Tomonori Tsukahara

Prevention of Adult T-Cell Leukemia-Like Lymphoproliferative Disease in Rats by Adoptively Transferred T Cells from a Donor Immunized with Human T-Cell Leukemia Virus Type 1 Tax-Coding DNA Vaccine

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) in infected individuals after a long incubation period. To dissect the mechanisms of the development of the disease, we have previously established a rat model of ATL-like disease which allows examination of the growth and spread of HTLV-1 infected tumor cells, as well assessment of the effects of immune T cells on the development of the disease. In the present study, we induced HTLV-1 Tax-specific cytotoxic T lymphocyte (CTL) immunity by vaccination with Tax-coding DNA and examined the effects of the DNA vaccine in our rat ATL-like disease model. Our results demonstrated that DNA vaccine with Tax effectively induced Tax-specific CTL activity in F344/N Jcl-rnu/+ (nu/+) rats and that these CTLs were able to lyse HTLV-1 infected syngeneic T cells in vitro. Adoptive transfer of these immune T cells effectively inhibited the in vivo growth of HTLV-1-transformed tumor in F344/N Jcl-rnu/rnu (nu/nu) rats inoculated with a rat HTLV-1 infected T cell line. Vaccination with mutant Tax DNA lacking transforming ability also induced efficient anti-tumor immunity in this model. Our results indicated a promising effect for DNA vaccine with HTLV-1 Tax against HTLV-1 tumor development in vivo.

NF-κB Activity Regulates Mesenchymal Stem Cell Accumulation at Tumor Sites

Mesenchymal stem cells (MSC) accumulate at tumor sites when injected into tumor-bearing mice, perhaps offering cellular vectors for cancer-targeted gene therapy. However, the molecular mechanisms involved in MSC targeting the tumors are presently little understood. We focused on MSC-endothelial cell (EC) adhesion following TNF-α stimulation in an attempt to elucidate these mechanisms. Interestingly, stimulation of MSCs with TNF-α enhanced the adhesion of MSCs to endothelial cells in vitro. This adhesion was partially inhibited by blocking antibodies against vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4). It is well known that TNF-α induces VCAM-1 expression via the NF-κB signaling pathway. Parthenolide has an anti-inflammatory activity and suppressed NF-κB activity by inhibition of IκBα phosphorylation after TNF-α stimulation and strongly inhibited TNF-α-induced VCAM-1 expression on MSCs. In vivo imaging using luciferase-expressing MSCs revealed that the bioluminescent signal gradually increased at tumor sites in mice injected with untreated MSCs. In contrast, we observed very weak signals at tumor sites in mice injected with parthenolide-treated MSCs. Our results suggest that NF-κB activity regulates MSC accumulation at tumors, by inducing VCAM-1 and thereby its interaction with tumor vessel endothelial cells. These findings have implications for the ongoing development of efficient MSC-based gene therapies for cancer treatment.

Molecular Mechanisms of Promoter Regulation of the gp34 Gene That Is Trans-activated by an Oncoprotein Tax of Human T Cell Leukemia Virus Type I

We investigated the molecular mechanism of transcriptional activation of the gp34 gene by the Tax oncoprotein of human T cell leukemia virus type I (HTLV-I). gp34 is a type II transmembrane molecule belonging to the tumor necrosis factor family and is constitutively expressed on HTLV-I-producing cells but not normal resting T cells. The transcriptional regulatory region of the gp34 gene was activated by HTLV-I Tax in the human T cell line Jurkat, in which endogenous gp34 is induced by Tax. Sequence analysis demonstrated that two NF-κB-like elements (1 and 2) were present in the regulatory region. Both NF-κB-like elements were able to bind to NF-κB or its related factor(s) in a Tax-dependent manner. Chloramphenicol acetyltransferase assays indicated that NF-κB-like element 1 was Tax-responsive, although the activity was lower than that the native promoter. NF-κB-like element 2 elevated promoter activity when combined with NF-κB-like element 1, indicating cooperative function of the elements for maximum promoter function. Unlike typical NF-κB elements, the NF-κB-like elements in gp34 were not activated by treatment of Jurkat cells with phorbol ester despite induction of the NF-κB-like binding activity. Chloramphenicol acetyltransferase reporter assays using the region upstream of the NF-κB-like elements identified an upstream region that reduced transcription from cognate and noncognate core promoters in a Tax-independent manner. Our results imply complex regulation of expression of the gp34 gene and suggest implication of gp34 in proliferation of HTLV-I infected T cells.

Cancer gene therapy using mesenchymal stem cells

Abstract Cellular and gene therapies represent promising treatment strategies at the frontier of medicine. Hematopoietic stem cells, lymphocytes, and mesenchymal stem cells (MSCs) can all serve as sources of cells for use in such therapies. Strategies for gene therapy are often based on those of cell therapy, and it is anticipated that some examples will be put to practical use in the near future. Given their ability to support hematopoiesis, MSCs may be useful for the enhancement of stem cell engraftment, and the acceleration of hematopoietic reconstitution. Furthermore, MSCs may advance the treatment of severe graft-versus-host disease, based on their immunosuppressive ability. This application is also based on the homing behavior of MSCs to sites of injury and inflammation. Interestingly, MSCs possess tumor-homing ability, opening up the possibility of applications in the targeted delivery of anti-cancer genes to tumors. Many reports have indicated that MSCs can be utilized to target tumors and to deliver anti-cancer molecules locally, as tumors are recognized as non-healing wounds with inflammatory tissue. Here, we review both the potential of MSCs as cellular vehicles for targeted cancer therapy and the molecular mechanisms underlying MSC accumulation at tumor sites.

Human T-cell leukemia virus type 1 Tax protein induces the expression of STAT1 and STAT5 genes in T-cells.

