Genetics of the mammalian phenylalanine hydroxylase system. IV. Evidence of phenylalanine hydroxylase in a cultured human hepatoma cell line

We report here the identification of a cultured human hepatoma cell line which possesses an active phenylalanine hydroxylase system. Phenylalanine hydroxylation was established by growth of cells in a tyrosine-free medium and by the ability of a cell-free extract to convert [¹⁴C]phenylalanine to [¹⁴C]tyrosine in an enzyme assay system. This enzyme activity was abolished by the presence in the assay system of p-chlorophenylalanine but no significant effect on the activity was observed with 3-iodotyrosine and 6-fluorotryptophan. Use of antisera against pure monkey or human liver phenylalanine hydroxylase has detected a cross-reacting material in this cell line which is antigenically identical to the human liver enzyme. Phenylalanine hydroxylase purified from this cell line by affinity chromatography revealed a multimeric molecular weight (estimated 275,000) and subunit molecular weights (estimated 50,000 and 49,000) which are similar to those of phenylalanine hydroxylase purified from a normal human liver. This cell line should be a useful tool for the study of the human phenylalanine hydroxylase system.     

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.     

P-chlorophenylalanine does not inhibit production of recombinant human phenylalanine hydroxylase in NIH3T3 cells or E. coli

P-chlorophenylalanine is an irreversible inhibitor of rat phenylalanine hydroxylase in vivo and in rat hepatoma cells and is frequently administered to rodents to create an animal model for phenylketonuria. We investigated the effect of p-chlorophenylalanine on production of human phenylalanine hydroxylase in human hepatoma cells and cells transformed with the recombinant human phenylalanine hydroxylase gene. P-chlorophenylalanine inhibited production of the human enzyme in human hepatoma cells and transformed mouse hepatoma cells but had no effect on the production of the enzyme in transformed NIH3T3 cells or in E. coli. Thus, phenylalanine hydroxylase inhibition does not result from a simple interaction between the drug and enzyme.     

Hormonal induction of tyrosine aminotransferase and RNA synthesis in the cells of Morris hepatoma strain 7777

The transfer of Morris hepatoma cells induced by the hormone within 10-60 min in to a hormone-free medium is associated with the augmentation of tyrosine aminotransferase synthesis. The kinetics of this process does not differ from that of the hormone-induced enzyme. The return of tyrosine aminotransferase synthesis to the basal level occurs 15-20 hours after the hormone withdrawal from the medium, although the concentration of the intranuclear hormone sharply decreases already after 3 hours. It was demonstrated that the presence in the hepatoma cell nuclei of 20-25% of the initially bound hormone for at least 20 hours after the cell transfer to the hormone-free medium is not sufficient for maintaining a high level of tyrosine aminotransferase gene expression. Using two-dimensional electrophoresis of 3H-labeled hepatoma cell proteins, it was demonstrated that the observed high activity of tyrosine aminotransferase is due to the de novo synthesis of enzyme molecules rather than to the existence of preformed long-living tyrosine aminotransferase molecules inside the cell. Study of [14C]uridine incorporation into non-ribosomal nuclear RNA of hepatoma cells showed a long-term presence of the label in the RNA throughout the chase experiment. It was assumed that the high activity of the enzyme for 10-15 hours after the hormone release from the hepatoma cell nuclei is due to the accumulation in the nuclei of long-living pre-mRNA molecules synthesized after the hormone addition to the cells and during the first hours after the cell transfer to the hormone-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA from rat hepatoma cells can activate phenylalanine hydroxylase gene of mouse erythroleukemia cells

Mouse erythroleukemia (MEL) cells do not synthesize any detectable level of phenylalanine hydroxylase and thus do not grow in Tyr- medium. Rat hepatoma cells that constitutively express phenylalanine hydroxylase were treated prior to fusion with MEL cells with biochemical inhibitors to inactivate different macromolecular components of the cells, and the fusion products were selected in Tyr- medium. Continuously growing populations of cells resembling the parental MEL cells and expressing mouse phenylalanine hydroxylase were obtained only when rat hepatoma cells treated with mitomycin or iodoacetamide, which inactivate DNA and SH proteins, respectively, were fused with MEL cells. Fusion of MEL cells with UV-treated rat hepatoma cells did not result in the activation of the mouse phenylalanine hydroxylase gene. UV treatment damages both DNA and RNA. These data suggested that RNA was involved in the regulation of phenylalanine hydroxylase gene. Additional evidence for the role of RNA in the phenylalanine hydroxylase gene regulation was obtained from RNA transfection studies. RNA only from cells which express phenylalanine hydroxylase, such as rat hepatoma cells and MEL cybrids, when introduced into MEL cells by the CaPO4 coprecipitation method, resulted in the permanent activation of the mouse phenylalanine hydroxylase gene.     

