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Biomedical subjects

J I Goodman

Publications and source records attributed to J I Goodman.

At least 55 records · Page 3Linked to original sources
PubMed1993

Comparative analysis of the methylation status of the 5' flanking region of Ha-ras in B6C3F1, C3H/He and C57BL/6 mouse liver.

We examined the methylation status of the 5' flanking region of Ha-ras in the liver of the liver tumor-prone B6C3F1 male (C57BL/6 female x C3H/He male) and C3H/He male, plus the relatively resistant C57BL/6 male mouse strains. Southern analysis revealed the presence of CCGG sites methylated at the internal cytosine, as well as unmethylated CCGG sites in all three strains. Digestion with StyI and XhoI revealed an unmethylated XhoI site in the C57BL/6 male. This pattern is not obvious in the B6C3F1 or C3H/He, indicating sequence variation and/or less methylation of Ha-ras in those strains of mice that exhibit a high propensity towards development of liver tumors.

5-Methylcytosine↗
PubMed1993

Hypomethylation of DNA: a possible nongenotoxic mechanism underlying the role of cell proliferation in carcinogenesis.

DNA methylation (i.e., the 5-methylcytosin content of DNA) plays a role in the regulation of gene activity. There is a persuasive body of evidence indicating that differential methylation of DNA (i.e., 5-methylcytosine versus cytosine) is a determinant of chromatin structure and that the methyl group provides a chemical signal that is recognized by trans-acting factors that regulate transcription. Hypomethylation (i.e., low levels of DNA 5-methylcytosine) of a gene is necessary but not sufficient for its expression, and, therefore, a hypomethylated gene can be considered to possess an increased potential for expression as compared to a hypermethylated gene. Cell proliferation is a fundamental component of carcinogenesis. It plays a key role in expanding clones of initiated cells and, in addition, cell replication may contribute to carcinogenesis by facilitating mutagenesis. This can occur either by causing the fixation of promutagenic DNA-damage before repair or as a consequence of a "normal" error occurring during DNA replication. During periods of cell proliferation the established pattern of DNA methylation is maintained by the action of a maintenance methylase following DNA replication. Changes in the methylation status of a gene provide a mechanism by which its potential for expression can be altered in an epigenetic heritable manner, and it is expected that modifications in DNA methylation would result from threshold-exhibiting events.

5-Methylcytosine↗
PubMed1991

Hypomethylation of ras oncogenes in chemically induced and spontaneous B6C3F1 mouse liver tumors.

The male hybrid B6C3F1 mouse exhibits a 30% spontaneous hepatoma incidence, and both males and females of this strain are sensitive to chemical induction of liver tumors. The Ha-ras, Ki-ras, and myc oncogenes have been implicated in a variety of solid tumors. Specifically, Ha- and, less frequently Ki-ras have been reported to be activated in B6C3F1 mouse liver tumors, and such activated oncogenes frequently contain a particular point mutation. In light of indications that the transforming capacity of some oncogenes is directly related to the level of the gene product, we hypothesized that transcriptional control of Ha-ras, Ki-ras, and myc is compromised in B6C3F1 mouse liver tumors. A positive correlation has been established between gene expression and hypomethylation. Therefore, the methylation states of these genes were examined in spontaneous liver tumors and in tumors induced by two diverse hepatocarcinogens: phenobarbital and chloroform. Ha-ras was found to be hypomethylated in all tumors examined, whereas Ki-ras was sometimes hypomethylated; such hypomethylation might play a role in the promotion stage of carcinogenesis. The methylation state of myc was unaltered, although this gene appeared to be amplified in tumors. These results suggest that a component of the mechanism by which these oncogenes are activated in B6C3F1 mouse liver tumors involves loss of stringent control of expression, via hypomethylation of the ras oncogenes and, possibly, amplification of myc. These results support the assertion that tumors induced by different classes of carcinogens or arising spontaneously share common biochemical pathways of oncogene activation during tumorigenesis.

Animals↗
PubMed1991

Differential DNase I hypersensitivity of ras oncogenes in B6C3F1, C3H/He, and C57BL/6 mouse liver.

