Complete down-regulation of low-density-lipoprotein-receptor activity in the human hepatoma cell line Hep G2 by beta-migrating very-low-density lipoprotein and non-lipoprotein cholesterol. Different cellular regulatory pools of cholesterol.

Regulation of low-density-lipoprotein-receptor activity by low-density lipoprotein (LDL), cholesteryl-ester-rich beta-migrating very-low-density lipoprotein (beta-VLDL) and non-lipoprotein cholesterol was investigated in the human hepatoma cell line Hep G2. Competition studies indicate that LDL and beta-VLDL are bound to the same recognition site, tentatively the LDL receptor. The regulatory response of the LDL receptor upon prolonged incubation with LDL or beta-VLDL was, however, markedly different. 22 h preincubation of Hep G2 cells with excess LDL caused a partial down regulation to 31% of the initial level of the high-affinity association of LDL and 26% of the high-affinity degradation of LDL, while with beta-VLDL a complete down regulation of the LDL-receptor activity is observed. Preincubation of Hep G2 cells with beta-VLDL for 22 h led to a fourfold increase in intracellular cholesterol esters and a twofold increase in acyl-coA:cholesterol acyltransferase activity. With LDL, the amount of intracellular cholesterol esters is increased 1.6-fold. The more effective down regulation of LDL receptors by beta-VLDL as compared to LDL can be explained by the more effective intracellular cholesterol delivery with beta-VLDL than with LDL. Preincubation of Hep G2 cells for 22 h with acetylated LDL hardly influenced the LDL-receptor activity. Non-lipoprotein cholesterol, however, caused a complete down regulation of LDL-receptor activity at even lower extracellular cholesterol concentrations than with beta-VLDL. The complete down regulation of LDL receptors by non-lipoprotein cholesterol is not accompanied by a significant increase in acyl-coA:cholesterol acyltransferase activity, while the intracellular cholesterol ester concentration is only increased 1.6-fold. It is suggested that the effectiveness of non-lipoprotein cholesterol to regulate LDL receptors is caused by its efficiency to reach the sterol regulatory site. The inability of LDL to down regulate its receptor completely can thus be explained by the inability of LDL to deliver cholesterol adequately at the intracellular regulatory site of the LDL receptor. The observed complete down regulation of the LDL receptor by beta-VLDL may be responsible for the cholesterol-rich-diet induced, complete down regulation of LDL-receptor-mediated clearance of LDL in vivo.     

Estrogens induce low-density lipoprotein receptor activity and decrease intracellular cholesterol in human hepatoma cell line Hep G2

Administration of estrogens in pharmacologic doses to rats and rabbits induces hepatic low-density lipoprotein (LDL) receptor activity. To determine if estrogens can regulate LDL receptor activity in human cells, 125I-LDL binding and ligand blotting studies were performed with the cell line Hep G2, well-differentiated cells derived from a human hepatoma, and with normal human fibroblasts. Addition of estradiol to Hep G2 cells growing in lipoprotein-deficient medium increased cell surface receptor activity by 141%, whereas fibroblast receptors were slightly reduced. Measurement of LDL internalization and degradation showed that estradiol induced the entire LDL receptor pathway and not simply surface receptors for LDL. Scatchard analysis of specific binding data in Hep G2 cells revealed that increased LDL receptor activity was due to high-affinity binding. When Hep G2 cells were incubated with LDL as well as estradiol, estradiol induction of LDL receptor activity did not occur. Estrogen treatment reduced Hep G2 free cholesterol content by 24% as determined by gas-liquid chromatography but had no significant effect on fibroblast free cholesterol, suggesting that estrogens may induce Hep G2 LDL receptor activity indirectly by lowering intracellular cholesterol. LDL receptor activity in Hep G2 cells grown in the absence of estradiol was resistant to down-regulation by LDL; incubation of cells with LDL for 48 h reduced receptor activity by only 25.8% in Hep G2 cells compared to 80.3% in fibroblasts. The Hep G2 LDL receptor was shown to be biochemically similar to the fibroblast receptor by ligand blotting and immunoblotting with IgG-C7, a monoclonal antibody to the extrahepatic LDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Verapamil enhances high-density lipoprotein processing in Hep G2 cells preloaded with cholesterol

