Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human mannan-binding proteins (MBPs) occur in two forms, serum MBP (S-MBP) and liver MBP (L-MBP), both of which are synthesized in the liver from a single form of human MBP mRNA. To investigate further the mechanisms of post-translational modification, molecular assembly and differentiation of S-MBP and L-MBP in vitro, we expressed a full-length human MBP cDNA in three human hepatoma cell lines, using the vaccinia virus expression system. The expression of human MBP cDNA reproduced the native MBP differentiation of S-MBP and L-MBP in human hepatoma cells. The recombinant S-MBP was secreted into the medium, and the recombinant L-MBP retained in the cells. The former had the ability to activate the complement through the classical or lectin pathway but the latter did not. Furthermore, one notable difference between the two MBPs was the degree of oligomerization through interchain disulfide bonds between subunits. In addition, we showed that both S-MBP and L-MBP undergo hydroxylation of lysine and proline residues in collagen-like sequences, and that the hydroxylysine is glycosylated to form glucosylgalactosylhydroxylysine (GluGalHyl) and galactosylhydroxylysine (GalHyl). Hydroxylation was required for S-MBP to be assembled into large complexes, the apparent molecular sizes of which were estimated to be 200-1,300 kDa by SDS-PAGE under non-reducing conditions and gel filtration under non-denaturing conditions. The hydroxylation and subsequent glycosylation and oligomerization were inhibited by alpha,alpha'-dipyridyl, an inhibitor of collagen lysyl and prolyl hydroxylases. These results suggested that newly synthesized lectins undergo post-translational modifications unique to the two forms of MBP, S-MBP, and L-MBP, in human hepatocytes and hepatoma cells, and that the collagen-like domains of the MBPs play an important role in promoting molecular assembly.
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PMID:Functional expression of human mannan-binding proteins (MBPs) in human hepatoma cell lines infected by recombinant vaccinia virus: post-translational modification, molecular assembly, and differentiation of serum and liver MBP. 939 86

Antisense oligonucleotides have been covalently attached to asialoglycoprotein (ASGP) via disulfide bond conjugation chemistry. These conjugates were characterized extensively by an array of chemical, chromatographic, and spectroscopic means. Multiple (approximately six) oligonucleotides can be conjugated to each ASGP molecule. The molecular conjugates were used to deliver antisense oligonucleotides complementary to the mRNA of the interleukin 6 signal transduction protein (gp130) to modulate the acute phase response of hepatoma (HepG2) cells in vitro. These conjugates were biologically active, as measured by inhibition of the cytokine-stimulated up-regulation of the acute phase protein haptoglobin. The level of inhibition was comparable to that found with previous technology featuring noncovalent complexes of ASGP-poly(L-lysine) and oligonucleotide. Because of the ability to control the stoichiometry of the conjugate and its unimolecular nature (as opposed to bimolecular for the noncovalent conjugates), this methodology holds great promise for further development and application.
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PMID:Covalent protein-oligonucleotide conjugates for efficient delivery of antisense molecules. 940 69

The rat hepatoma cell line H4-II-E was found to express much higher activities of Na+-dependent glutamine and aspartate transport than those observed in normal cultured hepatocytes, in agreement with previous work of others on human hepatocytes. Na+-dependent glutamine transport in rat hepatoma cells could be resolved into two components. One was pH-dependent, tolerated Li+ for Na+ substitution and was inhibited only by asparagine and histidine; characteristics similar to those of transport System N in hepatocytes. The other transport system had a similar Km for glutamine but was pH independent, did not accept Li+ ions and was completely inhibited by excess concentrations of lysine, histidine, leucine, serine and cysteine, but not by methyl-aminoisobutyrate or phenylalanine. This pattern of inhibition is distinct from that of any transporter occurring in normal hepatocytes and may indicate the presence of a new transporter isoform. Similar results were obtained with the cell line HTC. Na+-dependent aspartate transport in H4 hepatoma cells was mediated by a high-affinity system (Km 5 microM) and was inhibited by D-aspartate and L-glutamate but not by d-glutamate-properties characteristic of the high-affinity glutamate transporter EAAC1. C-terminal antibodies to the EAAC1 protein recognized a single band of 58 kDa in hepatocyte membranes, but an additional strong band of 60 kDa was present in H4 hepatoma cells. These results provide further evidence for the view that tumour cells may express additional isoforms of amino acid transport systems which are not present in non-transformed cells.
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PMID:Rat hepatoma cells express novel transport systems for glutamine and glutamate in addition to those present in normal rat hepatocytes. 946 18

