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.     

Wound-induced phenylalanine ammonia-lyase in potato tuber tissue. Development of enzyme activity and effects of antibiotics.

Phenylalanine ammonia-lyase [EC 4.3.1.5.] activity increased rapidly after a 3-hr lag period in potato tuber (Solanum tuberosum L. cv. May Queen) disks incubated in a suitable medium in the dark at 25 degrees. The activity reached a maxinum after incubation for about 40 hr. The effects of actinomycin D, 6-methylpurine, cycloheximide, chloramphenicol, and mitomycin C on the induction of phenylalanine ammonia-lyase were investigated during incubation of the disks. Actinomycin D, 6-methylpurine, and cycloheximide all inhibited the formation of phenylalanine ammonia-lyase, though cycloheximide was the most effective at low concentrations. Application of actinomycin D for the initial lag period (3 hr) caused strong inhibition; however, if it was supplied later it did not inhibit but actually increased phenylalanine ammonialyase formation. In contrast, cycloheximide was effective over most of the incubation period. Chloramphenicol and mitomycin C did not inhibit phenylalanine phenylalanine ammonialyase induction, but markedly stimulated it. Light was not an essential factor for phenylalanine ammonia-lyase induction in the wounded tissue.     

A tyrosine-free medium for the selective growth of cells expressing phenylalanine hydroxylase activity

Tyrosine-free modified Ham's F-12 medium supplemented with charcoal-extracted serum permits the exclusive propagation of cultured cells containing detectable levels of phenylalanine hydroxylase. The selective properties of this medium have been verified with two phenylalanine hydroxylase-positive (H4-II-E-C3 and MH₁C₁) and two phenylalanine hydroxylase-negative (HTC and BRL) cell lines. Although cocultivation experiments revealed that BRL cells could replicate slowly in the selective medium in the presence of sufficient numbers of H4-II-E-C3 cells, the former cells did not survive subcultivation in this medium at low cell inoculum (< 10³ cells per 60 × 15-mm Petri dish). This tyrosine-free medium should be particularly effective in selecting for phenylalanine hydroxylase-positive somatic-cell hybrids under conditions of double selection where the phenylalanine hydroxylase-positive parental cell would not survive.     

Rapid response of protein synthesis to insulin in 3T3 cells: Effects of protein kinase C depletion and differences from the response to serum repletion

Quiescent 3T3 cells grown in media containing 4% foetal calf serum showed different responses to insulin and to serum repletion (to 12%). Insulin stimulated protein synthesis within 1 h and this early response was insensitive to actinomycin D. The later insulin response showed progressive sensitivity to actinomycin D. The serum response was slower, not occurring until 1 h, and was inhibited by actinomycin D. Depletion of cell protein kinase C by pre-treatment with phorbol ester caused a total block of the immediate response to insulin but had little effect on the response to serum or the later response to insulin. Acute phorbol ester treatment stimulated protein synthesis.     

Possible role of H1 histone in the differentiation of mouse erythroleukemia cells

Serum-starved mouse erythroleukemia cells, stationary phase cells or cells cultured in dibutyryl cAMP (1 mM) can be induced to differentiate by addition of 20% fetal calf serum plus cycloheximide. Culturing unstarved log phase cells in 20% fetal calf serum plus low levels of cycloheximide and histone H1 also causes a significant level of differentiation. These same concentrations of cycloheximide and H1 histone employed separately with 20% fetal calf serum do not induce differentiation. The role these procedures may have in causing an accumulation of histone H1 and cell differentiation is discussed.     

The regulation of cell growth: II. Some characteristics of a fetal calf serum factor (FF2) stimulating ornithine decarboxylase in mouse liver

A heat stable, non-dialysable fetal calf serum factor (FF₂), capable of stimulating ornithine decarboxylase in mouse liver, kidney and spleen, has been detected in fetal calf serum and commercial preparations of 81% pure fetuin.The factor has a molecular weight of approx. 17 500, contains sulfhydryl groups necessary for its activity, and is protease resistant.Stimulation of hepatic ornithine decarboxylase by the fetal calf serum factor is dose and time dependent and is blocked by both cycloheximide and by actinomycin D, if the latter is administered within 1 h of the factor. Theophylline enhances the effect of the fetal calf serum factor on ornithine decarboxylase in the liver and the factor stimulate ornithine decarboxylase in hypophysectomized and adrenalectomized rats.     

