Establishment of a rat hepatoma cell line which has ornithine carbamoyltransferase activity and grows continuously in arginine-deprived medium

The epithelial cell line, H4-II-E derived from Reuber hepatoma H35 has no significant activity of ornithine carbamoyltransferase (OCT, EC 2.1.3.3) and is not able to grow in arginine-deprived medium. A multi-step selection procedure is described which selects from H4-II-E populations, cells with OCT activity which can grow in arginine-deficient, ornithine-supplemented media.     

Properties of the H-4-II-E tumor cell system. I. Growth and cell proliferation kinetics of an experimental hepatoma.

Growth and cell proliferation kinetics of hepatoma H-4-II-E and its tissue culture derivative have been studied to establish the characteristics of an in vivo--in vitro solid tumor model. The H-4-II-E line, originating from the Reuber H-35 hepatoma, can be maintained and studied either in cell culture or as a transplantable solid tumor in ACI male rats. In addition it allows for the in vitro assay of cell survival following treatment of animal tumors in situ. In vivo, hepatoma H-4-II-E is rapidly growing tumor with a mean doubling time of 49-2 hr. The cell cyle time is 39-1 hr with a cell loss factor of 0-32. Retrospective examination of tumor specimens obtained during the establishment of the H-4-II-E tumor system demonstrates that both structural as well as cell population changes have occurred. The biological characteristics of the primary tumor (H-35) and an early intermediate stage (H-35tc2) are compared with H-4-II-E and the histopathological, growth and cell kinetic changes are discussed.     

Inhibition by ultraviolet irradiation of the glucocorticoid induction of tyrosine aminotransferase in bromodeoxyuridine-treated H-35 hepatoma.

Low doses of ultraviolet irradiation at 254 nm inhibited the dexamethasone induction of tyrosine aminotransferase (E.C. 2.6.1.5) in H-35 Reuber (H-4-II-E) hepatoma cells exposed to bromodeoxyuridine for approximately two generations of growth. The cells were transferred to a serum-free medium for the hormone induction studies so that growth inhibition would not be a factor in the experiments. Doses of 90 ergs/mm² inhibited the glucocorticoid induction and resulted in a decline in tyrosine aminotransferase in both basal- and steroid-induced levels of the enzyme. Comparison of the inhibition of the glucocorticoid induction by low doses of ultraviolet irradiation at 254 nm in thymidine-treated versus bromodeoxyuridine-treated cells suggested that bromodeoxy-uridine-treated cells were more sensitive to low doses of irradiation. The basal activities of two other enzymes, alkaline phosphatase and leucine aminopeptidase, were only slightly inhibited 6 hours following irradiation of bromodeoxy-uridine-treated cells. These results suggest that studies using ultraviolet irradiation of monolayer cell cultures to study different mechanisms of hormonal regulation of cellular processes may provide an alternative approach for studies on modulation of gene expression.     

PROPERTIES OF THE H-4-II-E TUMOR CELL SYSTEM

Growth and cell proliferation kinetics of hepatoma H-4-II-E and its tissue culture derivative have been studied to establish the characteristics of an in vivo—in vitro solid tumor model. The H-4-II-E line, originating from the Reuber H-35 hepatoma, can be maintained and studied either in cell culture or as a transplantable solid tumor in ACI male rats. In addition it allows for the in vitro assay of cell survival following treatment of animal tumors in situ. In vivo, hepatoma H-4-II-E is a rapidly growing tumor with a mean doubling time of 49.2 hr. The cell cycle time is 39.1 hr with a cell loss factor of 0.32. Retrospective examination of tumor specimens obtained during the establishment of the H-4-II-E tumor system demonstrates that both structural as well as cell population changes have occurred. The biological characteristics of the primary tumor (H-35) and an early intermediate stage (H-35_tc2) are compared with H-4-II-E and the histopathological, growth and cell kinetic changes are discussed.     

