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Background & Aims: Hepatic steatosis, characterized by lipid accumulation in hepatocytes, is a key diagnostic feature in patients with chronic hepatitis C virus (HCV) infection. This study aimed to clarify the involvement of phospholipid metabolic pathways in the pathogenesis of HCV-induced steatosis., Methods: The expression and distribution of lipid species in the livers of human liver chimeric mice were analyzed using imaging mass spectrometry. Triglyceride accumulation and lipid droplet formation were studied in phospholipase A2 group 4C ( PLA2G4C ) knockout or overexpressing cells., Results: Imaging mass spectrometry of the infected mouse model revealed increased lysophosphatidylcholine levels and decreased phosphatidylcholine levels in HCV-positive regions of the liver. Among the transcripts associated with phosphatidylcholine biosynthesis, upregulation of PLA2G4C mRNA was most pronounced following HCV infection. Activation of the transcription factor NF-κB and upregulation of c-Myc were important for activation of PLA2G4C transcription by HCV infection and expression of the viral proteins Core-NS2. The amount and size of lipid droplets were reduced in PLA2G4C -knockout cells. Inhibition of NF-κB or c-Myc activity suppressed lipid droplet formation in HCV-infected cells. HCV infection promoted the stabilization of lipid droplets, but this stability was reduced in PLA2G4C -knockout cells. Overexpression of PLA2G4C decreased the levels of phosphatidylcholine species in the lipid droplet fraction and led to lower levels of key factors involved in lipolysis (breakdown of triglycerides into glycerol and free fatty acids), such as ATGL, PLIN1 and ABHD5 on the lipid droplets., Conclusions: HCV infection markedly increases PLA2G4C expression. This may alter the phospholipid composition of the lipid droplet membrane, leading to stabilization and enlargement of the droplets., Impact and Implications: The involvement of phospholipid metabolism pathways in the pathogenesis of hepatitis C virus (HCV)-related liver diseases remains unclear. We found that PLA2G4C expression is upregulated through NF-κB and c-Myc activation upon HCV infection, and this upregulation is associated with a decrease in phosphatidylcholine species. The increased expression of PLA2G4C resulted in changes in the phospholipid composition of lipid droplets, led to the dissociation of lipolysis-related factors from the lipid droplet surface and the accumulation of lipid content within the droplets. These findings suggest that the disruption of the phospholipid metabolism pathway caused by HCV infection may contribute to the development of HCV-associated fatty liver. It would be interesting to determine whether alcohol- and/or metabolic dysfunction-associated steatohepatitis are also associated with increased PLA2 activity, altered phospholipid composition and decreased levels of ATGL and its cofactors in lipid droplet membranes., Competing Interests: The authors have no conflicts to report. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2024 The Author(s).)

