All positive control mice implanted with 5 106 NHA/TSR cells, which is a transformed astrocytes, developed tumors at 4 6 weeks with 100% http://www.selleckchem.com/products/Temsirolimus.html incidence, whereas any of the mice injected with NHA/TS or NHA/TSE2 cells could not grew as tumors at least by 6 weeks. These data indicate that Ets2 is not sufficient to fully transform normal human astrocytes, and thus the acquisition of tumor stem Inhibitors,Modulators,Libraries like characters may be accomplished by synergistic actions of multiple factors. Human astrocytes transformed by oncogenic Ras can form neurosphere like colonies with the increased expression of CD133 mRNA NHA/TSR cells form malignant tumors with features of human anaplastic astrocytoma, the WHO classification Grade III in mice and malignant astrocytoma has been well known to display a stem cell signature.

To confirm whether Ras mediated transformation in human astrocytes could confer stem like features in parallel with the acquisition of tumorigenicity, we examined the expression level of CD133 and the activity of neuro sphere formation in NHA/TSR cells. In Inhibitors,Modulators,Libraries the absence of 5Aza dC/TSA, the more substantial level of CD133 mRNA Inhibitors,Modulators,Libraries was detected in NHA/TSR cells comparing to NHA/TSE2 cells, suggesting that Ras might play an additional role to modulate chromatin structure of CD133 promoter region. Actually, CD133 expression is regulated by promoter DNA methylation. How ever, the protein level of CD133 Inhibitors,Modulators,Libraries could not be increased, suggesting that Ras activation is also not sufficient to completely restore CD133 expression in human astrocytes.

The generation of neurospheres is considered as an important symbol of in vitro neural stem cell culture, as well as an exhibi tion of self renewal capacity. After culturing in NSC medium containing bFGF and EGF for 2 weeks, NHA/TSR cells generated significantly greater neuro sphere like colonies Inhibitors,Modulators,Libraries than NHA/ TS cells, and this capacity was maintained to generate secondary neurospheres. To except the pos sibility of cellular aggregates, single NHA/TSR cell was also sorted using flow cytometry and deposited into 48 well plates containing NSC medium. NHA/TSR had a forming efficiency of 12. 7 1. 5 single spheres per 48 well plate, as opposed to zero for NHA/TS.

A single sphere could also maintain the capa city to form secondary spheres, as well as differentiate into adherent cells morphologically identical to original NHA/TSR cells in 10% FBS containing medium, indicating that constitutive activation of the Ras pathway can trigger the development of stem like popu lation with self renewing ability to retain stemness, and possibly INCB028050 that human astrocytes could dedifferentiate to premature state following to the Ras mediated transfor mation and/or its sequential combination with immorta lization. Side population size was not increased in NHA/TSR cells.