The separate roles of glucose and insulin in the induction of glucokinase in hepatocytes isolated from neonatal rats.

1. The specificity of the effect of glucose on the induction of glucokinase activity that occurs when hepatocytes freshly isolated from 13-day-old rats are incubated in Medium 199 together with insulin [Wakelam & Walker (1980) FEBS Lett. 111, 115-119] was examined. A pattern that is different from other known effects of glucose is found, and metabolism of this compound is not necessarily to account for this particular effect. 2. The effects of a raised glucose concentration and of insulin on the induction can be separated. The hexose initiates the process in the absence of insulin in a manner that is sensitive to actinomycin D but not to cycloheximide. The subsequent effect of insulin is dependent on the prior effect of glucose or other positive analogue, does not require the presence of glucose and is inhibited by cycloheximide but not by actinomycin D. 3. Induction of glucokinase in vitro in hepatocytes from neonatal animals is inhibited by adrenaline, glucagon and dibutyryl cyclic AMP, but not by vasopressin or angiotensin II. The inhibition by cyclic AMP is on the stage requiring insulin and is comparatively specific, because total protein synthesis is not apparently diminished. 4. The implications of these results are discussed with reference to possible mechanisms of induction and to the situation in vivo.     

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.     

Effect of serum on phenylalanine hydroxylase levels in cultured hepatoma cells.

Continued high levels of phenylalanine hydroxylase in cultured H4-II-E-C3 rat hepatoma cells require either serum or glucocorticoids in the culture medium. Upon withdrawal of serum, cellular phenylalanine hydroxylase levels decay exponentially with a half-life of 22 hours for about 60 hours, after which time a low, constant enzyme content persists for at least 96 hours. This decline of phenylalanine hydroxylase is fully reversible; normal enzyme levels are restored in a time- and dosage-dependent fashion upon addition of serum to basal cultures. The serum factor is nondialyzable and moderately heat-stable. The stimulation by serum of the phenylalanine hydroxylas content of basal cultures is blocked by 3-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide and requires ongoing cellular protein synthesis. When added to the enzyme-assay mixture in vitro, serum does not alter the phenylalanine hydroxylase activity of extracts from basal cultures. Three lines of evidence suggest that serum contains a nonsteroidal phenylalanine hydroxylase stimulatory components(s): (a) glucocorticoid antagonists inhibit less than one-half of the biological activity of serum; (b) exhaustive extraction of endogenous serum glucocorticoids with charcoal reduces the activity of serum to about one-half of control values; and (c) the stimulatory effects of charcoal reduces the values; and (c) the stimulatory effects of charcoal-extracted serum and hydrocortisone are additive. The phenylalanine hydroxylase stimulatory activities of the charcoal-extracted sera from four mammalian species and from three stages in development in one mammalian species are comparable. A survey of partially purified preparations of a number of known hormones failed to reveal any one capable of elevating the phenylalanine hydroxylas levels of basal cultures in a manner comparable to that of charcoal-extracted serum.     

Effect of environmental stress of low pressure on tyrosine aminotransferase and phenylalanine 4-hydroxylase activities in the rat.

1. Tyrosine aminotransferase activity in the liver increased about fourfold after 9h, on exposure of rats to stress of low pressure. 2. The phenylalanine hydroxylase activity increased about 60% on exposure for 24h or more. 3. An environmental pressure decrease of about 0.033 MN/m2 is needed to increase the activity of tyrosine aminotransferase. 4. Adrenalectomy completely abolished the increase in activity of tyrosine aminotransferase obtained on exposure to low pressure. 5. Treatment with cycloheximide or actinomycin D prevented the increase in activity of tyrosine aminotransferase. 6. Treatment with cycloheximide at the early part of exposure to stress prevented the increase in activity of phenylalanine hydroxylase obtained after 24h.     

