Kazunori Kato

Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy

We developed human mesenchymal stem cell (MSC) lines that could differentiate into various tissue cells including bone, neural cells, bone marrow (BM) stromal cells supporting the growth of hematopoietic stem cell (HSC), and so‐called ‘tumor stromal cells’ mixing with tumor cells. We investigated the applicability of MSC as therapeutic cell transplanting reagents (cytoreagents). Telomerized human BM derived stromal cells exhibited a prolonged lifespan and supported the growth of hematopoietic clonogenic cells. The gene transfer of Indian hedgehog (Ihh) remarkably enhanced the HSC expansion supported by the human BM stromal cells. Gene‐modified MSC are useful as therapeutic tools for brain tissue damage (e.g. brain infarction) and malignant brain neoplasms. MSC transplantation protected the brain tissue from acute ischemic damage in the midcerebral artery occlusion (MCAO) animal model. Brain‐derived neurotrophic factor (BDNF)‐gene transduction further enhanced the protective efficacy against the ischemic damage. MSC possessed excellent migratory ability and exerted inhibitory effects on the proliferation of glioma cells. Gene‐modification of MSC with therapeutic cytokines clearly augmented the antitumor effect and prolonged the survival of tumor‐bearing animals. Gene therapy employing MSC as a tissue‐protecting and targeting cytoreagent would be a promising approach. (Cancer Sci 2005; 96: 149–156)

The soluble CD40 ligand sCD154 in systemic lupus erythematosus

We found that the plasma of patients with active systemic lupus erythematosus (SLE) could induce a human B-cell line (Ramos) to express high levels of immune accessory molecules that are commonly found on blood B cells of patients with active SLE. The ability of SLE plasma to induce such phenotypic changes could be abrogated by neutralizing antibodies specific for the CD40 ligand (CD154) but not by antibodies to TNF-alpha. Immunoprecipitation studies with anti-CD154 identified a 20-kDa protein in the plasma of SLE patients with active disease, but not in plasma of normal donors, indicating that such plasma contained soluble CD154 (sCD154). Using a quantitative ELISA method, we found that the plasma of patients with active disease had levels of sCD154 that were significantly higher than those found in plasma of normal donors. Levels of CD154 transcripts in SLE blood lymphocytes correlated with the relative concentrations of sCD154 found in SLE plasma. Furthermore, plasma levels of sCD154 correlated with the titers of anti-double-stranded DNA autoantibody and with clinical disease activity. These studies indicate that sCD154 of patients with SLE may act as a functional ligand for CD40 that is associated with SLE disease activity.

Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells.

OBJECTIVE To compare the hematopoietic support provided by telomerized human mesenchymal stem cells (MSCs) and telomerized MSC-derived stromal cells. METHODS We transfected the human telomerase catalytic subunit (hTERT) gene into primary MSCs to establish hTERT-transduced MSCs (hTERT-MSCs). Stromal induction of hTERT-MSCs was performed by replacing the culture medium with Dexter-type culture medium. Hematopoietic support was examined by coculture with cord blood CD34(+) cells. RESULTS The hTERT-MSCs were morphologically identical with the primary MSCs and expressed surface antigens including CD105, CD73, and CD166. hTERT-MSCs showed a similar doubling time as primary MSCs and continued to proliferate to over 80 population doublings (PD), although the primary MSCs underwent crisis in vitro at 16 PD. The osteogenic, chondrogenic, adipogenic, neurogenic, and stromal differentiation potential of hTERT-MSCs were maintained up to at least 40 PD. The degree of expansion of CD34(+) cells and total number of colony-forming units in culture (CFU-C) upon 12-day coculture with the hTERT-MSC-derived stromal cells were nearly the same as those upon 12-day coculture with hTERT-MSCs (CD34, 33.0-fold+/-2.8-fold vs 36.1-fold+/-1.7-fold of the initial cell number; CFUs, 344.4-fold+/-62.5-fold vs 239.3-fold+/-87.0-fold; CFU-mix, 368.4-fold+/-113.7-fold vs 341.3-fold+/-234.3-fold). However, on day 18 of coculture, the number of cobblestone areas (CA) observed beneath the stromal cells was 15 times higher than that beneath hTERT-MSCs (CA, 146.9+/-54.6 vs 9.4+/-8.1, p<0.01). CONCLUSION Stromal induction of hTERT-MSCs exclusively enhanced the support of CA formation provided by hTERT-MSCs. Our human hTERT-MSCs will be useful for elucidating the mechanism of the formation of CAs.