Human T-cell leukemia virus type 1 (HTLV-1) Tax transforms normal T-cells in the presence of interleukin (IL)-2 in vitro. STAT is a family of transcription factors that play a pivotal role in cytokine-induced functions of a various type of cells. We investigated the involvement of STATs in the transformation of T-cells by HTLV-1. HTLV-1-transformed T-cell lines expressed higher amounts of STAT1, STAT3 and STAT5 RNA and proteins than virus-negative T cells. The expression of STAT1 and STAT5 in a human T-cell line was induced by Tax. IL-2 induced the DNA binding activity of STAT3 and STAT5 of a HTLV-1-transformed cell line and then stimulated its proliferation. In contrast, IL-2 did neither in a cell line lacking STAT3 and STAT5. The expression of STAT1, STAT3 and STAT5 mRNAs were also induced by a T-cell mitogen in normal human peripheral blood mononuclear cells. Our results suggest that the induction of STAT1 and STAT5 by Tax enhances cytokine-induced functions of virus-infected T-cells, hence the induction may play a role in IL-2-dependent transformation steps of T-cells by HTLV-1.

Ubiquitin-independent binding of Hrs mediates endosomal sorting of the interleukin-2 receptor β-chain

Several lines of evidence have revealed that ubiquitylation of membrane proteins serves as a signal for endosomal sorting into lysosomes or lytic vacuoles. The hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) interacts with ubiquitylated cargoes through its ubiquitin-interacting-motif domain (UIM domain), and plays an essential early role in endosomal sorting. Here, we show that the C-terminal region of Hrs, which does not contain the UIM domain, can bind to interleukin-2 receptor β (IL-2Rβ). We found a direct interaction between bacterially expressed IL-2Rβ and Hrs in GST pull-down assays, indicating that their binding is independent of ubiquitin. Trafficking and degradation assays revealed that, similarly to wild-type IL-2Rβ, an IL-2Rβ mutant lacking all the cytoplasmic lysine residues is sorted from Hrs-positive early endosomes to LAMP1-positive late endosomes, resulting in degradation of the receptor. By contrast, an IL-2Rβ mutant lacking the Hrs-binding region passes through early endosomes and is mis-sorted to compartments positive for the transferrin receptor. The latter mutant exhibits attenuated degradation. Taken together, these results indicate that precise sorting of IL-2Rβ from early to late endosomes is mediated by Hrs, a known sorting component of the ubiquitin-dependent machinery, in a manner that is independent of UIM-ubiquitin binding.

Mechanisms of resistance to azacitidine in human leukemia cell lines

The DNA methylation inhibitor azacitidine (5-azacytidine) is used against myelodysplastic syndrome and acute myeloid leukemia, but drug resistance is an ongoing, intractable problem. To investigate resistance mechanisms, we generated two azacitidine-resistant cell lines, THP-1/AR and HL60/AR, and studied genetic disparities between them and their corresponding parental lines. In cells treated with azacitidine, significant mitotic variations were noted in parental cells which were absent in resistant cells, suggesting that resistance arises from negating azacitidine-mediated activation of apoptosis signaling and reestablishing G2/M checkpoint. Importantly, both resistant cell lines have common point mutations in the uridine-cytidine kinase 2 (UCK2) gene, which encodes the rate-limiting enzyme of the azacitidine activation pathway. Forced expression of mutated UCK2 in parental THP-1 cells abrogated azacitidine-induced apoptosis, whereas overexpression of wild type UCK2 in resistant THP-1/AR cells restored sensitivity to azacitidine, implying that UCK2 gene mutations perturb azacitidine activation and advance azacitidine resistance. Our study provides new insights into azacitidine resistance and establishes models useful in developing effective strategies to overcome it.

Characterization of Envelope Glycoprotein Mutants for Human T-Cell Leukemia Virus Type 1 Infectivity and Immortalization

ABSTRACT The human T-cell leukemia virus type 1 (HTLV-1) envelope protein is required for virus spread. This study further characterizes the role of the envelope protein in HTLV-1 immortalization. Viruses with single amino acid substitutions within the SU protein at residue 75, 81, 95, 101, 105, or 195 or with a C-terminal cytoplasmic domain truncation (CT), as well as an envelope-null (EN) virus, were generated within an infectious molecular clone, ACH. Transfection of 293T cells resulted in the release of similar amounts of virus particles from all of the mutants as determined by p19 enzyme-linked immunosorbent assay and immunoblot analysis of Gag in cell lysates and supernatants. The virus particles from all mutants except ACH-101, ACH-CT, and ACH-EN were infectious for B5 macaque cells in cell-free and cell-to-cell transmission assays and were capable of immortalizing transfected CD4+ lymphocytes. These results indicate that HTLV-1 spread is required for immortalization.

The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3+ T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19+ human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2−/−γc−/− immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

Identification of a high incidence region for retroviral vector integration near exon 1 of the LMO2 locus

Therapeutic retroviral vector integration near the oncogene LMO2 is thought to be a cause of leukemia in X-SCID gene therapy trials. However, no published studies have evaluated the frequency of vector integrations near exon 1 of the LMO2 locus. We identified a high incidence region (HIR) of vector integration using PCR techniques in the upstream region close to the LMO2 transcription start site in the TPA-Mat T cell line. The integration frequency of the HIR was one per 4.46 × 104 cells. This HIR was also found in Jurkat T cells but was absent from HeLa cells. Furthermore, using human cord blood-derived CD34+ cells we identified a HIR in a similar region as the TPA-Mat T cell line. One of the X-linked severe combined immunodeficiency (X-SCID) patients that developed leukemia after gene therapy had a vector integration site in this HIR. Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis.