Introduction II: American Cancer Society Research Workshop on Cancer and Nutrition.
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American Cancer Society↗
Biomedical subjects
E Bresnick
Publications and source records attributed to E Bresnick.
Kinetic and DNA-binding properties of recombinant human O6-methylguanine-DNA methyltransferase.
O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that plays an important role in chemotherapy, mutagenesis, and carcinogenesis. Recombinant human MGMT was isolated from an Escherichia coli high performance expression system and purified to homogeneity. The kinetic and DNA-binding properties of the recombinant human MGMT were studied. The purified human MGMT reacted stoichiometrically with methylated DNA under second-order rate kinetics. The rate constant with normal methylated DNA was 1 x 10(9) M-1 min-1 at 37 degrees C. The binding to DNA was the rate determining step in the repair process. Approximately eight base pairs of the DNA substrate were covered by the human MGMT protein. The affinity constant for interaction of DNA to MGMT was approximately 4.7 x 10(5) M-1. The binding to methylated DNA was also examined; the binding affinity to methylated DNA was two times higher than that to unmodified DNA. The interaction with DNA induced a conformational change in the human MGMT protein as monitored by circular dichroism and fluorescence analysis. A similar conformational change was induced by both methylated and unmodified DNA.
Base Sequence↗
Induction of cytochrome P450IA1 and its recombinant construct in H4IIE rat hepatoma cells.
1. Our previous studies have shown that benzo(a)pyrene (BP), 3-methylcholanthrene (3MC) and tetrachlorodibenzofuran (TCDBF) can induce the expression of the cytochrome P450IA1 mRNA in the rat hepatoma cell line, H4IIE, although the kinetics of induction differed. 2. In the present study, by using biochemical, immunochemical and recombinant DNA approaches, the effects of these inducers have been examined on the steady state level of endogenous cytochrome P450IA1 protein and on induction of chloramphenicol acetyltransferase activity (CAT) in the H4IIE cells transfected with pMC1CAT (a recombinant construct consisting of CAT linked to 5' upstream DNA sequence of the rat cytochrome P450IA1 gene). 3. From 7-ethoxyresorufin O-deethylase activity (EROD) and immunochemical analysis of cytochrome P450IA1, the optimal concentrations of BP, 3MC and TCDBF for induction in the H4IIE cells were determined as 1, 0.1-1 and 0.1 microM, respectively. 4. The elevated expression of the protein was more sustained in the TCDBF-exposed cells than in the BP or 3MC-treated cells. 5. After 1.5 hr of treatment, little if any detectable P450IA1 protein was observed in the H4IIE cells although a considerable amount of mRNA was present. 6. In addition, no cytochrome P450IA2 protein was detected in the control or induced H4IIE cells. 7. H4IIE cells were transfected by pMC1CAT, and the induction ratio of CAT expression in the transfected H4IIE cells after BP, 3MC or TCDBF treatment was 10-, 17- and 40-fold, respectively. 8. These results indicate that the rat H4IIE cell line offers a valid homologous system for studies of the regulation of the rat cytochrome P450IA1 gene.
Animals↗
Rat CYP1A1 negative regulatory element: biological activity and interaction with a protein from liver and hepatoma cells.
Rat CYP1A1 promoter activity was suppressed by the presence of a cis negative regulatory element (NRE) at position -843 to -746 in transiently transfected rat H4IIE and human HepG2 hepatoma cells. Removal of the NRE from the promoter-fusion gene constructs caused an increase in the basal promoter activity of 2-6-fold. Co-transfection of the NRE-containing or non-NRE-containing CYP1A1 promoter-fusion gene constructs with a cloned rat NRE, i.e., pNRE, into HepG2 cells caused a 2-fold or greater reduction in constitutive and induced promoter activities. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced expression of the endogenous human CYPA1 was also inhibited by transfection of pNRE into HepG2 cells. Deletion of the sequence from base pairs (bp) -658 to -269 in the NRE-containing construct caused a dramatic decrease of constitutive expression in transiently transfected HepG2 cells, compared with an identical construct that lacked the NRE. Deletion of the sequences between bp -658 and -158 in the CYP1A1 promoter did not affect reporter gene activity, indicating a second site of interaction. At least three different rat liver nuclear proteins bound to the rat NRE, as determined by gel mobility shift and DNase I footprinting assays. A 32-bp sequence within the rat NRE, with significant sequence identity to the 26-bp c-myc, fos/jun-octamer-binding, NRE, was protected from DNAse I cleavage by rat liver nuclear extracts. These data suggested a role for this region in the negative regulation of rat CYP1A1.
