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O Hankinson

Publications and source records attributed to O Hankinson.

At least 55 records · Page 3Linked to original sources
PubMed1986

DNA-mediated restoration of aryl hydrocarbon hydroxylase induction in a mouse hepatoma mutant defective in nuclear translocation of the Ah receptor.

In order to study the induction mechanism of aryl hydrocarbon hydroxylase (AHH), non-inducible mutants have previously been isolated from the mouse hepatoma cell line, Hepa-1. With the ultimate goal of isolating the corresponding genes, restoration of AHH inducibility to representative mutants by means of DNA-mediated gene transfer has been set out. The successful transfection of a C- mutant, which is defective in nuclear translocation of the Ah receptor-inducer complex, is described here, using rat genomic DNA as donor material. Primary and secondary rat transfectants were obtained, and they were assayed for hydroxylase activity, receptor translocation, and homology with a rat repetitive DNA sequence.

Animals↗
PubMed1985

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↗
PubMed1985

Transfection by genomic DNA of cytochrome P1-450 enzymatic activity and inducibility.

An aryl hydrocarbon hydroxylase (AHH)-deficient gene A- mutant of the mouse line Hepa-1 was treated with calcium phosphate precipitates of DNA from Hepa-1, the rat line H4IIEC3, or an A- -human hybrid in which the A- mutation is complemented by the corresponding human gene. AHH+ transfectants were isolated by selection with benzo[ghi]perylene plus near UV. In addition, a gene A- mutant which also carries a mutation for hypoxanthine phosphoribosyltransferase deficiency was treated with the above genomic DNAs together with pSV2-gpt DNA, and cotransfectants were isolated after treatment with both benzo[ghi]pereylene and HAT. All transfectants and cotransfectants were inducible for AHH by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Both transfectants and cotransfectants were unstable during culture, rapidly losing AHH activity. Rat DNA-derived transfectants were probed in Southern blots with a cDNA probe to mouse cytochrome P1-450 that cross-hybridizes to the corresponding rat gene. All rat DNA-derived transfectants contained the rat P1-450 gene. In half of the transfectants, the rat gene was amplified four- to sevenfold. In one transfectant, the rat gene was truncated at the 3' end. The proportion of rat DNA in different transfectants, as determined by hybridization to a rat repetitive sequence, ranged from less than 1% to 5%. AHH activity and the rat P1-450 gene segregated together in subclones of one of the transfectants. These results demonstrate that the A gene is either the structural gene for cytochrome P1-450, or another very closely linked gene. Previous results (O. Hankinson et al., J. Biol. Chem. 260:1790-1795, 1985) favor the former alternative.

Animals↗
PubMed1984

The Ah receptor: binding specificity only for foreign chemicals?

The murine Ah locus controls the induction of at least four drug-metabolizing enzymes: cytochromes P1-450, P2-450, and P3-450, and UDP-glucuronosyltransferase. The Ah gene codes for a cytosolic receptor. It is known that the induction response includes: (i) high-affinity binding of specific foreign chemicals to the Ah receptor; (ii) temperature-dependent translocation of the "activated" inducer-receptor complex into the nucleus; (iii) binding of the complex presumably to chromatin components; (iv) transcriptional activation of specific genes; (v) maximal increases in intranuclear high-molecular-weight precursor mRNA (pre-mRNA) that precede by several hours the maximal increases in cytoplasmic mRNA; (vi) translation of the mRNA principally on membrane-bound polysomes; and (vii) increases in the specific membrane-bound proteins (including architectural arrangement with other membrane-bound moieties) that reflect enhanced specific drug-metabolizing activities. It is not known how many of the other drug metabolism induction responses are also governed by receptors. The Ah locus studies have been chiefly unraveled in the mouse, due to several inbred strains having a receptor defect. In addition to "classical" pharmacologic methods (such as structure-activity studies) and standard biochemical techniques, the newer methods of recombinant DNA technology and somatic-cell genetics in culture are shown to be important in understanding the Ah receptor and its induction response. It is possible that this receptor is required for endogenous functions critical to life processes, as well as its function in the induction of drug metabolism by certain polycyclic aromatic compounds.