Synthesis of a liver enzyme in hybrid cells.

Rat hepatoma cells were fused with cells of an established mouse lymphoma line, with normal diploid mouse macrophages, lymphocytes and fibroblasts and with normal diploid rat macrophages and lymphocytes. The liver-specific enzyme tyrosine aminotransferase was produced by almost all the hybrid cells, but usually at a lower level than in the parental hepatoma cells. Most of the hybrids also showed increased levels of this enzyme after exposure to dexamethasone. In the rat x mouse hybrids, the electrophoretic mobility of the enzyme indicated that only the rat hepatoma enzyme was produced. The findings are difficult to explain in terms of simple models involving a single diffusible repressor or activator of tyrosine aminotransferase synthesis.     

Tyrosine aminotransferase induced in cells genetically and epigenetically deficient for this enzyme

Mice homozygous for lethal deletions around the albino (c) locus are deficient for several liver-specific enzymes, including tyrosine aminotransferase (TAT). The structural gene coding for this enzyme appears to be intact in these mutants and can be “activated” in homozygous mutant mouse liver-rat hepatoma cell hybrids. The present study demonstrates that the mouse form of TAT can also be induced in both normal and mutant mouse skin-rat hepatoma cell hybrids. Thus, a liver-specific enzyme is expressed in skin cells, both normal and mutant, the normal differentiated state of which excludes TAT expression.     

Enhancement of prostaglandin synthesizing activity by the treatment with sodium n-butyrate on cloned mastocytoma cells and various other tissue cell lines

We have compared the effects of n-butyrate on the prostaglandin synthesizing activities of cloned mouse mastocytoma cells, and various other tissue culture cell lines. Cells were treated with 1 mM n-butyrate for 40 hrs before harvesting. Prostaglandin synthesizing activities of the treated and the control cells were examined in a cell-free assay system. The treatment of some of the cloned mastocytoma cells with n-butyrate brought about the synthesis of prostaglandin D₂, E₂ and F_2α that were not synthesized by the control cells. The treatment of epithelial liver cells (BC-90) also resulted in the formation of 6-keto-prostaglandin F_1α which was not formed by the control cells. However, n-butyrate caused relatively small changes in the prostaglandin synthesizing activities of other clones of mastocytoma cells, mouse hepatoma cells, HeLa cells, rat granuloma cells and human embryonic fibroblasts. These data suggest differential effects of n-butyrate on different types of cultured cells.     

Phenylalanine hydroxylase in melanoma cells.

A pigmented subclone of Cloudman S91 melanoma cells, PS1-wild type, can grow in medium lacking tyrosine. This ability is conferred by phenylalanine hydroxylase activity, and not by tryptophan hydroxylase, tyrosine hydroxylase or tyrosinase activities, although the latter activity is also present in these cells. Conversion of phenylalanine to tyrosine was measured in living cells by chromatographic identification of the metabolites of [14C]phenylalanine and in cell extracts using a sensitive assay for phenylalanine hydroxylase. Phenylalanine hydroxylase activity in melanoma cell extracts was identified by its inhibition with p-chlorophenylalanine and not with 6-fluorotryptophan, 3-iodotyrosine, phenylthiourea, tyrosine or tryptophan; and by adsorption with antiserum prepared against purified rat liver phenylalanine hydroxylase, and migration of immunoprecipitable activity with authentic phenylalanine hydroxylase subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Expression and amplification of cloned rat liver tyrosine aminotransferase in nonhepatic cells

A full-length cDNA for the rat liver enzyme tyrosine aminotransferase has been used to construct mammalian expression vectors by recombinant DNA techniques. These vectors, which have employed either a simian virus 40 or a Rous sarcoma virus promoter, were transfected into a variety of nonhepatic mammalian cell lines in culture. Transient expression of tyrosine aminotransferase was readily observed after transfection into monkey COS cells and mouse L cells. Stable clones that express cloned tyrosine aminotransferase have been isolated from mouse L cells, hamster Wg1a fibroblasts, and Chinese hamster ovary (CHO) cells. A vector capable of expressing both tyrosine aminotransferase and dihydrofolate reductase was stimulated to undergo amplification by treatment with methotrexate in a CHO cell line deficient in the latter enzyme. Levels of tyrosine aminotransferase as much as 50-fold higher than typically seen in glucocorticoid-induced hepatoma cells were achieved in some CHO clones by this technique. The tyrosine aminotransferase produced at these highly amplified levels appeared structurally normal and had no major harmful effects on the cells.     