Animals↗
Irradiation-induced expression of O6-methylguanine-DNA methyltransferase in mammalian cells.
O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein which plays an important role in chemotherapy, mutagenesis, and carcinogenesis. The specific activity of MGMT in female rat liver can be induced by approximately 20-fold by treatment of the rats with gamma-irradiation. Maximum response occurred 48 h after 15 Gy irradiation. MGMT levels in male rats were induced by only 3-fold. MGMT activity was also induced by irradiation of rat hepatoma H4IIE cells with a 3-fold increase noted after treatment with 3 Gy. Northern analysis and nuclear run-on assays indicated that the induction of MGMT was regulated at the transcriptional level. The radiation-mediated increase in MGMT was blocked by H7, a protein kinase inhibitor, but not by H89, an inhibitor of protein kinase A. Hydroxyl radicals may play a role in the induction mechanism since dimethyl sulfoxide, a radical scavenger, blocked the radiation-mediated increase in MGMT. MGMT activity was also increased by treatment of the cells with H2O2, in accordance with the involvement of activated oxygen species in the induction of MGMT. Finally, the addition of cycloheximide, an inhibitor of protein synthesis, prior to but not after irradiation, abolished the increase in MGMT activity.
Animals↗
Expression of human O6-methylguanine-DNA methyltransferase in a DNA excision repair-deficient Chinese hamster ovary cell line and its response to certain alkylating agents.
A plasmid has been constructed in which the expression of human O6-methylguanine-DNA methyltransferase (MGMT) complementary DNA is driven by the Rous sarcoma virus promoter sequence. We had previously shown that transfection of this plasmid into Chinese hamster ovary (CHO) cells results in the expression of MGMT and in increased cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine and 1-(2-chloroethyl)-1-nitrosourea (CNU) but not N-nitroso-N-ethylurea (ENU). In the present study, the Rous sarcoma virus promoter-MGMT was transfected into DNA excision repair-deficient CHO UV41 cells to investigate the phenotype associated with MGMT expression in the absence of DNA excision repair. Both the UV41/MGMT and CHO/MGMT cells expressed similar levels of MGMT and exhibited a similar increased resistance to N-methyl-N'-nitro-N-nitrosoguanidine. The UV41 cells were 20-fold more sensitive to CNU than the wild-type CHO cells. Expression of MGMT increased the resistance to CNU about 6-fold in both cell lines, but the difference between the two cell lines attributable to the excision repair defect still persisted. The UV41 cells were 2- to 3-fold more sensitive than the wild-type CHO cells to the monofunctional alkylating agents 1-(2-hydroxyethyl)-1-nitrosourea and ENU, but the MGMT phenotype did not alter sensitivity. This suggests that alkylation at the O6 position of guanine has no role in cytotoxicity of ethylating agents and that monofunctional DNA damage has little role in the cytotoxicity of CNU. Since MGMT can prevent the formation of G-C interstrand cross-links formed by CNU, other excision repair-sensitive DNA adducts must play a major role in the sensitivity of UV41 cells to this bifunctional alkylating agent. These results suggest that DNA intrastrand cross-links may be major contributors to the cytotoxicity of CNU.
Animals↗
Induction of cytochromes P450IA1 and P450IA2 as determined by solution hybridization.
Two oligodeoxyribonucleotides were synthesized that were specific for the messenger RNAs for the polycyclic hydrocarbon-inducible cytochromes P450IA1 and P450IA2. The solution hybridization technique was modified for the use of these oligodeoxyribonucleotide probes so as to increase the sensitivity and specificity of this method. Using this technique, the steady-state levels of the mRNAs for cytochromes P450IA1 and P450IA2 in control rat liver were determined to be less than 3 and 6 molecules/cell, and 1.8 and 4.0 attomol/micrograms poly (A)+ RNA, respectively. At 15 hr after induction with 3-methylcholanthrene, the steady-state levels of the mRNAs for P450IA1 and P450IA2 were 68 and 200 molecules/cell, and 41.6 and 123 attomol/micrograms poly (A)+ RNA.
