Functional consequences of WNT3/Frizzled7-mediated signaling in non-transformed hepatic cells.

We have previously demonstrated that WNT3 and Frizzled7 (FZD7) expression levelswere upregulated in hepatocellular carcinoma (HCC) and that they directly interact to activate the canonical Wnt/ß-catenin pathway in HCC cell lines. In this study, we investigated the functional consequences of WNT3 and FZD7 expression levels in non-transformed hepatic cells to address the question of whether WNT3/FZD7-mediated signal transduction could be involved in cellular transformation. After stable transfection of WNT3 and FZD7, the activation of the Wnt/ß-catenin pathway was confirmed by western blot, immunostaining and quantitative real-time reverse transcriptase-PCR (qRT-PCR) analysis in two non-transformed hepatocyte-derived cell lines. In vitro characteristics of the malignant phenotype were measured, including cell proliferation, migration, invasion and anchorage-independent growth in soft agar. Stable expression of WNT3 and FZD7 in the two cell lines led to cellular accumulation of ß-catenin and expression of downstream target genes activated by this pathway. In the stable WNT3/FZD7-expressing clones, hepatic cell proliferation, migration, invasion as well as soft agar colony formation were enhanced compared with the non-transformed control cells. The epithelial-mesenchymal transition (EMT) factors, Twist, Snail and Vimentin, were increased in cells expressing WNT3 and FZD7. However, the WNT3/FZD7-expressing cells did not form tumors in vivo. We conclude that activation of the WNT3/FZD7 canonical pathway has a role in the early stages of hepatocarcinogenesis by promoting the acquisition of a malignant phenotype with features of EMT.

Generation of cellular immune responses to HCV NS5 protein through in vivo activation of dendritic cells.

Chronic hepatitis C (HCV) infection is a substantial medical problem that leads to progressive liver disease, cirrhosis, and hepatocellular carcinoma (HCC). The aim of this study was to achieve sustained cellular immune responses in vivo to a HCV nonstructural protein using dendritic cell (DC)-based immunization approach. We targeted the HCV NS5 protein to DCs in vivo by injecting microparticles loaded with this antigen. The DC population was expanded in BALB/C mice (H-2(d) ) by hydrodynamic injection of a plasmid pUMVC3-hFLex expressing the secreted portion of the human Fms-like tyrosine kinase receptor-3 ligand (hFlt3). Mice were subsequently injected with microparticles coated with HCV NS5 protein via the tail vein. Cellular immune responses were determined with respect to secretion of INFγ and IL2 by CD4(+) cells and cytotoxic T-lymphocyte (CTL) assays in vitro; inhibition of tumour cell growth was employed for the assessment of CD8(+) generated activity in vivo. We found that Flt3L treatment expanded the DC population in the spleen to 43%, and such cells displayed a striking upregulation of CD86 as well as CD80 and CD40 co-stimulating molecules. Viral antigen-specific T(H) 1 cytokine secretion by splenocytes was generated, and CTL activity against syngeneic NS5 expressing myeloma target cells was observed. In addition, these cells inhibited tumour growth indicating that NS5-specific robust CTL activity was operative in vivo. Thus, the capability of activating DCs in vivo using the methods described is valuable as a therapeutic vaccine strategy for chronic HCV infection.

Recent advances in the natural history of hepatocellular carcinoma.

Ongoing advances in liver disease management and basic research in recent years have changed our knowledge of the natural history of hepatocellular carcinoma (HCC). Indeed, the natural history of this tumor is fairly long and covers a preclinical and a clinical phase. Some of the biological steps involved in cell transformation and different carcinogenic pathways have been identified, disclosing potential novel markers for HCC. Following the progress in surveillance and early diagnosis, much more is now known about precancerous lesions and the process leading to overt HCC, including growth patterns, dedifferentiation and neoangiogenenesis. In particular, research has focused on clinical and biological factors predicting tumor aggressiveness and patients' prognosis. Lastly, clinical studies have described tumor presentation, evolution and causes of patients' death and how the new knowledge has influenced clinical management and patients' survival in recent years. By addressing 10 key questions, this review will summarize well-established and novel features of the natural history of HCC.

