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H C Pitot

Publications and source records attributed to H C Pitot.

At least 127 records · Page 7Linked to original sources
PubMed1993

Multistage carcinogenesis--genetic and epigenetic mechanisms in relation to cancer prevention.

The prevention of cancer is, at our present state of knowledge, the most effective and inexpensive mode of controlling this disease. Passive cancer prevention is practiced voluntarily or, in part, through governmental regulation in many parts of the world. However, active cancer prevention by the administration of vaccines, dietary factors, antihormones, or other agents is likely to be the most effective mechanism of cancer prevention in humans. Carcinogenesis develops in three defined stages--initiation, promotion, and progression--the first and third resulting from irreversible genetic changes in the cell, whereas the intermediate stage of promotion involves an epigenetic alteration of the expression of the genome and cell division. Thus, the stage of promotion is the most effective site to target for both active and passive cancer prevention. A review of the modalities presently used in cancer prevention, both active and passive, demonstrates that all such methods involve an inhibition and/or reversal of this intermediate stage, thus preventing the development of the malignant process. Future advances in cancer prevention will depend on better knowledge of the mechanisms of the stages of carcinogenesis in order that rational preventive agents and formats may be used in altering or modifying the appropriate stage(s) of neoplastic development.

Humans↗
PubMed1993

Reestablishment of cell polarity of rat hepatocytes in primary culture.

The cell-basement membrane interaction is an important determinant of epithelial cell polarity. Although hepatocytes in situ are polarized, no morphologically identifiable basement membrane is found at their basal surface. However, several studies have demonstrated immunoreactivity to basement membrane proteins in the space of Disse, indicating the existence of an extracellular matrix, albeit of low density. Therefore we hypothesized that the interaction of hepatocytes with this matrix may determine their polarity and asked whether a basement membrane-like substrate could reestablish hepatocyte polarity in vitro. For this purpose, established monolayers of primary rat hepatocytes were cultured overlaid with a basement membrane-like matrix extracted from the Engelbreth-Holm-Swarm mouse tumor, mimicking thus the in situ tissue architecture. The hepatocytes in this culture configuration, unlike hepatocytes in classic cultures, developed distinct membrane domains, as demonstrated by the reformation of gamma-glutamyltranspeptidase, Mg(2+)-ATPase-positive bile canalicular networks and intercellular gap junctions immunolocalized to the lateral membrane with antibodies to connexin 32. The actin cytoskeleton of these cells reorganized into pericanalicular webs, and no accumulation of "stress" filaments was found beneath the membrane facing the medium. Golgi complexes appeared to be preferentially located in mitochondria-poor pericanalicular cytoplasm, indicating the polarized distribution of these organelles. Together, these data indicate that a basement membrane-like substrate present between hepatocytes and nutrient medium restores the polarity of these cells in culture. Extrapolation of these findings to the intact liver suggests that the matrix in Disse's space governs the development of hepatocyte polarity.

Animals↗
PubMed1992

Regulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible expression of aldehyde dehydrogenase in hepatoma cells.

The environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces the expression of a number of genes. The biochemical process of the induction of aldehyde dehydrogenase (ALDH-3) was investigated in rat H4IIE hepatoma cells in culture. The kinetics of ALDH-3-induction exhibited parallel increases in the rate of transcription, mRNA, protein, and enzyme activity, all reaching a plateau at 36-48 h after addition of TCDD. Half maximal and maximal inductions occurred at 0.1 and 1 nM of TCDD, respectively. No significant changes in the half-life of ALDH-3 mRNA (14 h) were observed in the cells exposed to three different concentrations of TCDD. Other inducers of xenobiotic metabolism, such as 3-methylcholanthrene and beta-naphthoflavone, also induced ALDH-3 mRNA to a similar level as TCDD, whereas antioxidants or electrophiles, such as tert-butylhydroquinone and dimethyl fumarate, did not show any induction of ALDH-3 mRNA. To examine the involvement of the aryl hydrocarbon receptor (Ah receptor) in the induction of ALDH-3, mouse variant cell lines defective in cytochrome P450IA1-induction and a parental wild type cell line (Hepa1c1c7) were studied. ALDH-3 mRNA and the transcription of its gene were detected in TCDD-treated wild type cells, but not in the treated and untreated variant cells. These results demonstrate that TCDD induces transcription of the ALDH-3 gene via its binding to the Ah receptor.