Wnt3/RhoA/ROCK signaling pathway is involved in adhesion-mediated drug resistance of multiple myeloma in an autocrine mechanism

Adhesion of myeloma cells to bone marrow stromal cells is now considered to play a critical role in chemoresistance. However, little is known about the molecular mechanism governing cell adhesion–mediated drug resistance (CAM-DR) of myeloma cells. In this study, we focused our interests on the implication of the Wnt signal in CAM-DR. We first screened the expression of Wnt family in myeloma cell lines and found that Wnt3 was overexpressed in all the myeloma cells examined. KMS-5 and ARH77, which highly expressed Wnt3 protein, tightly adhered to human bone marrow stromal cells, and accumulation of β-catenin and GTP-bounded RhoA was observed in these myeloma cell lines. Conversely, RPMI8226 and MM1S, which modestly expressed Wnt3 protein, rather weakly adhered to human bone marrow stromal. We then examined the relevance of Wnt3 expression to adhesive property to stromal cells and to CAM-DR of myeloma cells. KMS-5 and ARH-77 exhibited apparent CAM-DR against doxorubicin. This CAM-DR was significantly reduced by anti-integrin β1 antibody, anti-integrin α6 antibody and a Wnt-receptor competitor, secreted Frizzled-related protein-1, and Rho kinase inhibitor Y27632, but not by the specific inhibitor of canonical signaling (Dickkopf-1), indicating that Wnt-mediated CAM-DR that is dependent on integrin α6/β1 (VLA-6)–mediated attachment to stromal cells is induced by the Wnt/RhoA/Rho kinase pathway signal. This CAM-DR was also significantly reduced by Wnt3 small interfering RNA transfer to KMS-5. These results indicate that Wnt3 contributes to VLA-6–mediated CAM-DR via the Wnt/RhoA/ROCK pathway of myeloma cells in an autocrine manner. Thus, the Wnt3 signaling pathway could be a promising molecular target to overcome CAM-DR of myeloma cells. [Mol Cancer Ther 2007;6(6):1774–82]

Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides

Epithelial carcinoma and leukemia cells express sialyl Lewis x oligosaccharides as tumor-associated carbohydrate antigens. To determine the role of sialyl Lewis x oligosaccharides in tumor dissemination, human melanoma MeWo cells, which do not express sialyl Lewis x, were transfected with α1,3-fucosyltransferase III (FTIII), and cell lines expressing different amounts of sialyl Lewis x were isolated. When these cells were injected into the tail vein of nude mice, cells expressing moderate amounts of sialyl Lewis x (MeWo-FTIII⋅M) produced a significantly greater number of lung tumor foci than did parental MeWo cells. In contrast, cells expressing large amounts of sialyl Lewis x (MeWo-FTIII⋅H) produced few lung tumor foci in nude mice but were highly tumorigenic in beige mice, which have defective natural killer (NK) cells. In vitro assays demonstrated that MeWo-FTIII⋅H cells are much more sensitive to NK cell-mediated cytotoxicity than are MeWo-FTIII⋅M cells or parental MeWo cells and the susceptibility of MeWo-FTIII⋅H cells to NK cell-mediated cytolysis can be inhibited by preincubating MeWo-FTIII⋅H cells with anti-sialyl Lewis x antibody. Moreover, we discovered that NK cell-mediated cytolysis of MeWo-FTIII⋅H cells can be inhibited by the addition of an antibody against the NK cell receptor CD94 or sialyl Lewis x oligosaccharides. These results, combined with structural analysis of MeWo-FTIII⋅H cell carbohydrates, indicate that moderate amounts of sialyl Lewis x lead to tumor metastasis, whereas expression of high levels of sialyl Lewis x leads to an NK cell attack on tumor cells, demonstrating that expression of different amounts of sialyl Lewis x results in entirely different biological consequences.