Dual-specificity tyrosine-regulated kinase 2 (DYRK2) is a protein kinase that phosphorylates p53-Ser46 and induces apoptosis in response to DNA damage. However, the relationship between DYRK2 expression and chemosensitivity after DNA damage in colorectal cancer has not been well investigated. The aim of the present study was to examine whether DYRK2 could be a novel marker for predicting chemosensitivity after 5-fluorouracil- and oxaliplatin-induced DNA damage in colorectal cancer. Here we showed that DYRK2 knockout decreased the chemosensitivity to 5-fluorouracil and oxaliplatin in p53 wild-type colorectal cancer cells, whereas the chemosensitivity remained unchanged in p53-deficient/mutated colorectal cancer cells. In addition, no significant differences in chemosensitivity to 5-fluorouracil and oxaliplatin between scramble and siDYRK2 p53(-/-) colorectal cancer cells were observed. Conversely, the combination of adenovirus-mediated overexpression of DYRK2 with 5-fluorouracil or oxaliplatin enhanced apoptosis and chemosensitivity through p53-Ser46 phosphorylation in p53 wild-type colorectal cancer cells. Furthermore, DYRK2 knockout decreased chemosensitivity to 5-fluorouracil and oxaliplatin in p53 wild-type xenograft mouse models. Taken together, these findings demonstrated that DYRK2 expression was associated with chemosensitivity to 5-fluorouracil and oxaliplatin in p53 wild-type colorectal cancer, suggesting the importance of evaluating the p53 status and DYRK2 expression as a novel marker in therapeutic strategies for colorectal cancer., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Hepatitis C virus (HCV) is a pathogen characterized not only by its persistent infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC), but also by metabolic disorders such as lipid and iron dysregulation. Elevated iron load is commonly observed in the livers of patients with chronic hepatitis C, and hepatic iron overload is a highly profibrogenic and carcinogenic factor that increases the risk of HCC. However, the underlying mechanisms of elevated iron accumulation in HCV-infected livers remain to be fully elucidated. Here, we observed iron accumulation in cells and liver tissues under HCV infection and in mice expressing viral proteins from recombinant adenoviruses. We established two molecular mechanisms that contribute to increased iron load in cells caused by HCV infection. One is the transcriptional induction of hepcidin, the key hormone for modulating iron homeostasis. The transcription factor cAMP-responsive element-binding protein hepatocyte specific (CREBH), which was activated by HCV infection, not only directly recognizes the hepcidin promoter but also induces bone morphogenetic protein 6 (BMP6) expression, resulting in an activated BMP-SMAD pathway that enhances hepcidin promoter activity. The other is post-translational regulation of the iron-exporting membrane protein ferroportin 1 (FPN1), which is cleaved between residues Cys284 and Ala285 in the intracytoplasmic loop region of the central portion mediated by HCV NS3-4A serine protease. We propose that host transcriptional activation triggered by endoplasmic reticulum stress and FPN1 cleavage by viral protease work in concert to impair iron efflux, leading to iron accumulation in HCV-infected cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ohta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Amyotrophic lateral sclerosis (ALS) is a disease that affects motor neurons and has a poor prognosis. We focused on TAR DNA-binding protein 43 kDa (TDP-43), which is a common component of neuronal inclusions in many ALS patients. To analyze the contribution of TDP-43 mutations to ALS in human cells, we first introduced TDP-43 mutations into healthy human iPSCs using CRISPR/Cas9 gene editing technology, induced the differentiation of these cells into motor and sensory neurons, and analyzed factors that are assumed to be altered in or associated with ALS (cell morphology, TDP-43 localization and aggregate formation, cell death, TDP-43 splicing function, etc.). We aimed to clarify the pathological alterations caused solely by TDP-43 mutation, i.e., the changes in human iPSC-derived neurons with TDP-43 mutation compared with those with the same genetic background except TDP-43 mutation. Oxidative stress induced by hydrogen peroxide administration caused the death of TDP-43 mutant-expressing motor neurons but not in sensory neurons, indicating the specific vulnerability of human iPSC-derived motor neurons with TDP-43 mutation to oxidative stress. In our model, we observed aggregate formation in a small fraction of TDP-43 mutant-expressing motor neurons, suggesting that aggregate formation seems to be related to ALS pathology but not the direct cause of cell death. This study provides basic knowledge for elucidating the pathogenesis of ALS and developing treatments for the disease., (© 2023. The Author(s).)

Colorectal cancer is one of the most common gastrointestinal tumors with good outcomes; however, with distant metastasis, the outcomes are poor. Novel treatment methods are urgently needed. Our in vitro studies indicate that dual-specificity tyrosine-regulated kinase 2 (DYRK2) functions as a tumor suppressor in colorectal cancer by regulating cell survival, proliferation, and apoptosis induction. In addition, DYRK2 expression is decreased in tumor tissues compared to nontumor tissues in colorectal cancer, indicating a correlation with clinical prognosis. In this context, we devised a novel therapeutic strategy to overexpress DYRK2 in tumors by adenovirus-mediated gene transfer. The present study shows that overexpression of DYRK2 in colon cancer cell lines by adenovirus inhibits cell proliferation and induces apoptosis in vitro. Furthermore, in mouse subcutaneous xenograft and liver metastasis models, enforced expression of DYRK2 by direct or intravenous injection of adenovirus to the tumor significantly inhibits tumor growth. Taken together, these findings show that adenovirus-based overexpression of DYRK2 could be a novel gene therapy for liver metastasis of colorectal cancer., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Genome editing techniques such as CRISPR/Cas9 have both become common gene engineering technologies and have been applied to gene therapy. However, the problems of increasing the efficiency of genome editing and reducing off-target effects that induce double-stranded breaks at unexpected sites in the genome remain. In this study, we developed a novel Cas9 transduction system, Exci-Cas9, using an adenovirus vector (AdV). Cas9 was expressed on a circular molecule excised by the site-specific recombinase Cre and succeeded in shortening the expression period compared to AdV, which expresses the gene of interest for at least 6 months. As an example, we chose hepatitis B, which currently has more than 200 million carriers in the world and frequently progresses to liver cirrhosis or hepatocellular carcinoma. The efficiencies of hepatitis B virus genome disruption by Exci-Cas9 and Cas9 expression by AdV directly (Avec) were the same, about 80-90%. Furthermore, Exci-Cas9 enabled cell- or tissue-specific genome editing by expressing Cre from a cell- or tissue-specific promoter. We believe that Exci-Cas9 developed in this study is useful not only for resolving the persistent expression of Cas9, which has been a problem in genome editing, but also for eliminating long-term DNA viruses such as human papilloma virus., (© 2021. The Author(s).)

