The effect of population density on phenylalanine hydroxylase activity in rat-hepatoma cells in culture

The expression of phenylalanine hydroxylase activity in rat-hepatoma cells in culture (line H4-II-E-C3) is a function of culture density: under normal growth conditions in the presence or in the absence of exogenously added hydrocortisone, the levels of this enzyme are low in subconfluent cell populations, but increase steeply as cultures attain confluency. This phenomenon (i) is not an artifact of the subcultivation process and (ii) is not produced by some alteration in the growth medium effected by high-density cultures. The levels of phenylalanine hydroxylase in high-density cultures of H4-II-E-C3 cells in the presence of serum plus added hydrocortisone are at least 80% of those seen in adult-rat liver. It is concluded that this density-associated phenomenon is the result of an intrinsic property of H4-II-E-C3 cells and possibly constitutes a form of epigenetic control governing the sensitivity of these cells to stimulation by serum or by serum plus hydrocortisone.     

Control of phenylalanine hydroxylase synthesis in tissue culture by serum and insulin.

Stationary-phase, minimal deviation hepatoma H4-II-E-C3 cell cultures that are serum-deprived respond with a biphasic time course of phenylalanine hydroxylase induction when dialyzed fetal calf serum or insulin is added. These two agents induce phenylalanine hydroxylase additively, during both the initial 3-hour and the delayed 24-hour phases. The initial phase of induction by insulin is inhibited by cycloheximide but not by actinomycin D. The delayed induction by both dialyzed fetal calf serum and insulin is inhibited by 10(-6) M cycloheximide and 0.20 mug/ml actinomycin D. H4-II-E-C3 cells in culture do not synthesize the factor(s) in serum that induce phenylalanine hydroxylase.     

Glucocorticoid stimulation of tetrahydrobiopterin levels and phenylalanine hydroxylase activity in rat hepatoma cells

Incubation of H4-II-E-C3 rat hepatoma cells with either hydrocortisone or dexamethasone resulted in 3- to 5-fold increases in the levels of both phenylalanine hydroxylase and its essential cofactor, tetrahydrobiopterin. Maximum elevation of phenylalanine hydroxylase was noted after 24 h of incubation, whereas significant increases in tetrahydrobiopterin were found only after 48 h exposure of the cells to glucocorticoids. Removal of hormone from the culture medium resulted in rapid loss of cell tetrahydrobiopterin, but a much slower decline in the level of phenylalanine hydroxylase. Thus, although the levels of both phenylalanine hydroxylase and tetrahydrobiopterin in rat hepatoma cells are regulated by glucocorticoids, this regulation is apparently not strictly coordinated. Nevertheless, control of cellular tetrahydrobiopterin levels may be an important regulator of hepatic phenylalanine catabolism since significant increases in the ability of intact rat liver cells to hydroxylate phenylalanine were observed only after 48 h exposure to glucocorticoids, in correlation with increases in cell tetrahydrobiopterin content.     

Insulin inhibition of protein degradation in cell monolayers

Protein degradation has been measured in confluent monolayers of eleven lines of contact-inhibited cells and ten transformed lines as the rate of release of trichloroacetic acid-soluble radioactivity after prelabeling cell protein with [³H]leucine. Insulin, at concentrations from 10^?12 M to 10^?6 M, has been added at the beginning of the 4-hour degradation period to detect selective effects of this hormone as an inhibitor of the inducible proteolysis occurring in serumfree medium. In addition insulin binding measurements have been performed on selected cell lines in an attempt to relate receptor properties to insulin action. Substantial effects of insulin are found in most cells with a selective inhibition at low insulin concentrations noted in several of the transformed lines. The difference in insulin sensitivity is not entirely definitive because temperature-sensitive transformation mutants of NRK cells are not more sensitive to insulin at a temperature where they show the transformed phenotype. Although insulin receptors on different cell lines have similar binding properties, two of the hepatomas used, H35 and MH₁C₁, show inhibition of protein degradation at insulin concentrations where receptor occupancy is extremely low. Calvarial osteoblast-like cells have a high rate of protein degradation which can be reduced by growth factors but not by insulin. The lack of an insulin response is a consequence of poor insulin binding to the cells. Insulin binds to the osteogenic sarcoma cells in substantial amounts. However, its normal action to inhibit the induced proteolysis is restricted because with these cells no increase of proteolysis occurs in serum-free medium. Generally higher rates of protein degradation are observed in the contact-inhibited lines than the transformed cells. We suggest that this difference may provide a selective growth advantage to transformed cells.     