Inhibition of thymidylate synthase by hydroxyurea in rapidly proliferating P815 mastocytoma cells

The incorporation of [¹⁴C]deoxycytidine, [³H]deoxyuridine, and [³H]thymidine, respectively into pyrimidine bases of DNA has been measured in rapidly proliferating P815 mouse mastocytoma cells in the presence of hydroxyurea. The incorporation of [¹⁴C]deoxycytidine-derived radioactivity into DNA cytosines is increased when compared to the incorporation into DNA thymines. The [³H]deoxyuridine-derived radioactivity is incorporated solely into DNA thymines and this incorporation is inhibited by hydroxyurea in a dose-dependent manner. This suggests an inhibitory effect of hydroxyurea on the thymidylate synthase which was proved in experiments in which the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate catalysed by a crude enzyme preparation from P815 cells was inhibited in the presence of hydroxyurea. Enzymatic DNA methylation as measured by the conversion of incorporated [¹⁴C]deoxycytidine into 5-methylcytosines was not affected by hydroxyurea.     

Cell density dependent regulation of phenylalanine hydroxylase activity in hepatoma cells : Evidence for both an active and inactive enzyme form

The cell density dependent regulation of phenylalanine hydroxylase activity in Reuber hepatoma (H4) cells growing in monolayer culture has been examined in detail. We found that 48 h or more after subculture phenylalanine hydroxylase activity in the cells is an exponential function of cell density (cells/cm²). No discontinuity in the relationship is seen with the formation of a confluent monolayer.A rapid loss or a rapid gain in enzyme activity in the cells is observed after diluting or concentrating the cell cultures. The two processes appear qualitatively different. The loss in activity is a first order process which starts at the time of subculture with the rate of loss dependent on the density of subculture. The gain in activity begins 6–8 h after subculture to a higher density; it can be blocked by cycloheximide and has a maximum rate of increase that is about 10% of the maximum rate of loss of activity.Using immunochemical procedures, we found the same amount of phenylalanine hydroxylase associated antigen in Reuber cells from low density as from high density cultures, over a range of phenylalanine hydroxylase specific activities from 0.2 to 4.2. After concentrating cells to a higher density, no increase in enzyme antigen was observed, despite a several-fold increase in enzyme activity and a requirement for protein synthesis during the process. These observations imply the presence of an active and inactive phenylalanine hydroxylase with the relative amounts of each determined by the cell density. The effects of db-cAMP are discussed. Evidence is presented here that the hydrocortisone stimulation of phenylalanine hydroxylase activity works through a different mechanism than the cell density dependent process.     

Measurement of phenylalanine hydroxylase turnover in cultured hepatoma cells.

A substantially new method has been developed to measure protein turnover. Its basis is the notion that in labeling experiments a secreted protein can be used to determine the specific radioactivity of the intracellular amino acid precursor pool. To measure protein turnover in the Reuber hepatoma H4 cell line, cultures were labeled with [3H]leucine for specified periods after which phenylalanine hydroxylase was isolated and its leucine specific radioactivity determined. Serum albumin secreted by the cultures was also isolated and used to estimate the leucine precursor pool specific radioactivity. The protein half-life of phenylalanine hydroxylase could them be calculated. Experiments performed at long and short labeling times and with high and low concentrations of leucine in the medium yielded equivalent results. Phenylalanine hydroxylase half-life in the H4 cells was investigated under both normal and hydrocortisone-induced growth conditions. Average half-lives of 7.4 and 8.2 h were found for induced and uninduced cultures, respectively. Although these measured enzyme half-lives were not essentially different, the steady state level of phenylalanine hydroxylase was increased 6.2-fold upon hydrocortisone induction, from 0.076 to 0.47 microgram/10(6) cells. The results demonstrated that hydrocortisone induces phenylalanine hydroxylase in the H4 cells by causing an increase in the rate of enzyme synthesis.     

