Inhibition of B16 melanoma growth and metastasis in C57BL mice by vaccination with a syngeneic endothelial cell line.

BACKGROUND: Key role of angiogenesis in tumor growth and metastasis based on accumulating evidence and recent progress of immunotherapy have led us to investigate vaccine therapy targeting tumor angiogenesis. METHODS: C57BL/6J mice were vaccinated with a syngeneic endothelial cell line Tpit/E by subcutaneous injection once a week. Prior to ninth vaccination, the mice were challenged with B16/F10 melanoma cells by subcutaneous inoculation on the back for the tumor growth model or by tail venous injection for the lung metastasis model. Development of subcutaneous tumor and lung metastasis was monitored by computed tomography scanning, which enabled accurate evaluation with the minimized sacrifice of mice. RESULTS: Vaccination with Tpit/E cells inhibited subcutaneous tumor growth and appearance of lung metastasis compared to control. Survival period was elongated in the Tpit/E vaccination in both of the two models. We also obtained hybridomas secreting specific antibodies to Tpit/E cells from a mouse vaccinated with the cells, indicating that specific immune response to the syngeneic endothelial cells was elicited. CONCLUSION: These results suggest that vaccination with an autologous endothelial cell line may be effective against melanoma.

Carbonic anhydrase II is a tumor vessel endothelium-associated antigen targeted by dendritic cell therapy.

Tumor-associated antigens are promising candidates as target molecules for immunotherapy and a wide variety of tumor-associated antigens have been discovered through the presence of serum antibodies in cancer patients. We previously conducted dendritic cell therapy on 10 malignant melanoma patients and shrinkage or disappearance of metastatic tumors with massive necrosis occurred in two patients. In this study, we found a 29-kDa protein against which antibody was elicited by dendritic cell therapy in one of the two patients. Matrix-assisted laser desorption ionization-time of flight/mass spectrometry analysis of the protein isolated by two-dimensional electrophoresis combined with Western blots revealed that the 29-kDa protein was carbonic anhydrase II (CA-II). Immunohistochemistry of the tumors and normal tissues showed that CA-II was expressed in the tumor vessel but not in normal vessel endothelium. CA-II expression in tumor endothelium was observed as well in other cancers including esophageal, renal, and lung cancers. In an in vitro angiogenesis model, CA-II expression of normal human vein endothelial cells was significantly up-regulated when cells were cultured in the acidic and hypoxic conditions indicative of a tumor environment. These findings suggest that CA-II is a tumor vessel endothelium-associated antigen in melanoma and other cancers, and elicitation of serum anti-CA-II antibody by dendritic cell therapy may be associated with good clinical outcome including tumor reduction.

Identification of MAGI-3 as a transforming growth factor-alpha tail binding protein.

The cytoplasmic domain of the transforming growth factor-alpha precursor (proTGFalpha) contains a C-terminal PSD-95/SAP90, Discs Large, and Zona Occludens-1 (PDZ) recognition motif (TVV). By yeast two-hybrid screening of a mouse embryo library, we have found that a third member of a family of PDZ-containing proteins, membrane associated guanylate kinase inverted-3 (MAGI-3), binds to TGFalpha's TVV. MAGI-3 is widely expressed in multiple mouse tissues, including brain. Immunolocalization showed that MAGI-3 and TGFalpha were colocalized in neurons in the cortex and dentate gyrus, as well as in ependymal cells and some astrocytes. In vitro, proTGFalpha bound the PDZ-1 domain of MAGI-3 and MAGI-2, but not MAGI-1. MAGI-3 and the 17-kDa cell surface form of proTGFalpha interact transiently in MDCK cells stably transfected with both MAGI-3 and human proTGFalpha cDNAs. MAGI-3 and wild-type proTGFalpha colocalize at the cell surface. In contrast, MAGI-3 forms a stable complex with membrane-fixed TGFalpha early in the secretory pathway and interacts with immature and cell surface forms of membrane-fixed TGFalpha. Overexpression of MAGI-3 resulted in increased levels of TGFalpha in the basolateral medium of polarized MDCK cells, suggesting that MAGI-3 has a role in efficient trafficking of TGFalpha to the cell surface in polarized epithelial cells.

A potential pro-angiogenic cell therapy with human placenta-derived mesenchymal cells.

Recently several strategies to treat ischemic diseases have been proposed but the ideal way has to be determined. We explored whether human placenta-derived mesenchymal cells (hPDMCs) can be used for this purpose because placenta is very rich in vessels. First, production of human vascular endothelial growth factor (hVEGF) from hPDMCs was examined. The amount of hVEGF secreted by hPDMCs was similar to the amount produced by HeLa cells. hVEGF was barely detected in human umbilical vein endothelial cells (hUVECs) or human peripheral blood mononuclear cells. hVEGF secreted from hPDMCs stimulated the proliferation of hUVECs, indicating its biological activity. Transplantation of hPDMCs to the ischemic limbs of NOD/Shi-scid mice significantly improved the blood flow of the affected limbs. Blood vessel formation was more prominently observed in the limbs of treated mice as compared to the control mice. Real-time RT-PCR revealed that hPDMCs produced hVEGF for at least 7 days after transplantation. Thus, transplantation of hPDMCs could potentially be a promising treatment for human ischemic diseases.

Human T-cell leukemia virus type 1 Tax oncoprotein induces and interacts with a multi-PDZ domain protein, MAGI-3.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), whereas the closely related virus HTLV-2 has not been associated with such malignant conditions. HTLV-1 Tax1 oncoprotein transforms a rat fibroblast cell line (Rat-1) much more efficiently than does HTLV-2 Tax2. By using a differential display analysis, we isolated MAGI-3 as a Tax1-inducible gene in Rat-1 cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that Tax1 induced MAGI-3 in Rat-1 cells. MAGI-3 has multiple PDZ domains and interacted with Tax1 but not Tax2 in 293T cells. The interaction of Tax1 with MAGI-3 was dependent on a PDZ domain-binding motif, which is missing in Tax2. The interaction of Tax1 with MAGI-3 altered their respective subcellular localization, and moreover, the interaction correlated well with the high transforming activities of Tax1 in Rat-1 cells relative to Tax2. MAGI-3 mRNA and the allied MAGI-1, but not MAGI-2, were expressed in HTLV-1-infected T-cell lines. Our results suggest that the interaction of Tax1 and MAGI-3 alters their respective biological activities, which may play a role in transformation by Tax1 as well as in the pathogenesis of HTLV-1-associated diseases.