The male hybrid B6C3F1 mouse exhibits a 30% spontaneous hepatoma incidence, whereas the paternal C3H/He strain and the maternal C57BL/6 strain exhibit a 60% and a negligible incidence, respectively. In addition, both male and female B6C3F1 mice are extremely sensitive to chemical induction of hepatocarcinogenesis. The Ha-ras, Ki-ras, and myc oncogenes have been implicated in a variety of solid tumors. Specifically, Ha- and, less frequently, Ki-ras have been reported to be activated in B6C3F1 mouse liver tumors. The objective of this study was to examine a possible point of transcriptional control of Ha-ras, Ki-ras, and myc in all three mouse strains, our hypothesis being that these oncogenes may be primed for expression in the nascent liver of those strains exhibiting a high spontaneous hepatoma incidence. A positive correlation has been established between gene expression and the presence of DNase I hypersensitive sites. DNase I hypersensitive sites were observed in the Ha-ras and myc oncogenes in the three mouse strains. However, Ha-ras appears to possess an additional site in B6C3F1 and C3H/He as compared to C57BL/6. Similarly, the Ki-ras oncogene exhibited a DNase I hypersensitive site only in B6C3F1 and C3H/He mouse liver. These results indicate that the hepatoma-prone strains (B6C3F1 and C3H/He) may have a greater potential for Ha- and Ki-ras expression than does the non-hepatoma-prone strain (C57BL/6).

Animals↗
PubMed1990

Infection of rat liver epithelial cells with v-Ha-ras: correlation between oncogene expression, gap junctional communication, and tumorigenicity.

The role of v-Ha-ras oncogene in tumorigenesis in an in vitro/in vivo model system was studied by investigating the expression of the Ha-ras gene, gap junctional intercellular communication, and tumorigenicity as endpoints. Infection of a Fischer 344 rat liver epithelial cell line (WB 344) with a retrovirus containing the v-Ha-ras oncogene resulted in altered cell morphology and decreased contact sensitivity. Gap junctional intercellular communication in v-Ha-ras infected WB cells (WBHa-ras), assessed by fluorescence redistribution after photobleaching (FRAP), microinjection/dye transfer, and scrape-loading/dye transfer techniques, was markedly decreased compared with the level in control WB cells. Injection of 10(7) WBHa-ras cells into the portal vein of male F344 rats caused multiple focal hepatic lesions within 1 and 2 wk, merging to large invading tumors after 3 and 4 wk. Examination of the methylation pattern of the Ha-ras gene in WBHa-ras and control WB cells showed that the infected Ha-ras gene was relatively hypomethylated in comparison to the normal cellular Ha-ras gene, indicating a greater potential for expression. There was an increased level of Ha-ras mRNA in hepatomas as compared with both adjacent nontumor liver tissue and liver tissue obtained from normal animals. Three cell lines derived from three different primary hepatic tumors induced by an injection of WBHa-ras cells in a F344 rat displayed similar growth characteristics, levels of gap junctional communication, and methylation patterns as the original WBHa-ras cells. The results of these studies have established a strong positive correlation between expression of the Ha-ras oncogene, reduced gap junctional intercellular communication, decreased contact sensitivity, and tumorigenicity of the v-Ha-ras-infected rat liver epithelial cells.

Animals↗
PubMed1989

Altered methylation of ras oncogenes in benzidine-induced B6C3F1 mouse liver tumors.

The B6C3F1 mouse is a hybrid strain which exhibits a high (30%) spontaneous hepatoma incidence and sensitivity to chemical induction of liver tumors. The spontaneous hepatoma incidence of the paternal C3H/He strain is approximately 60%, while that of the maternal C57BL/6 strain is very low. The presence of activated oncogenes, primarily Ha-ras, and to a lesser extent, Ki-ras, has been reported in B6C3F1 mouse liver tumors. Because alterations in a gene's capacity for expression, as well as mutation, may be involved in oncogene activation, this investigation was directed toward an examination of a putative control point for transcription, i.e., the methylation state of a gene. Hypomethylation is believed to be necessary, but not sufficient, for transcription. It was therefore hypothesized that alterations in the methylation state of the Ha-ras and Ki-ras oncogenes may facilitate the aberrant expression of these genes in B6C3F1 mouse liver. Restriction enzyme analysis (MspI, HpaII, and HhaI) was used to assess the extent of DNA methylation. MspI digestion of B6C3F1 and C3H/He DNA revealed the absence of a 15-kb Ha-ras band present in MspI-digested C57BL/6 DNA, suggesting that the Ha-ras oncogene of B6C3F1 and C3H/He mouse liver lacks a methylated site. In other respects, the Ha-ras and Ki-ras oncogenes are methylated to a degree which suggests that these oncogenes have a low potential for expression in normal mouse liver. The methylation state of the Ha-ras and Ki-ras oncogenes was also assessed in benzidine-induced hepatomas and adjacent nontumor tissue from B6C3F1 mice. In four out of four cases, the Ha-ras oncogene was hypomethylated in tumor as compared to nontumor tissue and increased expression of the gene was detected in three out of four hepatomas; the Ki-ras oncogene was hypomethylated in two out of four cases. These results suggest that hypomethylation of oncogenes may provide an epigenetic mechanism for facilitating their aberrant expression. The lack of a methylated site observed in the Ha-ras oncogene in B6C3F1 and C3H/He mouse liver may indicate an increased potential for its expression which could, in part, account for the high propensity for hepatoma development in these two strains.