Aldehyde Dehydrogenase↗
PubMed1992

Regulatory elements for the tissue-specific expression of the rat serine dehydratase-encoding gene.

L-Serine dehydratase (SDH; EC 4.2.1.13), the key enzyme for serine utilization in the rat, is synthesized primarily in the liver. Cis-acting DNA elements required for liver-specific expression of the SDH gene were identified by two approaches: (1) transient expression assays in primary cultured rat hepatocytes, and in rat fibrosarcoma and normal rat kidney epithelial (NRK-52E) cell lines; and (2) in vitro transcription assays with nuclear extracts prepared from rat liver and spleen. Deletion analyses of the 5' flanking sequences of the gene have defined two functionally different regions: (a) a cell-type-specific promoter located between positions -62 and +10, which is sufficient for liver-specific expression; and (b) a distal promoter region between bp -133 and -63 containing positive cis-acting elements that regulate the promoter activity in a non-tissue-specific fashion. No other cis-acting elements essential for liver-specific expression were found in the region of -134 to 2.1 kb upstream relative to the cap site of SDH.

Animals↗
PubMed1992

Isolation and characterization of the rat gene encoding ornithine aminotransferase.

Herein we describe the isolation and characterization of the rat gene encoding ornithine aminotransferase (rOAT). Six unique genomic clones were characterized and assigned to two nonoverlapping contigs representing approx. 33 kb of the rat genome. The 5' contig contains the rOAT promoter, exons 1 and 3, and a portion of exon 4; an exon corresponding to exon 2 of the human OAT gene (hOAT) was not identified. The rOAT promoter contains several putative regulatory elements in positions similar to hOAT. The 3' contig contains exons 7 through 11 in their entirety. Data presented and discussed herein suggest that approx. 3.0 kb of uncloned genomic DNA, containing the remainder of exon 4 and all of exons 5 and 6, separate the two contigs. Together, these data suggest that rOAT extends over approx. 20 kb and is organized into at least 10 exons, thereby closely resembling hOAT in size and exon/intron organization. Isolation of rOAT provides an important tool for examining the molecular mechanisms through which estrogen and thyroid hormone regulate transcription of this gene in a cell-type specific manner.

Animals↗
PubMed1992

Ploidy and specific karyotypic changes during promotion with phenobarbital, 2,5,2',5'-tetrachlorobiphenyl, and/or 3,4,3'4'-tetrachlorobiphenyl in rat liver.

Polychlorinated biphenyls are a group of industrial chemicals that are widely distributed in the environment. Since these compounds occur as mixtures, studies of their possible interactive effects are important. In order to determine whether an interaction of 2,5,2',5'-tetrachlorobiphenyl (TCB) with 3,4,3',4'-TCB occurs during multistage hepatocarcinogenesis in vivo, like that previously observed in lymphocytes in vitro (L. M. Sargent et al., Mutat. Res., 224: 79-88, 1989), we exposed rats to a single initiating dose of diethylnitrosamine (DEN), 10 mg/kg after a 70% partial hepatectomy, and subsequently to 0.1 ppm 3,4,3',4'-TCB and/or 10 ppm 2,5,2',5'-TCB in the diet for 1 year. Administration of each of the TCBs alone after DEN initiation resulted in a low incidence of chromosomal damage in hepatocytes; but when the two were given together after DEN initiation, there was a more than additive effect on this parameter at both 7 and 12 months which was highly significant. Administration of the TCBs alone or in combination in the absence of DEN initiation also resulted in chromosomal damage, approaching that seen in livers of animals initiated with DEN when sacrificed at 12 months. In animals receiving 0.05% phenobarbital for a 12-month period after initiation with DEN, a significant degree of chromosomal breakage and fragment formation occurred both in hepatocytes expressing the ectoenzyme gamma-glutamyltranspeptidase (GGT) and in those that were GGT negative. However, the GGT-negative cells showed a significantly lower incidence of chromosomal damage than the GGT-positive hepatocytes. Exposure to phenobarbital for 7 months after DEN initiation resulted in no significant chromosomal damage in hepatocytes, whether GGT positive or GGT negative. Some degree of specificity in chromosomal alterations was seen in hepatocytes of animals initiated with DEN and promoted either with a combination of TCBs or with phenobarbital. The most frequent alterations seen were a trisomy of chromosome 1 or of its long arm and a monosomy of chromosome 3 or its short arm. Some chromosome 7 aberrations were also seen. The highest frequency of specific aberrations occurred in hepatocytes from rats that also bore hepatocellular carcinomas, suggestive of the hypothesis that genes involved in the development of hepatic carcinoma may reside in chromosome 1 and/or 3 of the rat.

