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Biomedical subjects

H C Pitot

Publications and source records attributed to H C Pitot.

At least 235 records · Page 13Linked to original sources
PubMed1986

Oncogenes and human neoplasia.

Genes whose products are directly involved in the transformation of a normal to a neoplastic cell are present in most but not all oncogenic RNA viruses (retroviruses) have genes found in oncogenic RNA viruses (retroviruses) have also been found in normal cells, their coding sequences highly conserved in evolution. Such viral genes (v-onc) are expressed at high levels in infected cells. "Activation" of cellular homologues of v-onc genes (c-onc) may occur by a variety of mechanisms leading to an abnormal and/or increased expression of such activated c-onc genes in malignant cells. Although oncogene activation appears to be a critical step in the neoplastic transformation induced by oncogenic viruses, the role of this process in the development of chemical and radiation-induced neoplasia is not yet clear.

Cell Transformation, Neoplastic↗
PubMed1985

Sequence of the precursor to rat ornithine aminotransferase deduced from a cDNA clone.

The nucleotide sequence of ornithine aminotransferase mRNA from rat liver, including the entire coding and 3' untranslated regions, was determined from two overlapping cDNA clones. The mRNA encodes a precursor polypeptide of 439 amino acid residues with a molecular weight of 48,332. The deduced amino acid composition of the proposed mature enzyme sequence (residues 35 through 439) was in good agreement with that reported for the purified protein. The amino-terminal segment of the precursor corresponding to residues 1 through 34 has an overall positive charge, containing 6 basic residues and only a single acidic residue, and is postulated to be the mitochondrial leader sequence. The first 22 amino acid residues of the proposed leader sequences share 54% homology with the leader peptide of rat ornithine transcarbamylase precursor and more limited homology to the leader peptides of other nuclear-encoded mitochondrial matrix proteins. Homology was also observed between residues 286 through 362 ornithine aminotransferase precursor and a region containing the pyridoxyl phosphate binding domain of mitochondrial aspartate aminotransferase.

Amino Acid Sequence↗
PubMed1985

Levels of ornithine aminotransferase messenger RNA under conditions of cyclic AMP induction in cultured hepatocytes.

In primary cultures of adult rat hepatocytes, the relative rate of ornithine aminotransferase synthesis is increased approximately 7- to 8-fold in response to dibutyryl cyclic AMP. The corresponding increase in ornithine aminotransferase messenger RNA under these conditions was observed to be only 2- to 3-fold. The levels of ornithine aminotransferase mRNA were measured by hybridization of cellular RNA to a cloned cDNA probe. The ribosomal transit time (20-25 min) along ornithine aminotransferase mRNA was the same in both control and induced cultures. The level of ornithine aminotransferase nascent chains in cAMP-induced cultures was increased 7-fold over the level in control cultures. In the face of a 2- to 3-fold increase in the amount of ornithine aminotransferase mRNA, this suggests an approximately 3-fold increase in the rate of initiation per copy of ornithine aminotransferase messenger RNA. These results indicate that cAMP may regulate ornithine aminotransferase synthesis at both transcriptional and translational levels in cultured hepatocytes.

Animals↗
PubMed1985

Regulation of ornithine aminotransferase by cyclic AMP and glucose in primary cultures of adult rat hepatocytes.

Hepatic ornithine aminotransferase (EC 2.6.1.13) (OAT) is a mitochondrial matrix enzyme that plays a role in amino acid catabolism and in gluconeogenesis. In rats, the synthesis of hepatic OAT is regulated by glucagon, dietary protein, and glucose. Serum-free primary cultures of adult rat hepatocytes were used to demonstrate that glucagon, cyclic AMP, and glucose are able to alter OAT synthesis by a direct action on hepatocytes. The rates of OAT synthesis were measured by immunoprecipitation of pulse-labeled OAT with an affinity-purified monospecific antibody. Ten hours after cyclic AMP addition to the culture medium, the relative rate of OAT synthesis reached a peak value that was six- to eightfold above the control rate. OAT activity accumulated more slowly, reaching a level that was approximately threefold above the control by 24 h. The inclusion of glucose in the culture medium inhibited the increases in OAT synthesis and activity in a dose-dependent manner. Although synthesized as a precursor (pOAT), no pOAT was detected under control, induced, or carbohydrate-inhibited conditions; this suggests that pOAT processing may not be a regulatory site of OAT expression. By following the loss of labeled OAT, a half-life of 34 h in these cultures under all of the above conditions was observed. Regulation of OAT levels in cultured hepatocytes appears to be achieved primarily through changes in the rate of OAT synthesis.

Animals↗
PubMed1985

The significance of selected biochemical markers in the characterization of putative initiated cell populations in rodent liver.