Effects of protein kinase C activation on prostaglandin production and cyclooxygenase mRNA levels in ovine astroglia

We examined effects of protein kinase C (PKC) activation by phorbol dibutyrate (PDB) on prostaglandin production in astroglia. Astroglia were cultured from sheep fetal cortex and grown in Eagle's basal media supplemented with 10% fetal calf serum (BME-C). Prostaglandin F_2a (PGF_2α) levels in media were determined at 2–24 hours after exposure to PDB. PDB increased production of PGF_2α at 10⁻⁸M and 10⁻⁶M. In addition, PDB increased the ratio of membrane to cytosolic PKC. Coapplication of H7 [1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine] (10⁻⁴M) with PDB (10⁻⁶M) inhibited PDB-induced PGF_2a production. To investigate the role of protein synthesis in increased prostaglandin production by PDB, astroglia were coincubated with actinomycin D (1 mg/ml) or cycloheximide (10 mg/ml). At 4 hrs, both actinomycin D and cycloheximide inhibited increases in PGF_2a in response to PDB application. In addition, COX-2 mRNA levels and COX activity levels were examined. PDB increased COX-2 mRNA levels by 2 hours, and COX activity tripled after 12 hr exposure to PDB. In addition, the increase in COX activity was blocked by cycloheximide. In summary, PKC activation promotes enhanced prostaglandin production via an increase in COX synthesis.     

Nutrition and fetal brain maturation : I. Responses in vitro and in vivo

The glycolytic enzyme enolase increases during the perinatal period of brain development and was utilized as a marker for examining the effect of culture environment on differentiation of cells from 20-day fetal rat brain. Enolase activity in cell cultures increased from 0.91 +/- 0.03 (Day 0) to 2.11 +/- 0.10 mumol/min/mg protein (Day 6). Comparable levels were not reached in vivo until neonatal pups were 15 days old. The in vitro increase was inhibited by both cycloheximide and actinomycin D. Enolase activity in the cells responded to alterations in both incubation media and homologous serum. After 6 days in culture, cells incubated in rat serum (10%) added to MEM or RPMI produced twice as much enolase activity as cells incubated similarly in Ham's medium, i.e., 1.96 +/- 0.09 and 1.85 +/- 0.21 vs 1.02 +/- 0.09, P less than 0.001. Results of a comparable magnitude were obtained when fetal calf serum replaced adult rat serum, but enolase production was somewhat lower when newborn calf serum replaced adult rat or fetal calf serum. When cells were incubated for 6 days with graded concentrations of adult rat serum (2.5-15%), enolase activity increased progressively. The pattern of enolase response suggests that the fetal rat brain cell model described herein will provide a sensitive probe with which to gain insight into nutrition and fetal brain development.     

Superinduction by cycloheximide of mitogen-induced secreted proteins produced by Balb/c 3T3 cells

We describe here some of the characteristics of the regulation of a group of secretory proteins whose secreted levels rise within 2-4 h of adding fibroblast growth factor (FGF), epidermal growth factor (EGF), or serum to quiescent Balb/c 3T3 cells. The levels of these secretory proteins are regulated similarly to the interferons. When cycloheximide is present during the induction period, the amounts of [35S]methionine incorporated into five of these proteins that we have called "superinducible proteins" (SIPs) is increased 2-5-fold. Superinduction of the SIPs is seen also in response to polyribol-polyriboC, the classical inducer of interferons. None of the SIPs, however, are immuno-precipitated by anti-beta-interferon antibody. Induction and superinduction of the SIPs is inhibited by actinomycin D. Superinduction occurs at concentrations of cycloheximide that inhibit protein synthesis by at least 85%. The SIPs are not major intracellular proteins; they are barely detectable in cellular fractions. Their induction is, however, correlated with the ability of the polypeptide growth factor to stimulate DNA synthesis; EGF, FGF, and serum induce the SIPs, whereas insulin does not, and insulin alone weakly stimulates DNA synthesis in these cells. Because FGF, EGF, and serum cause the SIPs to be produced at concentrations of cycloheximide that inhibit 85% of bulk protein and DNA synthesis, it follows that the SIPs are produced directly from the action of the growth factor and not as a consequence of increased growth. Although probably not interferons, in analogy to the lymphokines, the SIPs could be a set of autocrine or paracrine factors that rapidly convey the growth or differentiation signal between cells.     