Metabolism of [3H]gibberellin A4 in somatic suspension cell cultures of carrot

The native gibberellin A₄ (GA₄), in radioactive form ([1,2-³H]GA₄, 1.06 Ci/mmol), was fed to carrot somatic cell cultures (suspension and immobilized cell systems) and its metabolism over a 48 hr period was investigated. It was found that the [³H]GA₄ was metabolized to at least two GAs, [³H]GA₁ and [³H]GA₈, six GA glucosyl conjugates, [³H]GA₁-0(3)-glucoside, [³H]GA₁-0(13)-glucoside, [³H]GA₁-glucosyl ester, [³H]GA₄-glucoside, [³H]GA₄-glucosyl ester, a [³H]GA₈ glucosyl conjugate(s) and a previously unknown [³H]GA₁ glucosyl conjugate ([³H]GA₁-0(3,13)-diglucoside-like compound). The GA₁-diglucoside-like compound was found only in extracts of cells and was present in significant amounts (33 % of total extractable radioactivity). All other metabolites were present in both cells and medium. For extracts of the medium, no differences between the suspension and immobilized cultures existed in types of [³H]GA₄ metabolites although quantitative differences were apparent.     

The principal glucocorticoid binding macromolecule in hepatoma cells in culture is similar to corticosteroid Binder II of rat liver cytosol

Reuber H-35 hepatoma (H4-II-E-C3) and HTC cells are known to retain differentiated corticosteroid induced functions in cell culture and to bind corticosteroids to macromolecules in cytosol which subsequently enter the cell nuclear fraction. Using both cell types we have demonstrated the major macromolecular fraction in cytosol to have properties (elution position from DEAE columns, pI, ³H-dexamethasone binding), very similar to those of rat liver corticosteroid Binder II which may be the hormone receptor.     

Stereoselective hydrolysis of xenobiotic esters by different cell lines from rat liver and hepatoma and its application to chiral prodrugs for designated growth suppression of cancer cells

Stereoselectivity in the hydrolysis of racemic ethyl 2-phenylacetate derivatives by cultured cells of noncancerous cell lines from rat liver (BRL, BRL 3A, Clone 9, and ARLJ301–3), spontaneously or oncogene transformed rat liver cell lines (ARLJ301–3TR1, Anr4, Anr9–1, and Anr13–1), and cancer cell lines from rat hepatoma (H4-II-E, McA-RH7777, and MH₁C₁) and sarcoma (XC) was studied. A strong (R)-enantiomer preference was found in the hydrolysis of ethyl 2-hydroxy- ( 2c ) and 2-methoxy-2-phenylacetate ( 3c ) by the noncancerous and oncogene-transformed cells and an (S)-enantiomer preference for ethyl N-acylphenylalaninates with all the present cell lines. These inclinations were, however, not recognized with ethyl 2-methoxy-2-phenylpropanoate and ethyl N-difluoroacetyl-or N-trifluoroacetylphenylalaninate. Moreover, the R preference for 3c was reversed in the reaction by hepatoma cells. Thus, the stereoselectivity is influenced by both structure of acyl group and species of cell lines. The hepatoma cells were considerably different from the noncancerous or oncogene-transformed cells in stereoselectivity. This fact was consistent with the order of colony formation in soft agar cultures (index of malignancy) and the resemblance in actively stained esterase patterns in gel electrophoresis. The stereoselective hydrolysis leads to cell-specific activation of anticancer prodrugs. This has been confirmed for the first time by the stereoselectivity of Anr4 and H4-II-E cells in the hydrolysis of a chiral mustard ester, bis(2-chloroethyl)aminophenyl 2-methoxy-2-phenylacetate ( 14 ) and by the difference of IC₅₀ values of (R)- and (S)- 14 against the two cell lines. © 1995 Wiley-Liss, Inc.     

