Single-cell gel electrophoresis assays with human-derived hepatoma (Hep G2) cells.

The purpose of the present study was the development of a protocol for detecting chemically-induced DNA damage, using the alkaline single-cell gel electrophoresis (SCGE) assay with human-derived, metabolically competent hepatoma (Hep G2) cells. Previous studies indicated that Hep G2 cells have retained the activities of certain phase I and phase II enzymes and reflect the metabolism of genotoxins in mammals better than other in vitro models which require addition of exogenous activation mixtures. The optimal trypsin concentration for the removal of the cells from the plates were found to be 0.1%. Dimethylsulfoxide, at concentrations up to 2%, was an appropriate solvent for water-insoluble compounds. To determine the optimal exposure periods for mutagen treatment, the time kinetics of comet formation was investigated with genotoxic chemicals representing various classes of promutagens namely benzo[a]pyrene (B[a]P), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and N-nitrosodimethylamine (NDMA) and with N-nitrosomethylurea (NMU). All compounds caused a statistically significant induction in DNA damage. With the promutagens, comet formation increased gradually as a function of the exposure duration, and reached maximum values between 20-24 h. With NMU, comet induction maximized already after a short exposure (1 h) and remained at a constant level for up to 24 h. Based on these results, the Hep G2/SCGE assay appears to be a suitable approach for investigating DNA damaging potential of chemicals. Further experiments with IQ and B[a]P showed that the assays are highly reproducible. Comparisons of the present results with those from earlier experiments in which other endpoints (induction of sister chromatid exchanges, micronuclei and chromosomal aberrations) were measured in Hep G2 cells, indicated that the sensitivity of the SCGE assays is more or less identical. Since the SCGE assay is less time consuming than other genotoxicity assays we anticipate that it might be a suitable approach to investigate DNA damaging effects of chemicals in the human-derived, metabolically competent cell line.     

In vitro translation and processing of human hepatoma cell (Hep G2) gamma-glutamyl transpeptidase

Human hepatoma cell (Hep G2) gamma-glutamyl transpeptidase (gamma-GT), a 120 ka single-chain glycoprotein, is much larger than the expected precursor of the dimeric enzyme in other human tissues. However, the Hep G2 gamma-GT mRNA encodes a 63 kDa peptide, similar to that of rat gamma-GT mRNA product and to the predicted, unglycosylated precursor of the enzyme in human tissues. Translation in presence of dog pancreas microsomes results in processing of the 63 kDa to an 80 kDa core-glycosylated species which is subsequently cleaved to 58 and 22 kDa subunits resembling those in other human tissues. The unusually large Mr of gamma-GT in Hep G2 would thus seem to be due to further glycosylation and processing in the Golgi. A deficiency of the processing protease is the most likely reason for the persistence of the single-chain form of gamma-GT in Hep G2 cells.     

Drug metabolism by the human hepatoma cell, Hep G2

The human liver-derived cell line, Hep G2, has aryl hydrocarbon hydroxylase and 7-ethoxycoumarin o-de-ethylase activities. Partial purification of cytochrome P-450 from Hep G2 cells provided spectral evidence of this hemeprotein in the purified fraction. These results suggest that Hep G2 cells will be useful for the study of cytochrome P-450 and the regulation of mixed function oxidase activities in liver cells of human origin.     