Animals↗
PubMed1984

Isolation and characterization of revertants from four different classes of aryl hydrocarbon hydroxylase-deficient hepa-1 mutants.

Revertants were selected from aryl hydrocarbon hydroxylase (AHH)-deficient recessive mutants belonging to three complementation groups and from a dominant mutant of the Hepa-1 cell line. The recessive mutants had low spontaneous reversion frequencies (less than 4 X 10(-7] that were increased by mutagenesis. The majority of these revertants also had reacquired only partial AHH activity. Revertants of group A mutants were identical to the wild type with respect to both in vivo and in vitro enzyme stability and the Km for the substrate, benzo [alpha]pyrene, and therefore failed to provide evidence that gene A is the AHH structural gene. Group B and group C mutants are defective in the functioning of the Ah receptor required for AHH induction. Revertants of these groups were normal with respect to in vivo temperature sensitivity for AHH induction and for the 50% effective dose for the inducer, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and thus provided no evidence that the B and C genes code for components of the receptor. Two rare group C revertants possessed AHH activity in the absence of induction. The phenotype of one of these was shown to be recessive to the wild type. Spontaneous revertants of the dominant mutant occurred at a frequency 300-fold greater than those of the recessive mutants, and this frequency was not increased by mutagenesis. These revertants all displayed complete restoration of AHH activity to wild type levels. These observations and the results from cell hybridization studies suggest that the dominant revertants arose by a high frequency event leading to functional elimination of the dominant mutation.

Animals↗
PubMed1983

Effects of cytochrome P1-450 inducers on the cell-surface receptors for epidermal growth factor, phorbol 12,13-dibutyrate, or insulin of cultured mouse hepatoma cells.

Hepa-1c1c7, a mouse hepatoma cell line, was used to study the effect of cytochrome P1-450 inducers on the binding of 125I-epidermal growth factor (EGF), 125I-insulin, or [20-3H]phorbol 12,13-dibutyrate each to its specific cell-surface receptor. After a 24-h exposure to the cultured cells, several polycyclic hydrocarbon P1-450 inducers decrease the binding of EGF to EGF receptors much more than phenobarbital does. There appears to be a selectivity in the inhibitory effects: whereas EGF binding to EGF receptors is blocked, the binding of either phorbol ester or insulin each to its specific cell-surface receptors remains unaffected. The rank order of binding affinities of these chemicals to the cytosolic Ah receptor (2,3,7,8-tetrachlorodibenzo-p-dioxin much greater than benzo[a]pyrene greater than benzo[a]anthracene greater than 6-aminochrysene much greater than phenobarbital) is not correlated with their effects on EGF binding capacity. The effect of polycyclic hydrocarbons on EGF binding takes 24 h at 37 degrees C to be maximal, whereas phorbol 12-myristate 13-acetate, a potent tumor-promoting compound, inhibits EGF binding in less than 30 min. Removal of benzo[a]anthracene from the growth medium after 24 h results in a gradual recovery in EGF binding, indicating that the effect is reversible. Benzo[a]pyrene and benzo[a]anthracene are relatively ineffective at decreasing EGF binding to the EGF receptors in Hepa-1 mutant clones c2 and c4, which lack a normally functioning Ah receptor and inducible aryl hydrocarbon hydroxylase activity (P1-450). The very toxic metabolite (+)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, when added directly to the growth medium of c4 cells, however, is effective at decreasing EGF binding. These data suggest that electrophilic metabolites of polycyclic aromatic compounds, formed by P1-450 induced during the exposure of Hepa-1 cells to these chemicals, are important in decreasing EGF binding to the EGF cell-surface receptor. Occupancy of the Ah receptor per se does not affect EGF binding.

Animals↗
PubMed1983

Dominant and recessive aryl hydrocarbon hydroxylase-deficient mutants of mouse hepatoma line, Hepa-1, and assignment of recessive mutants to three complementation groups.