Bcl-xL Gene Transfer Inhibits Bax Translocation and Prolongs Cardiac Cold Preservation Time in Rats

Background—Apoptosis is an important cause of early graft loss after heart transplantation. Bcl-xL was reported to protect the heart against normothermic ischemia and reperfusion injury. In this study, we determined whether overexpression of Bcl-xL could inhibit tissue injury resulting from prolonged cold preservation followed by warm reperfusion of heart transplants. Methods and Results—Lewis rat hearts were transduced with an adenovirus vector harboring Bcl-xL cDNA (AxCAhBclxL) 4 days before collection of tissue. After preservation in University of Wisconsin solution at 4°C for 24 hours, the heart was either perfused with a Langendorff device ex vivo or used for heterotopic heart transplantation in vivo. Bcl-xL gene transfer significantly reduced the infarct size (23.0±2.6% versus 47.7±7.0% in saline control and 48.6±6.1% in vector control, P<0.01) after 2-hour reperfusion at 37°C with the Langendorff device and significantly decreased creatine kinase release (0.82±0.27 IU, versus 1.57±0.33 and 1.50±0.37 IU in saline and vector controls, respectively; P<0.05). In heart transplantation, overexpresson of Bcl-xL inhibited Bax translocation from the cytosol to the mitochondria, resulting in decreased cytochrome c release from the mitochondria; it also significantly decreased cardiac cell apoptosis and improved graft survival rate after long cold preservation, followed by warm reperfusion. Conclusions—Bcl-xL gene transfer inhibited the translocation of Bax and prolonged the cold preservation time of cardiac transplants. This may be a potential therapeutic method in clinical practice.

Carcinoembryonic Antigen–Targeted Selective Gene Therapy for Gastric Cancer through FZ33 Fiber-Modified Adenovirus Vectors

Purpose: A major problem when using the adenoviral vectors for gene therapy applications is thought to be related to low transduction efficiency in cancer cells or to side effects in normal cells. There is an urgent requirement to improve the specificity of gene delivery in the context of cancer gene therapy. Experimental Design: We constructed a genetically modified adenovirus incorporating an IgG Fc-binding motif from the Staphylococcus protein A, Z33, within the HI loop (Adv-FZ33). A remarkable degree of targeted gene delivery to gastric cancer cells was obtained with Adv-FZ33 with the fully human anti–carcinoembryonic antigen (CEA) monoclonal antibody, C2-45. Results:In vitro LacZ or EGFP gene expression after Adv-FZ33 infection via C2-45 was 20 times higher than control monoclonal antibody in MKN-45 at 1,000 viral particles/cell. We generated Ax3CAUP-FZ33 (UP-FZ33), which is an Adv-FZ33 derivative vector expressing a therapeutic gene (i.e., Escherichia coli uracil phosphoribosyltransferase), which converts 5-fluorouracil (5-FU) directly to 5-fluoro-UMP. UP-FZ33 with C2-45 enhanced the cytotoxicity of 5-FU by 10.5-fold in terms of IC50 against MKN-45 compared with control IgG4. In a nude mouse peritoneal dissemination model, tumor growth in mice treated with UP-FZ33/C2-45/5-FU was significantly suppressed, and tumor volumes were less than one-fourth of those of the control IgG4 group (P < 0.05). The median survival time of the UP-FZ33/C2-45/5-FU group was significantly longer than those treated with PBS or 5-FU only (P < 0.01). Conclusions: These data suggest that CEA-targeted FZ33 mutant adenovirus-mediated gene delivery offers a strong and selective therapeutic modality against CEA-producing cancers.

T-cell-conditioned medium efficiently induces the maturation and function of human dendritic cells

We present evidence that T‐cell‐conditioned media (TCCM) can efficiently induce human immature dendritic cells (DC) to express high levels of immune accessory molecules commonly found on mature DC. TCCM prepared from cell‐free supernatants of anti‐CD3‐activated T cells contained several soluble factors including CD40‐ligand (sCD40L), TNF‐α, and IFN‐γ. In contrast to moderate up‐regulation of costimulatory molecules by the addition of individual cytokines or monocyte‐conditioned medium, treatment of immature DC with TCCM induced a marked increase in the expression of costimulatory molecules in a dose‐dependent manner. The ability of TCCM to induce such phenotypic changes could be abrogated by neutralizing antibodies specific for CD40L, TNF‐α, and IFN‐γ, indicating that these factors present in TCCM are mainly implicated in the maturation of DC. Importantly, TCCM‐treated DC can produce significantly higher levels of IL‐12 and are highly effective stimulators in allogenenic and autologous mixed‐lymphocyte reactions. Overall, these findings show that cultivation with TCCM is an efficient approach for the induction of mature DC that should be useful in eliciting antigen‐specific immune responses against cancer and viruses.

