Mary J. Wilson

Ratliver cells in culture: Effect of storage, long-term culture, and transformation on some enzyme levels

SummaryAryl hydrocarbon hydroxylase (AHH) and tyrosine aminotransferase (TAT) activities were determined in rat liver cell lines after frozen storage, long-term culture, and transformation in vitro. Levels of AHH activity after 17 months in frozen storage were comparable to levels prior to freezing. During long-term culture the AHH levels of the cell lines tended to decrease. Transformed lines had variable levels of AHH activity. Cell lines retained measurable TAT activity following long-term culture and frozen storage. TAT activity of transformed cells was comparable to that of normal lines. Prolonged frozen storage did not induce transformation up to one year.

An increased requirement for methionine by transformed rat liver epithelial cells in vitro

Abstract The growth of two normal and four transformed rat liver epithelial cell lines in a methionine-containing medium and a methionine-deficient medium supplemented with homocysteine was examined. The growth rates of the normal cells on the homocysteine-supplemented medium were approximately one-half the growth rates shown by the same cells in the methionine-containing medium. In contrast, three of the four transformed cell lines studied showed virtually no growth on the homocysteine-supplemented medium, although they grew quite rapidly on the methionine-containing medium. The fourth, transformed by N-methyl-N-nitrosourea, was able to grow on the homocysteine-supplemented medium at about one-third the rate as on the methionine-containing medium. Thus, transformed rat liver epithelial cells resemble other malignant cells in their reduced capacity to grow on homocysteine in the absence of methionine.

Reduced cadmium-induced cytotoxicity in cultured liver cells following 5-azacytidine pretreatment☆

Recent work indicated that administration of the pyrimidine analog 5-azacytidine (AZA), either to cells in culture or to rats, results in an enhancement of expression of the metallothionein (MT) gene. Since MT is thought to play a central role in the detoxification of cadmium, the present study was designed to assess the effect of AZA pretreatment on cadmium cytotoxicity. Cultured rat liver cells (TRL 1215) in log phase of growth were first exposed to AZA (8 microM). Forty-eight hours later, cadmium (10 microM) was added. MT concentrations were then measured 24 hr after the addition of cadmium. A modest increase in MT amounts over control (1.7-fold) was detected after AZA treatment alone. Cadmium alone resulted in a 10-fold increase in MT concentrations. The combination of AZA pretreatment followed by cadmium exposure caused a 23-fold increase in MT concentrations over control. Treatment with the DNA synthesis inhibitor hydroxyurea (HU) eliminated the enhancing effect of AZA pretreatment on cadmium induction of MT, indicating that cell division is required. AZA-pretreated cells were also harvested and incubated in suspension with cadmium (250 microM, 37 degrees C) for 0 to 90 min. After incubation intracellular and extracellular fluids were separated by centrifugation through an oil layer. AZA-pretreated cells showed marked reductions in cadmium-induced cytotoxicity as reflected by reduced intracellular potassium loss, glutamic-oxaloacetic transaminase loss, and lipid peroxidation (assessed by thiobarbituric acid reactants) following cadmium exposure. AZA pretreatment had no effect on the cellular uptake of cadmium. Results suggest that AZA pretreatment induces tolerance to cadmium cytotoxicity which appears to be due to an increased capacity to synthesize MT rather than high quantities of preexisting MT at the time of cadmium exposure.

THE ELEVATED REQUIREMENT FOR METHIONINE BY TRANSFORMED RAT LIVER EPITHELIAL CELLS IN VITRO

Growth constants appear to constitute a useful tool in comparing the behavior for transformed and untransformed cells in culture. The substitution of homocysteine for methionine in culture medium caused a greater depression of growth with 3/4 of the transformed epithelial and with 10/14 other cell lines tested compared to the growth depression seen in similar untransformed lines. The greater growth depression exhibited by transformed lines on methionine-deficient, homocysteine-supplemented medium could generally be attributed to: 1. Greater growth of transformed cells in complete medium, and 2. The lower levels of methyltransferase generally found in the transformed lines. Finally methyltransferase appears to be a major determinant of growth of both normal an transformed cells growing in a methionine-deficient, homocysteine-supplemented medium.

Enhancement of cadmium-induced metallothionein synthesis in cultured trl 1215 cells by butyric acid pretreatment

Cultured TRL 1215 cells in log phase of growth were exposed first to butyric acid (BA; 0.1-2.0 mM) and then 48 h later, to cadmium (Cd; 10 microM). Metallothionein (MT) concentrations were measured 24 h after Cd addition. Cd alone caused approx. a 10-fold increase in cellular MT levels, while BA alone had no effect. BA pretreatment followed by Cd exposure, however, resulted in MT levels up to 25 times those in control cells. Concurrent exposure to hydroxyurea (HU) eliminated the enhancing effect of BA pretreatment on induction of MT by Cd, indicating that DNA synthesis is required. BA-pretreated cells did not show marked increases in cellular uptake of Cd. BA pretreatment enhances Cd induction of MT synthesis through a mechanism that is dependent on the synthesis of DNA.

Aminoacylation of ethionine to rat liver tRNAMet and its incorporation into protein.

