Novel anti-tumor mechanism of galanin receptor type 2 in head and neck squamous cell carcinoma cells.

Galanin and its receptors, GALR1 and GALR2, are known tumor suppressors and potential therapeutic targets in head and neck squamous cell carcinoma (HNSCC). Previously, we demonstrated that, in GALR1-expressing HNSCC cells, the addition of galanin suppressed tumor proliferation via upregulation of ERK1/2 and cyclin-dependent kinase inhibitors, whereas, in GALR2-expressing cells, the addition of galanin not only suppressed proliferation, but also induced apoptosis. In this study, we first transduced HEp-2 and KB cell lines using a recombinant adeno-associated virus (rAAV)-green fluorescent protein (GFP) vector and confirmed a high GFP expression rate (>90%) in both cell lines at the standard vector dose. Next, we demonstrated that GALR2 expression in the presence of galanin suppressed cell viability to 40-60% after 72 h in both cell lines. Additionally, the annexin V-positive rate and sub-G0/G1 phase population were significantly elevated in HEp-2 cells (mock vs GALR2: 12.3 vs 25.0% (P < 0.01) and 9.1 vs 32.0% (P < 0.05), respectively) after 48 h. These changes were also observed in KB cells, although to a lesser extent. Furthermore, in HEp-2 cells, GALR2-mediated apoptosis was caspase-independent, involving downregulation of ERK1/2, followed by induction of the pro-apoptotic Bcl-2 protein, Bim. These results illustrate that transient GALR2 expression in the presence of galanin induces apoptosis via diverse pathways and serves as a platform for suicide gene therapy against HNSCC.

CD19 target-engineered T-cells accumulate at tumor lesions in human B-cell lymphoma xenograft mouse models.

Adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) is promising for treatment of advanced B-cell malignancies. Tumor targeting of CAR-modified T-cells is likely to contribute therapeutic potency; therefore we examined the relationship between the ability of CD19-specific CAR (CD19-CAR)-transduced T-cells to accumulate at CD19(+) tumor lesions, and their ability to provide anti-tumor effects in xenograft mouse models. Normal human peripheral blood lymphocytes, activated with immobilized RetroNectin and anti-CD3 antibodies, were transduced with retroviral vectors that encode CD19-CAR. Expanded CD19-CAR T-cells with a high transgene expression level of about 75% produced IL-2 and IFN-γ in response to CD19, and lysed both Raji and Daudi CD19(+) human B-cell lymphoma cell lines. Furthermore, these cells efficiently accumulated at Raji tumor lesions where they suppressed tumor progression and prolonged survival in tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared to control cohorts. These results show that the ability of CD19-CAR T-cells to home in on tumor lesions is pivotal for their anti-tumor effects in our xenograft models, and therefore may enhance the efficacy of adoptive T-cell therapy for refractory B-cell lymphoma.

[A case of advanced adenocarcinoma of esophagogastric junction with severe esophageal invasion effectively treated by chemoradiotherapy using paclitaxel and cisplatin, and S-1 after chemoradiotherapy].

The patient was a 66-year-old male with adenocarcinoma of the esophagogastric junction and severe esophageal invasion, which was diagnosed as cType 3, cT4a (SE) cN3cM1 (LYM), cStage IV(histopathology: por 1). We tried concurrent chemoradiotherapy consisting of PTX 60 mg/m(2) and CDDP 25 mg/m(2), respectively (once a week), and a total of 45 Gy of radiotherapy treatment. Then, for effective continuation, chemotherapy using S-1 was performed as second-line therapy. A complete response was achieved and continued for more than 2 years after initial chemoradiotherapy; his complaints abated and his quality of life improved. Although gastro-intestinal symptoms and bone marrow suppression were observed as adverse effects, they were within a tolerable range and did not interfere with the concurrent chemoradiotherapy. This regimen appears to be feasible and effective for advanced gastric carcinoma refractory to other regimens.

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 x 10(4) 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.

Eradication of cervical cancer in vivo by an AAV vector that encodes shRNA targeting human papillomavirus type 16 E6/E7.

The major causative agent of cervical cancer is human papilloma virus (HPV); the viral proteins E6 and E7 induce carcinogenesis through the inactivation of the host tumor-suppressor gene. Therefore, the stable expression of specific inhibitors of E6 and E7 in cancer cells is expected to provide effective treatment for cervical cancer without affecting normal tissue. In this study, we propose a novel therapeutic approach using an adeno-associated virus (AAV) vector encoding short hairpin RNA (shRNA) against the oncoproteins E6 and E7 (shE6E7) of HPV type 16 (HPV­16), termed AAV­shE6E7. Three different HPV­16-positive cervical cancer cell lines (BOKU, SiHa and SKG-IIIa cells) were tested for gene transfer efficiency using serotypes of AAV vectors. For in vitro analysis, the cells were transduced AAV­shE6E7; alternatively, in vivo studies were performed via the administration of a direct injection of AAV­shE6E7 into cervical cancer cell-derived tumors in mice. The high gene transfer efficiency was observed using AAV2 in all three cervical cancer cell lines. Following transduction, we observed apoptosis, G1 phase arrest and cell growth inhibition. Additionally, in the transduced cells, the E6, E7 and p16 expression levels decreased, whereas the expression levels of p53, p21 and pRb levels were enhanced. The growth of subcutaneously transplanted tumors was markedly inhibited by the single administration of AAV2­shE6E7, and the tumors were almost completely eradicated without any adverse effects. These results provided evidence of the utility of AAV2­shE6E7 as a novel treatment approach for cervical cancer.