Common dysregulation of Wnt/Frizzled receptor elements in human hepatocellular carcinoma.

Dysregulation of growth factors and their receptors is central to human hepatocellular carcinoma (HCC). We previously demonstrated that the Frizzled-7 membrane receptor mediating the Wnt signalling can activate the beta-catenin pathway and promotes malignancy in human hepatitis B virus-related HCCs. Expression patterns of all the 10 Frizzled receptors, and their extracellular soluble autoparacrine regulators (19 Wnt activators and 4 sFRP inhibitors) were assessed by real-time RT-PCR in 62 human HCC of different etiologies and their matched peritumorous areas. Immunostaining was performed to localise Frizzled on cell types in liver tissues. Regulation of three known Frizzled-dependent pathways (beta-catenin, protein kinase C, and C-Jun NH(2)-terminal kinase) was measured in tissues by western blot. We found that eight Frizzled-potentially activating events were pleiotropically dysregulated in 95% HCC and 68% peritumours as compared to normal livers (upregulations of Frizzled-3/6/7 and Wnt3/4/5a, or downregulation of sFRP1/5), accumulating gradually with severity of fibrosis in peritumours and loss of differentiation status in tumours. The hepatocytes supported the Wnt/Frizzled signalling since specifically overexpressing Frizzled receptors in liver tissues. Dysregulation of the eight Frizzled-potentially activating events was associated with differential activation of the three known Frizzled-dependent pathways. This study provides an extensive analysis of the Wnt/Frizzled receptor elements and reveals that the dysregulation may be one of the most common and earliest events described thus far during hepatocarcinogenesis.

Impaired placentation in fetal alcohol syndrome.

Intrauterine growth restriction (IUGR) is one of the key features of fetal alcohol syndrome (FAS), and IUGR can be mediated by impaired placentation. Insulin-like growth factors (IGF) regulate placentation due to stimulatory effects on extravillous trophoblasts, which are highly motile and invasive. Previous studies demonstrated that extravillous trophoblasts express high levels of aspartyl-(asparaginyl) beta-hydroxylase (AAH), a gene that is regulated by IGF and has a critical role in cell motility and invasion. The present study examines the hypothesis that ethanol impaired placentation is associated with inhibition of AAH expression in trophoblasts. Pregnant Long Evans rats were fed isocaloric liquid diets containing 0% or 37% ethanol by caloric content. Placentas harvested on gestation day 16 were used for histopathological, mRNA, and protein studies to examine AAH expression in relation to the integrity of placentation and ethanol exposure. Chronic ethanol feeding prevented or impaired the physiological conversion of uterine vessels required for expansion of maternal circulation into placenta, a crucial process for adequate placentation. Real-time quantitative RT-PCR analysis demonstrated significant reductions in IRS-1, IRS-2, and significant increases in IGF-II and IGF-II receptor mRNA levels in ethanol-exposed placentas. These abnormalities were associated with significantly reduced levels of AAH expression in trophoblastic cells, particularly within the mesometrial triangle (deep placental bed) as demonstrated by real time quantitative RT-PCR, Western blot analysis, ELISA, and immunohistochemical staining. Ethanol-impaired placentation is associated with inhibition of AAH expression in trophoblasts. This effect of chronic gestational exposure to ethanol may contribute to IUGR in FAS.

Isolation and characterization of human antibodies targeting human aspartyl (asparaginyl) beta-hydroxylase.