The hydroxylation of phenylalanine and tyrosine by tyrosine hydroxylase from cultured pheochromocytoma cells.

Pheochromocytoma tyrosine hydroxylase was reported to have unusual catalytic properties, which might be unique to the tumor enzyme (Dix, T. A., Kuhn, D. M., and Benkovic, S. J. (1987) Biochemistry 24, 3354-3361). Two such properties, namely the apparent inability to hydroxylate phenylalanine and an unprecedented reactivity with hydrogen peroxide were investigated further in the present study. Tyrosine hydroxylase was purified to apparent homogeneity from cultured pheochromocytoma PC12 cells. The purified tumor enzyme was entirely dependent on tetrahydrobiopterin (BH4) for the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine and hydrogen peroxide could not substitute for the natural cofactor. Indeed, in the presence of BH4, increasing concentrations of hydrogen peroxide completely inhibited enzyme activity. The PC12 hydroxylase exhibited typical kinetics of tyrosine hydroxylation exhibited typical kinetics of tyrosine hydroxylation, both as a function of tyrosine (S0.5 Tyr = 15 microM) and BH4 (apparent Km BH4 = 210 microM). In addition, the enzyme catalyzed the hydroxylation of substantial amounts of phenylalanine to tyrosine and 3,4-dihydroxyphenylalanine (apparent Km Phe = 100 microM). Phenylalanine did not inhibit the enzyme in the concentrations tested, whereas tyrosine showed typical substrate inhibition at concentrations greater than or equal to 50 microM. At higher substrate concentrations, the rate of phenylalanine hydroxylation was equal to or exceeded that of tyrosine. Essentially identical results were obtained with purified tyrosine hydroxylase from pheochromocytoma PC18 cells. The data suggest that the tumor enzyme has the same substrate specificity and sensitivity to hydrogen peroxide as tyrosine hydroxylase from other tissues.     

Regulation of expression of tyrosine aminotransferase in somatic cell hybrids between rat hepatoma cells and mouse fibroblasts

Somatic cell hybrids between rat hepatoma cells and mouse 3T3 fibroblasts fail to produce the liver-specific enzyme tyrosine aminotransferase. A novel approach using gamma-irradiation to induce chromosome loss from the non-expressing parent cell was applied to dissect genetically the factors in 3T3 cells that interact with the regulation of expression of tyrosine aminotransferase in these hybrids. Suppression of basal and steroid-inducible tyrosine aminotransferase activities was progressively relieved with increasing dose of radiation. The wide range in degree of reexpression suggests a complex of regulatory mechanisms. Suppression of steroid-inducibility was not linked to the mouse X-chromosome. Nor did the mouse genome affect the modulation of enzyme activity induced by insulin and by serum.     

p-Chlorphenylalanine effect on phenylalanine hydroxylase in hepatoma cells in culture.

We have investigated the p-chlorophenylalanine-dependent loss of phenylalanine hydroxylase activity in cultured hepatoma cells. The similarity of the effect of p-chlorophenylalanine on phenylalanine hydroxylase in the hepatoma cells and that reported from studies in vivo indicates that the loss of phenylalanine hydroxylase activity is due to a direct interaction of the amino acid analogue with the liver. We can find no evidence that the loss of phenylalanine hydroxylase activity is due to: a direct inactivation of the hydroxylase by p-chlorophenylalanine or an inhibitor produced by p-chlorophenylalanine treatment; an effect similar to that of p-fluorophenylalanine; or leakage of enzyme from the cells during p-chlorophenylalanine treatment. The data presented indicate: (a) the p-chlorophenylalanine effect is rather specific for phenylalanine hydroxylase; (b) following p-chlorophenylalanine removal, new protein synthesis is necessary for restoration of the hydroxylase activity; (c) the rate of loss of phenylalanine hydroxylase activity after the addition of p-chlorophenylalanine is much faster than the rate of restoration of the hydroxylase activity after removal of p-chlorophenylalanine; (d) even in the presence of p-chlorophenylalanine, hydrocortisone greatly stimulates the hydroxylase activity; (e) the cell density-dependent increase of phenylalanine hydroxylase activity is blocked by p-chlorophenylalanine. A discussion of the possible mechanisms of p-chlorophenylalanine-dependent loss of phenylalanine hydroxylase is presented. To measure very low leanine-dependent loss of phenylalanine hydroxylase is presented. To measure very low levels of phenylalanine hydroxylase activity, a new procedure, based on isotope dilution, was developed for isolating the tyrosine formed during the enzymatic reaction.     