Animals↗
PubMed1987

Investigation of parameters associated with activity of the Kirsten-ras, Harvey-ras, and myc oncogenes in normal rat liver.

Oncogenes have been implicated in the mechanism(s) involved in the different stages of carcinogenesis. Point mutations have been found to activate the ras family of oncogenes. However, these genes must also be expressed in order for phenotypic alterations to be seen. In addition, overproduction of the normal product of some oncogenes may be a factor in the development of cancer. This study was therefore designed to investigate the potential for activity that the Kirsten (Ki)-ras, Harvey (Ha)-ras, and myc oncogenes possess in normal rat liver; activation of these genes has previously been observed in hepatocellular carcinomas. The serum albumin gene was used as a positive control. Two possible control points of gene expression were examined: (1) gene methylation state, as there is an established positive correlation between hypomethylation and gene activity; and (2) DNase I sensitivity because transcriptionally active genes are preferentially sensitive to digestion by this enzyme. It was found that the serum albumin gene is hypomethylated and preferentially sensitive to DNase I, both states being consistent with gene activity. In contrast, Ki-ras is highly methylated and relatively resistant to DNase I digestion. The Ha-ras and myc oncogens exhibit a degree of methylation and DNase I sensitivity intermediate between the serum albumin gene and the Ki-ras oncogene. It thus appears that Ki-ras is not expressed in normal rat liver, whereas Ha-ras and myc may have the potential for activity. In addition, a correlation was found between the methylation state of a gene and its sensitivity to DNase I. These findings are compatible with the view that carcinogenesis is a multistage process. Our results indicate that possible control points for oncogene expression, as well as mutation, should be considered in investigations of the role these genes play in carcinogenesis.

Animals↗
PubMed1985

Methylation of the serum albumin gene as compared to the Kirsten-ras oncogene in hepatocytes and non-parenchymal cells of rat liver.

The extent of methylation of a gene, i.e. percent of cytosine present as 5-methylcytosine, is correlated with its activity. Hypermethylation is associated with non-expression, whereas hypomethylation is a necessary but not sufficient condition for expression. In this study, the methylation state of the serum albumin gene as compared to the Kirsten-ras (Ki-ras) oncogene was assessed in hepatocytes and non-parenchymal cells (NPC) isolated from rat liver. The results of this investigation indicate that the serum albumin gene is hypomethylated in hepatocytes and hypermethylated in NPC. This is consistent with expression of the gene in the former cell type, and non-expression in the latter. In contrast, the Ki-ras oncogene is hypermethylated in both hepatocytes and NPC, suggesting that it is, at most, minimally expressed in normal rat liver.

Animals↗
PubMed1984

DNA-purine methylation in hepatic chromatin following exposure to dimethylnitrosamine or methylnitrosourea.

The investigations reported in this paper were designed to analyze the patterns of DNA-purine methylation in hepatic chromatin following in vivo exposure to the carcinogenic alkylating agents dimethylnitrosamine (DMN) or methylnitrosourea (MNU). Male Sprague-Dawley rats were exposed to [14C]DMN (8 mumoles, 1.0 microCi per mumole per 100 g) or [3H]MNU (15 mumoles, 10 microCi per mumole per 100 g) via gastric intubation. Hepatic chromatin was fractionated into portions having characteristics of template-active euchromatin (S2) and template-repressed heterochromatin (P2) by digestion with DNase II followed by MgCl2 precipitation. Specific DNA purines were identified at 24 hr post-intubation using an isocratic high pressure liquid chromatographic system. A qualitatively similar pattern of 7-methylguanine, O6-methylguanine, 1-methyladenine and 3-methyladenine alkylation was observed in DNA from total chromatin versus heterochromatin at 24 hr following exposure to either carcinogen. These assessments were made at times following carcinogen exposure which produced maximal quantitative differences in alkylation of euchromatin versus heterochromatin DNA. Similar patterns of DNA purine alkylation were observed in total chromatin and heterochromatin. These observations suggest that, once the reactive species is generated and access to chromatin DNA occurs, a similar pattern of DNA-purine alkylation is produced in different regions of the genome.

Animals↗
PubMed1984

Non-random interaction of N-2-acetylaminofluorene and its N-hydroxy metabolite with DNA of rat hepatic parenchymal and non-parenchymal cell nuclei following in vivo administration of carcinogen.