Fifty-four benzo[a]pyrene (BP)-resistant, aryl hydrocarbon hydroxylase (AHH)-deficient mutants were found to be recessive, while five were dominant. Hybrids between the former mutants and the wild-type were killed by BP, and possessed AHH activities of at least 0.5 (relative to the wild-type). Dominant-mutant--wild-type hybrids were resistant to BP and had activities of about 0.05. Additional experiments assigned the recessive mutants to three complementation groups, designated A through C. Group-B--group-C hybrids were exceptional in possessing a mean AHH activity (0.36), less than the value (0.5) expected from gene dosage. This deficiency was probably due, in part, to instability of AHH activity in these hybrids. However, all hybrids tested retained stable DNA complements, equal to the sum of those of their parents, for 140 days in culture. Previous studies have shown that group B and group C mutations both affect the functioning of a cytosolic receptor required for AHH induction (1).

Animals↗
PubMed1983

The defects in all classes of aryl hydrocarbon hydroxylase-deficient mutant of mouse hepatoma line, Hepa-1, are restricted to activities catalyzed by cytochrome P-450.

The extent of phenotypic derangement was investigated in representative aryl hydroxylase-deficient mutants of Hepa-1. One mutant is dominant while the others are recessive and are mutated in 3 different genes. All the mutants lacked ethoxyresorufin-O-deethylase activity as well as aryl hydrocarbon hydroxylase. However all had normal activities of NADPH-cytochrome P-450 reductase, epoxide hydrolase and ornithine decarboxylase. They also retained 2 liver-specific functions possessed by the parental line, namely albumin secretion and transferrin secretion. The phenotypic alterations in the mutants were therefore restricted to the cytochrome P-450 activities measured.

Animals↗
PubMed1983

Characterization of NADPH-cytochrome P-450 reductase in a mouse hepatoma cell line.

NADPH-cytochrome c reductase in Hepa-1 cells was induced 2-fold by phenobarbital, but was not induced by benz[a]anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The apparent Km of the enzyme for NADPH was 0.57 microM; the activity was inhibitable by NADP; and segregated primarily to the microsomal fraction. Cytoplasm of Hepa-1 cells bound antibody to rabbit cytochrome P-450 reductase. 3T3 cells, which possessed one sixth of the cytochrome c reductase activity of Hepa-1 cells, bound correspondingly less cytochrome P-450 reductase antibody. This supports the notion that cytochrome P-450 reductase was responsible for the cytochrome c reductase activity that was measured.

Animals↗
PubMed1983

Single-step phototoxic selection procedure for isolating cells that possess aryl hydrocarbon hydroxylase.

The development of a selection procedure is described which utilizes the fact that certain polycyclic aromatic hydrocarbons are rendered highly cytotoxic when illuminated with near-ultraviolet light. Twenty polycyclic aromatic hydrocarbons were screened for phototoxicity and toxicity in the absence of light in the mouse hepatoma line Hepa1c1c7, which has high inducible aryl hydrocarbon hydroxylase (AHH) activity, and in AHH-deficient mutants derived from this line. In the assessment of phototoxicity, a period of time was allowed for the metabolism of these compounds prior to illumination. Benzo(g,h,i)perylene had the greatest phototoxicity in cells lacking AHH but was not toxic to cells possessing AHH either in the presence or absence of light. We show that, under conditions of our selection procedure, cells which possess AHH and thus are capable of metabolizing, and therefore of eliminating, benzo(g,h,i)perylene (as determined by the magnitude of the fluorescence of the compound in the cells) are resistant to subsequent exposure to near-ultraviolet light, while cells which lack AHH and are thus unable to eliminate the compound are killed by subsequent illumination. We show, furthermore, that cells possessing AHH can be selected from a majority population of cells lacking the enzyme by this procedure.

Animals↗
PubMed1981

Evidence that Benzo(a) pyrene-resistant, aryl hydrocarbon hydroxylase-deficient variants of mouse hepatoma line, Hepa-1, are mutational in origin.

