Joyce E. Becker

A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture.

Abstract A technique is described by which the intracellular water space can be determined in cells which are attached to a culture dish. A major advantage of this procedure is that the cells maintain their normal morphology. This technique takes advantage of the transport of the nonmetabolizable hexose, 3- O -methyl- d -glucose, to an intracellular concentration equal to the extracellular concentration, and the inhibition of 3- O -methyl- d -glucose efflux by phloretin. It requires only a determination of the nanomoles of hexose taken up at equilibrium plus a protein or cell number determination.

Primary monolayer cultures of adult rat liver parenchymal cells suitable for study of the regulation of enzyme synthesis

SummaryParenchymal cells from adult rat liver which had been fully regenerated were isolated and cultured in nonproliferating monolayers in vitro. The optimum conditions for attachment of these cells to Falcon plastic dishes were determined. When approximately 1.0×105 nuclei per cm2 suspended in Hams F-12 medium with 0.5 μg of insulin per ml and 25% fetal calf serum were incubated at 37°C for 24 hr, about 50% became attached and contiguous. When the above medium was supplemented with synthetic buffers 2-(N-morpholino) ethanesulfonic acid (MES) andN-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), the presence of 15% fetal calf serum also allowed an attachement effiency of 50%. Tyrosine aminotrasferase activity in the cells was elevated when the culture medium was supplemented with hydrocortisone or dexamethasone. The largest increases were observed after 72 hr of culture. Cycloheximide prevented the increase.

Induction of tyrosine aminotransferase by dibutyryl cyclic-AMP employing hepatoma cells in tissue culture☆

Abstract Tyrosine aminotransferase (TAT) activity in monolayers of H-35 cells was elevated in response to dibutyryl cyclic-AMP (DBC). Hydrocortisone appeared to potentiate the effect of DBC on TAT. On the basis of cycloheximide studies it appeared that continued protein synthesis was required in order for DBC to elevate TAT enzyme activity. Low levels of actinomycin D (0.2 μg/ml), which inhibited RNA synthesis by 60%, only partially blocked the induction of TAT by DBC. This same level of actinomycin D had no effect on the induction of TAT by insulin. Actinomycin D (5 μg/ml) inhibited RNA synthesis 95% but did not alter the elevation of TAT activity in response to DBC. These results suggest that insulin and DBC act at some post-transcriptional event. It is also suggested that comparative studies with the H-35 cells and HTC cells might provide some insight into the mechanism of cyclic-AMP action in enzyme induction since the H-35 cells respond to DBC and the HTC cells do not.

Hormonal effects on enzyme activities in tissue culture and in whole animals

Abstract Studies on the activity of tyrosine transaminase in whole animals and in tissue culture show that there are multiple factors involved in the regulation of this enzyme. The most likely factors so far implicated are cortisone, insulin and tryptophan and its metabolites.

Effect of glucocorticoids on fetoprotein production by an established cell line from Morris hepatoma 8994

Abstract Glucocorticoids at concentrations equal to or higher than 10−7M lead to an increase of alpha-fetoprotein production by an established cell line from Morris hepatoma 8994. These cells also secreted alphaM-fetoprotein into the culture medium but only after addition of at least 4×10−7M hydrocortisone or 5×10−8M dexamethasone. The effects on both fetoproteins were observed in spite of a decrease of cell multiplication and an increase of cell detachment.

Effect of cordycepin on induction of tyrosine aminotransferase employing hepatoma cells in tissue culture

Abstract Cordycepin acts like actinomycin in that it prevents the induction of tyrosine aminotransferase (TAT) by cortisol in the H-35 or HTC cells when added 1 2 h before cortisol. Unlike actinomycin, cordycepin did not further increase the levels of TAT which had been induced by cortisol in either the H-35 or HTC cells. Also in contrast to actinomycin, cordycepin did not interfere with the decline in TAT following removal of cortisol from the H-35 or HTC cells which had been induced with cortisol overnight. The effect of cordycepin on TAT was not a result of an effect at the translational level. Moreover when actinomycin D and cordycepin were added to the same plates the level of TAT increased thus indicating that cordycepin did not interfere with the action of actinomycin D. Cordycepin, like actinomycin D, did not block the induction of TAT by insulin in the H-35 or HTC cells, nor did it block the induction of TAT by DBC in the H-35 cells. The H-35 cells have about ten times more TAT activity than have HTC cells when both are induced with cortisol, and both lines can be induced to proportionately higher levels of activity by insulin in the presence of cortisol. Both lines respond similarly to cordycepin and to actinomycin D. Since both compounds inhibit RNA synthesis, while high levels of actinomycin D produce ‘superinduction’ of TAT with or without the presence of cordycepin it is difficult to see how actinomycin can produce this phenomenon purely in terms of the inhibition of RNA synthesis, as the Tomkins model proposes, without several additional ad hoc hypotheses.