Targeted inhibition of transferrin-mediated iron uptake in Hep G2 hepatoma cells

We have used a model system consisting of two human hepatoma cell lines, Hep G2, representing well differentiated normal hepatocytes, and PLC/PRF/5, representing poorly differentiated malignant hepatocytes, to demonstrate that the differential presence of asialoglycoprotein receptor activity in these cell lines can be used to influence transferrin-mediated iron uptake. We based our experiments on the following facts: Hep G2 cells possess receptors that bind, internalize, and degrade galactose-terminal (asialo-)glycoproteins; PLC/PRF/5 cells have barely detectable asialoglycoprotein receptor activity; both cell lines possess active transferrin-mediated iron uptake; transferrin releases iron during acidification of intracellular vesicular compartments; primary amines, e.g. primaquine, inhibit acidification and iron release from transferrin. When added to culture medium, [55Fe]transferrin delivered 55Fe well to both cell lines. As expected, in the presence of [55Fe]transferrin, free primaquine caused a concentration-dependent decrease in 55Fe uptake in both cell lines. To create a targetable conjugate, primaquine was covalently coupled to asialofetuin to form asialofetuin-primaquine. When PLC/PRF/5 (asialoglycoprotein receptor (-)) cells were preincubated with this conjugate, transferrin-mediated 55Fe uptake was unaffected. However, transferrin-mediated 55Fe uptake by Hep G2 (asialoglycoprotein receptor (+)) cells under identical conditions was specifically decreased by 55% compared to control cells incubated without the conjugate.     

2- and 4-Aminobiphenyls induce oxidative DNA damage in human hepatoma (Hep G2) cells via different mechanisms

4-Aminobiphenyl (4-ABP) and its analogue, 2-aminobiphenyl (2-ABP), were examined for their ability to induce oxidative DNA damage in Hep G2 cells. Using the alkaline comet assay, we showed that 2-ABP and 4-ABP (25-200 microM) were able to induce the DNA damage in Hep G2 cells. With both compounds, formation of intracellular reactive oxygen species (ROS) was detected using flow cytometry analysis. Post-treatment of 2-ABP and 4-ABP-treated cells by endonuclease III (Endo III) or formamidopyrimidine-DNA glycosylase (Fpg) to determine the formation of oxidized pyrimidines or oxidized purines showed a significant increase of the extent of DNA migration. This indicated that oxidative DNA damage occurs in Hep G2 cells after exposure to 2-ABP and 4-ABP. This assumption was further substantiated by the fact that the spin traps, 5,5-dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN), decreased DNA damage significantly. Furthermore, addition of the catalase (100 U/ml) caused a decrease in the DNA damage induced by 2-ABP or 4-ABP, indicating that H(2)O(2) is involved in ABP-induced DNA damage. Pre-incubation of the cells with the iron chelator desferrioxamine (DFO) (1mM) and with the copper chelator neocupronine (NC) (100 microM) also decreased DNA damage in cells treated with 200 microM 2-ABP or 200 microM 4-ABP, while the calcium chelator {1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester}(BAPTA/AM) (10 microM) decreased only DNA strand breaks in cells exposed to 4-ABP. This suggested that ions are involved in the formation of DNA strand breaks. Using RT-PCR and Western blotting, lower inhibition of the expression of the OGG1 gene and of the OGG1 protein was observed in cells treated with 4-ABP, and 2-ABP-treated cells showed a marked reduction in the expression of OGG1 gene and OGG1 protein. Taken together, our finding indicated the mechanisms of induced oxidative DNA damage in Hep G2 cell by 2-ABP and 4-ABP are different, although both tested compounds are isomers.     

Evidence for targeted gene delivery to Hep G2 hepatoma cells in vitro

We have developed a system for targeting foreign DNA to hepatocytes in vitro using a soluble DNA carrier that takes advantage of receptor-mediated endocytosis to achieve internalization. The idea is based on the fact that hepatocytes possess a unique receptor that binds and internalizes galactose-terminal (asialo)glycoproteins. To create a targetable carrier system that could bind DNA in a nondeforming manner, we used poly(L-lysine) to bind DNA in a strong but noncovalent interaction. An asialoglycoprotein, asialoorosomucoid (AsOR), was chemically coupled to poly(L-lysine) to form an asialoorosomucoid-poly(L-lysine) conjugate. Various proportions of conjugate to DNA were tested to determine conditions that maximized DNA content in a soluble complex and that limited solubility of complexes. To test the targetable gene delivery system, AsOR-poly(L-lysine) conjugate was complexed to the plasmid pSV2 CAT containing the gene for chloramphenicol acetyltransferase (CAT) driven by an SV-40 promoter. We tested this complex using a model system consisting of human hepatoma cell line Hep G2 [asialoglycoprotein receptor (+)], hepatoma SK-Hep 1, IMR-90 fibroblasts, and uterine smooth muscle [receptor (-)] cells. Each cell line was incubated with 0.2 micron filtered AsOR-poly(L-lysine)-DNA complex or controls consisting of DNA plus AsOR, DNA plus poly(L-lysine), or DNA alone. Cells were assayed for the presence of CAT activity as a measure of gene transformation. SK-Hep 1, IMR-90, and smooth muscle [receptor (-)] cells produced no detectable acetylated chloramphenicol derivatives under any of these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of tunicamycin and monensin on secretion of thyroxine-binding globulin by cultured human hepatoma (Hep G2) cells

