, benzo(a)pyrene. We have studied the structure of these complexes, using transient electric dichroism, circular dichroism, fluorescence quenching, thermal denaturation, polyacrylamide gel electrophoresis, and nuclease digestion. Our observations suggest that th covalently bound carcinogen is intercalated within the helix, forming a wedge-shaped complex. Binding of the carcinogen distorts the structure of the DNA over a region extending beyond the immediate binding site. The most striking aspect of this distortion is that it produces a bend in the helix.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Novel variants in benzo(a)pyrene metabolism. Isolation by fluorescence-activated cell sorting.
We have used the fluorescence-activated cell sorter to measure the metabolism of the carcinogenic polycyclic aromatic hydrocarbon, benzo(a)pyrene (BP), in individual, viable mouse hepatoma (Hepa 1c1c7) cells and to physically select variants with either markedly elevated or markedly decreased ability to metabolize to low, nontoxic concentrations of BP (25 nM) and measuring the rate of disappearance of BP fluorescence. We show by several criteria that the disappearance of BP fluorescence is due to the metabolism of the carcinogen. Cells sorted according to their rae of loss of BP fluorescence exhibit the predicted phenotypes in BP-metabolizing activity, as measured by the aryl hydrocarbon hydroxylase assay.
Animals↗
Calcium-dependent phosphorylation of histone H3 in butyrate-treated HeLa cells.
Ca2+ is prominant in the control of cell proliferation and function. However, the biochemical mechanism(s) mediating its effects on nuclear events is unknown. We report here that Ca2+, at physiological concentrations, stimulates the phosphorylation of histone H3 by an endogenous protein kinase in HeLa cell nuclei. Also, pretreatment of cells with Na butyrate, which increases histone acetylation, selectively increases the susceptability of histone H3 to phosphorylation by the protein kinase. Our results reveal a potential link between histone H3 acetylation and phosphorylation, modifications which are thought to have important effects on chromatin structure and function and suggest a possible mechanism whereby stimuli at the cell surface (such as hormones, mitogens and drugs) may influence biochemical events at the nuclear level; changes in the intracellular Ca2+ concentration may influence the phosphorylation of chromosomal proteins, mediated by Ca2+ -dependent kinases in th nucleus.
Acetylation↗
Aryl hydrocarbon hydroxylase and benzo[a] pyrene metabolism in rodent liver and human cels.
Explore the source record for details and available documents.
Animals↗