Animals↗
PubMed1992

Small cell colonies appear in the primary culture of adult rat hepatocytes in the presence of nicotinamide and epidermal growth factor.

Colonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum-free modified Dulbecco's modified Eagle's medium containing 10 mmol/L nicotinamide and 10 ng/ml epidermal growth factor. Each colony consisted of cells that had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically these cells were stained with albumin and transferrin. Ultrastructurally these cells had mitochondria, peroxisomes and desmosomes, indicating that they were derived from hepatocytes. When 6 x 10(5) cells were plated on 35-mm dishes, about 5.5 colonies/mm2 were observed. This result suggested that about 1.5% of adult rat hepatocytes has the potential for multiple replications and of forming a focal colony. These cell populations had higher proliferative activities than surrounding hepatocytes. DNA synthetic activity could not be inhibited by 2% dimethyl sulfoxide. Flow cytometric analysis showed that both 2N and 4N nuclei synthesized their DNA until day 4 but that the number of 2N nuclei rapidly increased at day 5. This result correlated with the observation of the appearance of small cell populations indicating that the cells of these focal colonies were predominantly diploid.

Animals↗
PubMed1992

Characteristics of small cell colonies developing in primary cultures of adult rat hepatocytes.

Phenotypes of the cells developing into small colonies after days of primary culture of adult rat hepatocytes in serum-free modified Dulbecco Modified Eagles' medium containing 10 mM nicotinamide and 10 ng/ml epidermal growth factor were analyzed immunocytochemically, cytochemically and ultrastructurally. Albumin, cytokeratin 8 and 18 were seen by immunocytochemical techniques in the cells of the small colonies at Day 6. Transferrin, alpha 1-antitrypsin, ceruloplasmin, and haptoglobin, proteins secreted by mature hepatocytes, were faintly stained in these cells as was alpha-fetoprotein. These proteins were secreted into the culture medium as evidenced by immunoblot analysis. gamma-Glutamyltransferase, alkaline phosphatase and glucose 6-phosphatase were not present in the cells of the small colonies as well as the surrounding hepatocytes at Day 6 of culture. In addition, ultrastructural examinations of the cells in the small colonies indicated that these cells not only had many characteristic mitochondria and desmosomes, but also a few small peroxisomes. Such cells, even after 20 days in culture were proliferating, as evidenced by the intranuclear presence of the proliferating cell nuclear antigen. The potential relation of these cells to hepatocytes which may serve as the principal reserve for replicating hepatocytes is discussed.

Albumins↗
PubMed1992

Location and characterization of multiple glucocorticoid-responsive elements in the rat serine dehydratase gene.

Transcription of the gene coding for serine dehydratase (SDH, EC 4.2.1.13) in the rat in vivo is dramatically increased by glucocorticoid hormones. To identify DNA elements mediating the glucocorticoid-regulated expression of the SDH gene, we transiently transfected 7AD-7 rat hepatoma cells with fusion genes consisting of various regions of the SDH 5' flanking sequence linked to the coding sequence of the gene for chloramphenicol acetyltransferase (CAT). Analysis of the CAT activities from these 5' deletion mutants identified three closely associated glucocorticoid-responsive elements (GREs), located more than 5 kb upstream relative to the cap site. Two distal GREs act synergistically to confer strong glucocorticoid inducibility to the gene, whereas the proximal GRE functions independently of the distal GREs and confers only a weak hormone response to the gene. The purified DNA-binding domain of rat glucocorticoid receptor binds to the sequence of each GRE as shown by footprinting experiments. However, only one of these sequences contains the TGTTCT consensus sequence reportedly associated with many other GREs.

