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.     

Genotoxic and antigenotoxic effects of catechin and tannins from the bark of Hamamelis virginiana L. in metabolically competent,human hepatoma cells (Hep G2) using single cell gel electrophoresis

The genotoxic and antigenotoxic activities of catechin, hamamelitannin and two proanthocyanidin fractions prepared from the bark of Hamamelis virginiana L. were investigated in a human derived, metabolically competent hepatoma cell line (Hep G2) using single cell gel electrophoresis (SCGE) for the detection of DNA-damage. DNA-migration was calculated as Olive tail moment (OTM). Catechin and a low-molecular weight proanthocyandin fraction (W(M)) caused only slight increases of OTM up to concentrations of 166 microg/ml whereas hamamelitannin and the proanthocyandin fraction with higher molecular weight (W(A)) led to a two-fold enhancement of OTM at the same concentrations. These effects were dose-independent. Treatment of the cells with the test compounds in a dose-range of 2-166 microg/ml prior to the exposure to benzo(a)pyrene (B(a)P, 10 microM, 2.5 microg/ml) led to a significant reduction of induced DNA damage which was dose-dependent for all test compounds, except for hamamelitannin. The inhibitory effects of proanthocyanidins were stronger than those of catechin and hamamelitannin; the lowest effective concentrations were about 2 microg/ml. In order to clarify the mechanisms of protection, possible effects of the test compounds on enzymes involved in toxification and detoxification of B(a)P were investigated. While B(a)P toxification by cytochrome P450 was not inhibited by the test compounds, detoxification by glutathion-S-transferase (GST) was induced by catechin and W(M). Combination experiments with the ultimate metabolite of B(a)P, (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE; 5 microM, 1.5 microg/ml), revealed strong inhibitory effects, indicating that the observed protective effects were caused by scavenging of the ultimate mutagen by the test compounds. Exposure of Hep G2 cells to the test compounds after B(a)P treatment did not influence B(a)P induced DNA damage, demonstrating that repair mechanisms were not affected.     

Carboxypeptidase H. A regulatory peptide-processing enzyme produced by human hepatoma Hep G2 cells

Human hepatoma (Hep G2) cells have been shown to secrete nanogram quantities of carboxypeptidase N (Grimwood, B. G., Plummer, T. H., Jr., and Tarentino, A. (1988) J. Biol. Chem. 263, 14397-14401). A second carboxypeptidase with an acidic pH optimum (pH 5.5) is also secreted at levels 2-3-fold greater than carboxypeptidase N. This enzyme was partially purified from the conditioned medium and compared with pure bovine pituitary carboxypeptidase H. The two enzymes behaved in a similar fashion in DE52 ion-exchange chromatography and on gel filtration, with the Hep G2 enzyme being slightly larger than the bovine pituitary enzyme (52-54 versus 50-52 kDa). Both enzymes hydrolyzed COOH-terminal basic amino acids from typical synthetic substrates as well as from natural leuenkephalin peptides and were identical based on pH activity profiles, inhibition by EDTA or guanidinoethyl mercaptosuccinic acid, and stimulation by Co2+ ions. Inhibition of enzyme secretion from Hep G2 cells by tunicamycin indicated that the Hep G2 enzyme was glycosylated. This finding was confirmed by a parallel deglycosylation of the Hep G2 and bovine pituitary carboxypeptidase H enzymes with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Immunoblots using mouse antiserum to bovine pituitary carboxypeptidase H revealed that the Hep G2 enzyme was immunocross-reactive with the bovine enzyme but was slightly larger in size (54 versus 52 kDa). Continuous [35S]methionine labeling and purification to near homogeneity using an affinity matrix corroborated the observations that the secreted Hep G2 carboxypeptidase H was slightly larger than bovine pituitary carboxypeptidase H. The Hep G2-secreted enzyme in pulse-chase experiments was initially detected intracellularly after a 15-min pulse as a single protein of about 54 kDa and was present in the 30-min chase medium with no evidence for pre- or postsecretion proteolytic processing. The human adrenergic cell line IMR-32 continuously labeled with [35S]methionine also secreted carboxypeptidase H of the same size as the Hep G2 enzyme.     

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)     

Human hepatoma (Hep G2) cultures contain salt-resistant triglyceridase ("liver lipase")

The culture fluid of Hep G2 human hepatoma cells contains triglyceridase activity resistant to high-salt concentrations. The lipase binds to Sepharose-heparin columns from which it can be eluted by 0.8 to 0.9 M NaCl. The nature of this lipase was studied using antibodies raised against "liver" lipases from human and rat origin. The anti-rat liver lipase inhibits both the postheparin human and rat plasma enzyme while the anti-human liver lipase has no effect on the rat enzyme. The lipase of the Hep G2 cultures showed affinity to the antibodies raised against rat as well as human "liver" lipase as shown by inhibition experiments. These results show that Hep G2 cells secrete "liver" lipase and that there seems to exist a structural homology between the lipases from rat and human origin.     