Biochemical 'markers' of neoplastic cells have been the subject of numerous investigations during the past several decades. Recently, studies from a number of laboratories have demonstrated that a very common biochemical marker for early 'preneoplastic' lesions occurring in several model systems of multistage carcinogenesis, especially hepatocarcinogenesis, is the enzyme, gamma-glutamyltranspeptidase (GGT). Despite the high frequency of this marker, especially in early hepatic lesions, an extensive degree of biochemical heterogeneity is evident when lesions are analyzed for the presence of multiple markers. Such markers have in the past been considered to be relatively stable. However, it is becoming increasingly apparent that environmental factors, drugs, diet, etc. may alter the phenotype of such lesions, especially in respect of GGT activity. Although various model systems have demonstrated different degrees of persistence of biochemically altered focal lesions induced during hepatocarcinogenesis, it is quite likely, but not yet proven, that the potential for the development of each focus remains in the tissue even on disappearance of the histochemical markers. Despite the relatively high frequency of the marker, GGT, in early focal lesions during hepatocarcinogenesis and the generally decreased level of xenobiotic metabolism in these lesions, no single marker, essential or critical for the neoplastic transformation in early or late lesions of hepatocarcinogenesis, has as yet been identified.

Adenosine Triphosphatases↗
PubMed1985

An approach to the development of a short-term whole-animal bioassay to distinguish initiating agents (incomplete carcinogens), promoting agents, complete carcinogens, and noncarcinogens in rat liver.

With increased knowledge of the stages of initiation and promotion in the natural history of neoplastic development and the multiple observations that such stages are likely to be ubiquitous in the natural history of all histogenetic types of neoplasms, it is becoming increasingly important to distinguish chemical agents that act selectively during one or the other of these stages. Utilizing data obtained from the published literature as well as further observations, the experimental findings reported in this paper serve to indicate an approach to the development of a short-term whole-animal bioassay capable of distinguishing agents that act at one or the other or both of the stages of initiation and promotion in rat liver. Because of the tissue specificity for both complete carcinogens and promoting agents, this bioassay system is not likely to identify the initiating and promoting capabilities of all carcinogenic agents and thus at present is not designed to replace the standard chronic bioassay systems. However, in view of the diverse metabolic capabilities of hepatic tissue, the system proposed in this paper may represent a model capable of distinguishing the stages of carcinogenesis at which many, if not most, specific carcinogenic chemicals exert their primary action.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1985

The quantitative analysis and stability of histochemical markers of altered hepatic foci in rat liver following initiation by diethylnitrosamine administration and promotion with phenobarbital.

The stability and response of histochemical phenotypes of altered hepatic foci (AHF) were studied both in the presence and following the withdrawal of 0.05% phenobarbital (PB) treatment in rats previously given a single dose of diethylnitrosamine (DEN) 20-24 h following partial hepatectomy (PH). AHF were scored by their expression of three biochemical markers: gamma-glutamyl transpeptidase (GGT), adenosine triphosphatase and glucose-6-phosphatase (G6P). AHF demonstrated significant heterogeneity with respect to the marker alterations. The use of three markers in the present study confirmed the findings of our earlier study, which showed the maximal response of GGT+ AHF to PB administration following PH/DEN initiation and the stability of GGT+/AHF induced by the PH/DEN/PB regimen after the withdrawal of PB. In the regimen employed, the GGT marker alone scored the great majority of the AHF detected by all three markers. The frequency distribution of histochemical phenotypes remained relatively constant in AHF during continuous PB administration and in AHF promoted by PB followed by a 6-month period of feeding a diet containing no PB. These findings suggest that individual AHF remain phenotypically stable throughout the PB promotion phase, i.e., do not progress from one phenotype to another. In every marker class, the mean volume of AHF increased during continuous PB administration. These data illustrate the enhancing effect of PB on the growth of the AHF. The size of AHF continued to increase following the withdrawal of PB in the 3-month PB treatment group, but not in the animals treated for 4 months. A mechanism that may account for the differences in these two treatment groups is discussed.

Adenosine Triphosphatases↗
PubMed1985

Transcriptional regulation of c-myc during chemically induced differentiation of HL-60 cultures.

Previous studies have demonstrated both an elevated level of cellular myc-homologous RNA in HL-60 cultures and a decrease in this messenger RNA following the chemically induced differentiation of HL-60 cells. A nuclear transcription system isolated from HL-60 cells was used to investigate whether an alteration in the rate of transcription of the c-myc gene was associated with this decrease in myc RNA. Five days after the addition of either 180 mM dimethyl sulfoxide or 60 nM 12-O-tetradecanoylphorbol-13-acetate to HL-60 cultures, transcription of the c-myc gene was markedly reduced when compared with control cultures. This specific decrease was not accompanied by an alteration in either the bulk rate of transcription of the c-myc copy number. This suggests that the decreased cellular myc RNA levels are due to decreased transcription of the myc protooncogene.