Treatment of Hepa-1 with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, UV light, or ICR-191G, increased the frequency of benzo(a)pyrene-resistant (BPr) clones. The relative efficacies of the mutagens at increasing the frequency of BPr clones correlated with their efficacies a inducing 6-thioguanine-resistant clones. Evidence was obtained that the mutagens induced the BPr variants rather than selecting for BPr cells that preexisted in the treated cultures. All variants tested had reduced AHH activities and phenotypes that were stable over time in culture. Certain "leaky" variants with residual AHH activities and low cloning efficiencies in BP were further selected in BP and subclones isolated. Half the subclones were indistinguishable from their parents, while the majority of the remainder retained at least half of their parents' AHH activity. The marked stability of the phenotype of the variants, and their induction by mutagens, suggests that they are bona fide mutants.

Animals↗
PubMed1980

Unstable aryl hydrocarbon hydroxylase-deficient variants of a rat hepatoma line.

The H-4-II-E-C3 line has high aryl hydrocarbon hydroxylase (AHH) activity. It was also found to be very sensitive to benzo(a)pyrene (BP) toxicity, and a single-step selection procedure for isolating BP-resistant (BPr) clones was designed. All the BPr variants tested had reduced AHH activities under both inducing and noninducing conditions. As H-4-II-E-C3 was propagated, it gradually declined in AHH activity and increased in cloning efficiency in BP. The spontaneous rate of origin of the BPr variants was approximately 3 x 10(-5) events per cell generation. However, the variants gradually increased in AHH activity and decreased in cloning efficiency in BP as they were propagated. Partially resistant variants also generated, at an appreciable rate, cells with even greater degrees of resistance to BP. The instabilities of the wild-type and variant cultures suggest that the variants arose by an epigenetic mechanism or by changes in gene dosage, rather than by point mutations.

Animals↗
PubMed1979

Single-step selection of clones of a mouse hepatoma line deficient in aryl hydrocarbon hydroxylase.

The mouse hepatoma line Hepa-1 has high and inducible aryl hydrocarbon (benzo[a]pyrene) hydroxylase[benzo[a]pyrene, reduced-flavoprotein:oxygen oxidoreductase (3-hydroxylating), EC 1.14.14.2] activity. Fourteen subclones of a clonal derivative of Hepa-1 were isolated and shown not to display heterogeneity in aryl hydrocarbon hydroxylase activity beyond what could be ascribed to experimental error. Hepa-1 was found to be very sensitive to benzo[a]pyrene (BaP) toxicity and a single-step selection procedure for isolating clones resistant to BaP at 4 microgram/ml was designed. Those BaP-resistant variants tested had much reduced aryl hydrocarbon hydroxylase activities under both inducing and noninducing conditions and they retained their resistance to BaP and low aryl hydrocarbon hydroxylase activities over considerable periods of time of culture in the absence of BaP. The spontaneous rate of origin of the BaP-resistant clones was estimated, by Luria-Delbrück fluctuation analysis, to be 2 X 10(-7) events per cell per generation. It is concluded that the properties of the variants are consistent with the notion that they are mutational in origin and that this system is a promising one for carrying out a somatic cell genetic analysis of aryl hydrocarbon hydroxylase.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1974

Mutants of the pentose phosphate pathway in Aspergillus nidulans.

Mutants of the pentose phosphate pathway have been isolated in Aspergillus nidulans. These fail to grow on a variety of carbohydrates that are catabolized through the pentose phosphate pathway. They also grow poorly on nitrate and nitrite as sole nitrogen sources. The pentose phosphate pathway mutations have been assigned to two unlinked genes. Mutants with lesions in the pppB locus have reduced activities of four enzymes of the pentose phosphate pathway, of glucose-phosphate isomerase, and of mannitol-1-phosphate dehydrogenase. pppA(-) mutants have elevated activities of these same enzymes except for transaldolase, for which they have much reduced activity. Both classes of mutants accumulate sedoheptulose-7-phosphate to an extent that is increased considerably when nitrate is present in the medium. Nitrate does not cause an increase in accumulation of sedoheptulose-7-phosphate in double mutants which, in addition to the pppA1 mutation, carry a mutation that leads to the lack of nitrate reductase activity. These last results suggest that nitrate stimulates the flux through the oxidative pentose phosphate pathway, but that this stimulation depends upon the metabolism of nitrate.

Alcohol Oxidoreductases↗