Animals↗
Expression of human O6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells and restoration of cellular resistance to certain N-nitroso compounds.
We have constructed a plasmid in which the expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA is driven by the Rous sarcoma virus promoter sequence. Transfection of this plasmid into Chinese hamster ovary (CHO) cells results in expression of MGMT and in cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 1-(2-chloroethyl)-1-nitrosourea (CNU), but not to N-nitroso-N-ethylurea. The specific activity of MGMT in transfected CHO cells correlated well with their resistance to MNNG and CNU. Southern analysis showed that the plasmid had been integrated into the CHO cell genome. Western analysis of extracts from transfected CHO cells using an antibody against a peptide corresponding to the carboxyl-terminal end of the human MGMT protein demonstrated a single band with a molecular size of 24-25 kDa; no such band was observed in extracts from wild-type CHO cells. These transfected cells may therefore be used to study the role of MGMT in the repair of alkylating DNA lesions and to determine its importance in carcinogenesis as well as in chemotherapy.
Alkylating Agents↗
Presence of the 4 S polycyclic hydrocarbon-binding protein in H4-II-E cells.
The 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein had been implicated in regulating the expression of rat cytochrome P450IA1 which is most closely associated with aryl hydrocarbon hydroxylase (AHH). We have now investigated the presence of both the 4 S PAH-binding protein and the 8 S Ah receptor in rat hepatoma H4-II-E cells as well as the induction of P450IA1 upon their exposure to PAH's such as benzo[a]pyrene (BP) and 3-methylcholanthrene (3MC), and halogenated dioxins such as 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDBF). Sucrose density gradient analyses and hydroxylapatite assays indicate that, in addition to the 8 S protein, the 4 S PAH binding protein is present in these cells. This protein interacts in a saturable and high affinity manner with BP and 3MC, but not with TCDD or TCDBF. Using a P450IA1 probe, the induction of gene expression was observed by Northern blot analysis of total cellular RNA after exposure of the H4-II-E cells to BP, 3MC, or TCDBF. Since the 4 S protein was observed to interact only with BP and 3MC, these results suggest that this protein may also play a role in the PAH-induced expression of cytochrome P450IA1 gene expression in H4-II-E.
Animals↗
Induction of cytochrome P450IA1 in rat hepatoma cell by polycyclic hydrocarbons and a dioxin.
In this study, the effects of benzo[a]pyrene (BP), 3-methylcholanthrene (3MC) and tetrachlorodibenzofuran (TCDBF) on the expression of the cytochrome P450IA1 gene in the rat hepatoma cell line H4IIE were examined. The initial rate of increase in the steady-state concentration of the mRNA for this gene was similar with each of these inducers; however, the elevated level of this mRNA was more sustained after TCDBF treatment. Nuclear run-off assays suggested that the elevated level of the mRNA was caused principally by an effect upon transcription.
Animals↗
Inhibitory effect of apigenin, a plant flavonoid, on epidermal ornithine decarboxylase and skin tumor promotion in mice.
This investigation studied the effect of topical application of apigenin on skin tumorigenesis initiated by 7,12-dimethylbenz(a)anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) in SENCAR mice. Apigenin was a potent inhibitor of epidermal ornithine decarboxylase induction by TPA in a dose-dependent manner from 1 to 20 mumol. Two tumorigenesis studies were conducted. In the first study, 20 mumol of apigenin was applied topically and no effect on body weight was observed. By week 33 after DMBA initiation, 48% of DMBA/TPA-treated mice developed carcinomas, while none occurred in DMBA/apigenin/TPA-treated groups. In the second study, doses of 5 and 20 mumol of apigenin were used. The papilloma incidence for 0, 5, and 20 mumol apigenin at 26 weeks after DMBA was 93.3, 58, and 39.3%, and papilloma numbers per mouse were 7.5, 2.5, and 1.8, respectively. Apigenin prolonged by 3 weeks the latency period of tumor appearance. In addition, apigenin significantly inhibited the incidence of carcinoma and the numbers of carcinomas. The incidence of carcinomas per tumor-bearing animal and the ratio of carcinomas/papillomas in two apigenin-treated groups decreased although there were no significant differences between the three groups. These data indicate that apigenin inhibited skin papillomas and showed the tendency to decrease conversion of papillomas to carcinomas.