We have reported in the preceding paper that human hepatoma (Hep G2) cells synthesize thyroxine-binding globulin (TBG). In this paper, we evaluated the kinetics of secretion of the protein and the effects produced by the ionophore monensin and the glycosylation inhibitor tunicamycin. Cells were pulse labeled with [35S]methionine and then chased after addition of excess unlabeled methionine. TBG appeared in the medium after 10 min, and 50% of the protein was secreted after 45 min. After 2 h, more than 85% of TBG had been released. The rate of secretion of TBG was much slower than that of albumin, 50% of which was secreted after 20 min. Monensin, 1 microM, caused a marked delay in TBG secretion, with 50% released after 80 min. After 2 h, less than 60% had been released and a plateau was approached. Endoglycosidase H (endo H) treatment of intracellular and secreted TBG showed no alteration in the rate of conversion of TBG oligosaccharide units from high-mannose type (endo H-sensitive) to complex type (endo H-resistant), thus suggesting that monensin impeded the exit of TBG from the Golgi apparatus without affecting the terminal glycosylation of the protein. Tunicamycin, 5 micrograms/ml, completely blocked glycosylation and markedly affected TBG secretion, almost doubling the time required for the secretion of 50% of the protein. The effect was specific for TBG, since it was not observed in the case of albumin. After 2 h, only 56% of the protein had been released. Analysis of intracellular and extracellular immunoprecipitated products revealed the presence of aggregates (Mr greater than 100,000). The lack of carbohydrates, although not preventing TBG secretion, had marked quantitative effects, and increased the susceptibility to aggregation.     

25-Hydroxylation of vitamin D3 in the human hepatoma cell lines Hep G2 and Hep 3B

Two human hepatoma cell lines, Hep G2 and Hep 3B, were screened for vitamin D3-25-hydroxylase enzyme activity by incubation with radioactive vitamin D3. A compound co-chromatographing with 25-OH-D3 was synthesized in both cell lines but its rate of synthesis was tenfold greater in Hep 3B than in Hep G2 cells. The identity of the compound was confirmed by comparing its chromatographic properties with authentic 25-OH-D3 on three different high pressure liquid chromatography systems. Its production was suppressed by adding fetal calf serum (10%), lipoprotein-deficient fetal calf serum, or pure vitamin D-binding globulin to the medium. The mechanism of action of these plasma proteins appears to involve retardation of uptake of the substrate. These two cell lines offer considerable potential as defined in vitro models for studying the effects of physiological factors on the 25-hydroxylation of vitamin D3.     

Subcellular localization of squalene synthase in human hepatoma cell line Hep G2.

Using the Hep G2 cell line as a model for the human hepatocyte the question was studied whether Hep G2-peroxisomes could be able to synthesize cholesterol. Hep G2 cell homogenates were applied to density gradient centrifugation on Nycodenz, resulting in good separation between the organelles. The different organelle fractions were characterized by assaying the following marker enzymes: catalase for peroxisomes, glutamate dehydrogenase for mitochondria and esterase for endoplasmic reticulum. Squalene synthase activity was not detectable in the peroxisomal fraction. Incubation of Hep G2 cells with U18666A, an inhibitor of the cholesterol synthesis at the site of oxidosqualene cyclase, together with heavy high density lipoprotein, which stimulates the efflux of cholesterol, led to a marked increase in the activity of squalene synthase as well as HMG-CoA reductase, whereas no significant effect on the marker enzymes was observed. Neither enzyme activity was detectable in the peroxisomal density gradient fraction, suggesting that in Hep G2-peroxisomes cholesterol synthesis from the water-soluble early intermediates of the pathway cannot take place. Both stimulated and non-stimulated cells gave rise to preparations where squalene synthase activity was comigrating with the reductase activity at the lower density side of the microsomal fraction; however, it was also present at the high density side of the microsomal peak, where reductase activity was not detected.     