Substitution of HIV Type 1 Nef with HTLV-1 p12.

Human retroviruses, such as HTLV-1 and HIV-1, encode accessory proteins, which regulate viral pathogenesis. The p12 protein of HTLV-1 is encoded from the pX-I open reading frame, and is critical for efficient virus replication in rabbits. Although dispensable for infection, replication, and immortalization of activated lymphocytes in culture, p12 expression is important for infection of quiescent lymphocytes. Similar to HTLV-1 p12, Nef is important for virus infectivity in SIV animal models. We questioned whether p12 could replace Nef in HIV-1, and reconstitute virus replication in culture. We found that p12 could complement for effects of Nef on HIV-1 infection of Magi-CCR5 cells or macrophages.

Cancer gene therapy using mesenchymal stem cells.

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.

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.

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.

An R132H mutation in isocitrate dehydrogenase 1 enhances p21 expression and inhibits phosphorylation of retinoblastoma protein in glioma cells.

Cytosolic isocitrate dehydrogenase 1 (IDH1) with an R132H mutation in brain tumors loses its enzymatic activity for catalyzing isocitrate to α-ketoglutarate (α-KG) and acquires new activity whereby it converts α-KG to 2-hydroxyglutarate. The IDH1 mutation induces down-regulation of tricarboxylic acid cycle intermediates and up-regulation of lipid metabolism. Sterol regulatory element-binding proteins (SREBPs) regulate not only the synthesis of cholesterol and fatty acids but also acyclin-dependent kinase inhibitor p21 that halts the cell cycle at G1. Here we show that SREBPs were up-regulated in U87 human glioblastoma cells transfected with an IDH1(R132H)-expression plasmid. Small interfering ribonucleic acid (siRNA) for SREBP1 specifically decreased p21 messenger RNA (mRNA) levels independent of the p53 pathway. In IDH1(R132H)-expressing U87 cells, phosphorylation of Retinoblastoma (Rb) protein also decreased. We propose that metabolic changes induced by the IDH1 mutation enhance p21 expression via SREBP1 and inhibit phosphorylation of Rb, which slows progression of the cell cycle and may be associated with non-aggressive features of gliomas with an IDH1 mutation.

Ubiquitin-independent binding of Hrs mediates endosomal sorting of the interleukin-2 receptor beta-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 beta (IL-2Rbeta). We found a direct interaction between bacterially expressed IL-2Rbeta 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-2Rbeta, an IL-2Rbeta 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-2Rbeta 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-2Rbeta 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.

Murine leukemia virus vector integration favors promoter regions and regional hot spots in a human T-cell line.

Genomic analysis of integration will be important in evaluating the safety of human gene therapy with retroviral vectors. Here, we investigated MLV vector integration sites in human T-cells, since they are amenable to gene transfer studies, and have been used therapeutically in clinical trials. We mapped 340 MLV vector integration sites in the infected human T-cell clones we established. The data showed that MLV preferred integration near the transcription start sites (+/-5kb), near CpG islands (+/-1kb), and within the first intron of RefSeq genes. We also identified MLV integration hot spots that contained three or more integrations within a 100kb region. RT-PCR revealed that mRNA-levels of T-cell clones that contained MLV integrations near transcription start sites or introns were dysregulated compared to the uninfected cells. These studies help define the profile of MLV integration in T-cells and the risks associated with MLV-based gene therapy.

In vivo analysis of replication and immunogenicity of proviral clones of human T-lymphotropic virus type 1 with selective envelope surface-unit mutations.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell lymphoma/leukemia (ATL). The HTLV-1 envelope gene exhibits limited variability when examined from infected individuals, but has not been tested using infectious clones of the virus in animal models. In vitro assays indicate that HTLV-1 envelope (Env) Ser75Ile, Asn95Asp, and Asn195Asp surface unit (SU) mutants are able to replicate in and immortalize lymphocytes. Herein, we examined the effects of these Env mutants in rabbits inoculated with HTLV-1 immortalized ACH.75, ACH.95, or ACH.195 cell lines (expressing full-length molecular clones with the SU mutations) or the ACH.1 cell line (expressing wild-type SU). All rabbits became infected, and the fidelity of the mutations was maintained throughout the 8-week study. However, SU point mutations resulted in decreased antibody responses to viral group-associated antigen (Gag) and Env antigens. ACH.195 rabbits had a selective decreased antibody response to SU, and one ACH.195 rabbit had an antibody response to both HTLV-1 and HTLV-2 SUs. Some mutant inoculation groups had altered proviral loads. However, peripheral-blood mononuclear cell (PBMC) proviral loads did not correlate with antibody responses. Our data are the first to demonstrate that mutations in critical determinants of HTLV-1 Env SU altered antibody responses and proviral loads, but do not prevent viral replication in vivo.