An investigation into the binding of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one to DNA in vitro.
After metabolic activation the carcinogen 15,16-dihydro-11-[3H]methylcyclopenta[a]phenanthren-17-one binds to DNA in vitro, and this binding is prevented by 7,8-benzoflavone. Radioactivity cannot be removed from the DNA with organic solvents or by chromatography on Sephadex G-50, even after heat denaturation of the DNA. Enzymatic hydrolysis yields radioactive fractions, which elute from a column of Sephadex LH-20 immediately after the natural nucleosides. At least two species of reactive metabolites are involved in this bending, those with a half-life of a few hr and others with greater stability. After extraction from the aqueous incubation mixture, they could be detected in discrete polar fractions from separations of the complex metabolite mixture by high-pressure liquid chromatography. Their ability to bind to DNA decreased with time at ambient temperature, and they were rapidly deactivated by acid. 7,8-Benzolflavone acted by suppressing the formation of polar metabolites derived from enzymatic oxidation of the aromatic double bonds. The inhibitor had no effect on the enzymes hydroxylating saturated carbon; hence it is unlikely that metabolism of the methyl group is important in conversion of this carcinogen to its proximate form, although the presence of the 11-methyl group is essential for carcinogenic activity in this series. (+info)
Effect of hepatocarcinogens on the binding of glucocorticoid-receptor complex in rat liver nuclei.
The effects of a number of carcinogens and hepatotoxins on the binding kinetics of the interactions of glucocorticoidcytosol receptor complex with nuclear acceptor sites in rat liver were investigated. Both the apparent sites in rat liver were investigated. Both the apparent concentration of nuclear binding sites and the Kd were significantly diminished following treatment of rats with sublethal doses of the carcinogens aflatoxin B1, diethylnitrosamine, dimethylnitrosamine, thioacetamide, 3'-methyl-4-dimethylaminoazobenzene, 4-dimethylaminoazobenzene, and 3-methylcholanthrene. Treatment with actinomycin D resulted in a slight reduction in the apparent concentration of nuclear acceptor sites but had no effect on the nuclear binding Kd. The hepatotoxic but noncarcinogenic analgesic, acetaminophen, as well as the weakly toxic aflatoxin B1 cognate, aflatoxin B2, were without effect on the kinetics or binding capacity of glucocorticoid-nuclear acceptor site interaction. These experiments suggest that chemically induced alteration of functional glucocorticoid binding sites on chromatin may be involved in the biochemical effects produced in liver by carcinogens of several chemical types. This experimental model may provide a useful approach for further elucidation of early events in carcinogenesis. (+info)
Assaying potential carcinogens with Drosophila.
Drosophila offers many advantages for the detection of mutagenic activity of carcinogenic agents. It provides the quickest assay system for detecting mutations in animals today. Its generation time is short, and Drosophila is cheap and easy to breed in large numbers. The simple genetic testing methods give unequivocal answers about the whole spectrum of relevant genetic damage. A comparison of the detection capacity of assays sampling different kinds of genetic damage revealed that various substances are highly effective in inducing mutations but do not produce chromosome breakage effects at all, or only at much higher concentrations than those required for mutation induction. Of the different assay systems available, the classical sex-linked recessive lethal test deserves priority, in view of its superior capacity to detect mutagens. Of practical importance is also its high sensitivity, because a large number of loci in one fifth of the genome is tested for newly induced forward mutations, including small deletions. The recent findings that Drosophila is capable of carrying out the same metabolic activation reactions as the mammalian liver makes the organism eminently suitable for verifying results obtained in prescreening with fast microbial assay systems. An additional advantage in this respect is the capacity of Drosophila for detecting short-lived activation products, because intracellular metabolic activation appears to occur within the spermatids and spermatocytes. (+info)
Carcinogenicity of triethanolamine in mice and its mutagenicity after reaction with sodium nitrite in bacteria.
Mice fed a diet containing 0.3 or 0.03% triethanolamine developed malignant tumors. Females showed a high incidence of tumors in lymphoid tissues, while this type was absent in males. Tumors in other tissues were produced at a considerable rate in both sexes, but no hepatoma was found. Triethanolamine was not mutagenic to Bacillus subtilis by itself, but it became mutagenic after reacting with sodium nitrite under acidic conditions or when the mixture was heated. Although N-nitrosodiethanolamine, a known carcinogen and mutagen, was detected in the reaction mixture by thin-layer chromatography, it may not be the main mutagenic product, because the product was a stable and direct mutagen and its mutagenic activity was destroyed by liver enzymes, unlike N-nitrosodiethanolamine. The lethal and mutagenic DNA damages produced by this unidentified product were susceptible to some extent to the repair functions of the bacteria. (+info)
The five amino acid-deleted isoform of hepatocyte growth factor promotes carcinogenesis in transgenic mice.