The functional expression of calcium-sensing receptors in BRL cells and related signal transduction pathway responsible for intracellular calcium elevation

The calcium-sensing receptors (CaSRs) exist in a variety of tissues and cells. In 2001, Canaff et al. first identified its expression in liver tissue and primary cultured hepatocytes, and demonstrated that GdCl₃ (a specific agonist of CaSR) can cause an increase in intracellular calcium and bile flow. However, authors did not elucidate its mechanisms. Therefore, this study sought to detect CaSR expression in BRL cell line, which is derived from buffalo rat liver, and to reveal the cellular signal transduction pathway by which the CaSR activation results in increased intracellular calcium by BRL cells. In this study, the expression and distribution of CaSR were detected by RT-PCR, Western blotting, and immunofluorescence, and the intracellular calcium concentration [Ca²⁺]_i was measured using LCSM. The results showed that CaSR mRNA and protein were expressed in BRL cells and mainly distributed in cell membrane and cytoplasm. Increased extracellular calcium or GdCl₃ could increase intracellular calcium concentration and CaSR expression. Moreover, this increase of [Ca²⁺]_i could be inhibited or even abolished by U73122 (a specific inhibitor of PLC), 2-APB (an inhibitor of IP₃ receptor), and thapsigargin (an inhibitor of endoplasmic reticulum calcium pump). In conclusion, CaSR is functionally expressed in BRL cells, and activation of CaSR involves in increased intracellular calcium through Gq–PLC–IP₃ pathway.     

Properties of phenylalanine hydroxylase of cultured hepatoma cells.

The kinetic and immunologic properties of phenylalanine hydroxylase of adult rat liver were compared to the properties of the similar enzyme present in cultured H4-II-E-C3 hepatoma cells. The enzymes from the two sources could not be distinguished by the Km values for either phenylalanine or 6,7-dimethyltetrahydropterin. Analysis by double immunodiffusion showed that phenylalanine hydroxylase from the two sources had identical immunologic determinants, but immunotitrations revealed a small but significant difference between the enzyme of the normal adult rat liver and the enzyme of cultured hepatoma cells. The results of double immunodiffusion and immunotitration experiments indicated also that the increased levels of phenylalanine hydroxylase seen in the hepatoma cells grown in the presence of hydrocortisone resulted from the accumulation of enzyme protein, but it could not be decided whether this accumulation resulted from an increased rate of synthesis or decreased rate of degradation.     

Effect of dexamethasone,ammonium lons,and serum-deprivation on intracellular proteolysis in cultured muscle cells