Immunoaffinity purification and characterization of lipoprotein-associated coagulation inhibitors from Hep G2 hepatoma, Chang liver, and SK hepatoma cells. A comparative study

A polyclonal antibody against a synthetic peptide corresponding to amino acids 3-25 of mature lipoprotein-associated coagulation inhibitor (LACI) was raised in rabbits. The antibody was used to study the production of LACI by Hep G2 hepatoma, Chang liver, and SK hepatoma cells, and to purify LACI from the culture media. By using an amidolytic assay for factor Xa, it was found that the culture media from these liver-derived cell lines contain inhibitors of factor Xa. In Hep G2 hepatoma culture medium, approximately 50% of Xa inhibitory activity was due to LACI. In the Chang liver and SK hepatoma culture media over 95% of the Xa inhibitory activity was due to LACI. The LACIs were purified from these media by immunoaffinity chromatography on an anti-LACI-lg-Sepharose 4B column and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified LA-CIs varied in molecular weight depending on whether the media were concentrated before chromatography. An Mr approximately 38,000 LACI was obtained by chromatography of unconcentrated media. Chromatography of concentrated media yielded a LACI of Mr approximately 35,000 with the same amino-terminal sequence, suggesting partial proteolysis in the carboxyl-terminal region. In addition, an Mr approximately 25,000 form of LACI was also present. The purified Mr approximately 38,000 and approximately 35,000 LACI species from the above cells possess similar specific activities when measured by an anti-Xa/amidolysis assay. To study the role of LACI in the control of coagulation, pooled human plasma was depleted of LACI antigen by immunoaffinity absorption and reconstituted with varying amounts of purified LACI to examine the effect on tissue factor (TF)-induced coagulation. LACI depletion shortens the time of TF-induced clotting of plasma and the clotting time is linearly related to the LACI concentration after reconstitution. These results suggest that LACI plays an important role in limiting TF-induced coagulation in human plasma. Comparison of the potencies of various purified LACIs in the prolongation of TF-induced coagulation revealed that LA-CIs from different sources are not equivalent. The plasma LACI, SK hepatoma LACI, and Chang liver LACI are approximately 7-, 6-7, and 1.3-fold higher in specific activity than Hep G2 hepatoma LACI in the TF-induced clotting assay when compared on an anti-Xa/amidolysis unit basis, suggesting possible differences in post-translational modification of these LA-CIs.     

Inoculum-dependent division lag of Bacillus cultures and its relation to an endogenous factor(s) ("schizokinen")

Lankford, C. E. (The University of Texas, Austin), James R. Walker, James B. Reeves, N. H. Nabbut, B. R. Byers, and R. J. Jones. Inoculum-dependent division lag of Bacillus cultures and its relation to an endogenous factor(s) ("schizokinen"). J. Bacteriol. 91:1070-1079. 1966.-When cells of Bacillus megaterium, grown on Brain Heart Infusion Agar, were inoculated into a chemically defined medium, they exhibited a division lag which was an inverse function of inoculum size. The addition of filtrates of cultures from the same medium eliminated the inoculum-dependent component of lag, but not an inoculum-independent residual lag of constant duration. Culture filtrate of B. subtilis var. niger not only eliminated its inoculum-dependent lag but also was required to sustain exponential division. Dose-response and "growth time" bioassays, developed to measure lag-reducing activity of filtrates, demonstrated accumulation of active filtrate factor to a "critical" concentration prior to division initiation. Addition of this concentration to cultures eliminated the inoculum-dependent lag. Accumulation of the factor ceased temporarily at onset of division, but excretion was resumed later during exponential growth. Accumulation of a lag-reducing, cell-associated factor followed a similar course. Chromatographic and bioautographic analyses of culture filtrates of B. megaterium indicated that a single substance was primarily responsible for their activity. Results of dose-response tests for reciprocal activities of filtrates of different Bacillus species and strains suggested production of different factors by some, and of different quantities of similar factors by others. It is proposed that such endogenous factors which are synthesized and accumulate to a population-dependent concentration as a requisite to initiation and maintenance of division be designated as "schizokinens."     