Treatment of rats with the hepatocarcinogen, N-2-acetylaminofluorene (AAF) results in development of malignant tumors derived primarily from hepatic parenchymal cells. Following administration of AAF, or its N-hydroxy derivative, in vivo nuclei from parenchymal cell and non-parenchymal cell populations (NI and NII nuclei populations, respectively) were isolated and treated with the endonuclease DNase I. The binding of carcinogen residues to the DNA of nuclease-accessible vs. nuclease-resistant regions of chromatin was evaluated on the basis of the selectivity of DNase I for transcriptionally active DNA. Under the experimental conditions employed DNase I digested approx. 50-60% of the genome of NI nuclei while only 10-20% of the DNA from non-parenchymal cell nuclei (NII) was susceptible to this enzyme. When the DNA of NI and NII nuclei were nick translated following limited digestion with DNase I, a greater degree of transcriptional activity (nuclease accessibility) was found in parenchymal cell nuclei (NI). Following a single injection of rats with [ring-3H]AAF or its N-hydroxy metabolite (N-[ring-3H]-OH-AAF) (1.8 mumol carcinogen/100 g), adducts were preferentially associated with DNA of DNase I resistant regions of target cell nuclei (NI), while preferentially associated with nuclease-accessible regions of non-target cell nuclei (NII). Damage following a single injection persisted for up to 3 days in DNase I-resistant DNA of NI nuclei, carcinogen adducts were rapidly lost from DNase I-accessible DNA of NII nuclei. These studies stress the importance of investigating specificity of carcinogens for particular regions of the genome of cell subpopulations within the target organ.

2-Acetylaminofluorene↗
PubMed1984

Quantitative studies on the in vitro metabolic activation of dimethylnitrosamine by rat liver postmitochondrial supernatant.

The metabolic activation of dimethylnitrosamine (DMN) to mutagenic and/or cytotoxic intermediates in vitro has been characterized and the relationship between DMN demethylase and ethoxyresorufin-O-deethylase (EROD) or ethylmorphine-N-demethylase (EMND) has been evaluated. A mammalian assay system which uses the postmitochondrial supernatant (S-15 fraction) prepared from a rat liver homogenate as an enzyme source and V79 Chinese hamster cells as targets for chemically induced damage was used. The enzyme pattern of the S-15 fraction was altered by pretreatment of experimental animals in vivo and/or by the use of enzyme inhibitors in vitro. The results of these studies indicate that the concentration of S-15 fraction in the reaction mixture can markedly influence the degree of DMN-induced cytotoxicity when it is metabolized in vitro and that the degree of DMN-induced cytotoxicity and mutagenicity are linearly related. The degree of cytotoxicity and mutagenicity induced in V79 cells by DMN does not correlate with EROD activity (a measure of 3-methylcholanthrene-inducible mixed-function oxidases) nor with EMND activity (a measure of phenobarbital-inducible mixed function oxidases) in the S-15 fraction.

Animals↗
PubMed1983

Hepatic tumor-promoting ability of 3,3',4,4',5,5'-hexabromobiphenyl: the interrelationship between toxicity, induction of hepatic microsomal drug metabolizing enzymes, and tumor-promoting ability.

Female Sprague-Dawley rats were fed diets containing 0, 0.01, 0.1, or 1.0 mg/kg 3,3',4,4',5,5'-hexabromobiphenyl (345-HBB) for 140 days after a 70% partial hepatectomy and diethylnitrosamine administration (10 mg/kg body weight) to determine if 345-HBB had tumor-promoting ability in a two-stage hepatocarcinogenesis assay. Tumor-promoting ability was assessed by measuring enzyme-altered foci exhibiting gamma-glutamyl transpeptidase activity. Enhancement of enzyme-altered foci occurred only at a dietary concentration of 345-HBB (1.0 mg/kg) that was toxic. The toxic effects were decreased body weight gain, involution of the thymus, increased liver weight, histologic and ultrastructural alterations of the liver, and elevated serum concentrations of aspartate aminotransferase. 345-HBB is a strict 3-methylcholanthrene (MC) type of hepatic microsomal drug metabolizing enzyme inducer and caused a dose-related increase of cytochrome P-450. 345-HBB, at a dietary concentration of 0.1 mg/kg, caused a physiologic response in rats as determined by induction of hepatic microsomal drug metabolizing enzymes, but there was minimal evidence of toxicity and no evidence of tumor-promoting ability. Results indicate that there can be induction of MC type of hepatic microsomal drug-metabolizing enzymes without toxicity or tumor-promoting ability and that the tumor-promoting ability of 345-HBB was most likely the result of hepatic degeneration and necrosis. This finding is in contrast to previous studies in which a closely related congener, 2,2',4,4',5,5'-hexabromobiphenyl, enhanced the development of enzyme-altered foci at dietary concentrations that were not hepatotoxic.

Animals↗