Epigenetic Modulation Enhances the Therapeutic Effect of Anti–IL-13Rα2 Antibody in Human Mesothelioma Xenografts

Purpose: The interleukin-13 receptor α2 (IL-13Rα2) is expressed by a variety of human malignant cells. Here, we have examined the constitutive surface expression and the epigenetic regulation of IL-13Rα2 by human mesothelioma. We have also investigated the therapeutic effect of the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) and anti–IL-13Rα2 monoclonal antibody on mesothelioma xenografts. Experimental Design: Cell surface expression of IL-13Rα2 by various lung carcinomas was analyzed using flow cytometry. Therapeutic effects of anti–IL-13Rα2 and 5-aza-dC were investigated using antibody-dependent cellular cytotoxicity and proliferation assays and by monitoring the survival of mesothelioma-bearing mice. Results: We found that human malignant mesotheliomas expressed surface IL-13Rα2 on their surface and that it was upregulated by treatment with 5-aza-dC. This augmented expression of IL-13Rα2 resulted in growth inhibition of the mesothelioma cells when cocultured with anti-IL-13Rα2 and effector cells, such as splenocytes and peritoneal exudate cells. The growth inhibition of mesothelioma cells was mediated by IFN-γ that was only detected in the supernatant when effector cells were exposed to 5-aza-dC–treated tumors in the presence of anti–IL-13Rα2. Compared with the control or either regimen alone, in vivo administration of anti-IL-13Rα2 in combination with 5-aza-dC significantly prolonged the survival of mice with mesothelioma xenografts. Conclusions: These observations indicate a promising role for IL-13Rα2 as a target for antibody treatment in malignant mesothelioma, and, in combination with epigenetic regulation by a DNA methylation inhibitor, suggest the potential for a novel strategy to enhance therapeutic potency. Clin Cancer Res; 17(9); 2819–29. ©2011 AACR.

Gene transfer of the CD40-ligand to human dendritic cells induces NK-mediated antitumor effects against human carcinoma cells

The CD40‐ligand (CD40L) is a key molecule for the activation of dendritic cells (DCs), followed by the induction of DC maturation and cytokine production. Here we found that DC infected with adenovirus vector encoding human CD40L (CD40L‐DC) displayed significantly higher levels of immune accessory molecules and IL‐12 production than did uninfected cells, and that CD40L‐DC produced much higher levels of IFN‐γ. To investigate whether CD40L‐DC‐derived these soluble factors could stimulate NK cells without physical cell‐to‐cell contact, we cocultured NK cells with CD40L‐DC in transwell culture plates. NK cells showed up‐regulated cytotoxic activity toward various squamous oral cell carcinoma (OSC‐70, HSC‐2, HSC‐3), and we determined that both IL‐12 and IFN‐γ contributed to the CD40L‐DC‐mediated NK cell activation. NK cells stimulated with CD40L‐DC resulted in the induction of the cell surface expression of TRAIL, the production of IFN‐γ and intracellular accumulation of granzyme B. The cytotoxic activity of NK cells stimulated with CD40L‐DC could be mostly inhibited by neutralizing antibody for TRAIL and completely abrogated by the combination of antibody and exocytosis inhibitor, indicating that this was mainly mediated by a TRAIL‐TRAIL‐receptor interaction and granule exocytosis. Moreover, CD40L‐DC‐activated NK cells could induce up‐regulation of a death‐receptor TRAIL‐R2 (DR5) and down‐regulation of a decoy receptor TRAIL‐R3 (DcR1) on carcinoma cells. Overall, these results have revealed that CD40L‐DC could activate an innate immune reaction by stimulating NK cells followed by carcinoma cells, supporting that administration of CD40L‐DC may have potential as an anticancer therapy.