Kenji Tamayose

Hepatocyte Growth Factor Receptor Is a Coreceptor for Adeno-Associated Virus Type 2 Infection

ABSTRACT After the first attachment of virus to the cell surface through a primary receptor, efficient entry of virus requires the presence of a coreceptor. For adeno-associated virus type 2 (AAV2) infection, heparan sulfate proteoglycan is supposed as the primary receptor, and αvβ5 integrin and FGFR1 are reported to act as coreceptors. In this study, we were able to demonstrate that hepatocyte growth factor receptor, c-Met, is also a coreceptor for AAV2 infection. AAV2-mediated transgene analyses revealed that c-Met expression significantly up-regulated transgene expression without increasing AAV2 cell binding. Moreover, a viral overlay assay elucidated the physical interaction between AAV2 and the β subunit of c-Met. These data suggest that c-Met plays the role of coreceptor for AAV2 infection by facilitating AAV2 internalization into the cytoplasm.

An AAV-derived Apaf-1 dominant negative inhibitor prevents MPTP toxicity as antiapoptotic gene therapy for Parkinson's disease

Adeno-associated virus (AAV) vector delivery of an Apaf-1-dominant negative inhibitor was tested for its antiapoptotic effect on degenerating nigrostriatal neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinsons disease. The wild-type caspase recruitment domain of Apaf-1 was used as a dominant negative inhibitor of Apaf-1 (rAAV-Apaf-1-DN-EGFP). An AAV virus vector was used to deliver it into the striatum of C57 black mice, and the animals were treated with MPTP. The number of tyrosine hydroxylase-positive neurons in the substantia nigra was not changed on the rAAV-Apaf-1-DN-EGFP injected side compared with the noninjected side. We also examined the effect of a caspase 1 C285G mutant as a dominant negative inhibitor of caspase 1 (rAAV-caspase-1-DN-EGFP) in the same model. However, there was no difference in the number of tyrosine hydroxylase-positive neurons between the rAAV-caspase-1-DN-EGFP injected side and the noninjected side. These results indicate that delivery of Apaf-1-DN by using an AAV vector system can prevent nigrostriatal degeneration in MPTP mice, suggesting that it could be a promising therapeutic strategy for patients with Parkinsons disease. The major mechanism of dopaminergic neuronal death triggered by MPTP seems to be the mitochondrial apoptotic pathway.

Isolation of Bone Marrow Stromal Cell–Derived Smooth Muscle Cells by a Human SM22α Promoter In Vitro Differentiation of Putative Smooth Muscle Progenitor Cells of Bone Marrow

Background—Bone marrow stromal cells (BMSCs) have many characteristics of mesenchymal stem cells that can differentiate into smooth muscle cells (SMCs). However, there have been few studies closely following the cell development of smooth muscle lineage among BMSCs. Methods and Results—To investigate the possible existence of a cell population committed to the SMC lineage among bone marrow adhesion cells, we tried to detect and follow the in vitro differentiation of such a cell type by using a promoter-sorting method with a human SM22&agr; promoter (−480 bp)/green fluorescent protein (GFP) construct. The construct was transfected to adhesion cells that appeared 5 days after the seeding of mononuclear cells from bone marrow. GFP was first detectable 5 days after the transfection in a cell population [Ad(G) cells], which expressed PDGF-&bgr; but neither mature (calponin) nor immature (SMemb) SMC-specific proteins at that time. However, the cells were eventually grown into individual clones that expressed SMC-specific proteins (&agr;-smooth muscle actin, calponin, and SM-1), suggesting that Ad(G) cells have partly at least progenitor properties. Because early studies have reported that PDGF-&bgr; signaling plays pivotal roles in the differentiation of mesenchymal smooth muscle progenitor cells, Ad(G) cells might be putative mesenchymal smooth muscle progenitors expressing PDGF-&bgr;. Conclusions—We demonstrated the presence of a cell population fated to become SMCs and followed their differentiation into SMCs among BMSCs.

Epstein-Barr Virus Infection of Human Natural Killer Cell Lines and Peripheral Blood Natural Killer Cells