Lysosomal degradation plays a crucial role in the metabolism of biological macromolecules supplied by autophagy. The regulation of the autophagy-lysosome system, which contributes to intracellular homeostasis, chemoresistance, and tumor progression, has recently been revealed as a promising therapeutic approach for pancreatic cancer (PC). However, the details of lysosomal catabolic function in PC cells have not been fully elucidated. In this study, we show evidence that suppression of acid alpha-glucosidase (GAA), one of the lysosomal enzymes, improves chemosensitivity and exerts apoptotic effects on PC cells through the disturbance of expression of the transcription factor EB. The levels of lysosomal enzyme were elevated by gemcitabine in PC cells. In particular, the levels of GAA were responsive to gemcitabine in a dose-dependent and time-dependent manner. Small interfering RNA against the GAA gene (siGAA) suppressed cell proliferation and promoted apoptosis in gemcitabine-treated PC cells. In untreated PC cells, we observed accumulation of depolarized mitochondria. Gene therapy using adenoviral vectors carrying shRNA against the GAA gene increased the number of apoptotic cells and decreased the tumor growth in xenograft model mice. These results indicate that GAA is one of the key targets to improve the efficacy of gemcitabine and develop novel therapies for PC., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Shortening of mRNA poly(A) tails (deadenylation) to trigger their decay is mediated mainly by the CCR4-NOT deadenylase complex. While four catalytic subunits (CNOT6, 6L 7, and 8) have been identified in the mammalian CCR4-NOT complex, their individual biological roles are not fully understood. In this study, we addressed the contribution of CNOT7/8 to viability of primary mouse embryonic fibroblasts (MEFs). We found that MEFs lacking CNOT7/8 expression [ Cnot7/8 -double knockout (dKO) MEFs] undergo cell death, whereas MEFs lacking CNOT6/6L expression ( Cnot6/6l -dKO MEFs) remain viable. Co-immunoprecipitation analyses showed that CNOT6/6L are also absent from the CCR4-NOT complex in Cnot7/8 -dKO MEFs. In contrast, either CNOT7 or CNOT8 still interacts with other subunits in the CCR4-NOT complex in Cnot6/6l -dKO MEFs. Exogenous expression of a CNOT7 mutant lacking catalytic activity in Cnot7/8 -dKO MEFs cannot recover cell viability, even though CNOT6/6L exists to some extent in the CCR4-NOT complex, confirming that CNOT7/8 is essential for viability. Bulk poly(A) tail analysis revealed that mRNAs with longer poly(A) tails are more numerous in Cnot7/8 -dKO MEFs than in Cnot6/6l -dKO MEFs. Consistent with elongated poly(A) tails, more mRNAs are upregulated and stabilized in Cnot7/8 -dKO MEFs than in Cnot6/6l -dKO MEFs. Importantly, Cnot6/6l -dKO mice are viable and grow normally to adulthood. Taken together, the CNOT7/8 catalytic subunits are essential for deadenylation, which is necessary to maintain cell viability, whereas CNOT6/6L are not.

Selective autophagy ensures the removal of specific soluble proteins, protein aggregates, damaged mitochondria, and invasive bacteria from cells. Defective autophagy has been directly linked to metabolic disorders. However how selective autophagy regulates metabolism remains largely uncharacterized. Here we show that a deficiency in selective autophagy is associated with suppression of lipid oxidation. Hepatic loss of Atg7 or Atg5 significantly impairs the production of ketone bodies upon fasting, due to decreased expression of enzymes involved in β-oxidation following suppression of transactivation by PPARα. Mechanistically, nuclear receptor co-repressor 1 (NCoR1), which interacts with PPARα to suppress its transactivation, binds to the autophagosomal GABARAP family proteins and is degraded by autophagy. Consequently, loss of autophagy causes accumulation of NCoR1, suppressing PPARα activity and resulting in impaired lipid oxidation. These results suggest that autophagy contributes to PPARα activation upon fasting by promoting degradation of NCoR1 and thus regulates β-oxidation and ketone bodies production.