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.     

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.     

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.     

Comparison of DNA damage in human-derived hepatoma line (HepG2) exposed to the fifteen drinking water disinfection byproducts using the single cell gel electrophoresis assay

Disinfection of drinking water reduces pathogenic infection, but generates disinfection by-products (DBPs) in drinking water. In this study, the effect of fifteen DBPs on DNA damage in human-derived hepatoma line (HepG2) was investigated by the single cell gel electrophoresis (SCGE) assay. These fifteen DBPs are: four trihalomethanes (THMs), six haloacetic acides (HAAs), three haloacetonitriles (HANs), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), and chloral hydrate (CH). Based on the minimal effective concentration (MEC) at which DBPs induced significant increase in olive tail moment (OTM), the rank order of DNA-damaging potency is: bromodichloromethane (BDCM)>dibromochloromethane (DBCM)>tribromomethane (TBM)>trichloromethane (TCM) of the four THMs; iodoacetic acid (IA)>bromoacetic acid (BA)>dibromoacetic acid (DBA)>dichloracetic acid (DCA)>trichloroacetic acid (TCA) of the five HAAs; dibromoacetonitrile (DBN)approximately dichloroacetonitrile (DCN)>trichloroacetonitrile (TCN) of the three HANs. The DNA damaging potency of MX and CH is similar to TCA and DCA, respectively. IA is the most genotoxic DBP in the fifteen DBPs, followed by BA. Chloroacetic acid (CA) is not genotoxic in this assay. Our findings indicated that HepG2/SCGE is a sensitive tool to evaluate the genotoxicity of DBPs and iodinated DBPs are more genotoxic than brominated DBPs, but chlorinated DBPs are less genotoxic than brominated DBPs.     

The study of nonspecific internalization of cholesteryl esters by the Hep G2 hepatoma cells

The cholesteryl oleate-POPC dispersions (1:3, mol/mol, mean particle size 110+/-20 nm) were taken up by the human hepatoma line Hep G2 cells via endocytosis. Internalization of the cholesteryl oleate-POPC dispersions by Hep G2 cells was dependent on the incubation time and dispersion concentration. At the cholesteryl oleate concentration 100 microM, its total uptake and internalization were found to be 1.5 nmol and 0.8 nmol per 1 mg of cell protein/24 h, respectively. Intracellular cleavage of the cholesteryl oleate incorporated in dispersions resulted in accumulation of free cholesterol capable of being released into the medium and metabolized to water-soluble polar products, presumably bile acids; oleic acid released is, apparently, involved in biosynthesis of triacylglycerides. The low-density lipoprotein receptor is not involved in internalization of lipid dispersions, and the presence of the cholesteryl oleate-POPC dispersions has no effect on the receptor-dependent internalization of cholesteryl esters of the low-density lipoproteins. The obtained data allow us to consider nonspecific internalization of cholesteryl esters by hepatocytes as a substantial part of the nonpolar lipid clearance.     

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.     

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.     

Long term and large-scale cultivation of human hepatoma Hep G2 cells in hollow fiber bioreactor

Long-term and large scale cultivation of an anchorage-dependent cell line using an industrial scale hollow fiber perfusion bioreactor is described. Hep G2 cells (a human hepatoma cell line) were cultivated in an Acysyst-P^® (Endotronic) with a total fiber surface area of 7.2 m² (6×1.2 m²) to produce Hep G2 crude conditioned medium (CCM). Pretreatment of the cellulose acetate hollow fibers with collagen enhances the attachment of the anchorage-dependent cells. We have succeeded in growing the Hep G2 cells in an antibiotics-and serum-free IMDM medium, supplemented with 50g/ml of Hep G2 CCM protein at inoculation. The Hep G2 cells replicate and secrete CCM protein in quantities comparable to those produced in DMEM containing 10% fetal calf serum (FCS). The highest CCM protein productivity during the 80-day cultivation was 1.1 g/day with a total of 30 g of protein accumulated. Hep G2 CCM (20–40 g protein/ml) was comparable to or even better than 10% FCS in supporting the growth of Molt-4 (a human T leukemia cell line) and FO (a mouse myeloma cell line) cells in vitro. The availability of this large amount of Hep G2 CCM will aid the further purification and characterization of growth factor(s) which could be used as serum substituents.     