9,10-Dimethyl-1,2-benzanthracene↗
Binding characteristics of 4S PAH-binding protein and Ah receptor from rats and mice.
Cytochrome P-450IA1 (Cyto-P450IA1) is the isozyme most closely associated with aryl hydrocarbon hydroxylase (AHH). At least two distinct high-affinity binding proteins may regulate its expression, the 4S protein that primarily binds polycyclic aromatic hydrocarbons (PAHs), and the 8S Ah receptor that binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and like congeners. The present study was conducted to investigate ligand binding characteristics of the 4S and 8S binding proteins before and after separation from liver cytosol in the presence and absence of sodium molybdate. Liver cytosol and 4S and 8S receptor-enriched fractions from livers of male Sprague-Dawley rats (AHH-responsive), and from C57BL/6N (AHH-responsive) and DBA/2N and AKR/N mice (AHH-nonresponsive) served as sources of these proteins. Competitive binding studies were performed using 10 nM [3H]benzo[a]pyrene (BaP) or [3H]-TCDD in the presence and absence of a 200-fold excess of BaP, 3-methylcholanthrene (3-MC), and tetrachlorodibenzofuran (TCDBF). Specific PAH-binding activity was assayed by using either sucrose density gradient analysis or a hydroxylapatite assay. Our results indicate that before and after the separation of liver cytosol into 4S and 8S fractions, ligand binding characteristics were relatively unaltered for the 4S protein in comparison to that for the Ah receptor, particularly in the presence of molybdate. The 4S protein had high affinity for BaP and 3-MC but very low affinity for TCDBF; the 8S protein had high affinity for TCDBF, lesser affinity for 3-MC, and low affinity for BaP. In the presence of sodium molybdate, the Ah receptor fractions were significantly stabilized, whereas the 4S protein was relatively unaffected. After the separation of Ah receptor fraction from liver cytosol in the presence of molybdate, 3-MC consistently bound to a greater extent. These results affirm the existence of two distinct PAH-binding proteins.
Animals↗
Reduction in mammary tumorigenesis in the rat by cabbage and cabbage residue.
Mammary cancer was induced in female Sprague-Dawley rats by a single injection of N-methyl-N-nitrosourea (MNU) and rats were randomized to control fat (5%) and high fat (24.6%) diets. In addition, dried cabbage (5 and 10%) and collards (5%) were included in the diets of some animals. No statistically significant differences were observed in food consumption, body wt gain and caloric intake between the MNU-treated and control groups in the rats fed the low-fat diet. However, the groups fed the high-fat diet consumed more than the rats maintained on the control diet. The rats on the control fat diet containing cabbage exhibited a significantly lower incidence of mammary cancer than rats that were fed the control-fat diet without cabbage. This effect was not observed in comparable rats on the high-fat diet. The inhibitory effect on mammary tumorigenesis was demonstrated using a residue obtained from cabbage by exhaustive extraction with methanol, methylene chloride and petroleum ether. These studies reinforce the efficacy of cabbage as a 'suppressor' of cancer in experimental model systems under control-fat dietary conditions.
Animals↗
Induction and purification of O6-methylguanine-DNA-methyltransferase from rat liver.
O6-Methylguanine-DNA-methyltransferase (OMMT) is a DNA repair protein that plays an important role in chemotherapy, mutagenesis and carcinogenesis. The sp. act. of OMMT in rat liver can be induced by approximately 12- to 20-fold by treatment of the rats with ionizing radiation. The effects of dose and time were investigated in this study. We have found that OMMT sp. act. can be increased, although to a lower extent, in kidney, spleen and brain in addition to liver. However, the sp. act. of OMMT in lung was reduced by irradiation. OMMT has been purified from the livers of irradiated rats by solubilization in high-salt-containing buffer, ammonium sulfate precipitation and a series of column chromatographic steps, including phenyl-Sepharose, heparin-agarose, double-stranded DNA-cellulose and FPLC. A 3000-fold enrichment of OMMT was achieved from the induced liver preparations. However, with regard to the sp. act. of this protein in normal rat liver, the fold purification was approximately 35,000. After methylation, OMMT during the course of its action exhibited a mol. wt of 28 kd under SDS-PAGE conditions.
