Drosophila RecQ5 is involved in proper progression of early spermatogenesis.

RecQ5, a member of the conserved RecQ DNA helicase family, is required for the maintenance of genome stability. The human RECQL5 gene is expressed ubiquitously in almost all tissues, with strong expression in the testes (Shimamoto et al., 2000). However, it remains to be elucidated in which cells RecQ5 is expressed and how RecQ5 functions in the testes. In this present study we analyzed the expression of RecQ5 in Drosophila testes. The RecQ5 protein was specifically expressed in germline cells in larval, pupal, and adult testes. Drosophila RecQ5 was localized in nuclei of male germline stem cells, spermatogoniablasts, spermatogonia, and early spermatocytes. As growth of the early spermatocyte proceeded, the amount of RecQ5 increased in the nuclei. However, before maturation of the spermatocyte, the level of RecQ5 declined. Thus, RecQ5 expression was regulated. Furthermore, we compared recq5 mutant testes with the wild-type ones. The most conspicuous alterations were swelling of the apical region of and an increase in the number of spermatocytes in the recq5 testis, suggesting a relative accumulation of spermatocytes in the recq5 mutant testes. Therefore, Drosophila RecQ5 may contribute to the proper progression from germline stem cells to spermatocytes for maintenance of genome stability.

RecQ5 protein translocation into the nucleus by a nuclear localization signal.

RecQ5, a member of the RecQ helicase family, maintains genome stability via participation in many DNA metabolic processes including DNA repair, DNA resolution, and RNA transcription, processes occurring in the nucleus. Previously, we reported that RecQ5 and Rad51, also involved in DNA repair, become co-localized in nuclei when co-expressed in cultured cells. Nuclear localization of RecQ5 appears to be important for cellular function along with Rad51. However, little is known about the nuclear localization of RecQ5. Here, we generated enhanced green fluorescent protein (EGFP)-tagged RecQ5 transgenic flies and analyzed localization of this protein in early embryos by live imaging. In syncytial embryos, RecQ5 was localized synchronously in interphase nuclei, and spread repeatedly over the embryos in mitosis. Thus, RecQ5 was transported into nuclei at the early interphase. Furthermore, we examined the subcellular localization of a series of truncated forms of Drosophila RecQ5 in cultured cells to determine the nuclear localization signal (NLS). Entire coding or deleted RecQ5 sequences of various sizes were ligated into EGFP vectors, which were then used to transfect cultured Drosophila cells. The region responsible for nuclear localization of Drosophila RecQ5 contained a short stretch of positively charged basic amino acids, 2 of which were particularly important for the nuclear localization. This stretch was sufficient for nuclear localization when fused with EGFP. Although the NLS of Drosophila RecQ5 was distinct from that of human RECQL5 in terms of position and amino acid sequence, this fly RecQ5 protein was translocated into the nucleus by an NLS.

[Role of RecQ5 in syncytial blastoderm of the Drosophila embryo].

RecQ5 belongs to the family of RecQ DNA helicases. There are 5 RecQ homologs in mammals, and defects in 3 of them (BLM, WRN, and RECQL4) give rise to cancer predisposition syndromes in humans. Although no human disease has yet been genetically linked to a mutation in RecQ5, this enzyme is thought to have unique functions, based on its ubiquitous expression profile and specific C-terminal amino acid sequence, both of which are very different from those of other RecQ DNA helicase family members. The analysis of MEF and ES cells derived from RecQ5-deficient mice investigated by Hu et al. suggested an important role for RecQ5 in the DNA metabolism of the early embryo. However, it is unknown how RecQ5 deficiency destabilizes DNA. To address the DNA instabilities in RecQ5-deficient animals, we chose Drosophila melanogaster which has simple checkpoint systems in its syncytial embryos. By analyzing Drosophila syncytial embryos, we demonstrated that the loss of RecQ5 increased the frequency of spontaneous mitotic defects such as anaphase bridge formation. A pair of daughter nuclei that had been linked by such DNA bridges was simultaneously eliminated via a Chk2-dependent pathway. These findings suggest that the lack of RecQ5 causes spontaneous double-strand DNA breaks. RecQ5 may thus function in the resolution of anaphase DNA bridges during mitosis in syncytial embryo.

Anaphase DNA bridges induced by lack of RecQ5 in Drosophila syncytial embryos.