Intracellular proteolysis was measured in primary cultures of newborn rat skeletal (gastrocnemius) and cardiac muscle cells by release to the medium of trichloroacetic acid-soluble label from cells grown in the presence of ¹⁴C-labeled phenylalanine. Exposure of the cultured cells to 10^?7M dexamethasone for 5 days starting at day 0 of culture resulted in an enhancement of proteolysis in skeletal muscle but not in cardiac muscle cells. Dexamethasone did not affect cell viability measured by release of label from cells preloaded with Na₂ ⁵¹CrO₄, release of creatine phosphokinase, and release of lactic dehydrogenase into the culture medium. Incorporation of ³H-thymidine into the cells increased during the first 3 to 4 days of culture and subsequently decreased, indicating that cell proliferation ceases at that time. When the exposure to dexamethasone was started on day 4 of culture, i.e., at a postmitotic stage, and continued for 4 days, proteolysis was again found to increase in skeletal but not cardiac cells, thereby suggesting that the response to the hormone is independent of the proliferative state of the culture. Ammonium chloride at a concentration of 10 mM produced a 50% reduction of the basal proteolysis in cultures of skeletal muscle cells and did not affect proteolysis in cardiac muscle cells. Exposure to ammonium chloride did not prevent the dexamethasone-induced increase of proteolysis in skeletal muscle cells. Serum-deprivation induced enhanced proteolysis which was not affected by NH₄Cl in both cell types. It is concluded that the differential responses of the two cultures to dexamethasone reflects the sparing of heart proteins and concomitant wasting of skeletal muscle proteins by glucocorticoid hormones in vivo, and that the enhancement of proteolysis by the glucocorticoid hormone or by serum-deprivation is not sensitive to the lysosomotropic agent NH₄Cl. Thus, while a lysosomal-autophagic enzyme system is responsible for almost half of the basal proteolysis, the accelerated proteolysis occurs via ammonium chloride-insensitive enzymes.     

Evidence for novel linkages in a glycoprotein involving beta-hydroxyphenylalanine and beta-hydroxytyrosine.

Cutinase, an extracellular enzyme from Fusarium solani f. pisi contains about 4% covalently attached carbohydrates. Treatment of the enzyme with alkali resulted in β-elimination and generation of dehydroamino acids absorbing at 241 nm. NaB³H₄ treatment in 0.1 N KOH followed by hydrolysis of the labeled protein gave rise to tritiated alanine, α-aminobutyric acid, phenylalanine, and tyrosine. Chemical and enzymatic degradation of the labeled phenylalanine showed that this amino acid was a 1:1 mixture of D- and L-stereo-isomers and that³H was equally distributed between the α- and β-positions. Therefore it is concluded that this glycoprotein contained 0-glycosidic linkages not only at serine and threonine residues but also at β-hydroxyphenylalanine and β-hydroxytyrosine; the latter two have not been found heretofore.     

Binding of thyrotropin releasing hormone and prolactin release by synchronized GH3 rat pituitary cell line.

GH3 cells were synchronized by growing them in a low serum concentration (1%). They were thereafter put back in normal medium (17.5% serum) (time 0 of synchronization). Four parameters were then examined every two hours for up to 40 hours : rate of [³H] thymidine incorporation, cell number, binding of [³H] Thyrotropin Releasing Hormone (TRH) after a 30 min exposure, and prolactin (PRL) content of culture medium and cell extract.The rate of thymidine incorporation presented a 10–20 fold increase in S phase, beginning on 12–16 hours and lasting at 26 hours. The cell population was doubled at 28 hours. [³H] TRH binding to attached cells was observed throughout the cell cycle, but presented a significant increase (40–80%) during the S phase. In contrast, the % increase of PRL release in response to TRH was optimum (300% of control) in G₁ phase. Variations of the PRL cell content as well as of the PRL spontaneous release ability of the cell do not account for the variations of TRH responsiveness. The discrepancy between the two parameters of the TRH-GH3 cells interaction strongly suggest a morphological or functional heterogeneity of the TRH-binding sites.     

The rat hepatoma cell line H4-II-E-C3 expresses high activities of the high-affinity glutamate transporter GLT-1A

The rate of uptake of aspartate into the rat hepatoma cell line H4-II-E-C3 is very much higher than that exhibited by normal rat hepatocytes. Using an RT-PCR-based strategy, a glutamate transporter resembling mouse liver GLT-1A has been cloned from H4-II-E-C3 cells. Northern blotting confirmed that relatively high levels of mRNA for GLT-1A are expressed in hepatoma cells compared with negligible levels in rat hepatocytes. To our knowledge, this is the first report of the cloning of a high-affinity glutamate transporter from a transformed cell line and also the first demonstration of functional expression of GLT-1 outside the central nervous system.     

