Autocrine netrin function inhibits glioma cell motility and promotes focal adhesion formation.

Deregulation of mechanisms that control cell motility plays a key role in tumor progression by promoting tumor cell dissemination. Secreted netrins and their receptors, Deleted in Colorectal Cancer (DCC), neogenin, and the UNC5 homologues, regulate cell and axon migration, cell adhesion, and tissue morphogenesis. Netrin and netrin receptor expression have previously been shown to be disrupted in invasive tumors, including glioblastoma. We determined that the human glioblastoma cell lines U87, U343, and U373 all express neogenin, UNC5 homologues, and netrin-1 or netrin-3, but only U87 cells express DCC. Using transfilter migration assays, we demonstrate DCC-dependent chemoattractant migration of U87 cells up a gradient of netrin-1. In contrast, U343 and U373 cells, which do not express DCC, were neither attracted nor repelled. Ectopic expression of DCC by U343 and U373 cells resulted in these cells becoming competent to respond to a gradient of netrin-1 as a chemoattractant, and also slowed their rate of spontaneous migration. Here, in addition to netrins' well-characterized chemotropic activity, we demonstrate an autocrine function for netrin-1 and netrin-3 in U87 and U373 cells that slows migration. We provide evidence that netrins promote the maturation of focal complexes, structures associated with cell movement, into focal adhesions. Consistent with this, netrin, DCC, and UNC5 homologues were associated with focal adhesions, but not focal complexes. Disrupting netrin or DCC function did not alter cell proliferation or survival. Our findings provide evidence that DCC can slow cell migration, and that neogenin and UNC5 homologues are not sufficient to substitute for DCC function in these cells. Furthermore, we identify a role for netrins as autocrine inhibitors of cell motility that promote focal adhesion formation. These findings suggest that disruption of netrin signalling may disable a mechanism that normally restrains inappropriate cell migration.

CT-GalNAc transferase overexpression in adult mice is associated with extrasynaptic utrophin in skeletal muscle fibres.

Duchenne muscular dystrophy is a genetic muscle disease characterized by the absence of sub-sarcolemmal dystrophin that results in muscle fibre necrosis, progressive muscle wasting and is fatal. Numerous experimental studies with dystrophin-deficient mdx mice, an animal model for the disease, have demonstrated that extrasynaptic upregulation of utrophin, an analogue of dystrophin, can prevent muscle fibre deterioration and reduce or negate the dystrophic phenotype. A different approach for ectopic expression of utrophin relies on augmentation of CT-GalNAc transferase in muscle fibre. We investigated whether CT-GalNAc transferase overexpression in adult mice influence appearance of utrophin in the extrasynaptic sarcolemma. After electrotransfer of plasmid DNA carrying an expression cassette of CT-GalNAc transferase into tibialis anterior muscle of wild type and dystrophic mice, muscle sections were examined by immunofluorescence. CT-GalNAc transgene expression augmented sarcolemmal carbohydrate glycosylation and was accompanied by extrasynaptic utrophin. A 6-week time course study showed that the highest efficiency of utrophin overexpression in a plasmid harboured muscle fibres was 32.2% in CD-1 and 52% in mdx mice, 2 and 4 weeks after CT-GalNAc gene transfer, respectively. The study provides evidence that postnatal CT-GalNAc transferase overexpression stimulates utrophin upregulation that is inherently beneficial for muscle structure and strength restoration. Thus CT-GalNAc may provide an important therapeutic molecule for treatment of dystrophin deficiency in Duchenne muscular dystrophy.

Inhibition of MMP-1 expression by antisense RNA decreases invasiveness of human chondrosarcoma.

We previously reported that an elevated level of matrix metalloproteinase-1 (MMP-1) gene expression in patients with chondrosarcoma has a strong statistical correlation with recurrence and in vitro invasion. In the present study, we used an antisense RNA strategy for MMP-1 inhibition to determine if this would affect the invasive characteristics of the cells. We transfected a human chondrosarcoma cell line with a retroviral plasmid expressing a 770 bp genomic fragment of the human MMP-1 gene in the sense or antisense orientation. The results show that cells transfected with the MMP-1 antisense fragment had a significant decrease in both MMP-1 protein and enzyme activity (p<0.05) as compared to cells transfected with an empty plasmid or the parental cells. Cells transfected with the MMP-1 antisense fragment demonstrated a significant decrease in their ability to invade the collagen I barrier (p<0.05). The gene expression for MMP-8 and MMP-13 were unaffected in cells transfected with the MMP-1 antisense fragment, MMP-1 sense fragment, or empty plasmid. These results support the hypothesis that MMP-1 facilitates tumor cell egress from chondrosarcoma tissue and demonstrate the potential of MMP-1 as a promising target for a novel biologic therapy in chondrosarcoma.