Yumi Kanegae

Ligand-independent signaling by overexpressed CD30 drives NF-κB activation in Hodgkin–Reed-Sternberg cells

Overexpression of CD30 and constitutive NF-κB activation characterizes tumor cells of Hodgkins disease (HD), Hodgkin and Reed-Sternberg (H–RS) cells. We report that in H–RS cells overexpression of CD30 leads to self-aggregation, recruitment of TRAF2 and TRAF5, and NF-κB activation, independent of CD30 ligand. CD30 and TRAF proteins co-localized in H–RS cell lines and in lymph nodes of HD. An adenovirus-vector carrying a decoy CD30 lacking the cytoplasmic region or a dominant negative IκBα mutant blocks NF-κB activation, down regulates IL-13 expression and induces apoptosis. Thus, in H–RS cells, ligand-independent activation of CD30 signaling drives NF-κB activation and this leads to constitutive cytokine expression, which provides a molecular basis for HD. Inhibition of NF-κB activation by adenovirus vector-mediated gene transfer may provide a novel strategy of cell- and target molecule-specific therapy for patients with HD.

Induction of photoreceptor-specific phenotypes in adult mammalian iris tissue

We show that iris tissue in the adult rat eye, which is embryonically related to the neural retina, can generate cells expressing differentiated neuronal antigens. In addition, the Crx gene transfer induced the specific antigens for rod photoreceptors in the iris-derived cells, which was not seen in the adult hippocampus-derived neural stem cells. Our findings demonstrate a remarkable plasticity of adult iris tissue with potential clinical applications, as autologous iris tissue can be feasibly obtained with peripheral iridectomy.

EFFICIENT GENE ACTIVATION SYSTEM ON MAMMALIAN CELL CHROMOSOMES USING RECOMBINANT ADENOVIRUS PRODUCING CRE RECOMBINASE

To develop a method for activating genes located on cell chromosomes, an on/off switching unit regulated by the site-specific recombinase Cre was constructed. The switching unit was designed to express firstly the neo gene and secondly the reporter lacZ gene by Cre-mediated excisional deletion of the neo gene. CV1 cell lines bearing the switching unit on a cell chromosome were isolated and activation of the lacZ gene was examined after infection with a Cre-producing recombinant adenovirus. In one cell line virtually 100% of the cells stably expressed the lacZ gene, whereas in another cell line lacZ-expressing cell populations reached only to about 90% and decreased after cell divisions. The Southern blot analyses showed that the latter type of cells contained a head-to-tail array of the switching units, and that consequently the lacZ-expressing units were excised from a cell chromosome and present as extrachromosomal circular DNAs. These results showed that the system offers efficient activation of genes introduced into cell chromosomes and that the organization of the reporter units are important for efficiency and duration of the activated gene expression.

Efficient Conditional Transgene Expression in Hepatitis C Virus cDNA Transgenic Mice Mediated by the Cre/loxP System

Conditional gene expression has greatly facilitated the examination of the functions of particular gene products. Using the Cre/loxP system, we developed efficient conditional transgene activation of hepatitis C virus (HCV) cDNA (nucleotides 294–3435) in transgenic mice. Efficient recombination was observed in transgenic mouse liver upon intravenous administration of adenovirus that expresses Cre DNA recombinase. After transgene activation, most hepatocytes were stained with anti-core polyclonal antibody, and 21-, 37-, and 64-kDa proteins were detected by Western blot analysis in liver lysates using anti-core, E1, and E2 monoclonal antibodies, respectively. Serum core protein was detected in transgenic mice 7 days after transgene activation with concurrent increases in serum alanine aminotransferase levels. Subsequently, an anti-core antibody response was detected 14 days after infection. Furthermore, a CD4 and CD8 positive cell depletion assay normalized both the serum alanine aminotransferase increases and pathological changes in the liver. These results suggest that HCV proteins are not directly cytopathic and that the host immune response plays a pivotal role in HCV infection. Thus, this HCV cDNA transgenic mouse provides a powerful tool with which to investigate the immune responses and pathogenesis of HCV infection.