Incorporation of exogenous ganglioside GM1 into neuroblastoma membranes: Inhibition by calcium ion and dependence upon membrane protein

Since exogenous gangliosides are known to promote neuritogenesis, the incorporation of exogenous GM1 into neuroblastoma membranes was examined. Neuro-2A cells, synchronized in the G1/G0 phase, were suspended in HEPES buffered saline containing 10^–4 M [³H]GM1, and membrane incorporation was measured as radioactivity remaining with the cell pellet following incubation with serum-containing medium and trypsin. Calcium ion (0.01 to 10 mM) reduced incorporation of exogenous GM1, due to its interaction with GM1 micelles in solution. When cells were treated with proteases prior to incubation with GM1, the inhibitory effect of Ca²⁺ was lost and total incorporation into membranes was lowered by approximately one order of magnitude. Pretreatment of cells with 0.05% trypsin resulted in an inhibition of GM1 incorporation within 5 minutes. When trypsinized cells were resuspended in complete growth medium, the cells recovered the ability to incorporate GM1 with time, and this paralleled labeling of cellular protein with [³H]leucine. The role of membrane protein in the incorporation of exogenous GM1 could not be explained by the lytic release of cytosolic transfer proteins nor the artifactual coating of the cell surface by serum proteins. These results suggest that the incorporation of exogenous gangliosides into cellular membrane lipid bilayers cannot be fully explained by considerations of lipophilicity alone, and leads us to propose that initial recognition by membrane protein(s) is necessary.Abbreviations used GM1 H³NeuAc-GgOse₄Cer - HBS HEPES buffered saline - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum     

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

Non-confluent monolayers of H4-II-E-C3 cells were maintained in serum-free media. Dexamethasone alone (5 × 10^?7M) stimulated α-fetoprotein secretion 2- to 4-fold while insulin alone (8.7 × 10^?8M) inhibited α-fetoprotein secretion by 20%. When dexamethasone (5 × 10^?7 to 5 × 10^?9M) and insulin (8.7 × 10^?8 to 8.7 × 10^?11M) 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.     

Development of a selective and potent radioactive ligand for histamine H3 receptors: A compound potentially useful for receptor occupancy studies

Radioligands are powerful tools for examining the pharmacological profiles of chemical leads and thus facilitate drug discovery. In this study, we identified and characterized 3-([1,1,1-³H]methyl)-2-(4-{[3-(1-pyrrolidinyl)propyl]oxy} phenyl)-4(3H)-quinazolinone ([³H]1) as a potent and selective radioligand for histamine H₃ receptors. Radioligand [³H]1 exhibited appreciable specific signal in brain slices prepared from wild-type mice but not from histamine H₃ receptor-deficient mice, demonstrating the specificity and utility of [³H]1 as a selective histamine H₃ receptor radioligand for ex-vivo receptor occupancy assays.     

The estimation of phenylalanine ammonia-lyase (PAL)-activity in intact cells of higher plant tissue

Summary A procedure is described which permits the estimation of the relative activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5.) in intact plant cells, exemplified by buckwheat hypocotyls. Hypocotyl segments are incubated at pH 5.5 with L-[3-³H]phenylalanine. N³HH₂, which is liberated from phenylalanine by the action of phenylalanine ammonia-lyase, equilibrates with tissue water to yield ³HOH, which is recovered by sublimation. Participation of phenylalanine transaminase in the reactions leading to ³HOH formation is excluded, and it is conclusively shown that ³HOH is formed intracellularly and not by enzymatic activity leaking out of wounded tissue.Abbreviation PAL phenylalanine ammonia-lyase (E.C. 4.3.1.5.)     