Characterization of the free radical of mammalian ribonucleotide reductase

Mouse fibroblast 3T6 cells, selected for resistance to hydroxyurea, were shown to overproduce protein M2, one of the two nonidentical subunits of mammalian ribonucleotide reductase. Packed resistant cells gave an EPR signal at 77 K very much resembling the signal given by the tyrosine-free radical of the B2 subunit of Escherichia coli ribonucleotide reductase. Also, the M2-specific free radical was shown to be located at a tyrosine residue. Of the known tyrosine-free radicals of ribonucleotide reductases from E. coli, bacteriophage T4 infected E. coli and pseudorabies virus infected mouse L cells, the M2-specific EPR signal is most closely similar to the signal of the T4 radical. The small differences in the low temperature EPR signals between these four highly conserved tyrosine-free radical structures can be explained by slightly different angles of the beta-methylene group in relation to the plane of the aromatic ring of tyrosine, reflecting different conformations of the polypeptide chain around the tyrosines. The pronounced difference in microwave saturation between the E. coli B2 tyrosine radical EPR signal and the M2 signal could be due to their different interactions with unspecific paramagnetic ions or with the antiferromagnetically coupled iron pair, shown to be present in the E. coli enzyme and postulated also for the mammalian enzyme. A difference in the iron-radical center between the bacterial and mammalian ribonucleotide reductase is also observed in the ability to regenerate the free radical structure. In contrast to the B2 radical, the M2 tyrosine free radical could be regenerated by merely adding dithiothreitol in the presence of O2 to a cell extract where the radical had previously been destroyed by hydroxyurea treatment.     

Tyramine-O-sulfate, in addition to tyrosine-O-sulfate, is produced and secreted by HepG2 human hepatoma cells, but not by 3Y1 rat embryo fibroblasts

The spent media of HepG2 human hepatoma cells and 3Y1 rat embryo fibroblasts labeled with [35S]sulfate, upon ultrafiltration, were analyzed by a two-dimensional thin-layer separation procedure. Autoradiographs of the cellulose thin-layer plate revealed the presence of tyramine-O-[35S]sulfate in addition to tyrosine-O-[35S]sulfate in spent medium from human hepatoma cells. In contrast, only tyrosine-O-[35S]sulfate was observed in spent medium of 3Y1 rat fibroblasts. Using adenosine, 3'-phosphate, 5'-phospho[35S]sulfate as the sulfate donor, sulfotransferase(s) present in HepG2 cell homogenate catalyzed the sulfation of tyramine to tyramine-O-[35S]sulfate, but not the sulfation of tyrosine to tyrosine-O-[35S]sulfate. Endogenous aromatic amino acid decarboxylase present in HepG2 homogenate was shown to catalyze the decarboxylation of [3H]tyrosine to form [3H]tyramine while attempts to use it for the decarboxylation of tyrosine-O-sulfate to form tyramine-O-sulfate were unsuccessful. These results suggest that tyramine-O-sulfate may be derived from the de novo sulfation of tyramine, instead of the decarboxylation of tyrosine-O-sulfate.     

Explanation for the apparent inactivation of tyrosine aminotransferase in hepatoma cells by concanavalin A

Brief treatment of hepatoma cells in monolayer culture with concanavalin A causes a decrease in tyrosine aminotransferase specific activity that is thought to be a rapid, reversible inactivation of the enzyme (T.V. Gopalakrishnam and E.B. Thompson 1977 J. Biol. Chem. $\text{252}$, 2717–2725). We confirm this decrease, but attribute it to an increased leakage of cellular protein from concanavalin A-treated monolayer cultures during the harvesting procedure. If the cells are washed free of medium and lysed $\text{in}$,$\text{situ}$ by freezing and thawing them, or by treating them with buffer containing a nonionic detergent, equal amounts of tyrosine aminotransferase are found in concanavalin A-treated and untreated cells. If cells are harvested by scraping them from the substrate, some tyrosine aminotransferase is lost into the buffer used to collect the cells. Treatment of cells with concanavalin A markedly increases the amount of enzyme lost during this procedure, and results in a low enzyme content in the washed cells. No inactivation occurs, however, because the total amount of tyrosine aminotransferase present in the cell pellet and the wash buffer is equal for treated and untreated cells.