The blocking of capacitative calcium entry by 2-aminoethyl diphenylborate (2-APB) and carboxyamidotriazole (CAI) inhibits proliferation in Hep G2 and Huh-7 human hepatoma cells

Calcium entry is a component of the processes regulating the proliferative phenotype of some types of cancer. In non-excitable cells, capacitative calcium entry (CCE) and non-capacitative calcium entry (NCCE) are thought to be the main pathways of Ca2+ influx into cells. Thus, blocking calcium entry may prevent normal and pathological cell proliferation and there is evidence to suggest that molecules blocking calcium entry also have antiproliferative properties. Carboxyamidotriazole (CAI), a novel inhibitor of the non-voltage-dependent calcium entry has been shown to have such properties in model systems in vitro and in vivo. We used Hep G2 and Huh-7 human hepatoma cells to investigate the effects of calcium entry blockers on cell proliferation. CAI (10 microM) and 2-APB (20 microM) completely blocked CCE in thapsigargin-treated Huh-7, and CAI and 2-APB inhibited cell proliferation with IC50 of 4.5 and 43 microM, respectively. The plateau phase of the [Ca2+]i increases triggered by 10% FCS were abolished in the absence of external Ca2+ and in the presence of CAI or 2-APB. We, therefore, suggest that CCE is the main pathway involved in regulation of the processes leading to cell proliferation.     

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.     

Lipid and lipoprotein metabolism in Hep G2 cells

Lipid composition, lipid synthesis and lipoprotein secretion by the Hep G2 cell line have been studied with substrate and insulin supplied under different conditions. The lipid composition of Hep G2 cells was close to that of normal human liver, except for a higher content in sphingomyelin (P less than 0.005) and a lower phosphatidylcholine/sphingomyelin ratio. Most of the [14C]triacylglycerols secreted into the medium were recovered by ultracentrifugation at densities of 1.006 to 1.020 g/ml. The main apolipoproteins secreted were apo B-100 and apo A-I. Hep G2 mRNA synthesized in vitro the pro-apolipoproteins A-I and E. Triacylglycerol secretion was 7.38 +/- 1.04 micrograms/mg cell protein per 20 h with 5.5 mM glucose in the medium and increased linearly with glucose concentration. Oleic acid (1 mM) increased the incorporation of [3H]glycerol into the medium and cell triacylglycerols by 251 and 899%, with a concomitant increment in cell triacylglycerols and cholesterol ester. Insulin (1 mU or 7 pmol/ml) inhibited triacylglycerol secretion and [35S]methionine incorporation into secreted protein by 47 and 28%, respectively, with a corresponding increase in the cells. Preincubation of cells with 2.5-10 mM mevalonolactone decreased the incorporation of [14C]acetate into cholesterol 6.2-fold, indicating an inhibitory effect on HMG-CoA reductase. It is concluded that in spite of some differences between Hep G2 and normal human hepatocytes, this line offers an alternative and reliable model for studies on liver lipid metabolism.     

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.     

Single-cell A3243G mitochondrial DNA mutation load assays for segregation analysis.

Segregation of mitochondrial DNA (mtDNA) is an important underlying pathogenic factor in mtDNA mutation accumulation in mitochondrial diseases and aging, but the molecular mechanisms of mtDNA segregation are elusive. Lack of high-throughput single-cell mutation load assays lies at the root of the paucity of studies in which, at the single-cell level, mitotic mtDNA segregation patterns have been analyzed. Here we describe development of a novel fluorescence-based, non-gel PCR restriction fragment length polymorphism method for single-cell A3243G mtDNA mutation load measurement. Results correlated very well with a quantitative in situ Padlock/rolling circle amplification-based genotyping method. In view of the throughput and accuracy of both methods for single-cell A3243G mtDNA mutation load determination, we conclude that they are well suited for segregation analysis.     

Receptor-mediated endocytosis of tissue-type plasminogen activator by the human hepatoma cell line Hep G2

Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA) was characterized with the human hepatoma cell line Hep G2. At 4 degrees C binding of 125I-t-PA to Hep G2 cells is rapid, specific, saturable, and reflective of a homogeneous population of 76,000 high-affinity surface sites per cell (Kd = 3.7 nM). The kinetics of 125I-t-PA binding to its receptor are characterized by rate constants for association (k1 = 1.2 x 10(6) min-1 M-1) and dissociation (k-1 = 0.001 min-1). A specific glycosylation pattern does not appear to be required for binding. Binding does not appear to be mediated by other recognized hepatic receptor systems. At 37 degrees C a single cohort of bound 125I-t-PA molecules disappears rapidly from the cell surface. Ligand then accumulates intracellularly. Thereafter, the intracellular concentration of ligand declines simultaneously with the release of ligand degradation products into the media. In the continued presence of 125I-t-PA at 37 degrees C the concentration of cell-associated ligand plateaus after 30 min with the concomitant appearance of low molecular weight 125I-labeled fragments in the media. Cumulative degradation then increases linearly with time. Under steady state conditions half-maximal ligand uptake and degradation is 26.6 nM and maximal rate of catabolism is 1.2 pmol/10(6) cells/h. At saturating ligand concentrations uptake and degradation by Hep G2 cells continue linearly for at least 6 h even in the absence of protein synthesis. During this period the cumulative ligand uptake exceeds the total cellular capacity of binding sites, consistent with receptor recycling. We conclude that t-PA clearance in human Hep G2 cells involves ligand binding, uptake, and degradation mediated by a novel high-capacity, high-affinity specific receptor system.