BNL CL Research Articles

In vitro gene delivery to hepatocytes with galactosylated polyethylenimine.

A hepatocyte-directed vector has been developed; it includes several key features thought to favor in vivo gene delivery to the liver: electrostatically neutral particles which avoid nonspecific binding to other cells, to the extracellular matrix, and to complement proteins; asialoglycoprotein receptor-mediated endocytosis which may address the complexes to the perinuclear region; and polyethylenimine (PEI)-mediated endosome buffering and swelling as an escape mechanism to the cytoplasm. This system is based on a 5% galactose-bearing polyethylenimine (PEI-gal) polymer which is condensed with plasmid DNA to neutrality. Murine (BNL CL.2) and human (HepG2) hepatocyte-derived cell lines were transfected 10(4)-10(5)-fold more efficiently than murine fibroblasts (3T3), whether transfection was assessed globally (luciferase expression from the cell extract) or following histochemical staining (beta-galactosidase). Under these conditions, over 50% of the hepatocytes were selectively transfected in the presence of 10% serum. Transfection was suppressed by removal of the targeting galactose residues, by their replacement with glucose, or by the addition of excess asialofetuin. Thus, results from comparative and competitive experiments indicate the asialoglycoprotein receptor is involved in transfection of hepatocytes with neutral PEI-gal/DNA complexes.

In vivo distribution and gene expression of genetically modified hepatocytes after intrasplenic transplantation

To investigate the feasibility and efficacy of liver gene therapy mediated by intrasplenic transplantation of genetically modified hepatocytes, the normal mouse liver cell line BNL CL. 2 cells were introduced with Neo-resistant (NeoR) gene or interleukin-2 (IL-2) genein vitro, and transplanted intrasplenically into normal syngeneic mice (2 x 10(6) cell/mouse); subsequently, the expressions of the introduced genesin vim were detected. The RT-PCR results showed that NeoR mRNA expressions were detectable in livers 24 h after transplantation and lasted over 11 weeks. Moreover, The NeoR mRNA was detected to be expressed temporarily in spleens (24 h-1 week) and lungs (24-96 h) after transplantation. After intrasplenic transplantation of IL-2 gene-modified BNL CL.2 cells, the stable expressions of IL-2 mRNA in the livers of transplanted mice were detectable by RT-PCR (24 h-11 weeks), and certain levels of IL-2 (5-40 pg/mL) remained in the peripheral blood. When IL-2 gene-modified BNL CL. 2 cells were transplanted intrasplenically to treat the metastatic liver colon carcinoma-bearing mice, the survival time of the treated mice was significantly prolonged. The data indicate that intrasplenic transplantation of genetically modified hepatocytes could allow for oriental distribution in host livers and long-term survival of the transplanted liver cells, and effective expression of exogenous genesin vim, suggesting that this can be a candidate approach to liver-directed gene therapy.

The role of Src kinase in the potentiation by ethanol of cytokine- and endotoxin-mediated nitric oxide synthase expression in rat hepatocytes.

This study demonstrates that exposure of primary rat hepatocytes or mouse BNL Cl.2 liver cell line to ethanol causes potentiation of tumor necrosis factor-alpha (TNF-alpha)- and lipopolysaccharide (LPS)-stimulated nitrite accumulation. The potentiating effect of ethanol (0.02-2 mM) appears to be time and concentration dependent. Consistent with nitrite production, the amount of inducible nitric oxide synthase (iNOS) mRNA and protein is initially detected at 4 hr after treatment with TNF-alpha/LPS/ethanol. Furthermore, the capability of these agents to induce iNOS expression is primarily determined by the age of the animals. Interestingly, antioxidants such as N-acetylcysteine (NAC), ascorbic acid, or alpha-tocopherol fail to inhibit TNF-alpha/LPS/ethanol-induced increase in iNOS protein. In addition, several kinase inhibitors, including staurosporine, genistein, curcumin, and herbimycin A, were used to examine their effects on this induction. Among them, only herbimycin A potently inhibits the accumulation of nitrite and iNOS expression. In vitro kinase assay verifies that Src tyrosine kinase is rapidly activated with a peak at 1 hr after treatment with TNF-alpha/LPS/ethanol but is not activated by these agents singly or doubly. As expected, herbimycin A can block Src kinase activity under circumstances in which iNOS expression is also inhibited. However, our results do not indicate that the mitogen-activated protein kinase is activated after treatment with these agents. The study results suggest that Src tyrosine kinase plays a prominent role in transducing the signal to induce iNOS expression in hepatocytes treated with TNF-alpha/LPS/ethanol.