Animals↗
PubMed1992

Identification of regions in the rat serine dehydratase gene responsible for regulation by cyclic AMP alone and in the presence of glucocorticoids.

Transcription of the rat serine dehydratase (SDH) gene is induced by glucagon, mediated by the action of cAMP. To identify the nucleotide sequences in the SDH gene responsible for this regulation, we constructed chimeric genes containing different portions of the 5' flanking region of the rat SDH gene fused to the structural sequence encoding the bacterial reporter enzyme, chloramphenicol acetyltransferase (CAT). The transcriptional activities of the fusion genes introduced into the rat hepatoma cell line 7AD-7 were assayed by measuring CAT activity in the cell lysates. Chlorophenylthio-cyclic AMP (CPT-cAMP), a potent protein kinase A activating agent, stimulated the expression of SDH-CAT fusion genes, and these inductions could be enhanced further by the addition of dexamethasone, although the glucocorticoid alone had no effect on CAT activity. Deletion analysis demonstrated that an 80 bp region located approximately 3.5 kb upstream from the transcription initiation site of the rat SDH gene was responsible for stimulation of transcription by CPT-cAMP, whereas the 120 bp region immediately upstream of the cAMP responsive element (CRE)-containing sequences is essential for the enhancement of CPT-cAMP induction by the glucocorticoid.

Animals↗
PubMed1992

Effects of phenobarbital on I-compounds in liver DNA as a function of age in male rats fed two different diets.

The age-dependent effects of diet and of phenobarbital (PB), a known promoter of hepatocarcinogenesis, on indigenous DNA adducts (I-compounds) were studied by the 32P-post-labeling technique. Late-gestation female Sprague-Dawley rats were fed either AIN-76A semisynthetic or Teklad cereal-based diet. At 21 days after birth, the male pups were weaned and continued on the diets, with half of each dietary group receiving 0.05% PB mixed into the diet for 2, 4 and 8 months. Age-dependent increases in I-compound levels were observed. In addition, both the levels of individual I-compounds and the overall number of I-compounds were greater in rats fed the Teklad cereal-based diet than in those fed the AIN-76A diet. Independent of the parent diet, PB administration reduced the levels of the majority of I-compounds in a time-dependent manner. This effect of PB was detected earlier in the Teklad-fed than in the AIN-76A-fed group. In contrast to the I-compounds, a second group of spots, termed reverse I-compounds, declined between 2 and 4 months and, especially in AIN-76A-fed animals, tended to increase when PB was administered. It is hypothesized that alterations of DNA modification patterns may play a role in diet-modified hepatocarcinogenesis promoted by PB.

Age Factors↗
PubMed1992

The role of the stages of initiation and promotion in phenotypic diversity during hepatocarcinogenesis in the rat.

Differences in the distribution of phenotypic alterations in initiated and promoted cell populations reflect both the dose and the action of the specific initiating agent, as well as the mechanism of action of the promoting agent. The use of multiple phenotypic markers to characterize AHF should allow further experimentation on the characteristics of promoting agents in hepatocarcinogenesis, especially their ability to promote separate populations of initiated cells, and on those characteristics of initiated cells that enable certain ones to survive and multiply in the specific environment provided by a promoting agent. Studies of promoting agents with differing mechanisms of action should further allow questions of reversibility, substitution and additivity of the actions of promoting agents to be addressed. The possibility that individual promoting agents do not enhance the growth of the entire population of initiated cells indicates that study of combinations of promoting agents is an important future direction of research. Therefore, information on the characteristics of promoting agents, singly and in combination, is necessary to assess more accurately the contribution of promoting agents to the carcinogenic risk for humans distinct from the effects of initiating agents and complete carcinogens.

Animals↗
PubMed1992

Characterization of the promotion of altered hepatic foci by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the female rat.