Multiple-system atrophy (MSA) is a neurodegenerative disease characterized by autonomic failure with various combinations of parkinsonism, cerebellar ataxia, and pyramidal dysfunction. We previously reported that functionally impaired variants of COQ2, which encodes an essential enzyme in the biosynthetic pathway of coenzyme Q10, are associated with MSA. Here, we report functional deficiencies in mitochondrial respiration and the antioxidative system in induced pluripotent stem cell (iPSC)-derived neurons from an MSA patient with compound heterozygous COQ2 mutations. The functional deficiencies were rescued by site-specific CRISPR/Cas9-mediated gene corrections. We also report an increase in apoptosis of iPSC-derived neurons from MSA patients. Coenzyme Q10 reduced apoptosis of neurons from the MSA patient with compound heterozygous COQ2 mutations. Our results reveal that cellular dysfunctions attributable to decreased coenzyme Q10 levels are related to neuronal death in MSA, particularly in patients with COQ2 variants, and may contribute to the development of therapy using coenzyme Q10 supplementation.

The complicated replication mechanisms of hepatitis B virus (HBV) have impeded HBV studies and anti-HBV therapy development as well. Herein we report efficient genome replication of HBV applying adenovirus vectors (AdVs) showing high transduction efficiency. Even in primary hepatocytes derived from humanized mice the transduction efficiencies using AdVs were 450-fold higher compared than those using plasmids. By using an expression unit consisting of the CMV promoter, 1.03-copy HBV genome and foreign poly(A) signal, we successfully generated an improved AdV (HBV103-AdV) that efficiently provided 58 times more pregenomic RNA than previously reported AdVs. The HBV103-AdV-mediated HBV replication was easily and precisely detected using quantitative real-time PCR in primary hepatocytes as well as in HepG2 cells. Notably, when the AdV containing replication-defective HBV genome of 1.14 copy was transduced, we observed that HBV DNA-containing circular molecules (pseudo-ccc DNA) were produced, which were probably generated through homologous recombination. However, the replication-defective HBV103-AdV hardly yielded the pseudo-ccc, probably because the repeated sequences are vey short. Additionally, the efficacies of entecavir and lamivudine were quantitatively evaluated using this system at only 4 days postinfection with HBV103-AdVs. Therefore, this system offers high production of HBV genome replication and thus could become used widely., Competing Interests: The authors declare no competing financial interests.

Temporal genetic modification of mice using the ligand-inducible Cre/loxP system is an important technique that allows the bypass of embryonic lethal phenotypes and access to adult phenotypes. In this study, we generated a tamoxifen-inducible Cre-driver mouse strain for the purpose of widespread and temporal Cre recombination. The new line, named CM32, expresses the GFPneo-fusion gene in a wide variety of tissues before FLP recombination and tamoxifen-inducible Cre after FLP recombination. Using FLP-recombined CM32 mice (CM32Δ mice) and Cre reporter mouse lines, we evaluated the efficiency of Cre recombination with and without tamoxifen administration to adult mice, and found tamoxifen-dependent induction of Cre recombination in a variety of adult tissues. In addition, we demonstrated that conditional activation of an oncogene could be achieved in adults using CM32Δ mice. CM32Δ;T26 mice, which harbored a Cre recombination-driven, SV40 large T antigen-expressing transgene, were viable and fertile. No overt phenotype was found in the mice up to 3 months after birth. Although they displayed pineoblastomas (pinealoblastomas) and/or thymic enlargement due to background Cre recombination by 6 months after birth, they developed epidermal hyperplasia when administered tamoxifen. Collectively, our results suggest that the CM32Δ transgenic mouse line can be applied to the assessment of adult phenotypes in mice with loxP-flanked transgenes.

Hepatitis C virus (HCV) core plays a key role in viral particle formation and is involved in viral pathogenesis. Here, constructs for single-domain intrabodies consisting of variable regions derived from mouse mAbs against HCV core were established. Expressed single-domain intrabodies were shown to bind to HCV core, and inhibit the growth of cell culture-produced HCV derived from JFH-1 (genotype 2a) and a TH (genotype 1b)/JFH-1 chimera. Adenovirus vectors expressing intrabodies were also capable of reducing HCV propagation. Intrabody expression did not affect viral entry or genome replication of single-round infectious trans-complemented HCV particles. However, intrabody expression reduced intracellular and extracellular infectious titres in CD81-defective Huh7-25 cells transfected with the HCV genome, suggesting that these intrabodies impair HCV assembly. Furthermore, intrabody expression suppressed HCV core-induced NFκB promoter activity. These intrabodies may therefore serve as tools for elucidating the role of core in HCV pathogenesis.