Although considerable part of natural killer (NK) cell neoplasms possess EBV genome, there has been no direct evidence that EBV infects human NK cells in vitro. In this study, we demonstrated EBV entry into NK cells using a recombinant EBV, which contains enhanced green fluorescent protein (EGFP) gene in its genome (EGFP-EBV). After 48 h of exposure to EGFP-EBV, we detected EGFP signals in ∼30% of NK-92 and NKL cells and >40% of peripheral blood NK cells from three healthy volunteers. Reverse transcription-PCR analysis of various EBV-associated genes confirmed EBV infection. In situ hybridization for EBERs and BHLFs showed that latent and lytic infections coexisted at the early phase of EBV infection in two NK cell lines. Although BHLF-positive cells in the early lytic phase were round-shaped, EBER-positive cells in latent EBV infection tended to show a bizarre shape. Flow cytometric analysis of EGFP-EBV-exposed NK cell lines showed that most of EBV-infected cells entered early apoptosis after 72 h of EBV exposure, which explains the difficulties to establish EBV-carrying NK clones. Flow cytometry and reverse transcription-PCR analysis indicated that two NK cell lines may fuse with EBV using HLA class II after binding to the virus through a distinct molecule from CD21. We established two EBV-carrying NKL clones showing latency types I and II, both of which are recognized in EBV-associated NK cell neoplasms. Because EBV-infected NKL cells showed only type I latency during the early phase of infection, the temporal profile of latent gene expression is similar to that of T cells. We first report in vitro EBV infection of human NK cells and establishment of EBV-carrying NK clones, which should contribute to elucidate the role of EBV in the development of NK cell neoplasms.

Ancient Ubiquitous Protein 1 Binds to the Conserved Membrane-proximal Sequence of the Cytoplasmic Tail of the Integrin α Subunits That Plays a Crucial Role in the Inside-out Signaling of αIIbβ3

Modification of the cytoplasmic tails of the integrin αIIbβ3 plays an important role in the signal transduction in platelets. We searched for proteins that bind to the αIIb cytoplasmic tail using the yeast two-hybrid assay with a cDNA library of the megakaryocyte-derived cell line and identified a protein, ancient ubiquitous protein 1 (Aup1), that is ubiquitously expressed in human cells. Observation of UT7/TPO cells expressing a red fluorescent protein-tagged Aup1 indicated its localization in the cytoplasm. Immunoprecipitation of UT7/TPO cells by an antibody for Aup1 revealed that ∼40% of αIIb is complexed with Aup1. Binding study with an αIIb cytoplasmic tail peptide and glutathioneS-transferase-Aup1 fusion protein revealed a low affinity (K d = 90 μm). Subsequent yeast two-hybrid assay indicated binding of Aup1 to cytoplasmic tails of other integrin α subunits. Binding study with the purified Aup1 and various glutathione S-transferase-αIIbcytoplasmic tail peptides revealed specific binding of Aup1 to the membrane-proximal sequence (KVGFFKR) that is conserved among the integrin α subunits and plays a crucial role in the αIIbβ3 inside-out signaling. As Aup1 possesses domains related to signal transduction, these results suggest involvement of Aup1 in the integrin signaling.

Site-Specific Integration of an Adeno-Associated Virus Vector Plasmid Mediated by Regulated Expression of Rep Based on Cre-loxP Recombination

ABSTRACT Recombinant adeno-associated virus (AAV) type 2 has attracted attention because it appears to have the potential to serve as a vector for human gene therapy. An interesting feature of wild-type AAV is its site-specific integration into AAVS1, a defined locus on chromosome 19. This reaction requires the presence of two viral elements: inverted terminal repeats and Rep78/68. Accordingly, current AAV vectors lacking the rep gene lack the capacity for site-specific integration. In this report, we describe the use of Cre-loxP recombination in a novel system for the regulated, transient expression of Rep78, which is potentially cytotoxic when synthesized constitutively. We constructed a plasmid in which the p5 promoter was situated downstream of the rep coding sequence; in this configuration, rep expression is silent. However, Cre circularizes the rep expression unit, directly joining the p5 promoter to the 5′ end of the rep78 coding sequence, resulting in expression of Rep78. Such structural and functional changes were confirmed by detailed molecular analysis. A key feature of this system is that Rep expression was terminated when the circular molecule was linearized and integrated into the chromosome. Using this regulated expression system, we attempted site-specific integration of AAV vector plasmids. A PCR-based assay and analysis of fluorescence in situ hybridization showed that the AAV vector sequence was integrated into chromosome 19. Sequence analysis also confirmed that transient expression of Rep78 was sufficient for site-specific integration at the AAVS1 locus, as is observed with integration of wild-type AAV.