Roles of tyrosine kinases in the regulation of nitric oxide synthesis in murine liver cells: modulation of NF-kappa B activity by tyrosine kinases.

Nitric oxide (NO) synthesis is upregulated during chronic hepatic inflammation. The present study characterized the mechanisms involved in the induction of NO production and inducible NO synthase (iNOS) messenger RNA (mRNA) expression in murine embryonic liver cell line, BNL CL.2 cells. No production by BNL CL.2 cells was induced by interferon-r (IFN-r) plus lipopolysaccharide (LPS). However, other inflammatory cytokines such as interleukin (IL)-beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 had no additional effects on it. The stimulatory effects of IFN-r and LPS were time- and dose-dependent. NO secretion was inhibited by treatment with inducible NOS inhibitors such as NG-monomethyl L-arginine, NG-amino-L-arginine, and diphenylene iodonium. iNOS mRNA was induced 3 hours after IFN-r plus LPS treatment, and iNOS expression was maximal in the presence of IFN-r and LPS. The protein tyrosine kinase inhibitors such as genistein and tyrphostin reduced IFN-r plus LPS-induced iNOS mRNA expression and NO production. In contrast, the inhibitors of protein kinase C, protein kinase A, and protein phosphatases did not affect iNOS expression induced by IFN-r plus LPS. In addition, iNOS mRNA expression was completely blocked by treatment with tyrphostin. However, mRNA expression of an early response gene, JunB, and constitutively expressed genes beta-actin and GAPDH were not inhibited by tyrphostin. Furthermore, tyrphostin inhibited the promoter activation of iNOS gene induced by IFN-gamma plus LPS, and it also suppressed IFN-gamma plus LPS-induced nuclear factor-kappa B-binding activity but not AP-1-binding activity. These results suggest that NO production and iNOS mRNA expression in this cell line is dependent on protein tyrosine kinases but does not require protein kinase C, protein kinase A, or protein phosphatases.

Glycerol enhancement of ligand-polylysine/DNA transfection.

Primary human fibroblasts and a series of cell lines (A549, BNL CL.2, H225, NIH 3T3 and Rat-1) are efficiently transfected by using positively charged complexes of plasmid DNA and transferrin-polylysine or polylysine in the presence of glycerol (1 molar to 1.8 molar, depending on the cell type). An increase in gene expression of up to several-hundredfold (compared to complexes without glycerol) is obtained if the transfection mixture is incubated with the cells for 3-4 h at 37 degrees C. This simple method has been used for transient expression of luciferase, beta-galactosidase and interleukin-2, and also for the generation of stably transfected cells.

Selective tumorigenesis in non-parenchymal liver epithelial cell lines by hepatocyte growth factor transfection.

Hepatocyte growth factor (HGF) has been recently suggested to contribute to tumorigenesis by an autocrine mechanism in fibroblast cells overexpressing its receptor, the MET/HGFR protein. Since epithelial cells represent the primary physiologic target of HGF, we investigated whether inappropriate expression of HGF by epithelial cells which normally express MET/HGFR may also contribute to tumorigenesis. We have transfected a full length rat HGF gene into three mouse epithelial cell lines, one derived from breast (MM55) and two (BNL CL.2 and NMuLi) representing liver non-parenchymal epithelial cells (NPEC). We confirmed the presence of the transfected gene by Southern blot analysis, the production of HGF protein by immunofluorescence, and the preservation of HGF biologic activity by bio-assay. In comparison to untransfected cells, all three HGF-transfected cell lines displayed high level MET/HGFR autophosphorylation and increased ability to proliferate in media containing low serum. The two HGF-transfected liver NPEC lines, but not the HGF-transfected mammary cell line, displayed loss of cell contact growth-inhibition and acquired a markedly increased ability to form colonies in soft agar. Furthermore, the NPEC HGF-transfected cell lines formed much larger tumors in nude mice than the untransfected counterparts, with extensive invasion and sporadic lung metastases. These results demonstrate that overexpression of HGF in at least some epithelial cells contribute to tumorigenesis, and furthermore suggest a possible role for the HGF-MET/HGFR system in the progression of certain epithelial tumors.