The CCR4-NOT complex is conserved in eukaryotes and is involved in mRNA metabolism, though its molecular physiological roles remain to be established. We show here that CNOT3-depleted mouse embryonic fibroblasts (MEFs) undergo cell death. Levels of other complex subunits are decreased in CNOT3-depleted MEFs. The death phenotype is rescued by introduction of wild-type (WT), but not mutated CNOT3, and is not suppressed by the pan-caspase inhibitor, zVAD-fluoromethylketone. Gene expression profiling reveals that mRNAs encoding cell death-related proteins, including receptor-interacting protein kinase 1 (RIPK1) and RIPK3, are stabilized in CNOT3-depleted MEFs. Some of these mRNAs bind to CNOT3, and in the absence of CNOT3 their poly(A) tails are elongated. Inhibition of RIPK1-RIPK3 signaling by a short-hairpin RNA or a necroptosis inhibitor, necrostatin-1, confers viability upon CNOT3-depleted MEFs. Therefore, we conclude that CNOT3 targets specific mRNAs to prevent cells from being disposed to necroptotic death.

Non-coding small RNAs are involved in many physiological responses including viral life cycles. Adenovirus-encoding small RNAs, known as virus-associated RNAs (VA RNAs), are transcribed throughout the replication process in the host cells, and their transcript levels depend on the copy numbers of the viral genome. Therefore, VA RNAs are abundant in infected cells after genome replication, i.e. during the late phase of viral infection. Their function during the late phase is the inhibition of interferon-inducible protein kinase R (PKR) activity to prevent antiviral responses; recently, mivaRNAs, the microRNAs processed from VA RNAs, have been reported to inhibit cellular gene expression. Although VA RNA transcription starts during the early phase, little is known about its function. The reason may be because much smaller amount of VA RNAs are transcribed during the early phase than the late phase. In this study, we applied replication-deficient adenovirus vectors (AdVs) and novel AdVs lacking VA RNA genes to analyze the expression changes in cellular genes mediated by VA RNAs using microarray analysis. AdVs are suitable to examine the function of VA RNAs during the early phase, since they constitutively express VA RNAs but do not replicate except in 293 cells. We found that the expression level of hepatoma-derived growth factor (HDGF) significantly decreased in response to the VA RNAs under replication-deficient condition, and this suppression was also observed during the early phase under replication-competent conditions. The suppression was independent of mivaRNA-induced downregulation, suggesting that the function of VA RNAs during the early phase differs from that during the late phase. Notably, overexpression of HDGF inhibited AdV growth. This is the first report to show the function, in part, of VA RNAs during the early phase that may be contribute to efficient viral growth.

Several autophagy proteins contain an LC3-interacting region (LIR) responsible for their interaction with Atg8 homolog proteins. Here, we show that ALFY binds selectively to LC3C and the GABARAPs through a LIR in its WD40 domain. Binding of ALFY to GABARAP is indispensable for its recruitment to LC3B-positive structures and, thus, for the clearance of certain p62 structures by autophagy. In addition, the crystal structure of the GABARAP-ALFY-LIR peptide complex identifies three conserved residues in the GABARAPs that are responsible for binding to ALFY. Interestingly, introduction of these residues in LC3B is sufficient to enable its interaction with ALFY, indicating that residues outside the LIR-binding hydrophobic pockets confer specificity to the interactions with Atg8 homolog proteins.

First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

First-generation adenovirus vectors (FG AdVs) are widely used in basic studies and gene therapy. However, virus-associated (VA) RNAs that act as small-interference RNAs are indeed transcribed from the vector genome. These VA RNAs can trigger the innate immune response. Moreover, VA RNAs are processed to functional viral miRNAs and disturb the expressions of numerous cellular genes. Therefore, VA-deleted AdVs lacking VA RNA genes would be advantageous for basic studies, both in vitro and in vivo. Here, we describe an efficient method of producing VA-deleted AdVs. First, a VA RNA-substituted "pre-vector" lacking the original VA RNA genes but alternatively possessing an intact VA RNA region flanked by a pair of FRTs was constructed. VA-deleted AdVs were efficiently obtained by infecting 293hde12 cells, which highly express FLP, with the pre-vector. The resulting transduction titers of VA-deleted AdVs were sufficient for practical use. Therefore, VA-deleted AdVs may be substitute for current FG AdV.