DNA damage induced by nitropyrenes in primary mouse hepatocytes and in rat H4-II-E hepatoma cells

The capacity of nitropyrenes to cause DNA damage in primary mouse hepatocytes (C57BL/6N mice) and rat H4-II-E hepatoma cells was studied by estimating single-strand breaks using the alkaline elution technique. 1-Nitropyrene (10-200 microM) caused clear dose-dependent increases in DNA strand breaks in both cell types, whereas no increase in DNA strand breaks was observed in hepatocytes treated with 1.3-, 1,6-, 1,8-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene under standard assay conditions (5-20 microM 30-min incubation). However, 1,8-dinitropyrene (1,8-DNP) caused dose-dependent increases in DNA strand breaks when incubated with the H4-II-E cells for 48 h, while no single-strand breaks were observed following treatment with 1,6-dinitropyrene (1,6-DNP) under the same conditions. Neither 1,6-DNP nor 1,8-DNAP induced DNA crosslinks in the H4-II-E cells. These data indicate that substrate specificity exists in the metabolic activation of nitropyrenes in murine liver.     

Induction of the messenger ribonucleic acid coding for phosphoenolpyruvate carboxykinase in H4-II-E cells. Evidence for a nuclear effect of cyclic AMP

The effect of N6,O2'-dibutyryl cyclic adenosine monophosphate (Bt2cAMP) on the induction of the mRNA coding for the enzyme phosphoenolpyruvate carboxykinase was examined in H4-II-E cells. this mRNA comprised about 0.1% of total cellular poly(A)+RNA activity in uninduced cells and was increased 5- to 7-fold by the cyclic nucleotide. The maximal level was reached 3 h after addition of the nucleotide to the cell culture. This induction is attributed to cAMP since the nonmetabolizable analogs 8-bromocAMP and 8-(4-chlorophenylthio)cAMP produce inductions comparable to Bt2cAMP while sodium butyrate and dibutyryl cyclic GMP had little effect. The increased translational activity correlated well with a proportionate increase in the amount of phosphoenolpyruvate carboxykinase (P-enolpyruvate carboxykinase) mRNA sequences which were hybridizable to a specific cDNA probe. Blot hybridization of total nuclear RNA isolated from uninduced H4-II-E cells revealed eight P-enolpyruvate carboxykinase RNA sequence species ranging in size from 1.8 to 6.9 kilobases. Treatment with Bt2cAMP increased the amount of all eight of these forms. This increase became maximal by 45-60 min and was maintained for at least 1 h. In contrast, analysis of cytoplasmic RNA showed a single 3.2-kilobase (23 S) band, which was still increasing in amount 2 h after Bt2cAMP treatment. Thus, Bt2cAMP resulted in a sequential induction of nuclear P-enolpyruvate carboxykinase RNA sequences followed by an increase in cytoplasmic phosphoenolpyruvate carboxykinase mRNA. We conclude that cyclic AMP exerts its main effect on P-enolpyruvate carboxykinase induction at the nuclear level.     

Antagonizing effect of 3-aminoharman on induction of sister-chromatid exchanges by mutagens

3-Aminoharman (3AH, 3-amino-1-methyl-9H-pyrido[3,4-b]indole), which has been reported as a novel substance with an antagonistic effect on induction of sister-chromatid exchange (SCE) by polycyclic mutagens in the presence of the metabolic activation system, was examined with a cultured human lymphoblastoid cell line, NL3, for its effect on SCE induction by direct-acting mutagens such as mitomycin C (MMC), nitrogen mustard N-oxide (NMO), methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide (4NQO) and 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (OH-Trp-P-2), and also by ultraviolet light (UV) irradiation. The results obtained on simultaneous treatment with 3AH and mutagens were as follows: (1) 3AH suppressed more than 50% of SCEs induced by MMC, NMO and OH-Trp-P-2; (2) 4NQO- and MNNG-induced SCEs were also suppressed by 3AH but to a lesser degree; (3) MMS-induced SCEs were not, however, altered by 3AH; and (4) the suppression of SCE by 3AH was dose-dependent. Treatment of cells with 3AH for 2 h immediately before MMC exposure suppressed SCE induction to a significant degree similar to the simultaneous treatment, but post-treatment with 3AH was much less effective. 3AH inhibited SCE induction by NMO when 3AH treatment was carried out either before or after NMO treatment, to an extent similar to the simultaneous treatment. Treatments with 3AH either before or after UV exposure did not change the UV-induced SCEs. Results with these direct-acting mutagens ruled out the relevance of metabolic activation as a necessary step for the antagonizing effect of 3AH.     