Galactose was introduced to poly(L-lysine) (PLL) with an average molecular weight of 13,000 to develop a hepatocyte-specific carrier for gene drugs. The pharmacokinetic characteristics of a model plasmid, pCAT (plasmid DNA encoding chloramphenicol acetyltransferase reporter gene), complexed with galactosylated PLL (Gal-PLL) was studied in mice in relation to its physicochemical properties. pCAT/Gal-PLL complex at a ratio of 1:0.6 (w/w) has a zeta potential of -20 mV and a mean particle size of about 180 nm. After intravenous injection, [32P]pCAT/Gal-PLL was rapidly eliminated from the circulation and preferentially taken up by the liver's parenchymal cells. The hepatic uptake of [32P]pCAT/Gal-PLL was significantly inhibited by prior administration of Gal-bovine serum albumin, suggesting that the uptake was mediated by the asialoglycoprotein receptors on hepatocytes. In vitro transfection experiments using a hepatoma cell line expressing the asialoglycoprotein receptor revealed that pCAT/Gal-PLL gave a high CAT gene expression whereas pCAT complexed with unmodified PLL failed to transfect the cells.
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PMID:Targeted delivery of plasmid DNA complexed with galactosylated poly(L-lysine). 974 38

Hereditary tyrosinemia type I (HT1) is an autosomal recessive inborn error of metabolism caused by the deficiency of fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolism pathway. This defect results in accumulation of succinylacetone (SA) that reacts with amino acids and proteins to form stable adducts via Schiff base formation, lysine being the most reactive amino acid. HT1 patients surviving beyond infancy are at considerable risk for the development of hepatocellular carcinoma, and a high level of chromosomal breakage is observed in HT1 cells, suggesting a defect in the processing of DNA. In this paper we show that the overall DNA-ligase activity is low in HT1 cells (about 20% of the normal value) and that Okazaki fragments are rejoined at a reduced rate compared with normal fibroblasts. No mutation was found by sequencing the ligase I cDNA from HT1 cells, and the level of expression of the ligase I mRNA was similar in normal and HT1 fibroblasts, suggesting the presence of a ligase inhibitor. SA was shown to inhibit in vitro the overall DNA-ligase activity present in normal cell extracts. The activity of purified T4 DNA-ligase, whose active site is also a lysine residue, was inhibited by SA in a dose-dependent manner. These results suggest that accumulation of SA reduces the overall ligase activity in HT1 cells and indicate that metabolism errors may play a role in regulating enzymatic activities involved in DNA replication and repair.
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PMID:Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I. 977 May 34

To investigate the delivery of DNA into cells, lactose-poly(ethylene glycol)-grafted poly-L-lysine (Lac-PEG-PLL) polymers were synthesized as polymeric gene carriers. The new synthetic carriers, varying the substitution ratio of lactose-poly(ethylene glycol) (lactose-PEG), were characterized by NMR spectroscopy and size-exclusion chromatography. Electrophoretic mobility assay confirmed that the new gene carrier makes a complex with plasmid DNA. The attached poly(ethylene glycol) gives better solubility properties to gene/carrier complex. Transfection experiments showed that Lac-PEG-PLL efficiently delivers DNA to a hepatoma cell line in vitro; the best efficiency was achieved at a 1:3 weight ratio of DNA to carrier. As the lactose-PEG substitution content increased up to 30%, the transfection efficiency increased, which demonstrates that the lactose serves as a targeting moiety. No considerable cytotoxicity was observed due to Lac-PEG-PLL or its complex with DNA within the concentration range for this experiment. The use of chloroquine increased transfection efficiency that indicates the involvement of hydrolytic degradation of the system in lysosome. It is likely that plasmid DNA/Lac-PEG-PLL complexes enter the cells through a receptor-mediated endocytosis mechanism. These results show that Lac-PEG-PLL can form a complex with plasmid DNA and serve as an efficient gene delivery carrier with lower cytotoxicity compared to that of poly-L-lysine. Therefore, it is expected that our Lac-PEG-PLL carrier can be used as an in vivo gene delivery vector.
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PMID:Lactose-poly(ethylene glycol)-grafted poly-L-lysine as hepatoma cell-tapgeted gene carrier. 981 64