Hepatocyte growth factor (HGF) is a polypeptide with mitogenic, motogenic, and morphogenic effects on different cell types including hepatocytes. HGF is expressed as two biologically active isotypes resulting from alternative RNA splicing. The roles of each HGF isoform in development, liver regeneration and tumorigenesis have not yet been well characterized. We report the generation and analysis of transgenic mice overexpressing the five amino acid-deleted variant of HGF (dHGF) in the liver by virtue of an albumin expression vector. These ALB-dHGF transgenic mice develop normally, have an enhanced rate of liver regeneration after partial hepatectomy, and exhibit a threefold higher incidence of hepatocellular carcinoma (HCC) beyond 17 months of age. Moreover, overexpression of dHGF dramatically accelerates diethyl-nitrosamine induced HCC tumorigenesis. These tumors arise faster, are significantly larger, more numerous and more invasive than those appearing in non-transgenic littermates. Approximately 90% of female dHGF-transgenic mice had multiple macroscopic HCCs 40 weeks after injection of DEN; whereas the non-transgenic counterparts had only microscopic nodules. Liver tumors and cultured tumor cell lines from dHGF transgenics showed high levels of HGF and c-Met mRNA and protein. Together, these results reveal that in vivo dHGF plays an active role in liver regeneration and HCC tumorigenesis. (+info)
Expression of dominant negative Erk2 inhibits AP-1 transactivation and neoplastic transformation.
The mitogen activated protein (MAP) kinases or extracellular signal-regulated kinases (Erks) are activated in response to Ras expression or exposure to tumor promoters or to growth factors, and have been implicated in AP-1 transactivation in some models. We have shown that tumor promoter induced activation of the transcription factor AP-1 is required for induced neoplastic transformation in the Balb/C JB6 cell model. Jun and Fos family protein levels have been found not to be limiting for AP-1 response. The present study asks whether activation of Erks1 and 2 is required for AP-1 transactivation and transformation of JB6 cells and whether Erks might be targeted for cancer prevention. Expression of either of two different dominant negative kinase inactive Erk2 mutants in transformation sensitive (P+) JB6 cells substantially inhibited the tumor promoter induced activation of Erks1 and 2 and of AP-1 measured by a collagenase-luciferase reporter. Multiple mutant Erk2 expressing clonal lines were also rendered non-responsive to induced neoplastic transformation. These observations, together with our recent finding attributing AP-1 non-responsiveness to Erk deficiency in a clonal line of transformation resistant (P-) cells, argue for a requirement for Erks1 and/or 2 activation in AP-1 transactivation in the mouse JB6 neoplastic progression model, and suggest the utility of Erks as a prevention target. (+info)
Carboxyl-terminal phosphorylation regulates the function and subcellular localization of protein kinase C betaII.
Protein kinase C is processed by three phosphorylation events before it is competent to respond to second messengers. Specifically, the enzyme is first phosphorylated at the activation loop by another kinase, followed by two ordered autophosphorylations at the carboxyl terminus (Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403). This study examines the role of negative charge at the first conserved carboxyl-terminal phosphorylation position, Thr-641, in regulating the function and subcellular localization of protein kinase C betaII. Mutation of this residue to Ala results in compensating phosphorylations at adjacent sites, so that a triple Ala mutant was required to address the function of phosphate at Thr-641. Biochemical and immunolocalization analyses of phosphorylation site mutants reveal that negative charge at this position is required for the following: 1) to process catalytically competent protein kinase C; 2) to allow autophosphorylation of Ser-660; 3) for cytosolic localization of protein kinase C; and 4) to permit phorbol ester-dependent membrane translocation. Thus, phosphorylation of Thr-641 in protein kinase C betaII is essential for both the catalytic function and correct subcellular localization of protein kinase C. The conservation of this residue in every protein kinase C isozyme, as well as other members of the kinase superfamily such as protein kinase A, suggests that carboxyl-terminal phosphorylation serves as a key molecular switch for defining kinase function. (+info)
Peroxisome proliferator-activated receptors (PPARS) and carcinogenesis.
Peroxisome proliferators (PPs) are an important group of chemicals that include certain hypolipidemic drugs, plasticizers and pollutants. Many of these agents are known rodent liver tumor promoters and debate exists as to whether humans are at increased cancer risk following exposure to PPs. Research over the last decade has focused on determining the biochemical and molecular mechanisms by which peroxisome proliferators exert their effects, in the hope that this controversy will be settled. PPs regulate gene expression via a steroid hormone receptor, the peroxisome proliferator-activated receptor (PPAR). At least three subtypes of PPAR (alpha, beta and gamma) have been cloned from several species, including humans. These receptors have been implicated in tumor promotion, cellular differentiation, and apoptosis. In the present article, the current understanding of how PPARs are involved in tumorigenesis, and what this may mean to human risk assessment, will be discussed. (+info)