Presence of cytochrome P-450- and cytochrome P-448-dependent monooxygenase functions in hepatoma cell lines

Cell lines derived from Reuber H-4-II-E hepatoma cells and their hybrids that differ in the expression of liver-specific functions are shown to contain different forms of monooxygenases. According to 1) the specificity toward the substrates benzo(a)pyrene, aldrin and chenodexycholic acid, 2) the kinetics of the epoxidation of aldrin, 3) the response to inducers, such as benz(a)anthracene and dexamethasone, and 4) the $\text{in}$$\text{vitro}$ modifier 7,8-benzoflavone, the monooxygenases predominating in differentiated cell lines belong to the cytochrome P-450-dependent enzyme(s), those in the less differentiated lines belong to the cytochrome P-448-dependent form(s).     

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.     

Suppression of cell proliferation and deoxyribonucleic acid synthesis in the cloned rat hepatoma H4-II-E cells overexpressing regucalcin.

The role of endogenous regucalcin (RC) in the regulation of cell proliferation was investigated in the cloned rat hepatoma H4-II-E cells overexpressing RC stably. H4-II-E cells were transfected with RC/pCXN2 vector and the multiple neomycin-resistant clones which overexpress stably RC were selected. The RC content of RC/pCXN2-transfected cells used in this study was 19.7-fold as compared with that of the parental wild type H4-II-E cells. Wild type H4-II-E cells, pCXN2 vector-transfected cells (mock type), and RC/pCXN2-transfected cells (transfectants) were cultured for 24, 48, and 72 h in the presence of fetal bovine serum (10% FBS). Cell numbers of wild and mock type were significantly increased with the time course of culture. Cell numbers of transfectants was significantly suppressed as compared with that of wild and mock type. Deoxyribonucleic acid (DNA) synthesis activity in the nuclear fraction of H4-II-E cells was significantly suppressed in transfectants with culture for 12-48 h. The presence of anti-RC monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant increase in DNA synthesis activity in the nuclei of wild type and transfectants; this increase was remarkable in transfectants. The effect of anti-RC monoclonal antibody (50 ng/ml) in increasing DNA synthesis activity in transfectants was completely prevented by the addition of regucalcin (1 microM). This study demonstrates that cell proliferation is suppressed in the cloned rat hepatoma H4-II-E overexpressing RC stably.     

Expression of microsomal and cytosolic epoxide hydrolases in cultured rat hepatocytes and hepatoma cell lines

Microsomal epoxide hydrolase activity, determined using benzpyrene 4,5-oxide and styrene 7,8-oxide, increased in cultured hepatocytes compared to freshly isolated cells. In contrast, cytosolic epoxide hydrolase activity, assayed using trans-stilbene oxide, had decreased 80% by 24 hr and was barely detectable after 96 hr in culture. There was no difference in enzyme activity between freshly isolated hepatocytes and the two rat hepatoma cell lines McA-RH 7777 and H4-II-E, when styrene 7,8-oxide was used as substrate. However, benzpyrene 4,5-oxide hydrolase activity of the McA-RH 7777 and H4-II-E cell lines were 55 and 10%, respectively, of freshly isolated hepatocytes. These results show that hepatoma cell lines provide a suitable system for studying the regulation of both the microsomal and cytosolic epoxide hydrolase enzymes.     