The effects of the calcium channel blocker of the arylalkylamine series verapamil have been investigated on high-density lipoprotein (HDL3) catabolism in the human hepatoma cell line Hep G2. It was found that verapamil markedly enhanced HDL3 binding, uptake and degradation in Hep G2 cells preloaded with nonlipoprotein cholesterol. This effect was dose-dependent, and a 1.5-2-fold increase of the three studied parameters was observed in cells pretreated 24 h with 100 microM verapamil. No significant effect of the drug was found in cells not preincubated with cholesterol. Verapamil induced an increase in the cellular cholesterol content in preloaded cells. Other calcium antagonists such as diltiazem, nifedipine, nitrendipine or amphiphilic drugs such as phenothiazines and propranolol also enhanced HDL3 uptake by Hep G2 cells. These effects of verapamil on HDL3 metabolism could be related to its amphiphilic characteristics, and to its calcium antagonist properties.     

Effect of ethanol on cell growth and cholesterol metabolism in cultured Hep G2 cells.

The Hep G2 human hepatoma cell line has been recognized as an excellent in vitro human model system. For this reason, this line was used to study the effect of ethanol on HMG-CoA reductase activity concerning cell growth and cholesterol metabolism. Cells were incubated in ethanol-containing medium (0-400 mmol/L) for up to 102 h. Ethanol caused an inhibition in the growth rate and in HMG-CoA reductase activity that could be reverted by the removal of ethanol from the culture medium, indicating no cellular damage. These changes cannot be ascribed to the regulatory effect of cholesterol levels, since its content was not modified either in the cells or in the medium. The addition of mevalonate to the culture medium could not revert the growth rate inhibition evoked by ethanol. Moreover, ethanol produced an increment in the cholesterol efflux in [3H]cholesterol-prelabeled cells. We conclude that the decrease in HMG-CoA reductase activity evoked by ethanol treatment on Hep G2 cells would not be the cause but the consequence of the impairment in cellular growth, since this impairment could not be reverted by the addition of mevalonate to the culture medium.     

Effect of 25-hydroxycholesterol and bile acids on the regulation of cholesterol metabolism in Hep G2 cells.

The effect of 25-hydroxycholesterol (25-OH-cholesterol) and chenodeoxycholic (CDC) acid on apoprotein secretion, low-density lipoprotein receptor activity, and [3H]triacylglycerol secretion in Hep G2 cells was studied. Both 25-OH-cholesterol and CDC acid increased the secretion of apolipoprotein (apo) E by Hep G2 cells. The secretion of apo A-I was slightly lowered (less than 10% disease). The maximal increase in apo E secretion was observed in culture medium containing 2 micrograms of 25-OH-cholesterol/ml or 10 micrograms of CDC acid/ml plus 10% fetal calf serum. Cholesterol, 7-OH-cholesterol and other bile acids were ineffective in inducing increases in apo E secretion. Another cholesterol synthesis inhibitor, mevinolin, was also ineffective in generating an increase in apoprotein secretion. The data indicated a specific interaction between 25-OH-cholesterol or CDC acid and apo E secretion in Hep G2 cells. Cholesterol synthesis, as measured by the incorporation of [14C]acetic acid into sterols, was repressed in Hep G2 cells in the presence of 25-OH-cholesterol (17% of control value). CDC acid, on the other hand, increased [14C]acetic acid incorporation (156% of control value). The number of LDL receptors in Hep G2 cells was decreased after incubation with 25-OH-cholesterol (62% of control value), but increased significantly after incubation with CDC acid (149% of control value). The secretion of [3H]triacylglycerol by Hep G2 cells incubated with 25-OH-cholesterol was greatly increased (248% of control value). On the contrary, CDC acid did not cause any increase in [3H]triacylglycerol secretion. The above results suggest that 25-OH-cholesterol and CDC acid have different effects on lipid metabolism in Hep G2 cells. The mRNA levels of apo E increased in cells preincubated with 25-OH-cholesterol and CDC acid, which suggested that the increase in apo E secretion is at least partly due to an increase in synthesis.     

The effect of (-)-hydroxycitrate on the activity of the low-density-lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reductase levels in the human hepatoma cell line Hep G2.