Cell Differentiation↗
PubMed1985

Activities of benzphetamine N-demethylase and aryl hydrocarbon hydroxylase in cells isolated from gamma-glutamyl transpeptidase-positive foci and surrounding liver.

The levels of two cytochrome P-450-linked enzymes of xenobiotic metabolism, benzphetamine N-demethylase and aryl hydrocarbon hydroxylase, were determined in cells isolated from gamma-glutamyl transpeptidase (GGT)-positive foci and in cells from surrounding liver obtained from carcinogen-treated inbred F344 rats. Rat liver foci were initiated with diethylnitrosamine (CAS: 55-18-5) and promoted with sodium phenobarbital [(PB) CAS: 64038-21-7] for 4 1/2 or 12 months. The levels of both enzymes were relatively low in the GGT-positive hepatocytes, while the GGT-negative hepatocytes from the surrounding liver had elevated levels of both enzymes comparable to levels seen in rats treated with PB alone. After 12 months of promotion the PB was removed from the diet and the activities of both enzymes fell below the control levels in the GGT-positive hepatocytes and returned to the control levels in the surrounding GGT-negative hepatocytes. Therefore, the cells in the GGT-positive foci contained low levels of these two cytochrome P-450 enzymes in relation to the levels in GGT-negative cells. These levels were responsive to phenobarbital induction, although the induced levels in the GGT-positive cells were much lower than the induced levels in GGT-negative hepatocytes. The liver surrounding the foci responded to phenobarbital induction to the same degree as did the liver of noninitiated rats.

Animals↗
PubMed1985

Chromatin structure of the c-myc gene in HL-60 cells during alterations of transcriptional activity.

HL-60 cells have an elevated level of cellular myc RNA due to an amplified c-myc gene. Subsequent to chemically induced differentiation of HL-60 cells, both cellular myc RNA levels (Grosso, L. E., and Pitot, H. C., Biochem. Biophys. Res. Commun., 119: 473, 1984) and myc-specific transcription decrease (Grosso, L. E., and Pitot, H. C., Cancer Res., 45: 847-850, 1985). We have compared the primary DNA structure, DNA methylation, and S1 nuclease sensitivity of the myc protooncogene in HL-60 cells before and after chemically induced differentiation. We find no change in the structure or methylation of the c-myc gene. The protooncogene is hypomethylated at CCGG sequences in the 5' region but is extensively methylated at sites detected by sequences homologous to the 3' exon or 3' flanking sequences. Four S1 nuclease-sensitive sites are detected prior to differentiation. After the induction of either myeloid or monocytic differentiation, three of the S1 nuclease-sensitive sites are present. The presence of the fourth S1 nuclease-sensitive site correlates with the transcriptional activity of this gene.

Cell Line↗
PubMed1985

Growth of carcinogen-altered rat hepatocytes in the liver of syngeneic recipients promoted with phenobarbital.

An improved transplantation system for the study of carcinogen-altered hepatocytes is described. This system, which is based on that reported by Laishes and Farber (Cancer Res., 61: 507-512, 1978), involves the transfer of hepatocytes from male F344 animals to syngeneic adult hosts. Unlike the earlier protocol, the recipient rats were fed phenobarbital (PB) rather than the DNA-reactive agent, 2-acetylaminofluorene. gamma-Glutamyl transpeptidase (GGT)-positive hepatocytes were induced in the donor animals by one of three different hepatocarcinogenic treatment regimens. The recipient rats received 0.05% PB in the diet for 3 wk prior to the cell transfer and were maintained on the PB diet for 2 to 7 mo. Hepatocytes from a male F344 donor rat that had received a 70% hepatectomy and 30 mg of diethylnitrosamine per kg and had been maintained on 0.05% PB for 12 mo formed GGT-positive colonies and hepatocellular carcinomas in both male and female recipients. No GGT-positive colonies were formed when 0.05% PB was omitted from the diet of the recipients. A 70% partial hepatectomy of the recipients at the time of cell transfer was also essential for the development of colonies and tumors. The mean volume of the colonies was 5 times larger in female recipients than in males, occupying 38% of the total liver volume in the females. GGT-positive foci arose in recipient livers that had received hepatocytes from either a male F344 donor rat treated according to the Solt and Farber [Nature (Lond.), 263: 701-703, 1976] selection protocol or a male F344 donor rat that received a 70% hepatectomy and 30 mg of diethylnitrosamine per kg and was maintained on 0.05% PB for 5 mo. The recipient animals were treated with PB which the initial experiments showed was essential for the development of foci. The number and volume of the foci in the recipient varied according to the treatment regimen that the donor rat received. This system provides a method for analyzing the growth regulation of altered foci at various stages of neoplastic development during hepatocarcinogenesis in the rat in the absence of DNA-reactive selection agents.