Differences in the induction of SCEs between human whole blood cultures and purified lymphocyte cultures and the effect of an S9 mix

Studies for SCE induction are frequently performed on human blood cultures. Either whole blood cultures (WBC) or purified lymphocyte cultures (PLC) are employed. However, it has been shown that fundamental differences with respect to metabolic activity exist between these two systems. In order to further characterize the whole blood culture and the purified lymphocyte culture, differently acting substances were studied comparatively with and without an Aroclor-1254-induced S9 mix. Treatment with ethyl methanesulfonate (EMS), a direct mutagen, produced distinct SCE induction in both systems. Cyclophosphamide (CP) and benzo[a]pyrene (BP), two indirect mutagens, also led to a significant increase of SCEs both in WBC and PLC without S9 mix. Only with CP was this effect more pronounced after addition of S9 mix. Sodium selenite (Na2SeO3), which induced SCEs in WBC, did not show this effect in the PLC. After S9 mix was added to purified lymphocytes, an increase of SCEs by sodium selenite was observed as in WBC. H2O2, a radical former, led to SCE induction in purified lymphocytes but not in the whole blood culture. By adding S9 mix, a distinct reduction of the SCEs induced by H2O2 was established. These results show that human lymphocytes can metabolize indirect mutagens and that it should be kept in mind when using S9 mix that, besides mixed-function oxygenases, it also contains enzymes which influence the SCE-inducing effects of substances.     

The induction of ornithine decarboxylase by tumor promoting phorbol ester analogues in Reuber H35 hepatoma cells

The induction of ornithine decarboxylase activity was studied in a rat hepatoma cell line (Reuber H35) incubated with a group of structurally-related phorbol ester analogues. A single application of 1.6 μM of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to H35 cells caused a dramatic increase in the activity of ornithine decarboxylase. The stimulation of the enzyme activity was rapid but transient, peaking at 4 to 5 hr with a value which was 116-fold greater than control and then declining to the basal level after 8 hr. In addition, the increase in ODC activity was dependent upon the concentration of TPA added to the culture medium and the EC₅₀ was estimated to be about 2.63 × 10^?7 M. Our studies of the effect of various phorbol ester analogues on the H35 ODC activity indicated an apparent correlation between the ability of phorbol ester derivatives to induce ODC activity in the H35 cells and their activity to promote papilloma formation in the mouse skin in that the various derivatives possessed the following relative abilities to increase ODC activity: TPA > PDB > PDA > 4 α-P > 4 α-PDD. Concurrent addition of either actinomycin D or cycloheximide abolished the increase in ODC activity after TPA treatment. Changes of intracellular concentrations of polyamines, particularly putrescine, were in good agreement with the increase in ODC activity in response to TPA: a 10-fold increase in putrescine over the control level was observed at 6 hr. Our data suggest that cultured Reuber H35 hepatoma cells exhibit a marked and specific response to the phorbol ester tumor promoters and may be of great value in studying the biochemical mechanism of ODC induction by these agents.     

Evaluation of a human hepatoma cell line as a target cell in genetic toxicology

A cell line derived from a human hepatoblastoma, HepG2, was examined for its ability to activate cyclophosphamide (CY) to a genotoxic form. Metabolism of CY to genotoxic product(s) was determined by the induction of sister-chromatid exchanges (SCE). The dose-dependent response pattern in HepG2 was compared to the patterns obtained by three other mammalian cell lines. HepG2 and a rat hepatoma cell line, H4-II-E, show similar dose-dependent increases of induced SCE, whereas non-hepatic-derived fibroblast lines show little or no CY-induced SCE. Microsomal enzyme activities characteristic of cytochromes P450 and P448 and epoxide hydrolase were examined in the two hepatoma cell lines and compared to levels in rat liver microsomal preparations. Although no cultured cell line can be a universal surrogate for in vivo metabolism, we propose that HepG2 may be useful to determine in a qualitative manner whether human cells possess the ability to activate a chemical to a genetically damaging form.     