Pyruvate kinase, hexokinase, and aldolase isozymes in rat liver cells in culture

SummaryIsozyme patterns of the enzymes of ATP-hexose phosphotransferase, aldolase, and pyruvate kinase have been examined in a culture of cells derived from an adult rat liver. Normal adult liver characteristically contains glucokinase, aldolase B, and pyruvate kinase I isozymes. In contrast to this, the isozymes present in the cell line were found to be hexokinase, aldolase A, and pyruvate kinase III. Thus cells from an adult liver when taken into culture resemble fetal liver rather than adult liver. Attempts to induce in these cells the isozymes of ATP-hexose phosphotransferase and pyruvate kinase characteristic of the adult liver have been unsuccessful.The culture conditions were found to affect the nature of the kinetic properties of the pyruvate kinase isozyme in these cells. Cells maintained in the absence of glucose gave a sigmoidal-shaped substrate velocity curve for phosphoenolpyruvate, with aKm of 0.33mm. However, when the enzyme was extracted from cells grown in the presence of glucose, a hyperbolic-shaped substrate velocity curve was found, with aKm of 0.05mm. The two forms of this isozyme have been designated A and B, respectively. Form A can be converted to form B by preincubation with fructose diphosphate, whereas the B to A transition is mediated by ethylenediaminetetraacetate, ATP, or citrate. These two forms of the enzyme have distinctive properties in relation to inhibition by ATP, alanine, and phenylalanine, and to activation by fructose diphosphate. Their properties appear to resemble those of adipose tissue pyruvate kinase. The physiological significance of these properties is discussed.

The antagonistic effect of dexamethasone and insulin on α-fetoprotein secretion by cultured H4-II-E-C3 cells derived from the Reuber H-35 hepatoma

Abstract Non-confluent monolayers of H4-II-E-C3 cells were maintained in serum-free media. Dexamethasone alone (5 × 10 −7 M) stimulated α-fetoprotein secretion 2- to 4-fold while insulin alone (8.7 × 10 −8 M) inhibited α-fetoprotein secretion by 20%. When dexamethasone (5 × 10 −7 to 5 × 10 −9 M) and insulin (8.7 × 10 −8 to 8.7 × 10 −11 M) were added simultaneously, insulin diminished the stimulatory effect of dexamethasone. When α-fetoprotein secretion was elevated by dexamethasone and the medium was replaced by media containing either insulin or no hormones, the rate of α-fetoprotein secretion diminished more rapidly with the insulin-supplemented medium. Alone or in combination, insulin and dexamethasone had little effect on albumin secretion.

Amino acid transport in hepatoma cell cultures during tyrosine aminotransferase induction.

Alpha-aminoisobutyric acid transport in hepatoma cells in culture was increased by insulin but not by hydrocortisone. Both of these agents induce tyrosine aminotransferase activity in this system. The apparent increase in alpha-aminoisobutyric acid transport and tyrosine aminotransferase activity produced by glucagon is probably caused by insulin contamination. Insulin did not increase transport in this system until after tyrosine aminotransferase activity had reached maximum levels. The mechanisms underlying increased alpha-aminoisobutyric acid transport appear to differ from those for tyrosine aminotransferase induction with hydrocortisone despite their close association in previous whole animal experiments.

The role of messenger RNA in tyrosine aminotransferase superinduction: effects of camptothecin on hepatoma cells in culture.

Abstract Camptothecin inhibited the hydrocortisone but not the insulin induction of tyrosine aminotransferase activity in hepatoma cells in culture. However, camptothecin did not cause “superinduction” of tyrosine aminotransferase activity even though it reportedly inhibits messenger RNA synthesis. In hydrocortisone pre-induced cultures, camptothecin treatment caused a rapid decline in tyrosine aminotransferase activity suggesting it did not block degradation of the enzyme. A comparison of actinomycin D with camptothecin indicated that some of the effects of actinomycin D on tyrosine aminotransferase activity may not be mediated through inhibition of messenger RNA synthesis.