BabA-mediated Adherence Is a Potentiator of the Helicobacter pylori Type IV Secretion System Activity

Chronic infection of Helicobacter pylori in the stomach mucosa with translocation of the bacterial cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV secretion system (TFSS) into host epithelial cells are major risk factors for gastritis, gastric ulcers, and cancer. The blood group antigen-binding adhesin BabA mediates the adherence of H. pylori to ABO/Lewis b (Leb) blood group antigens in the gastric pit region of the human stomach mucosa. Here, we show both in vitro and in vivo that BabA-mediated binding of H. pylori to Leb on the epithelial surface augments TFSS-dependent H. pylori pathogenicity by triggering the production of proinflammatory cytokines and precancer-related factors. We successfully generated Leb-positive cell lineages by transfecting Leb-negative cells with several glycosyltransferase genes. Using these established cell lines, we found increased mRNA levels of proinflammatory cytokines (CCL5 and IL-8) as well as precancer-related factors (CDX2 and MUC2) after the infection of Leb-positive cells with WT H. pylori but not with babA or TFSS deletion mutants. This increased mRNA expression was abrogated when Leb-negative cells were infected with WT H. pylori. Thus, H. pylori can exploit BabA-Leb binding to trigger TFSS-dependent host cell signaling to induce the transcription of genes that enhance inflammation, development of intestinal metaplasia, and associated precancerous transformations.

INDUCTION OF APOPTOSIS AFTER SWITCH-ON OF THE HEPATITIS B VIRUS X GENE MEDIATED BY THE CRE/LOXP RECOMBINATION SYSTEM

The HBx protein of hepatitis B virus is a multifunctional protein that is implicated in the pathogenesis of hepatocellular carcinoma by regulating gene transcription, causing cell proliferation and, as shown recently, inducing cell death. However, analysis of the effects of HBx in stable cultured cell clones has been hampered because only cell lines that adapted to the effects of HBx were selected during the establishment of cell clones. Here, we describe a system in which transcription of the X gene of hepatitis B virus is switched on by the use of the site-specific Cre recombinase. Two human liver cell lines, HLF and HepG2, were used, the former with a mutant p53 allele and the latter with wild-type p53. The stable cell clones isolated, which carried the X gene in a transcriptionally silent state, were infected with recombinant adenovirus carrying Cre recombinase. Ninety-six hours after adenovirus infection, cell clones that expressed HBx had undergone TUNEL-positive cell death with characteristics of apoptosis. Apoptosis was induced despite concomitant inactivation of the p53 protein as a result of its cytoplasmic translocation by HBx. In contrast, neither the X gene-carrying cells infected with wild-type adenovirus nor various control cells infected with Cre-expressing adenovirus exhibited apoptosis. These results indicate that the expression of HBx protein leads to liver cell apoptosis independently of the p53 pathway. The significance of HBx-induced apoptosis in natural infection is unclear, but it may contribute to the development of hepatitis and serve to spread progeny virus to neighbouring cells while evading the host immune responses.

Percutaneous transluminal gene transfer into canine myocardium in vivo by replication-defective adenovirus

OBJECTIVE The aim was to examine the feasibility, efficiency and safety of adenovirus-mediated in vivo gene transfer into the canine myocardium by a percutaneous transluminal method using a needle-catheter. METHODS Either a replication-defective adenovirus (Adex1SRLacZL) or a plasmid (pSRLacZ), both expressing E. coli lacZ coding beta-galactosidase (beta-gal), was directly injected into the left ventricle of dogs through a needle-catheter inserted via a femoral artery. Expression of lacZ was examined by histochemical staining and quantified by measuring beta-gal activity. RESULTS Injections with Adex1SRLacZL induced lacZ expression as a result of 40 out of 41 injections; the expression level was 10 times higher than that obtained with pSRLacZ. Induced beta-gal activity was detected within 24 h, peaked at 7 days and retained for 2 weeks after gene transfer. A repetitive administration of the same adenovirus at 14 days after the first injection also evoked a reduced but significant level of expression despite neutralizing antibodies to adenovirus in serum. Although injection induced an inflammatory response that peaked at 3 days after injection and gradually subsided without a second peak, the temporal change and the extent of inflammation induced by adenovirus injection was not significantly different from those induced by injection with either saline or plasmid. Neither leakage of enzymes such as CPK or LDH nor alteration in the ECG was detected in the 30 days following gene transfer. CONCLUSIONS Our findings demonstrate that a catheter-mediated direct injection with an adenovirus can induce gene expression in the ventricle more efficiently without additional myocardial damage and inflammation compared with injection with a plasmid. A repeat dose of the same adenovirus elicited gene expression at an attenuated but significant level. This method may potentially have clinical applications: in modifying myocardial phenotype and/or improving general circulation under certain circumstances.