Hepatocyte growth factor induces proliferation and morphogenesis in nonparenchymal epithelial liver cells

Hepatocyte growth factor is the most potent mitogen known for hepatocytes, and increasing evidence suggests that hepatocyte growth factor plays an important role in liver regeneration. However, hepatocytes are not the only liver epithelial cell population that proliferates during regeneration. Experimental and clinical data indicate that the regenerative liver response also includes the participation of nonparenchymal epithelial cells. The possible role of hepatocyte growth factor in this nonparenchymal epithelial cell regenerative response has not been explored. We studied the effects of hepatocyte growth factor with a model of two normal mouse nonparenchymal epithelial cell-derived cell lines with varying differentiation potentials. Addition of hepatocyte growth factor induced mitogenesis and scattering of colonies growing on culture dishes in both cell lines. Furthermore, hepatocyte growth factor was found to exert a profound morphogenic effect on cells growing in collagen matrixes. Hepatocyte growth factor-treated embryonic BNL CL.2 cell colonies developed elaborately branching elongated cords with only minimal tubularization. Hepatocyte growth factor-treated postnatal NMuLi cell colonies developed a network of anastomosing tubules with wide lumens. These morphological changes were not inhibitable by transforming growth factor-beta and were not induced by other hepatocellular growth factors including epidermal growth factor, transforming growth factor-alpha, acidic fibroblast growth factor and insulin. Histological sections of the hepatocyte growth factor-treated BNL CL.2 cell colonies resembled neocholangioles, believed to include facultative stem cells, which proliferate after submassive and massive hepatic necrosis. Sections of hepatocyte growth factor-treated NMuLi colonies resembled ductules proliferating in biliary tract obstruction from a wide variety of causes. This work represents the first examples of hepatocyte growth factor-induced mitogenesis, scattering and morphogenesis in the same cell lines. More important, our data suggest that hepatocyte growth factor mediates liver response to injury not only by acting on hepatocytes but also by exerting mitogenic and morphogenic influences on nonparenchymal epithelial cell components.

Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system.

Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle.

Complexes containing plasmid DNA, transferrin-polylysine conjugates, and polylysine-conjugated peptides derived from the N-terminal sequence of the influenza virus hemagglutinin subunit HA-2 have been used for the transfer of luciferase or beta-galactosidase marker genes to K562 cells, HeLa cells, and BNL CL.2 hepatocytes. These DNA complexes mimic the entry of viruses into cells, as they contain functions for (i) the packaging of the nucleic acid with polylysine, (ii) the attachment to the cell and receptor-mediated endocytosis with transferrin as a ligand, and (iii) the release from endosomes by using membrane-disrupting influenza peptides. The presence of these influenza peptide conjugates in the DNA complexes renders the complexes active in membrane disruption in a liposome leakage assay and results in a substantial augmentation of the transferrin-polylysine-mediated gene transfer.

Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.

Bafilomycin A1 is known as a strong inhibitor of the vacuolar type H(+)-ATPase in vitro, whereas other type ATPases, e.g. F1,F0-ATPase, are not affected by this antibiotic (Bowman, E.M., Siebers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7972-7976). Effects of this inhibitor on lysosomes of living cultured cells were tested. The acidification of lysosomes revealed by the incubation with acridine orange was completely inhibited when BNL CL.2 and A431 cells were treated with 0.1-1 microM bafilomycin A1. The effect was revealed by washing the cells. Both studies using 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and fluorescein isothiocyanate-dextran showed that the intralysomal pH of A431 cells increased from about 5.1-5.5 to about 6.3 in the presence of 1 microM bafilomycin A1. The pH increased gradually in about 50 min. In the presence of 1 microM bafilomycin A1, 125I-labeled epidermal growth factor (EGF) bound to the cell surface at 4 degrees C was internalized normally into the cells at 37 degrees C but was not degraded at all, in marked contrast to the rapid degradation of 125I-EGF in the control cells without the drug. Immunogold electron microscopy showed that EGF was transported into lysosomes irrespective of the addition of bafilomycin A1. These results suggest that the vacuolar type H(+)-ATPase plays a pivotal role in acidification and protein degradation in the lysosomes in vivo.