Over-expression of the enzyme human aspartyl (asparaginyl) beta-hydroxylase (HAAH) has been detected in a variety of cancers. It is proposed that upon cellular transformation, HAAH is overexpressed and translocated to the tumor cell surface, rendering it a specific surface antigen for tumor cells. In this work, twelve human single-chain Fv fragments (scFv) against HAAH were isolated from a human non-immune scFv library displayed on the surface of yeast. Five of the twelve were reformatted as human IgG1. Two of the five IgGs, 6-22 and 6-23, showed significant binding to recombinant HAAH in ELISA, tumor cell lines, and tumor tissues. The apparent dissociation constants of 6-22 and 6-23 IgG were 1.0 +/- 0.2 nM and 20 +/- 10 nM respectively. These two antibodies were shown to target different domains of HAAH, with 6-22 targeting the catalytic domain of HAAH and 6-23 targeting the N-terminal non-catalytic domain of HAAH. 6-22 IgG was further characterized, as it had high affinity and targeted the catalytic domain. 6-22 IgG alone does not exhibit significant cytotoxicity toward the tumor cells. However, 6-22 internalizes into tumor cells and can therefore be employed to deliver cytotoxic moieties. A goat anti-human IgG-saporin conjugate was delivered into tumor cells by 6-22 IgG and hence elicited cytotoxicity toward the tumor cells in vitro. These tumor-binding human antibodies can potentially be used in both diagnosis and immunotherapy targeting HAAH-expressing tumor cells.

Chronic gestational exposure to ethanol causes insulin and IGF resistance and impairs acetylcholine homeostasis in the brain.

In fetal alcohol syndrome (FAS), cerebellar hypoplasia is associated with impaired insulin-stimulated survival signaling. This study characterizes ethanol dose-effects on cerebellar development, expression of genes required for insulin and insulin-like growth factor (IGF) signaling, and the upstream mechanisms and downstream consequences of impaired signaling in relation to acetylcholine (ACh) homeostasis. Pregnant Long Evans rats were fed isocaloric liquid diets containing 0%, 2%, 4.5%, 6.5%, or 9.25% ethanol from gestation day 6. Ethanol caused dose-dependent increases in severity of cerebellar hypoplasia, neuronal loss, proliferation of astrocytes and microglia, and DNA damage. Ethanol also reduced insulin, IGF-I, and IGF-II receptor binding, insulin and IGF-I receptor tyrosine kinase activities, ATP, membrane cholesterol, and choline acetyltransferase (ChAT) expression. In vitro studies linked membrane cholesterol depletion to impaired insulin receptor binding and insulin-stimulated ChAT. In conclusion, cerebellar hypoplasia in FAS is mediated by insulin/IGF resistance with attendant impairments in energy production and ACh homeostasis.

Ethanol inhibits insulin expression and actions in the developing brain.

Ethanol-induced cerebellar hypoplasia is associated with inhibition of insulin-stimulated survival signaling. The present work explores the mechanisms of impaired insulin signaling in a rat model of fetal alcohol syndrome. Real-time quantitative RT-PCR demonstrated reduced expression of the insulin gene in cerebella of ethanol-exposed pups. Although receptor expression was unaffected, insulin and insulin-like growth factor (IGF-I) receptor tyrosine kinase (RTK) activities were reduced by ethanol exposure, and these abnormalities were associated with increased PTP1b activity. In addition, glucose transporter molecule expression and steady-state levels of ATP were reduced in ethanol-exposed cerebellar tissue. Cultured cerebellar granule neurons from ethanol-exposed pups had reduced expression of genes encoding insulin, IGF-II, and the IGF-I and IGF-II receptors, and impaired insulin- and IGF-I-stimulated glucose uptake and ATP production. The results demonstrate that ethanol inhibits insulin-mediated actions in the developing brain by reducing local insulin production and insulin RTK activation, leading to inhibition of glucose transport and ATP production.

Neuronal thread protein regulation and interaction with microtubule-associated proteins in SH-Sy5y neuronal cells.

