Adhesamine is an organic small molecule that promotes adhesion and growth of cultured human cells by binding selectively to heparan sulfate on the cell surface. The present study combined chemical, physicochemical, and cell biological experiments, using adhesamine and its analogues, to examine the mechanism by which this dumbbell-shaped, non-peptidic molecule induces physiologically relevant cell adhesion. The results suggest that multiple adhesamine molecules cooperatively bind to heparan sulfate and induce its assembly, promoting clustering of heparan sulfate-bound syndecan-4 on the cell surface. A pilot study showed that adhesamine improved the viability and attachment of transplanted cells in mice. Further studies of adhesamine and other small molecules could lead to the design of assembly-inducing molecules for use in cell biology and cell therapy.

Altered expression of b-catenin, a key component of the Wnt signaling pathway, is involved in a variety of cancers because increased levels of b-catenin protein are frequently associated with enhanced cellular proliferation. Although our previous study demonstrated that gene silencing of b-catenin in melanoma B16-BL6 cells by plasmid DNA (pDNA) expressing short-hairpin RNA targeting the gene (pshb-catenin) markedly suppressed their growth in vivo, gene silencing of b-catenin could promote tumor metastasis by the rearranging cell adhesion complex. In this study, we investigated how silencing of b-catenin affects metastatic aspects of melanoma cells. Transfection of B16-BL6 cells with pshb-catenin significantly reduced the amount of cadherin protein, a cell adhesion molecule binding to b-catenin, with little change in its mRNA level. Cadherin-derived fragments were detected in culture media of B16-BL6 cells transfected with pshb-catenin, suggesting that cadherin is shed from the cell surface when the expression of b-catenin is reduced. The mobility of B16-BL6 cells transfected with pshb-catenin was greater than that of cells transfected with any of the control pDNAs. B16-BL6 cells stably transfected with pshb-catenin (B16/pshb-catenin) formed less or an equal number of tumor nodules in the lung than cells stably transfected with other plasmids when injected into mice via the tail vein. However, when subcutaneously inoculated, B16/pshb-catenin cells formed more nodules in the lung than the other stably transfected cells. These results raise concerns about the gene silencing of b-catenin for inhibiting tumor growth, because it promotes tumor metastasis by reducing the amount of cadherin in tumor cells. ' 2008 Wiley-Liss, Inc.Key words: b-catenin; cadherin; RNA interference; pulmonary metastasis; melanoma b-catenin is a key component of the Wnt signaling pathway, which transmits proliferative and survival signals to cells. 1 Aberrant Wnt signaling, including the stabilization and nuclear translocation of b-catenin, has been observed in various types of cancers, such as colon, lung, skin, breast, liver and pancreas cancers. 2 Nuclear translocation of b-catenin is followed by its cooperation with the T cell factor/lymphocyte-enhancer binding factor family and activation of the expression of genes related to cell proliferation and survival. Previous studies have shown that b-catenin plays an important role as an oncogene to promote tumor cell growth. 2,3 These lines of evidence support cancer therapy based on the suppression of b-catenin expression. In our previous study, we demonstrated that delivery of plasmid DNA (pDNA) expressing short-hairpin RNA (shRNA) targeting b-catenin (pshb-catenin) or hypoxia inducible factor-1a (HIF-1a; pshHIF-1a) to a murine melanoma B16-BL6 (B16) tumor could suppress the corresponding target gene expression. 4 Moreover, suppression of b-catenin expression in B16 tumor inhibited the tumor growth. To our surprise, however, we also noticed that the lungs of mice that had received intratumor...

Experimental results have suggested that transgene expression can be saturated when large amounts of plasmid vectors are delivered into cells. To investigate this saturation kinetic behavior, cells were transfected with monitoring and competing plasmids using cationic liposomes. Even although an identical amount of a monitoring plasmid expressing firefly luciferase (FL) was used for transfection, transgene expression from the plasmid was greatly affected by the level of transgene expression from competing plasmids expressing renilla luciferase (RL). Similar results were obtained by exchanging the monitoring and competing plasmids. The competing plasmid-dependent reduction in transgene expression from the monitoring plasmid was also observed in mouse liver after hydrodynamic injection of plasmids. On the other hand, the mRNA and protein expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an endogenous gene, in the liver hardly changed even when transgene expression process is saturated. The expression of FL from a monitoring plasmid was significantly restored by siRNA-mediated degradation of RL mRNA that was expressed from a competing plasmid. These results suggest that the efficiency of protein synthesis from plasmid vectors is reduced when a large amount of mRNA is transcribed with no significant changes in endogenous gene expression.