Adeno-associated virus-mediated human IL-10 gene transfer suppresses the development of experimental autoimmune orchitis

Testicular germ cell-induced autoimmune orchitis is characterized by inflammatory cell infiltration followed by disturbance of spermatogenesis. Experimental autoimmune orchitis (EAO) is an animal model for human immunological male infertility; delayed-type hypersensitivity (DTH) response plays a key role in its induction. Interleukin-10 (IL-10) is a regulatory cytokine that is critical in preventing organ-specific autoimmune inflammation. To determine the effects on EAO of human IL-10 (hIL-10) gene transfer, C3H/He mice immunized by unilateral testicular injury were administered intramuscular (i.m.) injections of adeno-associated viral (AAV) vector-encoding hIL-10 on the day of immunization. Serum hIL-10 was detected beginning at 1 week postinjection, and peaked at 3 weeks. Histological examinations showed a significantly low incidence of orchitis and disturbance of spermatogenesis in AAV hIL-10-treated mice, and the DTH response to autologous testicular cells was significantly suppressed. Immunohistochemical analysis of IFN-γ and IL-2, T-cell-associated cytokines, in the spleen and testes revealed significantly fewer cytokine-expressing cells after treatment. We conclude that a single i.m. administration of AAV hIL-10 significantly suppresses EAO and hypospermatogenesis by regulating cell-mediated immunity in the testes.Gene Therapy advance online publication, 26 May 2005; doi:10.1038/sj.gt.3302463

Improvement of idiopathic thrombocytopenic purpura by antithyroid therapy

Abstract:  Here we report a case of idiopathic thrombocytopenic purpura accompanied by Graves’ disease. Improvement in thyroid function with methimazole led to the spontaneous recovery of the platelet count from 8 × 109/L to 84 × 109/L. Furthermore, the second fall and recovery of the platelet count well coincided with the recurrence of hyperthyroidism after the discontinuation of methimazole and its normalization by resumption of the drug, respectively. These parallel fluctuations of platelet and thyrotropin because of the cessation and resumption of antithyroid therapy suggests that correction of hyperthyroidism may be beneficial to the control of an imbalance in the immune system which impairs not only thyroid but also the platelet.

Hsp90-inhibitor geldanamycin abrogates G2 arrest in p53-negative leukemia cell lines through the depletion of Chk1.

Checkpoint protein Chk1 has been identified as an Hsp90 client. Treatment with 100 nM geldanamycin (GM) for 24 h markedly reduced the Chk1 amount in Jurkat and ML-1 leukemia cell lines. Because Chk1 plays a central role in G2 checkpoint, we added GM to G2-arrested Jurkat and HL-60 cells pretreated with 50 nM doxorubicin for 24 h. GM slowly released both cell lines from doxorubicin-induced G2 arrest into G1 phase. GM also abrogated ICRF-193-induced decatenation G2 checkpoint in Jurkat and HL-60 cells. Western blot analysis showed that addition of GM attenuates doxorubicin- and ICRF-193-induced Chk1 phosphorylation at Ser345. GM, however, failed to abrogate G2 arrest in p53-positive ML-1 cells maybe due to the p21 induction. GM released HeLa cells from doxorubicin-induced G2 arrest but trapped them at M phase. Flow cytometric analysis showed that addition of GM converted doxorubicin-induced necrosis into apoptosis in Jurkat cells. Colony assay indicated that although GM has a weak cytotoxic effect as a single agent, it dramatically intensifies the cytotoxicity of doxorubicin and ICRF-193 in Jurkat and HL-60 cells. These results suggest that abrogation of G2 checkpoint by GM may play a central role in sensitizing p53-negative tumor cells to DNA-damaging and decatenation-inhibiting agents.

Low-dose doxorubicin-induced necrosis in Jurkat cells and its acceleration and conversion to apoptosis by antioxidants.

Summary. We treated rapidly growing Jurkat cells with 40 nmol/l of doxorubicin for 72 h. After 36 h, the G2‐arrested cells became larger and some of them started endoreplication. Nuclear staining with Hoechst 33342 combined with propidium iodide (PI) exclusion revealed that about 90% of the cells were necrotic at 72 h, although apoptotic cells accounted for only 8%. Incubation with 40 nmol/l of aclarubicin or cytosine β‐d‐arabinofuranoside for 60 h induced necrosis both in Jurkat and ml‐1 cells. Pre‐necrotic Jurkat cells incubated with 40 nmol/l of doxorubicin had much higher intracellular reactive oxygen species (ROS) levels than pre‐apoptotic ones. Addition of Tempol or Desferal accelerated doxorubicin‐induced necrosis and partially converted it into apoptosis. Both antioxidants reduced surviving colony numbers of prenecrotic Jurkat cells. n‐acetyl‐l‐cysteine had little effect on the apoptotic conversion but profoundly accelerated necrosis. Because an apoptosis‐resistant Jurkat subclone was also refractory to doxorubicin‐induced necrosis, apoptosis and necrosis might share some common pathways. Low‐dose doxorubicin increased micronuclei‐positive cell percentages and also suppressed high‐dose doxorubicin‐induced apoptosis in Jurkat and ml‐1 cells. Some of the prenecrotic cells, therefore, might survive and obtain genomic instability. Antioxidants may be useful to suppress, at least to some extent, this vicious consequence.