In Alzheimer's disease (AD), neuronal thread protein (NTP) accumulates in cortical neurons and colocalizes with phospho- tau-immunoreactive cytoskeletal lesions that correlate with dementia. To generate additional information about the potential role of NTP in AD, we characterized its expression and regulation in human SH-Sy5y neuronal cells. Quantitative real-time reverse transcription-polymerase chain reactin and Western blot analysis demonstrated prominent insulin, moderate insulin-like growth factor, type 1 (IGF-1) and minimal nerve growth factor stimulation of NTP expression. In addition, NTP protein was more stable and it progressively accumulated in cells that were stimulated with insulin for 24 or 48 h. Metabolic labeling and phospho-amino acid analysis demonstrated phosphorylation of NTP on Serine residues, 30-60 min after insulin or IGF-1 stimulation, when glycogen synthase kinase 3beta (GSK-3beta) activity would no longer have been suppressed. Kinase inhibitor and in vitro phosphorylation studies demonstrated a role for GSK-3beta in the positive regulation of NTP expression and phosphorylation. Coimmunoprecipitation studies demonstrated physical interactions between NTP and tau or microtubule-associated protein 1b (MAP-1b), and ubiquitin immunoreactivity in NTP immunoprecipitates. In summary, these studies showed that (i) NTP expression is regulated at the level of transcription by insulin and IGF-1 stimulation; (ii) NTP is phosphorylated by GSK-3beta; (iii) NTP can physically interact with tau and MAP-1b and (iv) NTP-MAP complexes are ubiquitinated. The results suggest a functional role for NTP in relation to the turnover or processing of neuronal cytoskeletal proteins, attributes that may be modulated by insulin/IGF-1-mediated signaling.

CpG immuno-stimulatory motifs enhance humoral immune responses against hepatitis C virus core protein after DNA-based immunization.

Chronic HCV infection is associated with a high morbidity and mortality rate, and currently a prophylactic or therapeutic vaccine is not available. DNA-based immunization is a powerful method to generate cellular and humoral immune responses. However, DNA immunization against HCV core results only in a weak humoral immune response demonstrated in several studies. Therefore, co-immunization with a novel adjuvant may enhance such potentially important immune responses. We examined whether unmethylated CpG motifs in the form of oligodeoxynucleotides (ODN) or E. coli DNA can act as adjuvants for a DNA vaccination approach, since CpG motifs have been shown to stimulate the innate immune system as well as B and T cell immune reactivity. The present study demonstrates that CpG motifs enhance in vivo antibody levels after DNA immunization against HCV core. However, despite some in vitro activity of CpG motifs, no enhancement of T cell responses in vivo was observed after immunization with HCV plasmid DNA and CpG motifs in mice. Our results suggest that co-immunization with CpG-ODN may strengthen humoral immune responses but show no potential effect as an adjuvant to induce cellular immunity against HCV core.

Chronic gestational exposure to ethanol impairs insulin-stimulated survival and mitochondrial function in cerebellar neurons.

Chronic gestational exposure to ethanol has profound adverse effects on brain development. In this regard, studies using in vitro models of ethanol exposure demonstrated impaired insulin signaling mechanisms associated with increased apoptosis and reduced mitochondrial function in neuronal cells. To determine the relevance of these findings to fetal alcohol syndrome, we examined mechanisms of insulin-stimulated neuronal survival and mitochondrial function using a rat model of chronic gestational exposure to ethanol. In ethanol-exposed pups, the cerebellar hemispheres were hypoplastic and exhibited increased apoptosis. Isolated cerebellar neurons were cultured to selectively evaluate insulin responsiveness. Gestational exposure to ethanol inhibited insulin-stimulated neuronal viability, mitochondrial function, Calcein AM retention (membrane integrity), and GAPDH expression, and increased dihydrorosamine fluorescence (oxidative stress) and pro-apoptosis gene expression (p53, Fas-receptor, and Fas-ligand). In addition, neuronal cultures generated from ethanol-exposed pups had reduced levels of insulin-stimulated Akt, GSK-3beta, and BAD phosphorylation, and increased levels of non-phosphorylated (activated) GSK-3beta and BAD protein expression. The aggregate results suggest that insulin-stimulated central nervous system neuronal survival mechanisms are significantly impaired by chronic gestational exposure to ethanol, and that the abnormalities in insulin signaling mechanisms persist in the early postnatal period, which is critical for brain development.