Hepatic arginine and lysine uptake is partly regulated by changes in the transport activity of a group of cell surface proteins exhibiting properties of the transport system y+. The Cat-1 gene encodes a sodium-independent high-affinity cationic amino acid transporter of the y+ system which is nearly undetectable in the quiescent liver. In this paper we investigate the regulation of expression of Cat-1 in the quiescent rat liver by glucocorticoids and insulin, two hormones which play a critical role in amino acid dependent pathways of hepatic metabolism. Injection of insulin and glucocorticoids resulted in a rapid (15-30 min, 4-5 fold) increase in transcription which returned to basal levels within 4 hours. In contrast to the rapid single peak of transcriptional induction of the Cat-1 gene, the accumulation of the Cat-1 mRNAs occurred transiently with two peaks, the first at 30 minutes and the second at 2-4 hours following hormone treatment. These data indicate that expression of the Cat-1 gene in the quiescent liver can be transiently induced by both transcriptional and post-transcriptional mechanisms. In FTO2B rat hepatoma cells, expression of the gene is constitutive and accumulation of Cat-1 mRNAs in response to dexamethasone and insulin was dependent on transcription and protein synthesis. Furthermore, the accumulation of the basal level of the Cat-1 mRNAs was reduced by 70%, upon treatment of cells with inhibitors of protein synthesis for 6 h, when the transcription rate of the gene did not decrease significantly. We conclude the following: (i) under normal physiologic conditions, expression of the Cat-1 gene in the quiescent liver is negligible, probably to prevent unnecessary transport and metabolism of arginine by the hepatic arginase in the hepatocytes. (ii) in the cases when hepatic cationic amino acid transport is needed, such as following feeding, cellular growth and illness, glucocorticoids and insulin induce expression of the Cat-1 gene in liver cells through induction of transcription and stabilization of the mRNA. (iii) constitutive Cat-1 mRNA accumulation in rat hepatoma cells depends on protein synthesis through a labile regulated factor. Overall, constitutive expression of Cat-1 is associated with hepatic cellular growth and transformation.
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PMID:Control of expression of the gene for the arginine transporter Cat-1 in rat liver cells by glucocorticoids and insulin. 989 57

We have targeted the serpin enzyme complex receptor for gene transfer in human hepatoma cell lines using peptides < 30 amino acids in length which contain the five amino acid recognition sequence for this receptor, coupled to poly K of average chain length 100 K, using the heterobifunctional coupling reagent sulfo-LC SPDP. The number of sulfo-LC SPDP modified poly-L-lysine residues, as well as the degree of peptide substitution was assessed by nuclear magnetic resonance spectroscopy. Conjugates were prepared in which 3.5%, 7.8% or 26% of the lysine residues contained the sulfo-LC SPDP moiety. Each of these conjugates was then coupled with ligand peptides so that one in 370, one in 1039, or one in 5882 lysines were substituted with receptor ligand. Electron microscopy and atomic force microscopy were used to assess complex structure and size. HuH7 human hepatoma cells were transfected with complexes of these conjugates with the plasmid pGL3 and luciferase expression measured 2 to 16 days after treatment. All the protein conjugates in which 26% of the K residues were modified with sulfo-LC SPDP were poor gene transfer reagents. Complexes containing less substituted poly K, averaged 17 +/- 0.5 nm in diameter and gave peak transgene expression of 3-4 x 10(6) ILU/mg which persisted (> 7 x 10(5) ILU) at 16 days. Of these, more substituted polymers condensed DNA into complexes averaging 20 +/- 0.7 nm in diameter and gave five-fold less luciferase than complexes containing less substituted conjugates. As few as eight to 11 ligands per complex are optimal for DNA delivery via the SEC receptor. The extent of substitution of receptor-mediated gene transfer complexes affects the size of the complexes, as well as the intensity and duration of transgene expression. These observations may permit tailoring of complex construction for the usage required.
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PMID:Ligand substitution of receptor targeted DNA complexes affects gene transfer into hepatoma cells. 1002 48