Serum-dependent induction of plasminogen activator in human fibroblasts by catecholamines and comparison with the effects of prostaglandin E1

Human foreskin fibroblasts produce the protease plasminogen activator, as shown by the ability of cell extracts to lyse 125I-labelled fibrin in the presence of plasminogen. Cellular plasminogen activator was stimulated up to 3-fold by 0.01-10 microM epinephrine, norepinephrine, or isoproterenol. Increases in plasminogen activator were slow in onset (24 h) and long-lived (greater than 48 h), and were abolished by 10 micrograms/ml of cycloheximide or 1 microgram/ml of actinomycin D, suggesting de novo synthesis of the protease. Stimulation of plasminogen activator by catecholamines was inhibited by 10 microM propranolol but not by 10 microM phentolamine, suggesting the involvement of beta-adrenergic receptors. Catecholamines stimulated plasminogen activator only in the presence of fetal bovine serum; under serum-free conditions they were inhibitory. Serum did not appear to alter the uptake and metabolism of epinephrine during incubation with fibroblasts. The ability of fetal bovine serum to support the induction of plasminogen activator by either 1 microM epinephrine or 3 microM prostaglandin E1 was maintained following dialysis but lost on heating (70 degrees C, 10 min) or acidification (pH 2.5). Human and calf sera supported the stimulatory effects of prostaglandin E1 but not of epinephrine. These results indicate that serum may influence the synthesis of plasminogen activator in cultured cells by modifying their response to vasoactive hormones.     

Specificity of deoxyribonucleic Acid intercalating compounds in the control of phenylalanine ammonia lyase and pisatin levels

Compounds with planar triple ring systems such as acridine orange, 9-amino acridine, 9-amino-1,2,3,4-tetrahydroacridine (tacrine), 6,9-diamino-2-ethoxyacridine lactate monohydrate (DE-acridine), 6-chloro-9-(3′-diethylamino-2′-hydroxypropylamino) -2-methoxyacridine·2 HCl (CDM-acridine), quinacrine, 6-chloro-9-(4′-diethylamino-1′-methylbutylamino) -2-methoxy-1,10-diazaanthracene (CDM 1,10-diazaanthracene), thionine, azure A, methylene blue, and pyronine Y when applied to excised pea pods were potent inducers of phenylalanine ammonia lyase or of pisatin, or of both. Compounds with an array of structural variation around the planar three-ring system were tested for their ability to induce these responses in pea tissue. In general, dimethylamino, diethylamino, or amino substitutions at position 2 and 6 or an amino (with or without an aliphatic side chain) substitution at position 9 of the three-ring system augmented induction potential. Methyl green, methylene blue, 2,7-diaminofluorene, nile blue, neutral red, pyrogallol red, ethidium bromide, nogalamycin, quinine, chloroquine, spermine, 8-azaguanine, gliotoxin, chromomycin A₃, actinomycin D, and mitomycin C were also potent inducers. The inhibition of phenylalanine ammonia lyase induction by the application of actinomycin D (300 micrograms per milliliter) or 6-methylpurine (1 milligram per milliliter) within 1 hour after inducer application indicated that newly synthesized RNA is necessary for induction. Phenylalanine ammonia lyase induction was also inhibited by cycloheximide (150 micrograms per milliliter).     

Prostaglandin production by methylcholanthrene-transformed mouse BALB/3T3. Requirement for protein synthesis

Stimulation of prostaglandin synthesis in transformed mouse fibroblasts by serum, thrombin, and bradykinin was blocked by actinomycin D and cycloheximide. These RNA and protein synthesis inhibitors did not affect prostaglandin synthetase in vitro or in vivo; nor did they affect the acylation of arachidonic acid into phospholipids. Serum-stimulated release of arachidonic acid and prostaglandins from [3H]arachidonic acid-labeled cells also was inhibited by actinomycin D and cycloheximide. RNA and protein synthesis appear to be required for expression of phospholipase activity; a prerequisite for prostaglandin synthesis by these cells.     

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.