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Previous study reported that the activation of Ras pathway cooperated with E6/E7-mediated inactivation of p53/pRb to transform immortalized normal human astrocytes (NHA/hTERT) into intracranial tumors strongly resembling human astrocytomas. The mechanism of how H-Ras contributes to astrocytoma formation is unclear. Using genetically modified NHA cells (E6/E7/hTERT and E6/E7/hTERT/Ras cells) as models, we investigated the mechanism of Ras-induced tumorigenesis. The overexpression of constitutively active H-RasV12 in E6/E7/hTERT cells robustly increased the levels of urokinase plasminogen activator (uPA) mRNA, protein, activity and invasive capacity of the E6/E7/hTERT/Ras cells. However, the expressions of MMP-9 and MMP-2 did not significantly change in the E6/E7/hTERT and E6/E7/hTERT/Ras cells. Furthermore, E6/E7/hTERT/Ras cells also displayed higher level of uPA activity and were more invasive than E6/E7/hTERT cells in 3D culture, and formed an intracranial tumor mass in a NOD-SCID mouse model. uPA specific inhibitor (B428) and uPA neutralizing antibody decreased uPA activity and invasion in E6/E7/hTERT/Ras cells. uPA-deficient U-1242 glioblastoma cells were less invasive in vitro and exhibited reduced tumor growth and infiltration into normal brain in xenograft mouse model. Inhibitors of Ras (FTA), Raf (Bay 54-9085) and MEK (UO126), but not of phosphatidylinositol 3-kinase (PI3K) (LY294002) and of protein kinase C (BIM) pathways, inhibited uPA activity and cell invasion. Our results suggest that H-Ras increased uPA expression and activity via the Ras/Raf/MEK signaling pathway leading to enhanced cell invasion and this may contribute to increased invasive growth properties of astrocytomas., (Copyright (c) 2008 Wiley-Liss, Inc.)

Low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytotic receptor that may modify the biological activity of reactive astrocytes in neuroplasticity and neurodegeneration and of malignant astrocytes in brain invasion. In this study, the regulation of LRP by epidermal growth factor receptor (EGFR) ligands in both cultured human fetal astrocytes and astrocytic tumor cell lines (U-251 MG and U-1242 MG) was investigated. All astrocytic cell types expressed LRP, as determined by the binding of activated alpha2-macroglobulin (alpha2M*) on intact cells and by Western and Northern blot analyses of cell extracts. Primary cultured astrocytes expressed the highest levels of alpha2M*-binding capacity (Bmax = 30 fmol/mg protein). This was twofold higher than for the U-1242 MG astrocytoma cells (Bmax = 15 fmol/mg protein) and fourfold greater than for the glioblastoma U-251 MG cells (7.0 fmol/mg protein). Receptor affinity (K(D)) ranged from 0.25 to 0.6 nM in all the astroglial cell types. Functional LRP at the surface was down-regulated by EGF, compared with controls, as indicated by a reduction of both Bmax and LRP-mediated endocytosis by approximately 50% and 60%, respectively. In comparison, EGF treatment of primary astrocytes did not down-regulate LRP expression or LRP-mediated endocytosis. Treatment of the tumor cells with EGF or TGFalpha (25 ng/ml) significantly down-regulated total cellular LRP. Receptor-associated protein (RAP) mRNA expression was not affected by EGF in either tumor cells or primary astrocytes. The reduction of LRP in the tumor cells resulted from a specific decrease in LRP mRNA transcription, as determined by Northern blot and nuclear run-on experiments. These data suggest that EGF mediates a functional down-regulation of LRP endocytotic activity in astrocytic tumor cells and that LRP expression is differentially regulated in neoplastic and non-neoplastic astrocytes.

Human alpha 2-macroglobulin (alpha 2M) is a broad spectrum proteinase inhibitor and cytokine carrier synthesized by a number of cell types including monocytes and macrophages. In this study, we report on the expression of alpha 2M by THP-1 cells. This monocytic cell line can be differentiated into a macrophage-like phenotype by treatment with interferon-gamma (IFN-gamma) or phorbol 12-myristate 13-acetate (PMA). alpha 2M was synthesized by THP-1 cells at a rate of 75 ng/10(6) cells/24 h, as determined by Western blot analysis. After treating the cells with 500 U/ml of IFN-gamma or with 100 ng/ml PMA, the synthesis rate increased to 219 ng/10(6) cells/24 h and to 179 ng/10(6) cells/24 h, respectively. The same agents also increased alpha 2M expression, as determined by Northern blot analysis. When the alpha 2M receptor antagonist, receptor associated protein (RAP), was included in the THP-1 medium, the amount of alpha 2M recovered in the conditioned medium increased. This result suggests that THP-1-secreted proteinases react with secreted alpha 2M and that the resulting complexes are catabolized by the alpha 2M receptor, which is also called low density lipoprotein receptor-related protein (LRP). We conclude that alpha 2M synthesis by THP-1 cells depends on the state of cellular differentiation. Reaction of alpha 2M with secreted proteinases may have minimized previous estimates of the rate of synthesis of alpha 2M by certain cells in culture.