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Analysis of transformation of mammary epithelial cells by activated M-Ras Ward, Katherine Rachel


The apparent absence of activating mutations within the p21 Ras genes in human breast cancer raised the possibility of involvement of other Ras family genes in this tumor type. It had been proposed that activating mutations in R-Ras2 (TC21) may have a role in breast tumors and work within the laboratory had identified elevated levels of R-Ras3 (M-Ras) in breast cancer cell-lines. To investigate this idea I expressed M-Ras in a functional murine mammary epithelial cell-line, scp2. Expression of activated mutants of M-Ras, but not wild type M-Ras, in scp2 cells resulted in epithelial to mesenchymal transition (EMT) and oncogenic transformation. Cells expressing constitutively active M-Ras exhibited a loss of epithelial markers and a gain of mesenchymal markers. These cells continued to grow in the absence of serum and gained the capacity for anchorage-independent growth in methylcellulose. Furthermore, unlike the parental cells, they failed to form differentiated mammospheres on Matrigel and instead formed branched networks of cells. When injected into mice scp2 cells expressing activated M-Ras rapidly formed tumors. Expression of activated p21 Ras also resulted in EMT and tumorigenesis, although there was evidence that high levels of expression were toxic to this cell type. Tumors derived from scp2 cells expressing activated M-Ras or p21 Ras exhibited activation of both PKB and ERK. When expressed at similar levels Q71L M-Ras and G12V H-Ras resulted in comparable rates of tumor formation, although higher levels of expression of the'weaker G22V M-Ras mutant were required. More abstract follow

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