Ethionine is the carcinogenic ethyl analogue of methionine. The mechanism of its carcinogenicity is of particular interest since carcinogenic doses of ethionine do not result in alkylation of DNA [ 11. The toxic and carcinogenic effects of ethionine can be completely prevented by elevated levels of methionine, therefore, ethionine apparently exerts its carcinogenic activity by interfering with some aspect of methionine metabolism. The ability of ethionine to replace methionine in protein synthesis has been studied extensively in procaryotic but not eucaryotic systems. Ethionine is aminoacylated to Escherichia coli tRNAMet [2] and incorporated at normal methionine sites in bacterial protein [3]. In E. coli, ethionyltRNAfMet is formylated by the bacterial transformylase enzyme [4] and formylethionyl-tRNAyet initiates polymethionine synthesis as directed by poly(AUG) [2]. Early reports of the incorporation of ethionine into eucaryotic protein relied on the precipitation of total protein from rat tissue [5]. While these studies may indicate that ethionine is incorporated into protein, they cannot rule out non-specific binding of ethionine to protein or ethylation of amino acid side chains. This report examines the ability of ethionine to participate in eucaryotic protein synthesis. Using tRNA and aminoacyl-tRNA synthetases from the liver of a strain of rat susceptible to the hepatocarcinogenicity of ethionine, we demonstrate that ethionine is aminoacylated to tRNAMet isoacceptors and is a competitive inhibitor of methionyl-tRNA synthetase. Further, we report similar rates of transfer of ethionine and methionine to protein from tRNA!et using a eucaryotic cell free protein synthesizing system.

Characterization of rat liver cells transformed in culture by DL-ethionine.

SummaryA rat liver-derived epithelial cell line transformed withdl-ethionine and the corresponding control cell line were characterized according to morphological and cytochemical criteria to establish their origin from liver epithelium and to identify cellular changes due to transformation bydl-ethionine. The presence of intermediate junctions confirms the epithelial nature; glycogen accumulation and glucose-6-phosphatase activity confirm the hepatic origin of the cells. Persistent alterations resulting from ethionine transformation were variations in cell shape and size, focal multilayered growth, an increase in the nucleolar: nuclear ratio, and a reduction in the number of cells displaying a primary cilium. Hyperplasia of the inner nuclear membrane, elongation and branching of mitochondria, and a reduction in the length and frequency of cell junctions were also characteristic of the transformed cells.

Carcinogenesis and DNA Hypomethylation in Methyl-Deficient Animals

The current increasing interest in the possible role of normal DNA methylation in carcinogenesis has sprung from three major sources: (1) cell culture studies on the role of DNA methylation in cell differentiation; (2) hypomethylation of specific genes in a variety of cancers; and (3) liver cancer causation in methyl-deficient rats. Studies on the role of DNA methylation in mammalian cell differentiation conducted by Christman et al., 1977, and Razin and Riggs, 1980, led to the postulate that undermethylation of the C5 position of cytosine may play a determining role in cancer causation (Holliday, 1979; Riggs and Jones, 1983). Feinberg and Vogelstein (1983b) showed that the genes coding for human growth hormone, α-globin, and γ-globin in human tumors were undermethylated compared to the same genes in the corresponding normal tissues. Subsequent studies have extended these observations to include other tumors and genes (Hoffman, 1984). Finally, dietary deprivation of the methyl donors methionine and choline had been shown to induce liver carcinomas in rats (Copeland and Salmon, 1946). Although these findings were accepted for a period of nearly 10 years, the subsequent demonstration of aflatoxin contamination in the peanut meal-based diets used to produce the methionine- and choline-deficiency, led credence in these findings to be suspended (Newberne, 1965). However, a second system to produce liver tumors in association with a methyl-deficient state was seen by the chronic administration of ethionine to rats (Farber, 1963).

Increased synthetic capacity for metallothionein in cultured liver cells following dimethyl sulfoxide pretreatment

Cultured TRL 1215 cells in log phase of growth were exposed to dimethyl sulfoxide (DMSO; 14-280 mM) followed 48 h later by cadmium (10 micron). Intracellular concentrations of metallothionein (MT) were measured 24 h after cadmium addition. Cadmium alone caused a 10-fold increase in the levels of MT, while DMSO alone had no effect on cellular MT levels. DMSO pretreatment followed by cadmium exposure, however, resulted in MT levels that were elevated by a factor of as much as 25-fold those observed in control cells. Concurrent treatment with the DNA synthesis inhibitor hydroxyurea (HU) eliminated the enhancing effect of DMSO pretreatment on cadmium induction of MT, indicating the requirement of DNA synthesis. An enhancement of the cellular accumulation of the metal ion did not account for the increased cadmium-induced MT synthesis in DMSO-pretreated cells as these cells did not show significantly increased uptake of cadmium during the initial period of exposure. DMSO pretreatment enhances cadmium induction of MT synthesis through a mechanism that appears to be dependent on the synthesis of DNA.

Formation and utilization of methionine by rat liver cells in culture.

SummaryThe enzymeN5-methyltetrahydrofolate: homocysteine methyltransferase (methionine synthetase) catalyzes the synthesis of methionine from homocysteine. Methylcobalamin is a cofactor for the reaction. The effects of methionine deprivation and methylcobalamin supplementation on the growth of normal and transformed rat liver epithelial cell lines were determined using growth constants to quantitate cell proliferation. No marked specific requirement by the transformed cell lines for methionine relative to leucine was observed. A sigmoidal relationship, however, was found to exist between growth constants and the logarithms of the amino acid concentrations for both normal and transformed cells. Methylcobalamin stimulated the growth rates of the normal and transformed liver cells in methionine-deficient, homocysteine-containing medium. Growth on methionine was not increased by the addition of methylcobalamin. The growth constants for two normal, two spontaneously transformed, one chemically transformed, and one tumor cell line grown in medium in which methionine was replaced by homocysteine were found to be proportional to the level of methionine synthetase. The results demonstrate the utility of growth quantitation to study the methionine dependency of transformed cells.