CD147 plays a critical role in the invasive and metastatic activity of malignant melanoma cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases and vascular endothelial growth factor. We developed a system that blocks CD147 in the human malignant melanoma cell line, A375, using RNA interference. By transfecting melanoma cells with the small interfering RNA (siRNA) that targets human CD147, we were able to establish two stable clones in which CD147 expression was significantly downregulated. This resulted in the decreased proliferation and invasion of A375 cells in vitro. CD147 siRNA also downregulated the expression of vascular endothelial growth factor in these cells and reduced the migration of vascular endothelial cells. The reduction in the CD147 level suppressed the size of s.c. tumors and the microvessel density in an A375 s.c. nude mouse xenograft model. In addition, the in vivo metastatic potential of A375 cells transfected with CD147 siRNA was suppressed in a nude mouse model of pulmonary metastasis. (Cancer Res 2006; 66(23): 11323-30)

Summary Xylem development is a process of xylem cell terminal differentiation that includes initial cell division, cell expansion, secondary cell wall formation and programmed cell death (PCD). PCD in plants and apoptosis in animals share many common characteristics. Caspase‐3, which displays Asp‐Glu‐Val‐Asp (DEVD) specificity, is a crucial executioner during animal cells apoptosis. Although a gene orthologous to caspase‐3 is absent in plants, caspase‐3‐like activity is involved in many cases of PCD and developmental processes. However, there is no direct evidence that caspase‐3‐like activity exists in xylem cell death. In this study, we showed that caspase‐3‐like activity is present and is associated with secondary xylem development in Populus tomentosa. The protease responsible for the caspase‐3‐like activity was purified from poplar secondary xylem using hydrophobic interaction chromatography (HIC), Q anion exchange chromatography and gel filtration chromatography. After identification by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS), it was revealed that the 20S proteasome (20SP) was responsible for the caspase‐3‐like activity in secondary xylem development. In poplar 20SP, there are seven α subunits encoded by 12 genes and seven β subunits encoded by 12 genes. Pharmacological assays showed that Ac‐DEVD‐CHO, a caspase‐3 inhibitor, suppressed xylem differentiation in the veins of Arabidopsis cotyledons. Furthermore, clasto‐lactacystin β‐lactone, a proteasome inhibitor, inhibited PCD of tracheary element in a VND6‐induced Arabidopsis xylogenic culture. In conclusion, the 20S proteasome is responsible for caspase‐3‐like activity and is involved in xylem development.

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Metabolic labeling of glycans with chemical reproters (e.g., alkyne or azide) in conjunction with bioorthogonal chemistry is a powerful tool for imaging glycome; however, this method lacks protein-specificity and therefore is not applicable to imaging glycosylation of a specific protein of interest (POI). Here we report the development of a cis-membrane FRET-based methodology that allows protein-specific imaging of glycans on live cells. We exploit metabolic glycan labeling in conjunction with site-specific protein labeling to simultaneously install a FRET acceptor and a donor onto the glycans and the extracellular terminal of the protein of interest, respectively. The intramolecular donor-acceptor distance for the POI falls within the range for effective FRET, whereas the intermolecular FRET is disfavored since the excess acceptors on other proteins are distant from the donor. We demonstrated the capability of this cis-membrane FRET imaging method by visualizing the sialylation of several important cell surface receptors including integrin αXβ2, epidermal growth factor receptor, and transforming growth factor-beta receptor type I. Furthermore, our imaging experiments revealed that the sialylation might be important for β2 integrin activation. Our methodology should enable the live-cell studies on how glycosylation regulates the functions and dynamics of various cell-surface proteins.