Drosophila melanogaster RecQ5, a member of the RecQ family, is expressed in early embryos. The loss of maternally-derived RecQ5 leads to spontaneous mitotic defects in syncytial embryos. We demonstrate that the mitotic defects are derived from anaphase DNA bridges. Pairs of daughter nuclei that had been linked by the bridges concurrently exited from the cycle and were eliminated by Chk2-dependent centrosome inactivation. These results suggest that the lack of RecQ5 leads to spontaneous double-stranded DNA breaks (DSBs). RecQ5 may function in the resolution of anaphase DNA bridges during mitosis or in DSB repair during interphase in syncytial Drosophila embryos.

Efficient osteoblast differentiation from mouse bone marrow stromal cells with polylysin-modified adenovirus vectors.

Bone marrow stromal cells (BMSCs) are expected to be a source for tissue regeneration because they can differentiate into multiple cell types. Establishment of efficient gene transfer systems for BMSCs is essential for their application to regenerative medicine. In this study, we compared the transduction efficiency in mouse primary BMSCs by using fiber-modified adenovirus (Ad) vectors, and demonstrated that AdK7, which harbors a polylysin (K7) peptide in the C-terminus of the fiber knob, could efficiently express a transgene in BMSCs. Notably, AdK7 robustly drove transgene expression in more than 90% of the BMSCs at 3,000 vector particles/cell. Furthermore, we showed that in vitro and in vivo osteogenic potential of BMSCs was dramatically promoted by the transduction of Runx2 gene using AdK7. These results indicate that this transduction system could be a powerful tool for therapeutic applications based on BMSCs.

Loss of RecQ5 leads to spontaneous mitotic defects and chromosomal aberrations in Drosophila melanogaster.

RecQ5 belongs to the RecQ DNA helicase family that includes genes causative of Bloom, Werner, and Rothmund-Thomson syndromes. Although no human disease has been genetically linked to a mutation in RecQ5, Drosophila melanogaster RecQ5 is highly expressed in early embryos, suggesting an important role for it in the DNA metabolism of the early embryo. In this present study, we generated RecQ5 mutants in D. melanogaster. Embryos lacking maternally derived RecQ5 contained irregular nuclei in early embryogenesis. These irregular nuclei emerged in nuclear cycle 11-13, lost cell-cycle markers, and were located below the surface monolayer of nuclei. By time-lapse microscopy, these irregular nuclei were observed not to divide, whereas all neighboring nuclei proceeded through normal mitotic division with synchrony. These data suggest that the irregular nuclei exited from the nuclear division cycle. This phenotype is reminiscent of the effect of X-ray irradiation on wild-type embryos and was rescued by expression of RecQ5. Thus, the maternal supply of RecQ5 is important for the nuclear cycles in syncytical embryos. Furthermore, the frequencies of spontaneous and induced chromosomal aberrations were increased in RecQ5 mutant neuroblasts. These data imply that DNA damage accumulates spontaneously in RecQ5 mutants. Therefore, endogenous genomic damage may be produced in Drosophila development, and RecQ5 would be involved in the maintenance of genomic stability by suppressing the accumulation of DNA damage.

Adenoviral expression of suppressor of cytokine signaling-1 reduces adenovirus vector-induced innate immune responses.

Adenovirus (Ad) vectors are among the most commonly used viral vectors in gene therapy clinical trials. However, the application of Ad vectors has been limited to local injection in many cases, because the systemic administration of Ad vectors triggers innate immune responses such as inflammatory cytokine production and tissue damage. To overcome this limitation, it will be necessary to develop safer Ad vectors less likely to induce the innate immune response. In the present study, we demonstrated that a suppressor of cytokine signaling-1 (SOCS1)-expressing Ad vector, Ad-SOCS1, reduces the innate immune response induced by Ad vectors. RAW264.7-SOCS1, a macrophage-like cell line that stably expresses SOCS1, was shown to produce lower levels of inflammatory cytokines after the transduction of Ad vectors. The systemic administration of Ad-SOCS1 into mice elicited the reduced production of inflammatory cytokines, as compared with that elicited by control Ad vectors, i.e., luciferase-expressing Ad vector, Ad-L2. Furthermore, the coadministration of Ad-L2 with Ad-SOCS1 attenuated inflammatory cytokine production and liver toxicity as compared with injection with Ad-L2 alone, and this was achieved without the suppression of luciferase production in various organs. The JAK/STAT pathway was involved in Ad vector-mediated cytokine production, which was impaired by the overexpression of SOCS1. These findings indicate that Ad-SOCS1 could be useful for reducing Ad vector-mediated innate immunity.

Efficient adenovirus vector-mediated PPAR gamma gene transfer into mouse embryoid bodies promotes adipocyte differentiation.