(-)-Hydroxycitrate, a potent inhibitor of ATP citrate-lyase, was tested in Hep G2 cells for effects on cholesterol homoeostasis. After 2.5 h and 18 h incubations with (-)-hydroxycitrate at concentrations of 0.5 mM or higher, incorporation of [1,5-14C]citrate into fatty acids and cholesterol was strongly inhibited. This most likely reflects an effective inhibition of ATP citrate-lyase. Cholesterol biosynthesis was decreased to 27% of the control value as measured by incorporations from 3H2O, indicating a decreased flux of carbon units through the cholesterol-synthetic pathway. After 18 h preincubation with 2 mM-(-)-hydroxycitrate, the cellular low-density-lipoprotein (LDL) receptor activity was increased by 50%, as determined by the receptor-mediated association and degradation. Measurements of receptor-mediated binding versus LDL concentration suggests that this increase was due to an increase in the numbers of LDL receptors. Simultaneously, enzyme levels of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase as determined by activity measurements increased 30-fold. Our results suggest that the increases in HMG-CoA reductase and the LDL receptor are initiated by the decreased flux of carbon units in the cholesterol-synthetic pathway, owing to inhibition of ATP citratelyase. A similar induction of HMG-CoA reductase and LDL receptor was also found after preincubations of cells with 0.3 microM-mevinolin, suggesting that the underlying mechanism for this induction is identical for both drugs.     

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.     

Hep G2 cells as a resource for metabolic studies: lipoprotein, cholesterol, and bile acids

Hep G2, a liver cell line derived from a human hepatoblastoma that is free of known hepatotropic viral agents, has been found to express a wide variety of liver-specific metabolic functions. Among these functions are those related to cholesterol and triglyceride metabolism. Confluent Hep G2 monolayers express normal low-density lipoprotein (LDL) receptors and continue to internalize and metabolize chylomicrons, very low-density lipoproteins (VLDL), LDL, and high-density lipoproteins. In lipoprotein-free medium, apolipoproteins A-I, A-II, B, C, and E accumulate in the medium together with cholesterol, cholesteryl ester, triglyceride, and all the primary bile acids. The regulation of their synthesis and secretion is not fully known and their interrelationships have not been established. Because Hep G2 cells express these and other components of cholesterol and triglyceride metabolism, they are a microcosm for studying the central role of the liver.     

Regulation of LDL receptor, apoB, and apoE protein and mRNA in Hep G2 cells.

The regulation of low-density lipoprotein (LDL) receptor activity, protein synthesis, and cellular mRNA content was evaluated in the human hepatoma cell line Hep G2. Incubation of the cells with LDL led to a complete downregulation of LDL receptor mRNA and LDL receptor protein synthesis. This LDL regulation of the LDL receptor and its mRNA was both time- and concentration-dependent. In contrast to protein synthesis and cellular mRNA concentrations of the LDL receptor, which were reduced to undetectable levels by prolonged incubation in the presence of LDL, LDL receptor activity was reduced to only 44% of preincubation levels. These findings support the presence of a second metabolic pathway for LDL uptake in human hepatocytic cells. The effect of LDL on cellular LDL receptor expression was specific for LDL because incubation in the presence of HDL did not affect any of these study end points. The potential coordinate regulation of the expression of the LDL receptor with its principal ligands, apolipoproteins (apo) B and E, was also investigated. In contrast to the LDL receptor mRNA downregulation with LDL incubation, cellular apoB and apoE mRNA concentrations were not affected by either LDL or HDL. Secretion of apoB, however, was significantly increased by incubating Hep G2 cells with LDL. These findings indicate that, in contrast to LDL receptor which is regulated at the mRNA level, the ligands for the LDL receptor are regulated either co- or post-translationally.     

