ABSTRACT
3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) is a potent hepatocarcinogen in rats and a weak carcinogen in mice, whereas o-aminoazotoluene (OAT) is a potent hepatocarcinogen in mice but weak hepatocarcinogen in rats. They significantly suppress glucocorticoid induction of tyrosine aminotransferase (TAT) in the liver of sensitive animals and have minor effect on the induction of this enzyme in the liver of resistant animals (3'-MeDAB-treated mice and OAT-treated rats). The inhibitory effect of these carcinogens is realized at the level of gene transcription (decreased accumulation of TAT mRNA). This effect is mediated via reduction of DNA-binding activity of transcription factor HNF3 (without decrease of its content) without any involvement of the glucocorticoid receptor. It was shown that carcinogens influence DNA-binding activity of HNF3 via an unknown nuclear factor.
Subject(s)
Carcinogens/pharmacology , Glucocorticoids/antagonists & inhibitors , Liver/drug effects , Liver/enzymology , Transcription Factors , Tyrosine Transaminase/genetics , Tyrosine Transaminase/metabolism , Animals , Binding, Competitive , DNA/metabolism , DNA-Binding Proteins/metabolism , Disease Models, Animal , Enzyme Induction/drug effects , Glucocorticoids/pharmacology , Hepatocyte Nuclear Factor 3-beta , Hepatocyte Nuclear Factor 3-gamma , Liver/metabolism , Liver Neoplasms, Experimental/pathology , Liver Neoplasms, Experimental/prevention & control , Male , Mice , Nuclear Proteins/metabolism , Rats , Rats, Wistar , Receptors, Glucocorticoid/metabolism , Species SpecificityABSTRACT
The sequence of K-ras intron 2, which has been associated with lung tumor susceptibility in inbred mouse strains, was analyzed in susceptible strain GR and in resistant strains PT and UT. In the latter case, the intron had a tandem repeat of a 37-bp sequence with variant GC of its two single-nucleotide polymorphisms (SNPs), as earlier reported for resistant strains AKR, C57BL/c, and C3H/A. Strain GR did not differ in intron structure from susceptible strains A/He and ICR, having one copy of the 37-bp sequence with SNP variant CA. By gel retardation assay, a DNA probe corresponding to "susceptible" allele CA of K-ras region 278-307 formed an additional complex with nuclear proteins extracted from the lungs, as compared with probes corresponding to the "resistant" GC and "intermediate" CC alleles. With specific antibodies, the protein binding to the susceptible allele was identified as transcription factor GATA-6. Reverse transcription with subsequent multiplex PCR did not reveal a significant difference in K-ras expression for susceptible and resistant strains. The results suggest that SNPs of K-ras intron 2 do not affect the level of K-ras expression but do control the binding of GATA-6, which plays an important role in lung differentiation.
Subject(s)
DNA-Binding Proteins/metabolism , Genes, ras , Genetic Predisposition to Disease , Lung Neoplasms/genetics , Transcription Factors/metabolism , Animals , Base Sequence , Cell Differentiation/genetics , DNA-Binding Proteins/genetics , GATA6 Transcription Factor , Gene Expression Regulation , Introns , Lung/cytology , Lung/physiology , Mice , Mice, Inbred Strains , Molecular Sequence Data , Polymorphism, Single Nucleotide , Transcription Factors/geneticsABSTRACT
In the rodent liver, hepatocarcinogens inhibit the glucocorticoid induction of several liver-specific genes, including tyrosine aminotransferase (TAT). A distinct positive correlation exists in mice between the extent of inhibition of TAT induction after acute administration of o-aminoazotoluene (OAT) and the frequency of liver tumors after chronic exposure to the carcinogen. To elucidate the mechanism of the carcinogenic action, the effects of OAT on the DNA-binding activity of several transcription factors participating in the glucocorticoid regulation of TAT gene expression were studied. The experimental inbred male mice were sensitive (A/He and SWR/J, tumor induction frequency of 75-100%, TAT induction inhibition of 35-50%) and resistant (CC57BR/Mv and AKR/J, 0-6% and 10-15%, respectively) to OAT. Gel retardation experiments showed that hepatocyte nuclear factor 3 (HNF3)gamma DNA-binding activity was strongly reduced in nuclear extracts from the livers of OAT-treated A/He and SWR/J mice but only slightly reduced in CC57Br/Mv and AKR/J mice. The DNA-binding activities of Ets, AP1 family members, and GME binding proteins were unaffected. HNF3gamma DNA-binding activity was reduced by 1 h after OAT administration and remained low for 1 mo, as did inhibition of TAT induction in the liver. These results suggested that the inhibitory effect of OAT on the glucocorticoid induction of TAT is mediated by reduced HNF3gamma DNA-binding activity.