BACKGROUND: Establishment of a transient gene delivery system, such as adenovirus (Ad) vectors, into embryonic stem (ES) cells and their aggregation form, embryoid bodies (EBs), is essential for its application in regenerative medicine because the transgene should not be integrated in the host genome. In this study, we optimized Ad vector-mediated transduction into EBs, and examined whether Ad vector-mediated transduction of adipogenesis-related gene into EBs could promote the adipocyte differentiation. METHODS: We prepared beta-galactosidase-expressing Ad vectors under the control of four different promoters (cytomegalovirus (CMV), rouse sarcoma virus, human elongation factor-1alpha, and CMV enhancer/beta-actin promoter (CA)) to estimate the transduction efficiency. Adipocyte differentiation efficiency by transduction of the PPAR gamma or C/EBP alpha gene into EBs was examined. RESULTS: Of the four promoters tested, the CA promoter exhibited the highest transduction efficiency in the EBs. However, Ad vector-mediated transduction was observed only in the periphery of the EBs. When repeated transduction by Ad vector was performed, gene expression was observed even in the interior of EBs as well. When EB-derived single cells were transduced by an Ad vector containing the CA promoter, more than 90% of the cells were transduced. Furthermore, Ad vector-mediated PPAR gamma gene transduction into EBs led to more efficient differentiation into adipocytes than could untransduced EBs, examined in terms of lipogenic enzyme activities and accumulation of the lipid droplets. CONCLUSIONS: Ad vector-mediated transduction into EBs could be a valuable tool for molecular switching of cell differentiation and could be applied to regenerative medicine.

Modification of pIX or hexon based on fiberless Ad vectors is not effective for targeted Ad vectors.

Adenovirus (Ad) vector application in gene therapy is limited by its naïve tropism. We previously developed protein IX (pIX)-modified and hexon-modified Ad vectors in order to alter Ad vector tropism. However, these modified Ad vectors failed to infect cells with the foreign ligands displayed in the pIX or hexon. We hypothesized that steric hindrance by fiber proteins might have prevented the ligand-mediated transduction, as fibers are the outmost capsid proteins of Ad vectors. Therefore, we generated a series of fiberless Ad vectors and investigated their gene expression properties. Unexpectively, however, pIX- or hexon-modified fiberless Ad vector did not achieve any gene expression (the gene expression level by these vectors was similar to the background level). These results might be caused by the fact that the fiberless particles were weaker against physical burdens. To the best of our knowledge, this study is the first reported attempt to develop fiberless Ad vectors containing foreign ligands in the pIX or hexon region. The drawback of the lower stability of fiberless Ad vectors must be overcome to develop targeted Ad vectors based on such vectors. This study could provide basic information for the development of effective targeted Ad vectors.

Innate immune response induced by gene delivery vectors.

Gene therapy is a clinical strategy that has the potential to treat an array of genetic and nongenetic diseases. Vectors for gene transfer are the essential tools of gene therapy. For gene therapy to be successful, an appropriate amount of the therapeutic gene must be delivered into the target cells without substantial toxicity. A major limitation of the use of gene therapy vectors is the innate immune responses triggered by systemic administration of such vectors. It is essential to overcome vector-mediated innate immune responses, such as production of inflammatory cytokines, the maturation of antigen-presenting cells and tissue damage, because the induction of these responses not only shortens the period of gene expression but also leads to serious side effects. Viral vectors (for example, adenovirus (Ad) vectors) have been assumed to be more potent in inducing innate immune responses in spite of their high transduction efficiency since they contain pathogenic proteins. However, recent studies have demonstrated that not only viral vectors but also nonviral vectors, such as lipoplex (liposome/plasmid DNA complex), can induce innate immune responses. Indeed, nonviral vectors including lipoplex induce comparable or larger levels of innate immune response than viral vectors. In this review, we present an overview of the innate immune responses induced by Ad vector and lipoplex, which are used primarily for in vivo gene transfer.

Role of MyD88 and TLR9 in the innate immune response elicited by serotype 5 adenoviral vectors.

A replication-incompetent adenoviral (Ad) vector is generating interest for both gene therapy and immunotherapy. A major limitation of the use of Ad vectors is the innate immune response, which causes inflammatory cytokine production and tissue damage; however, the precise mechanism of the innate immune response remains to be clarified. Here, we show that serotype 5 human Ad vectors elicit innate immune responses through a myeloid differentiating factor 88 (MyD88)/Toll-like receptor (TLR)-9-dependent and/or -independent manner according to cell type. After stimulation with Ad vectors, the production of interleukin (IL)-6 and IL-12 was significantly decreased in MyD88- or TLR9-deficient dendritic cells (DCs), compared with wild-type DCs. In addition, the surface expression of maturation marker proteins, such as CD40, CD80, CD86, and MHC class II, in MyD88- or TLR9-deficient granulocyte-macrophage colony-stimulating factor (GM-CSF)-DCs was similar to that in wild-type DCs. On the other hand, MyD88- or TLR9-deficient peritoneal macrophages produced the same level of IL-6 as wild-type macrophages after infection with Ad vectors. We did not find any differences in the mRNA expression levels of the molecules involved in innate immunity, such as MyD88, TLR3, TLR7, and TLR9, between DCs and macrophages. The intravenous injection of luciferase-expressing Ad vectors into MyD88- or TLR9-deficient mice resulted in almost comparable levels of IL-6 and IL-12 production and luciferase expression with wild-type mice. These results suggest that Ad vectors can activate innate immunity via MyD88/TLR9-dependent and -independent mechanisms.