Inhibition of hepatitis C virus NS3 function by antisense oligodeoxynucleotides and protease inhibitor.

Hepatitis C Virus (HCV) NS3 protease is an attractive target for antiviral agent development because it is required for viral replication. Because a stable cell culture system or small animal model to study HCV replication is not readily available, we constructed an in vitro model allowing the investigation of NS3 transcription, translation, and protease function. Sequences encoding for full length HCV genomes were cloned and transfected into HuH-7 human hepatocellular carcinoma cells to analyze NS3 transcription/translation. A plasmid pHCV ORF I luc that expresses the complete HCV coding region upstream of a luciferase reporter gene was designed to enable quantification of translated HCV proteins. Additionally, NS3 protease function was assessed by direct coexpression of NS3 and NS5 in HuH 7 cells, and the subsequent measurement of cleavage products. We found that antisense oligodeoxynucleotides (AS-ODN) interfered with NS3 translation in a dose dependent fashion; AS-ODN 5 cotransfection directed against NS3 sequences significantly inhibited protease activity as measured by cleaved NS5A levels. Finally, cleaved NS5A levels served as anindex of protease activity and Chymostatin, a protease inhibitor, almost completely blocked NS3 enzymatic activity. This cell culture system is useful in the assessment of potential antiviral agents on HCV NS3 expression and function.

A short sequence within domain C of duck carboxypeptidase D is critical for duck hepatitis B virus binding and determines host specificity.

Virus-cell surface receptor interactions are of major interest. Hepadnaviruses are a family of partially double-stranded DNA viruses with liver tropism and a narrow host range of susceptibility to infection. At least in the case of duck hepatitis B virus (DHBV), host specificity seems controlled partly at the receptor level. The middle portion in the pre-S region of the viral large envelope protein binds specifically to duck carboxypeptidase D (DCPD) but not to its human or chicken homologue. Although domain C of DCPD is implicated in ligand binding, the exact pre-S contact site remains to be determined. We prepared and tested a panel of chimeric constructs consisting of DCPD and human carboxypeptidase D (HCPD). Our results indicate that a short region at the N terminus of domain C (residues 920 to 949) is critical to DHBV binding and is a major determinant for the host specificity of DHBV infection. Replacing this region of the DCPD molecule with its human homologue abolished the DHBV interaction, whereas introducing this DCPD sequence into HCPD conferred efficient DHBV binding. Extensive analysis of site-directed mutants revealed that both conserved and nonconserved residues were important for the pre-S interaction. There were primary sequence variations and secondary structural differences that contributed to the inability of HCPD to bind the DHBV pre-S domain.

Adenovirus-mediated gene transfer to orthotopic hepatocellular carcinomas in athymic nude mice.

Gene therapy may become an option for the treatment of malignant tumors such as hepatocellular carcinoma (HCC), once safe and efficient vector systems have been established. Due to their stability in vivo, recombinant adenoviral vectors are promising vectors for gene delivery to HCC. To study the characteristics of gene delivery into HCCs by recombinant adenoviral vectors in vivo, we established an in situ HCC model in the livers of athymic nude mice by intrahepatic injection of human HCC cells. Recombinant adenovirus vectors expressing beta-galactosidase (Ad2CMV beta gal) were injected via the tail vein of mice bearing HCC or directly into intrahepatic tumors. Levels of beta-galactosidase expression in tumor tissue and surrounding normal liver were analyzed by histochemistry or for quantification by a chemiluminescence assay in tissue homogenates. Following tail vein injection, high levels of beta-galactosidase expression were found in the liver, but virtually no gene expression could be detected in the tumor tissue. In contrast, after direct injection of Ad2CMV beta gal into intrahepatic HCCs, high levels of beta-galactosidase expression were detected in the tumor tissue. However, single transduced hepatocytes scattered throughout the normal liver could also be identified. These results indicate that barriers such as the endothelial lining of the tumor vasculature impair the efficiency of adenoviral vectors for gene delivery into HCCs by intravenous administration, which can be overcome by direct injection into the tumor tissue. However, due to the observed transduction of disseminated hepatocytes following intratumoral administration, additional HCC-specific targeting to further enhance the safety of adenoviral vectors may be required.