Intracellular uptake of an antitumor-active azole-bridged dinuclear platinum(II) complex in cisplatin-resistant tumor cells

A cationic azolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH₃)₂}₂(μ-OH)(μ-methyl-pyrazolate)]²⁺ (4M-PzPt), was developed to overcome resistance to cisplatin (CDDP). This study aimed to assess the cytotoxicity of 4M-PzPt against a CDDP-resistant cell line, H4-II-E/CDDP, and compare the intracellular accumulation of CDDP and 4M-PzPt. H4-II-E and H4-II-E/CDDP displayed similar sensitivity to 4M-PzPt; however, the sensitivity of H4-II-E/CDDP to CDDP was approximately 19-fold lower than that of H4-II-E. The difference in the sensitivity to both platinum complexes corresponded with the difference in the amount of intracellular platinum accumulation after exposure to CDDP or 4M-PzPt in both cell lines. In H4-II-E, HepG2, and HuH-7 cells, the intracellular uptake of CDDP and 4M-PzPt occurred via active transport and passive transport. Results of co-exposure with the transport inhibitors ouabain, tetraethylammonium, and cimetidine indicated that the intracellular uptake of CDDP was dependent on Na⁺/K⁺-ATPase and that of 4M-PzPt was dependent on organic cation transporters (OCTs), probably OCT1. This study suggested that 4M-PzPt could inhibit the growth of a CDDP-resistant tumor via an intracellular uptake mechanism different from that of CDDP.     

Differences in murine procarcinogen activation enzymes are not accompanied by parallel differences in procarcinogen-induced sister-chromatid exchange

C57B1/6 and DBA/2 mice, strains in which there is marked induction of hepatic monooxygenase activity by phenobarbital, were tested for in vivo sister-chromatid exchange (SCE) formation in response to cyclophosphamide, an agent metabolized by this inducible enzyme system. Baseline SCE frequencies were between 4 and 6 SCEs/cell in regerating liver and bone marrow of both strains of mice. Administration of cyclophosphamide (5 mg/kg) led to an increase of nearly 8 SCEs/cell in both tissues of C57B1/6 mice and an increase of more than 10 SCEs/cell in DBA/2 mice. Prior exposure to phenobarbital induced p-chloromethylaniline demethylase activity in regenerating liver of both mouse strains approx. 6-fold, but the changes in measured SCE frequencies were not significantly different from those obtained in the absence of enzyme induction. These results, together with our previous observation that induction of by 3-methylcholanthrene of benzo[a]pyrene hydroxylase activity in the same mouse strains was not accompanied by a comparable change in benzo[a]pyrene-induced SCE formation, reinforce the impression that simple assays of differences in mixed function oxidase activities may not necessarily be good predictors of hereditary differences in the response to genetic damage by procarcinogens which are presumed to be metabolized by these enzymes.     

Mechanism of inactivation of phenylalanine hydroxylase by p-chlorophenylalanine in hepatome cells in culture. Two possible models.

The mechanism by which p-chlorophenylalanine specifically reduces phenylalanine hydroxylase activity in rat liver in vivo and in Reuber H4 hepatoma cells in culture has been investigated. Chromatography on hydroxylapatite of liver extract from rats injected with p-chlorophenylalanine showed that the compound differentially affected the three normal phenylalanine hydroxylase isoenzymes (I, II, and III); isoenzymes II and III were completely absent after the treatment, but isoenzyme I was only reduced in quantity compared with normal adult rats. Normal Reuber H4 cells only possess isoenzyme I; treatment with p-chlorophenylalanine yielded a reduced level of enzyme activity which appeared to be noraml isoenzyme I by both chromatographic and kinetic criteria. There is evidence, based on immunochemical techniques, that cultures grown in the presence of p-chlorophenylalanine have significantly reduced levels of phenylalanine hydroxylase antigen, and that p-chlorophenylalanine inactivates phenylalanine hydroxylase at or near the time of enzyme synthesis. The bulk of enzyme synthesized prior to the addition of the compound appears unaffected by it. There is no indication that protein synthesis itself is affected by p-chlorophenylalanine. In addition, p-chlorophenylacetate was found to inactivate phenylalanine hydroxylase in an apparently identical manner with p-chlorophenylalanine, which almost certainly eliminates from consideration any mechanism of inactivation specifically requiring an amino acid. Finally, effects of cycloheximide and chlorophenylalanine were compared. Taken together, the data lead to two possible models for the inactivation of the enzyme. The model most consistent with all data requires (predicts) the existence of a proenzyme form of phenylalanine hydroxylase which can be specifically inactivated by p-chlorophenylalanine.