As shown in Figure 3F, inhibition of EGFR kinase activity by 500 nM of Gefitinib or Erlotinib, demonstrated a 5 7 fold decrease www.selleckchem.com/products/17-AAG(Geldanamycin).html in the number of spheres. further the size of the spheres was also significantly reduced. A secondary point mutation in exon 20 of EGFR is associated with acquired resistance to gefiti nib or Erlotinib, but this can be overcome by the irre versible EGFR tyrosine kinase inhibitor BIBW2992. We tested the effect of 500 nM of gefitinib and 200 nM of BIBW on EGFR phosphorylation and Inhibitors,Modulators,Libraries self renewal growth of SP cells from H1975 cell line, which harbors gefitinib resistant Inhibitors,Modulators,Libraries T790M mutation along with Gefitinib responsive L858R mutation in exon 21. West ern blot analysis showed that tyrosine phosphorylation of EGFR was insensitive to 500 nM concentration of gefitinib, whereas significant downregulation occurred after treatment with 200 nM of BIBW in H1975 cells.

Consistent with this, BIBW could significantly inhibit the self renewal of SP cells from H1975 cells. Adherent cultures of SP Inhibitors,Modulators,Libraries cells maintain stem like properties To conduct further molecular studies on SP cells, we attempted to establish adherent cell cultures of isolated SP cells from A549, H1975 Inhibitors,Modulators,Libraries and H1650 cell lines, as sug gested for glioma stem cells. Isolated SP cells were plated on uncoated or Poly D Lysine Laminin coated culture plates in serum free, stem cell media. While A549 SP and H1975 SP cells detached from the surface, H1650 SP cells grew as an adherent culture. As shown in Figure 3A, H1650 SP cells cultured on uncoated sur face failed to maintain SP phenotype with high frequency but 80% of the cells main tained as SP cells even after 5 passages when plated on PDL laminin coated surface.

H1650 SPAdh cells. H1650 SPAdh cells cultured back in 5% FBS containing medium for 10 days could recapitulate the proportion of SP and MP cells found in parental H1650 cells with a concomitant reduc tion in expression of ABCG2, as well as Oct4, Sox2 and Nanog mRNA as seen by R PCR. Cell cycle analysis showed Inhibitors,Modulators,Libraries that H1650 SPAdh cells were slow cycling compared to parental cells, hav ing approximately 20% higher number of cells in G0 G1 phase. but upon serum induced differentiation, H1650 SPAdh cells acquired cell cycle phase distribution Imatinib Mesylate clinical com parable to H1650 parental cells. Treatment of H1650 SPAdh cells with 200 nM BIBW significantly suppressed the number as well as the size of spheres. at the same time, treatment with 30 uM cisplatin did not affect the number or the size of the spheres formed by H1650 SP cells, suggesting enhanced chemoresistance of these cells. Further, the sphere formation ability of SP was not altered by the ABCG2 inhibitor, FTC, suggesting that self renewal of SP cells was independent of ABCG2 activity.