Induction of cytotoxic T lymphocyte responses against hepatitis delta virus antigens which protect against tumor formation in mice.

The cellular immune response is a crucial defense mechanism against hepatotropic viruses and in chronic viral hepatitis prevention. Moreover, hepatitis delta virus (HDV) immunogenicity may be an important component in the development of prophylactic and therapeutic vaccines. Therefore, we evaluated the immunogenicity of the small (HDAg) or large delta antigen (LHDAg) to be used as a DNA-based vaccine. We immunized different mouse haplotypes, determined cellular immune responses, and tested protection of animals against tumor formation using syngeneic tumor cells stably expressing the delta antigens. Both LHDAg and HDAg primed CD4+ and CD8+ T cell immunity against both forms of delta antigens. CD8+ T cell frequencies were about 1% and antigen-specific CD8+ T cells remained detectable directly ex vivo for at least 35 days post-injection. No anti-delta antibody responses could be detected despite multiple detection systems and varied immunization approaches. We observed protection against syngeneic tumor formation and growth in mice immunized with DNA plasmids encoding secreted or intracellular forms of HDAg and LHDAg but not with recombinant HDAg establishing the generation of significant cellular immunity in vivo. Both CD4+ and CD8+ T cells were required for antitumoral activity as determined by in vivo T cell depletion experiments. The results indicate that DNA-based immunization with genes encoding LHDAg and HDAg induces strong T cell responses and, therefore, is an attractive approach for the construction of therapeutic and prophylactic T cell vaccines against HDV.

Mechanisms of cell death induced by suicide genes encoding purine nucleoside phosphorylase and thymidine kinase in human hepatocellular carcinoma cells in vitro.

For gene therapy of hepatocellular carcinoma (HCC), the Escherichia coli purine nucleoside phosphorylase (PNP)/fludarabine suicide gene system may be more useful than the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system as a result of a stronger bystander effect. To analyze the molecular mechanisms involved in PNP/fludarabine-mediated cell death in human HCC cells in comparison with HSV-tk/GCV, we transduced human HCC cells of the cell lines, HepG2 and Hep3B, with PNP or HSV-tk using adenoviral vectors, followed by prodrug incubation. Both systems predominantly induced apoptosis in HepG2 and Hep3B cells. PNP/fludarabine induced strong p53 accumulation and a more rapid onset of apoptosis in p53-positive HepG2 cells as compared with p53-negative Hep3B cells, but efficiency of tumor cell killing was similar in both cell lines. In contrast, HSV-tk/GCV-induced apoptosis was reduced in p53-negative Hep3B cells as compared with p53-positive HepG2 cells. HSV-tk/GCV, but not PNP/fludarabine, caused up-regulation of Fas in p53-positive HepG2 cells and of Fas ligand (FasL) in both HCC cell lines. These results demonstrate cell line-specific differences in response to treatment with PNP/fludarabine and HSV-tk/GCV, respectively, and indicate that PNP/fludarabine may be superior to HSV-tk/GCV for the treatment of human HCC because of its independence from p53 and the Fas/FasL system.

Neurodegeneration changes in primary central nervous system neurons transfected with the Alzheimer-associated neuronal thread protein gene.