Previous studies have suggested that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) acts as a promoting agent in various organ systems including the rat liver. Since a major characteristic of the stage of tumor promotion is its operational reversibility, we have assessed whether TCDD-induced promotion is reversible in a two-stage model of hepatocarcinogenesis. In this model, female Fischer F344 rats were administered a single, intragastric dose of the initiating agent, diethylnitrosamine (DEN, 10 mg/kg), at the peak of proliferation induced by a partial hepatectomy. TCDD was then administered biweekly (0.14 micrograms/kg, s.c.) for 1, 3 or 5 months. One group of animals was killed at each of these time points, while a second group was maintained for each time point for an additional 6 months in the absence of further TCDD. Four serial frozen sections of liver were each stained with a different enzyme marker of altered hepatic foci (AHF). The AHF were identified and the number and volume fraction determined by quantitative stereology. Exposure to TCDD resulted in an increase in the number and size of AHF in the initiated relative to the uninitiated rats. Increasing the duration of promotion with TCDD led to an increase in the number of AHF per liver, the volume fraction of the liver occupied by AHF and the number of markers expressed aberrantly by a single AHF. Discontinuation of TCDD administration for 6 months before killing the animals resulted in a decrease in the total number of AHF observed, but those AHF that remained increased in size with an overall increase in volume fraction of AHF. Analysis of the size class distribution for AHF for each of the periods of TCDD promotion revealed an increase in the larger AHF but a decrease in the smaller, thereby resulting in an overall decrease in number of AHF with an increase in the volume fraction of AHF. Increasing the duration of the TCDD exposure prior to its withdrawal led to an increased AHF size, phenotypic complexity and number of AHF remaining after cessation of TCDD administration. Although the levels of TCDD in livers of rats 6 months after cessation of TCDD administration were still greater than background, they were markedly reduced compared to immediately after administration. Thus, cessation of exposure to TCDD after a brief duration led to a reversal of its promotional effects on the majority of AHF, while prolonged exposure led to maintained promotion of a minority of AHF.

Adenosine Triphosphatases↗
PubMed1992

A phase II study of intravenous 6-thioguanine (NSC-752) in advanced colorectal carcinoma.

Fifteen patients with advanced measurable colorectal carcinoma were treated with intravenous 6-thioguanine (6-TG) at a dosage of 55 mg/m2 for 5 consecutive days every 5 weeks. Only one patient had received prior adjuvant chemotherapy. No responses were detected, and eight patients had stable disease for a medium duration of three treatment cycles. Toxicity was tolerable. We conclude that 6-TG given by this dosage schedule is ineffective in treating metastatic colorectal carcinoma.

Adult↗
PubMed1992

Ovariectomy promotes the growth of altered hepatic foci after withdrawal and reintroduction of phenobarbital during hepatocarcinogenesis in rats.

Female F344/N rats were given 70% partial hepatectomies and intubated with diethyl-nitrosamine (DEN, 10 mg/kg) 24 hours later. They were fed a cereal-based diet, NIH-07 (NIH) + 0.05% phenobarbital (PB) for 6 months, at which time NIH + PB was withdrawn and the rats were ovariectomized (OV) or sham-operated (SH). Groups of 7-10 rats were fed a semipurified diet (AIN-76) for 1 or 2 months after withdrawal of NIH + PB, or NIH + PB for 2 months, or AIN-76 diet for 1 month and subsequently NIH + PB for 1 month. Placental glutathione S-transferase (PGST)- and gamma-glutamyltransferase (GGT)-positive (+) altered hepatic foci (AHF) were analysed by quantitative stereology. Ovariectomy stimulated growth of AHF after withdrawal and reintroduction of NIH + PB. AHF, especially PGST+ AHF, continued to regress throughout the PB withdrawal period in rats fed AIN-76 diet. In most studies of chemical hepatocarcinogenesis, females have been shown to develop a greater volume of AHF than males. In our study, however, ovariectomy stimulated the growth of AHF after withdrawal and reintroduction of PB. Because AHF occurring spontaneously in male rats develop more rapidly than in female rats, the greater rate of growth of AHF in OV female rats may reflect a similar mechanism.

Animals↗
PubMed1992

Breast cancer.

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Breast Neoplasms↗