Regulation of low-density lipoprotein receptors in the human hepatoma cell line Hep G2

The human hepatoma cell line Hep G2 can be maintained in continuous culture and secretes numerous plasma proteins and lipoproteins into the medium. To better characterize cholesterol homeostasis in these cells we have examined the binding, internalization and degradation of [125I]LDL by cultured Hep G2 cells. Hep G2 cells express high-affinity low-density lipoprotein (LDL) receptors which facilitate the binding, internalization and degradation of [125I]LDL; these receptors can be induced by growth in LDL-depleted medium and repressed by further incubation in medium supplemented with LDL. The degradation of [125I]LDL by derepressed Hep G2 cells was inhibited by greater than 90% by monensin. Incubation of Hep G2 cells in the presence of increasing concentrations of LDL also inhibited cholesterol biosynthesis. Our results indicate that Hep G2 cells possess high affinity LDL receptors which are subject to metabolic regulation and suggest that this cell line affords a valuable model to further examine cholesterol and lipoprotein metabolism in human liver cells.     

Effect of 3-substituted Delta8(14)-15-ketosterols on cholesterol metabolism in hepatoma Hep G2 cells

The effects of 3-substituted Delta8(14)-15-ketosterols--3beta-(2-hydroxyethoxy)-, 3beta-(2-propenyloxy)-, 3beta-[2(R,S),2,3-oxidopropyloxy]-, 3beta-[2(R,S),2,3-dihydroxypropyloxy]-, 3beta-(2-oxoethoxy)-, 3beta-[2(R,S),2-acetoxy-3-acetamidopropyloxy]-, and 3beta-[2(R,S), 2-hydroxy-3-acetamidopropyloxy]-5alpha-cholest-8(14)-en-15-o nes--on cholesterol metabolism were studied in human hepatoma Hep G2 cells. 3beta-(2-Propenyloxy)-, 3beta-(2-oxoethoxy)-, and 3beta-[2(R,S),2, 3-oxidopropyloxy]-5alpha-cholest-8(14)-en-15-ones inhibited cholesterol biosynthesis without any effect on triglyceride biosynthesis, while 3beta-[2(R,S),2-acetoxy-3-acetamidopropyloxy]- and 3beta-[2(R,S), 2-hydroxy-3-acetamidopropyloxy]-5alpha-cholest-8(14)-en-15-o nes inhibited both cholesterol biosynthesis and triglyceride biosynthesis at concentrations exceeding 10 microM. 3beta-[2(R,S),2, 3-Dihydroxypropyloxy]-5alpha-cholest-8(14)-en-15-one, effectively inhibiting cholesterol biosynthesis, was found also to be toxic in Hep G2 cells at micromolar concentrations. 3beta-[2(R,S),2, 3-Oxidopropyloxy]-5alpha-cholest-8(14)-en-15-one effectively inhibited cholesterol acylation. All the tested compounds decreased the HMG-CoA reductase mRNA level at concentrations exceeding 10 microM; however, they did not affect the LDL receptor mRNA level. Among the compounds tested, only 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one decreased the uptake and internalization of LDL-associated cholesteryl esters, being as effective as 25-hydroxycholesterol.     

Amino acid transport systems in the human hepatoma cell line Hep G2.

The human hepatoma cell line Hep G2 was used to investigate amino acid transport systems in human liver tissue. The ubiquitous transport systems responsible for the uptake of most neutral amino acids (systems A, ASC and L) were found to be present. Transport system A was predominant for proline uptake but system ASC was the major Na(+)-dependent transport system, particularly for glutamine. The specific hepatic system N was functional, but only partially mediated glutamine uptake. The study of Na(+)-independent arginine uptake demonstrated the presence of the cationic transport system Y+, reflecting the transformed nature of Hep G2 cells.