The AD7c-NTP gene is over-expressed in brains with Alzheimer's disease (AD), and increased levels of the corresponding protein are detectable in cortical neurons, brain tissue extracts, cerebrospinal fluid, and urine beginning early in the course of AD neurodegeneration. In the present study, we utilized a novel method to transfect post-mitotic primary neuronal cell cultures, and demonstrated that over-expression of the AD7c-NTP gene causes cell death and neuritic sprouting, two prominent abnormalities associated with AD. These results provide further evidence that aberrantly increased AD7c-NTP expression may have a role in AD-type neurodegeneration. In addition, we demonstrate that primary post-mitotic neurons can be efficiently transfected with conventional recombinant plasmid DNA to evaluate the effects of gene over-expression in relevant in vitro models.

Mitochondrial dna damage and impaired mitochondrial function contribute to apoptosis of insulin-stimulated ethanol-exposed neuronal cells.

BACKGROUND: Ethanol inhibition of insulin signaling may contribute to impaired central nervous system development in fetal alcohol syndrome. An important consequence of ethanol inhibition of insulin signaling is increased apoptosis due to reduced levels of insulin-stimulated phosphoinositol-3-kinase activity. METHODS: We used viability assays, end-labeling, Western blot analysis, and MitoTracker (Molecular Probes, Eugene, OR) fluorescence labeling to determine whether ethanol-induced central nervous system neuronal cell death was mediated in part by increased mitochondrial (Mt) DNA damage and impaired Mt function. RESULTS: In ethanol-exposed, insulin-stimulated PNET2 central nervous system-derived human neuronal cells, reduced viability was associated with increased Mt DNA damage, reduced Mt mass (manifested by reduced Mt protein expression and MitoTracker Green fluorescent labeling), and impaired Mt function (manifested by reduced levels of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide activity, cytochrome oxidase-Complex IV, Subunit II expression, and MitoTracker Red fluorescence). The adverse effects of ethanol on Mt function were reduced by pretreating the cells with broad-spectrum caspase inhibitors and nearly abolished by nerve growth factor stimulation, with or without concomitant treatment with global caspase inhibitors. CONCLUSIONS: These results suggest that ethanol-induced death of insulin-stimulated immature neuronal cells is mediated in part by impaired Mt function associated with Mt DNA damage and reduced Mt mass, and therefore it is likely to contribute to neuronal loss associated with fetal alcohol syndrome. The findings also suggest that the adverse effects of ethanol on insulin-stimulated survival and metabolic function could be overcome by stimulating with growth factors that support Mt function through insulin-independent pathways.

Cationic liposome-mediated gene delivery to the liver and to hepatocellular carcinomas in mice.

The potential of cationic liposomes as nonviral vectors for in vivo gene delivery to the liver and to intrahepatic hepatocellular carcinoma (HCC) was investigated. Mice were injected via the tail vein or portal vein with a cationic lipid complexed to plasmid DNA (pDNA) encoding the chloramphenicol acetyltransferase (CAT) reporter gene at various cationic lipid:pDNA molar ratios to analyze the efficiency of gene delivery after intravenous administration. Tail vein injection resulted in high CAT expression levels in lung and spleen and low levels in the liver. Portal vein injection, by comparison, significantly enhanced hepatic reporter gene expression but also resulted in pronounced hepatic toxicity. Gene delivery to intrahepatic tumors produced by intrahepatic injection of human HCC cells was analyzed in nude mice. Tail vein injection as well as portal vein injection resulted in low levels of gene expression in intrahepatic tumors. By comparison, high levels of gene expression were achieved by direct, intratumoral injection of liposome-pDNA complexes, with only minimal expression in the surrounding normal liver. Therefore, direct liposome-pDNA complex injection appears far superior to systemic or portal intravenous administration for gene therapy of localized intrahepatic tumors, and may be a useful adjunct in the treatment of human HCCs.