Recent studies suggest that lysophosphatidic acid (LPA) and its G protein-coupled receptors (GPCRs) LPA 1, LPA2, or LPA3 may play a role in the development of several types of cancers, including colorectal cancer. However, the specific receptor subtype(s) and their signal-transduction pathways responsible for LPA-induced cancer cell proliferation have not been fully elucidated. We show by specific RNA interference (RNAi) that LPA 2 and LPA3 but not LPA1 are targets for LPA-induced proliferation of HCT116 and LS174T colon cancer cells. We determined that LPA-induced colon cancer cell proliferation requires the -catenin signaling pathway, because knockdown of -catenin by RNAi abolished LPA-induced proliferation of HCT116 cells. Moreover, LPA activates the main signaling events in the -catenin pathway: phosphorylation of glycogen synthase kinase 3 (GSK3), nuclear translocation of -catenin, transcriptional activation of T cell factor (Tcf)͞lymphoid-enhancer factor (Lef), and expression of target genes. Inhibition of conventional protein kinase C (cPKC) blocked the effects, suggesting its involvement in LPA-induced activation of the -catenin pathway. Thus, LPA 2 and LPA3 signal the proliferation of colon cancer cells through cPKC-mediated activation of the -catenin pathway. These results link LPA and its GPCRs to cancer through a major oncogenic signaling pathway.colon cancer cell ͉ G protein-coupled receptor
The factors controlling cationic liposome-DNA complex (CLDC)-based gene transfer in cells and in animals are poorly understood. We found that cell surface heparin/heparan sulfate-bearing proteoglycans mediate CLDC-based gene transfer and expression both in cultured cells and following intravenous gene delivery into animals. CLDC did not transfect Raji cells, which lack proteoglycans, but did efficiently transfect Raji cells stably transfected with the proteoglycan, syndecan-1. Fucoidan, heparin, or dextran sulfate, all of which are highly anionic polysaccharides, each blocked CLDC-mediated transfection both in cultured cells and following intravenous injection into mice, but had no effect on transfection by either recombinant adenovirus infection or electroporation. Intravenous pretreatment of mice with heparinases, which specifically cleave heparan sulfate molecules from cell surface proteoglycans, blocked intravenous, CLDC-mediated transfection in mice, confirming that proteoglycans mediate CLDC gene delivery in vivo. Modulation of proteoglycan expression may prove useful in controlling the efficiency of, as well as targeting the sites of, CLDC-based gene transfer in animals.
Systemic gene transfer provides new opportunities for the analysis of gene function and gene regulation in vivo, as well as for human gene therapy. We used the chloramphenicol acetyltransferase reporter gene to examine several parameters important for the development of efficient, cationic liposome-mediated, intravenous (IV) gene transfer in mice. We then demonstrated that this approach can produce high level expression of biologically important genes. Specifically, we assessed the relationship of expression vector design to the level of systemic gene expression produced, and compared transfection levels produced by intravenously injecting DNA alone versus DNA⅐liposome complexes. We found that both the position of the heterologous intron, and the promoter element used in the expression plasmid, significantly affected the level of systemic gene expression produced. Although intravenous injection of plasmid DNA alone transfected every tissue analyzed, liposome-mediated delivery was much more efficient. We also established that repeated IV injection of DNA⅐liposome complexes produced high level systemic transfection. The second injection of DNA⅐liposome complexes produced levels of gene expression at least as high as those following a single IV injection. Thus, unlike some viral vectors, a neutralizing host-immune response does not limit re-expression, following reinjection of DNA⅐liposome complexes.Finally, we showed that the expression vectors which produced the highest levels of chloramphenicol acetyltransferase reporter gene expression could also produce high level expression of two colony stimulating factor genes in mice. Specifically, IV injection of liposomes complexed to expression vectors into which we had inserted either the murine granulocyte-macrophagecolony stimulating factor cDNA or the human granulocyte-CSF cDNA, produced circulating levels of the corresponding colony stimulating factor gene product comparable to levels which have been shown previously to be both biologically and therapeutically significant.
Two radical-based approaches enable the efficient trifluoromethylation of aromatic sidechains in fully unprotected peptides under mild, biocompatible conditions.
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