Caspase cascade proceeds rapidly after cytochrome c release from mitochondria in tumor necrosis factor-alpha-induced cell death.

The caspase activation cascade and mitochondrial changes are major biochemical reactions in the apoptotic cell death machinery. We attempted to clarify the temporal relationship between caspase activation, cytochrome c release, mitochondrial depolarization, and morphological changes that take place during tumor necrosis factor (TNF)-alpha-induced cell death in HeLa cells. These reactions were analyzed at the single-cell level with 0.5 - 1 min resolution by using green fluorescent protein (GFP)-variant-derived probes and chemical probes. Cytochrome c release, caspase activation, and cellular shrinkage were always observed in this order within 10 min in all dying cells. This sequence of events was thus considered a critical pathway of cell death. Mitochondrial depolarization was also observed in all dying cells observed, but frequently occurred after caspase activation and cellular shrinkage. Mitochondrial depolarization is therefore likely to be a reaction that does not induce caspase activation and subsequent cellular shrinkage. Mitochondrial changes are important for apoptotic cell death; moreover, cytochrome c release, and not depolarization, is a key reaction related to cell death. In addition, we also found that the apoptotic pathway proceeds only when cells are exposed to TNF-alpha. These findings suggest that the entire cell death process proceeds rapidly during TNF-alpha exposure.

Comparison of gene expression efficiency and innate immune response induced by Ad vector and lipoplex.

Vectors for gene expression are the essential tools for both gene therapy and basic research. There are two groups of gene therapy vectors, viral and non-viral vectors. At present, toxicity triggered by vectors is one of the major concerns for clinical trials. In general, non-viral vectors, such as plasmid DNA-cationic liposome complex (lipoplex), are thought to be safer than viral vectors, such as adenovirus (Ad) vector, although lipoplex is less efficient in term of gene expression than the Ad vector. However, there has been no study directly comparing the gene expression efficiency and safety of viral and non-viral vectors. Here, we present evidence that the Ad vector shows much more efficient gene expression and is safer than lipoplex, at least with respect to the innate immune response. After being systemically administered to mice, the Ad vector showed a transduction efficiency that was 2 to 5 log orders higher than that of lipoplex, depending on the organ. On the other hand, surprisingly, the administration of lipoplex produced a greater amount of inflammatory cytokines such as interleukin-6, interleukin-12, and tumor necrosis factor-alpha than did the administration of the Ad vector, whereas a comparable level of hepatotoxicity was induced by these vectors. The production of inflammatory cytokines induced by the injection of lipoplex was reduced when the CpG motifs were removed completely from plasmid DNA. Thus, care should be taken to ensure the innate immune response induced by gene therapy vectors, especially lipoplex.

Simultaneous real-time detection of initiator- and effector-caspase activation by double fluorescence resonance energy transfer analysis.

Fluorescence resonance energy transfer (FRET) with green fluorescent protein (GFP) variants has become widely used for biochemical research. In order to expand the choice of fluorescent range in FRET analysis, we designed various color versions of the FRET-based probes for caspase activity, in which the substrate sequence of the caspase was sandwiched by donor and acceptor fluorescent proteins, and studied the potential of these color versions as fluorescent indicators. Six color versions were constructed by a combination of cyan fluorescent protein (CFP), GFP, yellow fluorescent protein (YFP), and DsRed. Real-time monitoring in single cells revealed that all probes could detect caspase activation during tumor necrosis factor (TNF)-alpha-induced cell death as a fluorescent change. GFP-DsRed and YFP-DsRed were as sensitive as CFP-YFP, and CFP-DsRed also showed a large fluorescent change. By using two probes, CFP-DsRed and YFP-DsRed, we carried out simultaneous multi-FRET analysis and revealed that the initiator- and effector-caspases were activated almost simultaneously in TNF-alpha-induced cell death. These findings may give experimental bases for the development of novel techniques to analyze multi-events simultaneously in single cells by using FRET probes in combination.