Inactivation of PTEN is responsible for the survival of Hep G2 cells in response to etoposide-induced damage

The chemo-resistance character of human hepatocellular carcinoma cells is well known but the anomalies associated with such resistance character are not completely understood. In this study, etoposide-induced signaling events in human hepatocellular carcinoma cell line, Hep G2 has been compared with Chang Liver cells, a normal human liver cell line. Hep G2 cells are resistant to etoposide when compared with Chang Liver cells. Etoposide-induced γH2AX foci in Hep G2 cells are persisted for a longer time without affecting cell cycle, indicating that Hep G2 cells are able to maintain its growth with damaged DNA. Further, Akt signaling pathway is deregulated in Hep G2 cells. The upstream negative regulator of Akt, PTEN remains inactive, as it is hyperphosphorylated in Hep G2 cells. Inhibition of PI-3K pathway by wortmannin partially reverses the etoposide-resistance character of Hep G2 cells. Either Hep G2 or Chang Liver cells when transfected with plasmid carrying active Akt (myr-Akt) become resistance towards etoposide compared to the cells transfected with empty vectors or kinase defective Akt. Transient transfection of wild type PTEN in Hep G2 cells does not change its response towards etoposide whereas Chang Liver cells become sensitive after transfection with same plasmid. These results suggest that inactivation of PTEN, which renders activation of Akt, may contribute largely for the etoposide-resistance character of Hep G2 cells.     

Resveratrol- induced apoptosis is mediated by p53-dependent pathway in Hep G2 cells

Resveratrol, a phytoalexin found in many plants, has been reported to possess a wide range of pharmacological properties and is one of the promising chemopreventive agents for cancer. Here, we examined the antiproliferation effect of resveratrol in two human liver cancer cell lines, Hep G2 and Hep 3B. Our results showed that resveratrol inhibited cell growth in p53-positive Hep G2 cells only. This anticancer effect was a result of cellular apoptotic death induced by resveratrol via the p53-dependent pathway. Here we demonstrated that the resveratrol-treated cells were arrested in G1 phase and were associated with the increase of p21 expression. In addition, we also illustrated that the resveratrol-treated cells had enhanced Bax expression but they were not involved in Fas/APO-1 apoptotic signal pathway. In contrast, the p53-negative Hep 3B cells treated with resveratrol did not show the antiproliferation effect neither did they show significant changes in p21 nor Fas/APO-1 levels. In summary, our study demonstrated that the resveratrol effectively inhibited cell growth and induced programmed cell death in Hepatoma cells on a molecular basis. Furthermore, these results implied that resveratrol might also be a new potent chemopreventive drug candidate for liver cancer as it played an important role to trigger p53-mediated molecules involved in the mechanism of p53-dependent apoptotic signal pathway.     

Apolipoprotein A-I gene expression is regulated by cellular zinc status in Hep G2 cells

The influence of Zn on the expression of the apolipoprotein A-I(apoA-I) gene in Hep G2 cells was examined. Zn depletion was achievedwith a low-Zn (ZD) medium prepared from Zn-free growth medium(Opti), a ZD medium containing Chelex 100-extracted fetal bovine serum (CHE), and a medium containing chelator1,10-phenanthroline (OP). Compared with those for their respectivecontrols, cellular Zn levels were reduced by 55, 48, and 46% andapoA-I mRNA abundances were reduced by 20, 29, and 28% in Opti, CHE,and OP systems, respectively, after one passage in ZD media or 24 h inOP medium. To establish the specificity of Zn treatment, groups of ZDcells were treated with their respective control media for the last 24 h (ZDA) or normal cells were cultured with OP medium supplemented withZn (OP-Zn). ZDA treatments partially normalized cellular Zn levels inthe Opti system and restored or elevated apoA-I mRNA levels in the Optior CHE system, respectively. Similarly, the OP-Zn treatment restoredthe cellular Zn and apoA-I mRNA levels. Furthermore, one passage ofculture with Zn-supplemented media in both the Opti and CHE systemsresulted in higher cellular Zn and apoA-I mRNA levels than those forcontrols. Most significantly, short-term high-Zn induction to normalcells markedly elevated the cellular Zn (3-fold) and apoA-I mRNA(5-fold) levels. Data derived from this study strongly suggest that theexpression of apoA-I is regulated by cellular Zn status.