Subject(s)
DNA, Neoplasm/metabolism , DNA-Binding Proteins/metabolism , Glucocorticoids/pharmacology , Hydrocortisone/pharmacology , Liver Neoplasms, Experimental/enzymology , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Tyrosine Transaminase/biosynthesis , o-Aminoazotoluene/pharmacology , Animals , Blotting, Western , DNA Primers/chemistry , Enzyme Induction/drug effects , Hepatocyte Nuclear Factor 3-gamma , Liver Neoplasms, Experimental/drug therapy , Male , Mice , Mice, Inbred A , Mice, Inbred AKR , Species Specificity , Trans-Activators/genetics , Tyrosine Transaminase/antagonists & inhibitorsABSTRACT
rSNP_Guide is a novel curated database system for analysis of transcription factor (TF) binding to target sequences in regulatory gene regions altered by mutations. It accumulates experimental data on naturally occurring site variants in regulatory gene regions and site-directed mutations. This database system also contains the web tools for SNP analysis, i.e., active applet applying weight matrices to predict the regulatory site candidates altered by a mutation. The current version of the rSNP_Guide is supplemented by six sub-databases: (i) rSNP_DB, on DNA-protein interaction caused by mutation; (ii) SYSTEM, on experimental systems; (iii) rSNP_BIB, on citations to original publications; (iv) SAMPLES, on experimentally identified sequences of known regulatory sites; (v) MATRIX, on weight matrices of known TF sites; (vi) rSNP_Report, on characteristic examples of successful rSNP_Tools implementation. These databases are useful for the analysis of natural SNPs and site-directed mutations. The databases are available through the Web, http://wwwmgs.bionet.nsc.ru/mgs/systems/rsnp/.
Subject(s)
DNA/genetics , Databases, Factual , Polymorphism, Single Nucleotide , Transcription Factors/metabolism , Binding Sites/genetics , DNA/metabolism , Humans , Internet , Mutagenesis, Site-Directed , Mutation , Protein Binding , Regulatory Sequences, Nucleic Acid/genetics , Transcription Factors/geneticsABSTRACT
The presence of receptors for oligodeoxynucleotides (OdN) on the surface of L929 cells has previously been described. To study the possible coupling of the receptor to cellular signal transducing systems, the effect of phosphodiester OdN of different sequences on cellular phospholipase C and protein kinase C (PKC) activities in L929 fibroblasts was studied. Treatment of cells with OdN induced an increase in 32P labeling of phosphatidic acid which was accompanied by a gradual decrease in diacylglycerol. These effects seem to be independent of the OdN sequence. PKC activity in membranes isolated from OdN-treated cells was found to be lower than that in membranes of control cells. SDS-PAGE of the 32P-labeled cellular proteins revealed that OdN treatment caused a decrease in phosphorylation of the 26 and 73 kDa cellular proteins in the cells.
Subject(s)
Oligodeoxyribonucleotides/pharmacology , Protein Kinase C/metabolism , Type C Phospholipases/metabolism , Animals , Cell Line , Cell Membrane/enzymology , Cell Membrane/metabolism , Diglycerides/metabolism , Mice , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/metabolism , Phosphatidic Acids/metabolism , Phosphorylation , Receptors, Cell Surface/metabolism , Signal TransductionABSTRACT
Estradiol receptors are discovered in nuclei, cytoplasm and plasmatic membranes of the target cells. Estradiol activates the transmembrane polyphosphoinositide system: it stimulates the protein kinase C translocation from cytosol to cell membranes, the membrane protein phosphorylation being elevated. Transmembrane adenylate cyclase system is also activated. The cAMP system stimulation by estradiol is mediated by protein kinase C, phosphorylating a protein of adenylate cyclase complex. Estradiol causes protein kinases A translocation into the cell nuclei and enhances the protein kinase NII activity. The role of protein phosphorylation, activated by steroid hormones, in the transcription and protein synthesis regulation, is discussed.
Subject(s)
Hormones/physiology , Second Messenger Systems/physiology , Adenylyl Cyclases/metabolism , Animals , Cell Membrane/physiology , Estradiol/metabolism , Female , Phosphatidylinositols/metabolism , Phosphorylation , Protein Kinases/metabolism , Receptors, Cell Surface/physiology , Receptors, Estradiol/physiologyABSTRACT
It was demonstrated that cell cytosol and nuclei of estradiol-dependent mammary tumours and kidney of the rat contain a polyamine-dependent protein kinase whose activity is stimulated 2-5 fold by 2 mM spermine and is inhibited by heparin (8-10 micrograms/ml). This protein kinase uses acid proteins (casein) as substrates and ATP and GTP as phosphate sources. The polyamine-dependent protein kinase is eluted from DEAE-cellulose with 0.22-0.24 M NaCl. In cell nuclei the enzyme activity is 3-5 times as high as that in the cytoplasm. In cell nuclei of hormone-dependent tumours and kidney estradiol increases the activity of the polyamine-dependent protein kinase 2-5 times within 30 minutes.