In this study, we compared the entry processes of trans-complemented hepatitis C virus particles (HCVtcp), cell culture-produced HCV (HCVcc) and HCV pseudoparticles (HCVpp). Anti-CD81 antibody reduced the entry of HCVtcp and HCVcc to almost background levels, and that of HCVpp by approximately 50%. Apolipoprotein E-dependent infection was observed with HCVtcp and HCVcc, but not with HCVpp, suggesting that the HCVtcp system is more relevant as a model of HCV infection than HCVpp. We improved the productivity of HCVtcp by introducing adapted mutations and by deleting sequences not required for replication from the subgenomic replicon construct. Furthermore, blind passage of the HCVtcp in packaging cells resulted in a novel mutation in the NS3 region, N1586D, which contributed to assembly of infectious virus. These results demonstrate that our plasmid-based system for efficient production of HCVtcp is beneficial for studying HCV life cycles, particularly in viral assembly and infection., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pulmonary fibrosis has high rates of mortality and morbidity, but there is no established therapy at present. We demonstrate here that bleomycin-induced pulmonary fibrosis in mice is ameliorated by intratracheal administration of keratinocyte growth factor (KGF)-expressing adenovirus vector. Progressive pulmonary fibrosis was created by continuous subcutaneous administration of 120 mg/kg of bleomycin subcutaneously using an osmotic pump twice from Day 1 to 7 and Day 29 to 35. The mice initially exhibited subpleural fibrosis and then exhibited advanced fibrosis in the parenchyma of the lungs. These histopathological changes were accompanied by reduced lung compliance (0.041 ± 0.011 versus 0.097 ± 0.004; P < 0.001), reduced messenger expression of surfactant proteins, and reduced KGF messenger expression in the lungs at 4 weeks compared with naive group. Intratracheal instillation of Ad-KGF at 1 week after the first administration of bleomycin increased KGF mRNA expression in the lungs compared with the fibrosis-induced mice that received saline alone. The phenotype was associated with alveolar epithelial cell proliferation, increased pulmonary compliance (0.062 ± 0.005 versus 0.041 ± 0.011; P = 0.023), and decreased mortality (survival rate on Day 56: 68.8% versus 0%; P = 0.002), compared with mice receiving only the saline vehicle. These observations suggest the therapeutic utility of a KGF-expressing adenoviral vector for pulmonary fibrosis.

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 (Le(b)) 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 Le(b) on the epithelial surface augments TFSS-dependent H. pylori pathogenicity by triggering the production of proinflammatory cytokines and precancer-related factors. We successfully generated Le(b)-positive cell lineages by transfecting Le(b)-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 Le(b)-positive cells with WT H. pylori but not with babA or TFSS deletion mutants. This increased mRNA expression was abrogated when Le(b)-negative cells were infected with WT H. pylori. Thus, H. pylori can exploit BabA-Le(b) 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.

Cre and FLP recombinases mediate not only specific deletions and insertions, but also the recombinase-mediated cassette exchange (RMCE) reaction, which is used in cell biotechnology including ES cells and mouse genetics. However, comparison of efficiencies for Cre and FLP in RMCE has not been made. We here examined the detailed process of RMCE with Cre and FLP in vitro using mutant loxP 2272 and three mutant FRTs (FRT G, FRT H, and FRT F3) and then quantitatively compared the RMCE reactions in vitro. Interestingly, in the in vitro reactions, the RMCE efficiency of Cre reached a plateau level of approximately 5% and did not proceed further, whereas that of FLPe reached approximately 12-13%, showing that FLPe reached a higher level of efficiency than Cre possibly when they were supplied at a very high concentration. Moreover, we quantitatively compared the production efficiency of E1-deleted adenovirus vector using the RMCE method with Cre or FLP. The results showed that FLPe was again found more efficient than Cre in RMCE reaction. Thus, although Cre is considered more active than, or similar to, FLPe, it may not be necessarily true for RMCE reaction. Possible reasons explaining these results are discussed., (© 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.)

Tissue-/cancer-specific promoters for use in adenovirus vectors (AdVs) are valuable for elucidating specific gene functions and for use in gene therapy. However, low activity, non-specific expression and size limitations in the vector are always problems. Here, we developed a 'double-unit' AdV containing the Cre gene under the control of an α-fetoprotein promoter near the right end of its genome and bearing a compact 'excisional-expression' unit consisting of a target cDNA 'upstream' of a potent promoter between two loxPs near the left end of its genome. When Cre was expressed, the expression unit was excised as a circular molecule and strongly expressed. Undesired leak expression of Cre during virus preparation was completely suppressed by a dominant-negative Cre and a short-hairpin RNA against Cre. Using this novel construct, a very strict specificity was maintained while achieving a 40- to 90-fold higher expression level, compared with that attainable using a direct specific promoter. Therefore, the 'double-unit' AdV enabled us to produce a tissue-/cancer-specific promoter in an AdV with a high expression level and strict specificity.