Recently, lamivudine used to treat patients with hepatitis B virus (HBV) infection was revealed to have potent antiviral activity. However, HBV resistance to lamivudine has been reported and shown to have amino acid substitutions in the methionine residue of the conserved tyrosine (Y), methionine (M), aspartate (D), aspartate (D) motif of RNA-dependent DNA polymerase. To explore the consequences of substitutions in this motif (YMDD), we made 7 variants by substituting the methionine of the YMDD motif with isoleucine (I), valine (V), alanine (A), leucine (L), lysine (K), arginine (R), and threonine (T). Replication ability of these variants was evaluated by transfection into human hepatoma cells. Sensitivity to lamivudine was tested for replication-competent variants. Four variants with hydrophobic substitutions (I, V, A, and L) remained replication-competent, whereas 3 others with hydrophilic substitutions (K, R, and T) exhibited impaired replication. Of the 4 replication-competent variants, 2 (I and V) were resistant, and 2 (A and L) were sensitive to lamivudine. Because the polymerase and the surface gene overlap, the introduction of these mutations affected the secretion of hepatitis B surface antigen (HBsAg), namely 4 variants (I, V, L, and R) secreted HBsAg, whereas 3 variants (A, K, and T) did not. Our study elucidated that only one amino acid substitution in the YMDD motif was sufficient to cause lamivudine resistance in vitro. As a result of replication competence and lamivudine sensitivity, only viruses having YIDD or YVDD sequences may appear during treatment with lamivudine. This in vitro system could be used to study HBV mutations, replication competence, and their susceptibility to antivirals.
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PMID:YMDD motif in hepatitis B virus DNA polymerase influences on replication and lamivudine resistance: A study by in vitro full-length viral DNA transfection. 1005 1

The expression of asialoglycoprotein receptor (ASGP-R) on human hepatocarcinoma cells might be exploited to reduce the extrahepatic toxicity of DNA synthesis inhibitors by their conjugation with galactosyl- terminating peptides. We conjugated 2',2'-difluorodeoxycytidine (dFdC), an inhibitor of DNA synthesis active on solid tumors, with lactosaminated poly-L-lysine (L-poly(LYS)). In experiments in vitro, L-poly(LYS)-dFdC inhibited proliferation of Hep G2 cells, a human hepatocarcinoma cell line which maintains the ASGP-R. Inhibition was rescued by asialofetuin. To study the pharmacological action of the conjugate in vivo, we used rats 18-24 hr after 2/3 hepatectomy and observed that regenerating hepatocytes expressed ASGP-R on their surface and internalized L-poly(LYS)-dFdC. Conjugate uptake by bone marrow, spleen, and intestine was negligible. We also found that L-poly(LYS)-dFdC inhibited [3H]thymidine incorporation into DNA of regenerating liver. These results indicated that hepatectomized rats were a suitable animal model to study the pharmacological action, on DNA-synthesizing hepatocytes, of conjugates binding to ASGP-R and carrying inhibitors of DNA synthesis. L-poly(LYS)-dFdC also inhibited [3H]thymidine incorporation in bone marrow, spleen, and intestine. Evidence was obtained that inhibition of DNA synthesis in extrahepatic tissues was a consequence of drug release from hepatocytes into blood-stream after the bond with the carrier has been broken down within liver cells. Possible ways of reducing the exit of dFdC from liver cells, thereby obtaining an inhibition of DNA synthesis restricted to dividing hepatocytes, were discussed.
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PMID:Inhibition of [3H]thymidine incorporation into DNA of rat regenerating liver by 2',2'-difluorodeoxycytidine coupled to lactosaminated poly-L-lysine. 1007 85


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