Sulfated glycoproteins and extracellular matrix of cultured human pulmonary endothelial cells

Endothelial cells derived from human pulmonary arteries incorporate (³H)-glucosamine and ³⁵SO₄ into glycosaminoglycans and into the carbohydrate side chains of glycoproteins. These ³H/³⁵S-carbohydrate chains were isolated from cells and culture medium after Pronase digestion. The ³H/³⁵S-glycosaminoglycans were separated from the ³H/³⁵S glycopeptides by chromatography on Sephadex G-50. The distribution of cellular glycosaminoglycans and glycopeptides indicated that 30–60% of the cellular ³⁵S-glycopeptides may be associated with the matrix components that are synthesized by the cell and attached to a plastic substratum. Human pulmonary arterial endothelial cells were grown on collagen or on a matrix derived from vascular smooth muscle cells in order to investigate how smooth muscle cell extracellular matrix components may regulate the synthesis of endothelial cell glycoconjugates. Endothelial cells grown on plastic release various proportions of the glycoconjugates they synthesize into the culture medium. However, these same cells, when grown on substratum composed of extracellular matrix materials, synthesized altered proportions of cell-associated glycosaminoglycans and reduced the levels of total glycosaminoglycans they released into the culture medium. Thus the growth of endothelial cells on a matrix of smooth muscle cell components indicates that the glycosaminoglycan materials released into the culture medium by cells grown on a plastic substratum may not be an accurate reflection of the levels or composition of extracellular matrix materials made by endothelial cells in vivo.     

Keratin-protein kinase C interaction in reactive oxygen species-induced hepatic cell death through mitochondrial signaling

Keratins (Ks), the intermediate filament (IF) proteins of epithelia, constitute at least 20 cytoskeletal proteins subdivided into type I (K9-20) and type II (K1-K8) and expressed as type I/type II pairs in a cell differentiation manner. Hepatocyte IFs are made only of K8/K18, the hallmark of simple epithelial cells. We have shown previously that a K8/K18 loss leads to a modulation of apoptosis in Fas-stimulated mouse hepatocytes. Here we report that K8-knockout mouse hepatocytes and K8-knockdown H4-II-E-C3 (shK8b1) rat hepatoma cells were much more resistant than their K8/K18-containing counterparts, wild-type hepatocytes, and H4ev hepatoma cells, in response to excess H2O2 or tert-butyl hydroperoxide, a ROS generator. While excess H2O2 altered glutathione (GSH) and ROS levels in H4ev versus shK8b1 cells, the differential death response was largely GSH level independent. Assessment of key cell death features revealed that hepatic cells exposed to H2O2 die through a mitochondrial involvement. Similarly, administration of the GSH depletor L-buthionine-sulfoximine to generate mitochondrial ROS-sensitized H4-II-E-C3 cells but not shK8b1 cells to death. Treatment with protein kinase C (PKC) inhibitors yielded a resistance of H2O2-treated H4-II-E-C3 cells comparable to that of nontreated shK8b1 cells, which in turn were not affected by the treatment. In addition, this differential death response was associated with altered PKCdelta activation and surface-membrane/mitochondria distribution in H2O2-treated shK8b1 cells. Together, these results point to a key regulatory function for K8/K18 in ROS-induced mitochondria-mediated death through PKCdelta involvement in hepatic cells.     

CELL CYCLE EVENTS IN THE HYDROCORTISONE REGULATION OF ALKALINE PHOSPHATASE IN HELA S3 CELLS

The increase in alkaline phosphatase in asynchronous cultures of HeLa S₃ cells grown in medium supplemented with hydrocortisone is characterized by a lag period of 10–12 hr. Present studies utilizing synchronous cell populations indicate: (a) a minimum of 8–10 hr of incubation with hydrocortisone is necessary for maximum induction of alkaline phosphatase; (b) the increase in enzyme activity produced by hydrocortisone is initiated exclusively in the synthetic phase of the cell cycle; (c) alkaline phosphatase activity does not vary appreciably over a normal control cell cycle. Radioactive hydrocortisone is rapidly distributed into HeLa cells irrespective of their position in the cell cycle, indicating that inductive effects are not governed by selective permeability during the cell cycle. Hydrocortisone-1,2-[³H] diffuses back from the cell into the medium when the cells are incubated in fresh medium containing no hydrocortisone, and the alkaline phosphatase induction, under these conditions, is completely reversible.