A loss-of-function mutation in the APC gene initiates colorectal carcinogenesis. Although the molecular mechanism of tumor initiation is complex, several modifier genes have been identified using mouse models, including the ApcMin mouse. Among the familial adenomatous polyposis mouse lines carrying a truncation mutation at codon 580 in Apc (Apc580D), one line (line19-Apc(580D/+)) showed a remarkably reduced incidence of intestinal adenomas (<5% compared with other lines). Extensive genetic analysis identified a deletion in the alpha-catenin (Ctnna1) gene as the cause of this suppression. Notably, the suppression only occurred when the Ctnna1 deletion was in cis-configuration with the Apc580D mutation. In all adenomas generated in line19-Apc(580D/+), somatic recombination between the Apc and Ctnna1 loci retained the wild-type Ctnna1 allele. These data strongly indicate that simultaneous inactivation of alpha-catenin and Apc during tumor initiation suppresses adenoma formation in line19-Apc(580D/+), suggesting that alpha-catenin plays an essential role in the initiation of intestinal adenomas. Although accumulating evidence obtained from human colon tumors with invasive or metastatic potential has established a tumor-suppressive role for alpha-catenin in late-stage tumorigenesis, the role of alpha-catenin in the initiation of intestinal tumorigenesis is not well documented, especially compared with that of beta-catenin. A mouse model used in this study focused on the early stage of tumor initiation and clearly indicated an essential role for alpha-catenin. Thus, alpha-catenin has dual roles in intestinal tumorigenesis, a supporting role in tumor initiation, and a suppressive role in tumor progression.

First-generation (FG) adenoviral vectors (AdVs) have been widely used not only for gene therapy but also for basic studies. Because vectors of this type lack the E1A gene that is essential for the expression of other viral genes, their expression levels in target cells have been considered low. However, we found that the viral pIX gene, located immediately downstream of the inserted expression unit of the transgene, was significantly coexpressed with the transgene in cells infected with FG AdV. Whereas CAG and SRalpha promoters activated the pIX promoter considerably through their enhancer effects, the EF1alpha promoter hardly did. Moreover, when the expression unit was inserted in the rightward orientation, not only the pIX protein but also a fusion protein consisting of the N-terminal part of transgene product and pIX were sometimes coexpressed with the transgene product through an aberrant splicing mechanism. In in vivo experiments, a LacZ-expressing AdV bearing the CAG promoter caused an elevation of alanine aminotransferase, but an AdV bearing the EF1alpha promoter produced no detectable levels. Whereas the FG AdV expressing human growth hormone under the control of the CAG promoter maintained a high hormone level for less than 1 month, the FG AdV under the control of the EF1alpha promoter maintained a high level for at least 6 months. These results suggest that pIX coexpression may be one of the main causes of AdV-induced immune responses, and that the EF1alpha promoter is probably valuable for the long-term expression of FG AdV. Thus, the in vivo utility of FG AdV should be reevaluated.

At present there is no known effective pharmacological therapy for acute lung injury (ALI). Because keratinocyte growth factor (KGF) promotes epithelial cell growth, intratracheal administration of KGF has the possibility of restoring lung tissue integrity in injured lungs and improving patient outcomes. However, treatment using recombinant KGF protein is limited by its short effective duration. Thus, we investigated the effectiveness of intratracheal KGF gene transduction using adenoviral vector in ALI. We constructed an adenoviral vector expressing mouse KGF (mKGF), and 1.0 x 10(9 ) plaque-forming units of mKGF cDNA-expressing (Ad-KGF) and control (Ad-1w1) adenoviral vector was intratracheally instilled, using a MicroSprayer, into anesthetized BALB/c mice. Three days later, the mice were exposed to >90% oxygen for 72 hr, and the effect of KGF on hyperoxia-induced lung injury was examined. In the Ad-KGF group, KGF was strongly expressed in the airway epithelial cells, while peribronchiolar and alveolar inflammation caused by adenoviral vector instillation was minimal. The KGF overexpression not only induced proliferation of surfactant protein C-positive cuboidal cells, especially in the terminal bronchiolar and alveolar walls, but also prevented lung injury including intraalveolar exudation/hemorrhage, albumin permeability increase, and pulmonary edema. The arterial oxygen tension and the survival rate were significantly higher in the KGF-transfected group. These findings suggest that KGF gene transduction into the airway epithelium is a promising potential treatment for ALI.

DNA viruses are often used as vectors for foreign gene expression, but large DNA region from cloned or authentic viral genomes must usually be handled to generate viral vectors. Here, we present a unique system for generating adenoviral vectors by directly substituting a gene of interest in a small transfected plasmid with a replaced gene in a replicating viral genome in Cre-expressing 293 cells using the recombinase-mediated cassette exchange (RMCE) reaction. In combination with a positive selection of the viral cis-acting packaging signal connected with the gene of interest, the purpose vector was enriched to 97.5 and 99.8% after three and four cycles of infection, respectively. Our results also showed that the mutant loxP V (previously called loxP 2272), a variant target of Cre used in the RMCE reaction, was useful as a non-compatible mutant to wild-type loxP. This method could be useful for generating not only a large number of adenovirus vectors simultaneously, but also other DNA virus vectors including helper-dependent adenovirus vector.