Animals↗
Nuclease hypersensitivity of the rat cytochrome P450IA1 gene.
The bovine pancreatic deoxyribonuclease I (DNAase I) hypersensitivity of the rat cytochrome P450IA1 gene was investigated. A nuclease-hypersensitive region was observed at approximately 3.2 to 5.1 kilobase pairs upstream of exon 1 in adult and fetal rat liver. This region did not necessarily correlate with gene expression following 3-methylcholanthrene induction, although it may determine the potential for inducibility of this gene.
Animals↗
Inducibility of rat liver cytochrome P-450IA1 (P-450c) mRNA during the partial inhibition of protein synthesis.
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Animals↗
Glucocorticoid regulation of the rat cytochrome P450c (P450IA1) gene: receptor binding within intron I.
The actions of polycyclic aromatic hydrocarbons and glucocorticoids to regulate the expression of cytochrome P450c were investigated using cultured fetal rat hepatocytes. Cytochrome P450c mRNA content, determined by Northern blot analysis, was induced in cells treated with 1,2-benzanthracene from levels undetectable in untreated cells. When dexamethasone was included in the culture medium together with 1,2-benzanthracene there was a further 2-fold increase in the induction of cytochrome P450c mRNA. The concentration of dexamethasone required for a half-maximal increase in cytochrome P450c mRNA content was approximately 10(-9) M. By nuclear run-on transcription assays, treatment with 1,2-benzanthracene induced cytochrome P450c transcription 5.3-fold over untreated cells. In the presence of dexamethasone and 1,2-benzanthracene, there was a further 2-fold increase in cytochrome P450c transcription. Southwestern blotting and exonuclease footprinting methods have identified binding interactions of a purified glucocorticoid receptor fraction with portions of the cytochrome P450c gene within the first intron. Using a chimeric plasmid containing the first intron, the first exon, and 824 bp of 5'-flanking region of the cytochrome P450c gene, chloramphenicol acetyltransferase activity was induced in transfected HepG2 hepatoma cells by the addition of 1,2-benzanthracene. The addition of dexamethasone induced a further 2.2-fold increase in activity. Deletion of the first intron within the chimeric plasmid abolished responsiveness to dexamethasone. It is concluded that glucocorticoids act together with polycyclic aromatic hydrocarbons to increase the levels of cytochrome P450c expressed in the fetal rat hepatocyte, and that this action is mediated by the glucocorticoid receptor. A glucocorticoid responsive element, which binds the glucocorticoid receptor, has been identified within the first intron of the cytochrome P450c gene. These results suggest that glucocorticoids play a significant role in the response of the hepatic cytochrome P450c gene to xenobiotics.
Actins↗
The effect of dietary fat on metastasis of the Lewis lung carcinoma and the BALB/c mammary carcinoma.
The effect of feeding mice diets high in beef tallow (high in saturated fat) or corn oil (high in polyunsaturated fat) on the production of lung metastases by the Lewis lung carcinoma and the BALB/c mammary tumor was determined. Diets were fed ad libitum, and the mice fed the high-fat (24.6%) diets consumed more calories and gained more weight than those fed the control (5%) diets. With the Lewis lung carcinoma, we found that both high-fat diets significantly increased the growth of the primary tumor in the footpad as well as the number of spontaneous metastases produced after the primary was removed; this was in comparison with results from the appropriate control diets. With the BALB/c mammary tumor, the high-fat beef tallow diet (but not the corn oil diet) significantly increased the number of lung metastases formed after tail vein injection. In addition, the group given the control corn oil diet had more metastases than the group given the control beef tallow diet. Overall, these studies showed that the consumption of high-fat/high-calorie diets increased metastasis compared to the consumption of high-fat/high-calorie diets increased metastasis compared to the consumption of low-fat diets. However, the results varied depending on the tumor model used and the type of fat.
Animals↗