2-Acetylaminofluorene↗
PubMed1984

Increased activities of liver cathepsins T and D in carbon tetrachloride-treated rats.

Changes in activities of a new proteinase cathepsin T as well as some other lysosomal acid proteinases and hydrolases were examined in liver homogenate from rats treated with a single hepatotoxic dose of carbon tetrachloride. The most striking changes were several-fold increases of liver cathepsin T and D activities over their levels in untreated rats 3 days after administration of the agent to rats. Increase of cathepsin T was greater than that of cathepsin D at all doses of the hepatotoxin examined. The activities of N alpha-benzoyl-DL-arginine 2-naphthylamide hydrolase, acid phosphatase, beta-galactosidase and beta-glucuronidase in poisoned rat liver were unchanged or only slightly increased. Cathepsin T and D activities were less enhanced in mitochondrial lysosomal fractions than in the homogenate, and were greatly elevated in the supernatant fractions of liver from the treated rats. As judged from the molecular weights, the elevated activities of cathepsins T and D in the treated rat liver could be attributable to the two cathepsins themselves and not to other proteinases. Administration to rats of other hepatotoxic agents, thioacetamide and dimethylnitrosamine, also induced the elevation of the two cathepsin activities in liver, but on partial hepatectomy the activities of liver cathepsins T and D did not show such marked increases. Nonparenchymal liver cell fractions were responsible for almost all the increased activities of liver cathepsins T and D. It is possible that cathepsins T and D play a role in the heterolytic breakdown of hepatocyte molecules following CCl4 poisoning.

Animals↗
PubMed1984

Alterations in the maturation and structure of ribosomal precursor RNA in Novikoff hepatoma cells induced by 5-fluorocytidine.

The effects of 5-fluorocytidine on ribosomal RNA maturation and structure in Novikoff hepatoma cells were investigated. Like other nucleic acid base analogues that are incorporated into RNA, this compound inhibits maturation of the 45S ribosomal RNA precursor. The 45S RNA precursor produced in the presence of 5-fluorocytidine has an abnormal electrophoretic mobility compared with that of the control precursor under nondenaturing conditions, but the two have identical mobilities under denaturing conditions. Under the conditions of these experiments, 5-fluorocytidine inhibited cellular protein synthesis only slightly, whereas equimolar concentrations of 5-azacytidine resulted in nearly 75% inhibition of this process. Despite this difference in the effects of the two analogues as well as the greater chemical lability of the 5-azacytidine, their effects on ribosomal RNA maturation are identical.

Animals↗
PubMed1984

Modulation of c-myc expression in the HL-60 cell line.

A decrease in the expression of the myc proto-oncogene of HL-60 cells has been reported as an accompaniment of myeloid differentiation induced by either dimethylsulfoxide or retinoic acid. We report herein that several inhibitors of poly(ADP-ribose)-polymerase induced myeloid differentiation in HL-60 cultures. Studies on the expression of the c-myc gene in total cell RNA populations indicate that expression of this gene is inversely correlated with the state of differentiation, either myeloid or monocytic, of the cultured cells independent of the inducer and the rate of cell proliferation.

Cell Differentiation↗
PubMed1984

Transcriptional control of ornithine aminotransferase synthesis in rat kidney by estrogen and thyroid hormone.

Ornithine aminotransferase, a mitochondrial matrix enzyme, is expressed constitutively in many mammalian tissues and is subject to complex hormonal and dietary regulation in rat liver and kidney. The kidney enzyme is induced by estrogen and thyroid hormone (3,3',5-triiodo-L-thyronine, T3). We demonstrated previously that the hormonal induction in kidney is the result of an increase in ornithine aminotransferase mRNA levels (Mueckler, M. M., and Pitot, H. C. (1983) J. Biol. Chem. 258, 1781-1784). In order to define further the level at which the enzyme is induced in the kidney, the transcriptional activity of the ornithine aminotransferase gene was measured after hormonal induction. This was accomplished by incubating purified kidney nuclei in an in vitro transcription reaction in the presence of [32P] UTP and hybridizing the nascent, 32P-labeled, nuclear RNA to cloned ornithine aminotransferase cDNA immobilized on nitrocellulose filters. Administration of estrogen resulted in an increase in the rate of ornithine aminotransferase gene transcription by 1 h, and the rate remained elevated until at least 20 h after administration. The activity of the gene was elevated 12 h after T3 administration. A lag time of at least 4 h was observed for the effect of T3 on ornithine aminotransferase gene transcription. When administered in combination, estrogen and T3 exerted a synergistic effect on the activity of the gene. The administration of estrogen or both estrogen and T3 also resulted in the increased steady state level of two putative ornithine aminotransferase nuclear precursor RNAs, 3190 and 4260 nucleotides in length.

Animals↗