The site-specific recombinase Cre has often been used for on/off regulation of expression of transgenes introduced into the mammalian chromosome. However, this method is only applicable to the regulation of a single gene and cannot be used to simultaneously regulate two genes, because site-specific recombination occurs from the target loxP sequence of one regulating unit to the loxP sequence of any other unit and would eventually disrupt the structure of both regulating units. We previously reported a mutant loxP sequence with a two base substitution called loxP V (previously called loxP 2272), with which wild-type loxP cannot recombine but with which the identical mutant loxP recombines efficiently. In the present study we isolated cell lines bearing two regulating units on a chromosome containing a pair of wild-type loxP sequences or mutant loxP V sequences. After infection with Cre-expressing recombinant adenovirus AxCANCre, expression of a gene in each regulating unit was simultaneously turned on and off. Southern analyses showed that both regulating units were processed simultaneously and independently, even after infection with a limited amount of AxCANCre. The results showed that simultaneous regulation of gene expression on a mammalian chromosome mediated by Cre can be achieved by using mutant loxP V and wild-type loxP. This method may be a useful approach for conditional transgenic/knockout animals and investigation of gene function involving two genes simultaneously. Another possible application is for preparation of a new packaging cell line of viral vectors through simultaneous production of toxic viral genes.

Overexpression of CD30 and constitutive NF-kappaB activation characterizes tumor cells of Hodgkin's 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-kappaB 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 IkappaBalpha mutant blocks NF-kappaB activation, down regulates IL-13 expression and induces apoptosis. Thus, in H-RS cells, ligand-independent activation of CD30 signaling drives NF-kappaB activation and this leads to constitutive cytokine expression, which provides a molecular basis for HD. Inhibition of NF-kappaB activation by adenovirus vector-mediated gene transfer may provide a novel strategy of cell- and target molecule-specific therapy for patients with HD.

We previously constructed an adenovirus vector carrying a gene encoding a soluble form of fusion protein, consisting of the extracellular portion of cytotoxic lymphocyte antigen 4 (CTLA4) and the Fc portion of human immunoglobulin G1 (Adex1CACTLA4IgG). Murine type II collagen-induced arthritis (CIA) was treated with Adex1CACTLA4IgG. A single intraarticular injection of 1 x 10(5) PFU was able to support serum CTLA4IgG at more than 10 microg/ml for at least 12 weeks and was able to inhibit the CIA clinically and histologically. In contrast, intravenous, intramuscular, or subcutaneous injection of 1 x 10(5) PFU was unable to support a significant level of serum CTLA4IgG and thus was unable to inhibit the development of arthritis. Thus, we demonstrated that (1) a low-dose intraarticular injection of Adex1CACTLA4IgG was effective in delaying the onset of CIA and reducing the severity of arthritis; (2) an intraarticular (knee joint) injection of Adex1CACTLA4IgG effectively blocked the development of arthritis in distal paws; (3) the inhibitory effect of Adex1CACTLA4IgG lasted at least up to 20 weeks; (4) although serum CTLA4IgG at more than 10 microg/ml persisted for at least 12 weeks, mice treated by intraarticular injection of Adex1CACTLA4IgG were not anergic to adenovirus and were able to mount antibody responses against various antigens.

CTLA4IgG was shown to inhibit the costimulatory signal for T cell activation by interfering with the ligation of CD28 and B7-1 or B7-2. To inhibit various immune responses including acute cellular rejection of allografts, a certain level of serum CTLA4IgG should be maintained for an appropriate period. We previously reported on an adenovirus vector containing CTLA4IgG, which we designated Adex1CACTLA4IgG. Adex1CACTLA4IgG was able to maintain a significant level of serum CTLA4IgG for a long period on intravenous injection, which in turn inhibited various immune responses including protective immunity against infectious agents. To overcome the inhibitory effect, we constructed a new adenovirus vector, Adex1CALoxCTLA4IgGLox, by cloning CTLA4IgG cDNA between two loxP sequences under the control of the CAG promoter. We demonstrated that the administration of adenovirus vector containing Cre recombinase gene (Adex1CACre) at the desired time induced Cre-mediated recombination within a gene derived from Adex1CALoxCTLA4IgGLox vector, and the cDNA of CTLA4IgG was excised from the transduced gene and terminated the expression of CTLA4IgG in vitro and in vivo. More importantly, we also demonstrated that the long-term acceptance of allografts was achieved after the termination of CTLA4IgG expression, while the immune response against adenovirus was restored.