     

PDEA-coated magnetic nanoparticles for gene delivery to Hep G2 cells

Poly(2-(diethylamino)ethyl methacrylate) coated magnetic nanoparticles (PDEA-MNPs) were synthesized as a new gene nanocarrier to delivery plasmids (pEGFPN1 and pRL-TK) into human hepatoma (Hep G2) cells. The PDEA-MNPs shows the pH-sensitive property. These nanoparticles are positively charged at acidic pH and negatively charged at neutral or alkaline pH. The PDEAMNPs exhibited a low cytotoxicity in Hep G2 cells. PDEA-MNPs could bind and protect DNA from DNase I degradation. The transfection study demonstrated that the PDEA-MNPs could carry plasmid into Hep G2 cells and exhibited a high gene transfection efficiency. These results indicated that the novel magnetic nanoparticles could enhance gene transfection in vitro and hold the potential to be a promising non-viral nanodevice.     

The protein-tyrosine kinase activity of the insulin receptor is necessary for insulin-mediated receptor down-regulation

We have previously demonstrated that the human insulin receptor, mutated in the ATP-binding domain of the beta-subunit, is kinase-defective and fails to mediate multiple post-receptor actions of insulin in stably transfected Chinese hamster ovary cells (Chou, C.-K., Dull, T. J., Russell, D. S., Gherzi, R., Lebwohl, D., Ullrich, A., and Rosen, O. M. (1987) J. Biol. Chem. 262, 1842-1847). This study addresses the role of protein-tyrosine kinase activity in insulin-mediated receptor down-regulation. Although the mutant insulin proreceptor was properly processed and able to bind insulin like the wild-type human receptor, it differed from the latter in the following respects: 1) it failed to mediate internalization of surface-bound radiolabeled ligand; 2) it did not undergo short- or long-term down-regulation in response to 1 microM insulin; 3) it did not exhibit ligand-promoted receptor turnover; and 4) it was not phosphorylated on either tyrosine or serine residues in response to insulin. Although the cells transfected with the mutant receptor failed to respond to insulin-mediated insulin receptor down-regulation, they were able to down-regulate their insulin-like growth factor I receptors in response to insulin-like growth factor I or high concentrations of insulin and were sensitive to monoclonal antibody-induced down-regulation of their insulin receptors. Antibody-mediated receptor internalization alone, however, was unable to mimic at least one action of insulin, thymidine incorporation into DNA, and did not lead to any phosphorylation of the receptor. It is concluded that either the protein-tyrosine kinase activity of the insulin receptor or its phosphorylation state is essential for ligand-mediated receptor down-regulation.     

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)     

Fatty acid uptake and metabolism in Hep G2 human-hepatoma cells

The aim of this work was to study the fatty acid metabolism of the human-hepatoma cell line Hep G2. The cultured cells were incubated with either a saturated (palmitic, stearic) or a polyunsaturated (linoleic, -linolenic, eicosatrienoic n-6) radioactive fatty acid. The fatty acids were incorporated into all the basic lipid classes as well as into the main phospholipid subclasses in the cellular membranes. All the fatty acids tested provided a source of carbon for lower members of the saturated fatty-acid family or for cholesterol through -oxidation and a new cycle ofde novo synthesis. Moreover, all radioactive fatty-acid precursors, whether saturated or unsaturated, were anabolized to higher derivatives within their own family. In the case of saturated fatty acids, palmitic and stearic, they were readily monodesaturated to their corresponding products, thus demonstrating the presence of a 9 desaturase. Linoleate and -linolenate were both desaturated and elongated to all the subsequent members of their respective n-6 and n-3 families. These latter observations provide evidence for the incidence of desaturation at the 6 and 5 positions along with the existence of an elongating capacity for fatty acids of all families and chain lengths. In addition, the cellular steady-state fatty-acid profile was seen to be significantly different from the spectrum of exogenous fatty acids available in the growth medium. We conclude that the Hep G2 human-hepatoma line represents an appropriate and relevant experimental model system for investigating the fatty-acid metabolism of adult human liverin vivo.     

Tetrandrine-induced cell cycle arrest and apoptosis in Hep G2 cells

The effects of tetrandrine in the human hepatoblastoma G2 (Hep G2) cell line were investigated in this study. The results showed that tetrandrine not only inhibited Hep G2 growth but also induced apoptosis and blocked cell cycle progression in the G1 phase. ELISA assay demonstrated that tetrandrine significantly increased the expression of p53 and p21/WAF1 protein, which caused cell cycle arrest. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by tetrandrine. Taken together, p53 and Fas/FasL apoptotic system possibly participated in the antiproliferative activity of tetrandrine in Hep G2 cells.     

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.