Quantitative Analysis of Repeat Adenovirus-Mediated Gene Transfer Into Injured Canine Femoral Arteries

We quantitatively evaluated the effectiveness of a repeat administration of a recombinant adenoviral vector expressing bacterial Escherichia coli lacZ into the same arterial site of a relatively large animal, the dog. The replication-defective adenoviral vector was introduced percutaneously into balloon-injured femoral arteries through a double-balloon catheter. After a single dose of adenoviral vector, up to 90% of surface (73 +/- 16%, n = 7) and smooth muscle cells in multiple layers of the media showed transgene expression as evaluated by 5-bromo-4-chloro-3-indoyl beta-D-galactopyranoside histostaining without extralocal expression, as assessed by polymerase chain reaction. High-level expression (measured as beta-galactosidase activity) peaked 7 days after transfer and was transient, although it was retained for a month. Second does of the same adenovirus to the same arterial site were given 1, 2, 5, or 8 weeks after the first administration. At 1 week the second dose significantly enhanced lacZ expression. At 2, 5, or 8 weeks the second dose reinduced lacZ expression at 25% to 30% of the full expression. lacZ expression was also detected in preimmuned dogs, although the expression levels correlated inversely to the titer of neutralizing antibodies in their serum. These results demonstrate that arterial gene expression can be enhanced by a second administration of the same adenovirus after a short interval and that a repeat dose after a long interval partially but significantly reinduces gene expression despite the presence of an immune response. These data may provide an additional scientific foundation for the use of adenovirus-mediated arterial gene transfer in future clinical practice.

Cancer gene therapy using a pro-apoptotic gene, caspase-3

Caspase-3 is a member of the cysteine protease family, which plays a crucial role in apoptosis. We applied the human caspase-3 gene as a novel form of anticancer gene therapy. Overexpression of human caspase-3 alone could not induce apoptosis of tumor cell lines, but apoptosis was markedly enhanced by the addition of etoposide. In an AH130 liver tumor model, transduction of human caspase- 3, but not the empty vector, induced extensive apoptosis and reduced tumor volume when combined with etoposide administration. However, this effect was not observed with a Bcl-2 overexpressing tumor. In conclusion, caspase-3 gene transduction accompanied by an additional death stimulus may be a useful method of anticancer gene therapy, except for Bcl-2 overexpressing tumors.

Complete rescue of lethal albino c(14CoS) mice by null mutation of 4- hydroxyphenylpyruvate dioxygenase and induction of apoptosis of hepatocytes in these mice by in vivo retrieval of the tyrosine catabolic pathway

Hereditary tyrosinemia 1 (HT1) is characterized by progressive liver damage, from infancy, and by a high risk for hepatocellular carcinoma. HT1 is due to mutations in the fumarylacetoacetate hydrolase gene Fah, encoding the last enzyme in the tyrosine catabolic pathway. Lethal albino deletionc 14CoS mice and mice with target-disruptedFah are models for HT1, but they die in the perinatal period, albeit with a different phenotype from that seen in HT1 in humans. We first asked whether homozygous null mutation of the 4-hydroxyphenylpyruvate dioxygenase gene Hpd could rescue the homozygous c 14CoS mice (c 14CoS /c 14CoS orFah −/−). The double mutantFah −/− Hpd −/− mice appeared normal, at least until age 18 months, and there was no evidence of liver disease, findings that facilitated examination of the effect of Fah −/− on mature and unmodified hepatocytes in vivo. The hepatocytes ofFah −/− undergo rapid apoptosis, and acute death follows. Essentially the same phenomena were observed whenFah −/− Hpd −/− mice were administered homogentisate intraperitoneally. These changes in liver pathology in Fah −/− Hpd −/− mice after the administration of homogentisate were associated with massive urinary excretion of succinylacetone. These results suggest that accumulation of fumarylacetoacetate, maleylacetoacetate, or succinylacetone seems to trigger the endogenous process of apoptosis in hepatocytes that lack fumarylacetoacetate hydrolase activity. This apoptosis may be related to the development of hepatocellular carcinomas seen in HT1 patients and pharmaceutically treated fumarylacetoacetate hydrolase-deficient mice.