Crucially, the effects of SNS 032 in AML cells were partially reversible after

At higher concentra tions, a change in the cell morphology was observed, suggesting that amlexanox concentrations higher than 25 uM might interfere with cellular metabolism. However, KU-55933 臨床試験 the cell viability even at 125 uM was comparable to that observed with the DMSO alone, Therefore, to avoid any confounding results, 25 uM was the highest concentration used. A faint band corresponding to p53 mRNA was detected in the DMSO sample. This residual expression of p53 in Calu 6 cells has already been observed previously and could represent a subpopulation of NMD protected p53 mRNAs or cytoplasmic untranslated p53 mRNAs.<br><br> The third cell line used is an immorta lized cystic fibrosis airway epithelial cell line derived from a population enriched for submucosal gland epithe lial cells, These cells are from a patient who is compound heterozygous: one allele is a CTT deletion spanning codons 507 and 508 of CFTR that results buy Linifanib in the in frame deletion of a phenyalanine, the other al lele is a CAG!TAG nonsense mutation at the codon 2 of the CFTR gene, A previous study indicated that there was little or no expression of the F508 nor of the Q2X alleles in the 6CFSMEo cells, When the cells were treated with increasing concentrations of amlex anox a 3 to 4 fold increase in the CFTR mRNA amount was detected at 25 uM of amlexanox, Even We then tested an immortalized myocyte cell line from a DMD patient with nonsense mutation in exon 71 at codon 3420 of the dystrophin gene, These cells were treated with amlexanox for 48 hours while they were exposed to conditions that pro mote differentiation and dystrophin expression.<br><br> As with the Calu 6 cells, increasing concentrations of amlexanox resulted in increased levels of the mutant mRNA, though we favor the hypothesis that LY3009104 1187594-09-7 amlexanox stabilized PTC containing mRNAs, we cannot exclude the possibility that amlexanox activated the expression of the F508 CFTR allele. As with Calu 6 cells, 25 uM was the most effective for facilitating CFTR mRNA increase in 6CFSMEo cells, Altogether results from Figures 1 and 2 confirm that mol ecule amlexanox blocks NMD in various PTCs and PTC environments. Cytotoxicity, translation efficiency and stabilization of natural NMD targets The specificity and cytotoxicity of the amlexanox were evaluated on Calu 6 cells. The effect of amlexanox on cell survival was evaluated after a 20 hours treatment at the working concentrations or at 125 uM of amlexanox.<br><br> The viability after amlexanox or DMSO in cubation was between 95 and 98%, indicating that amlexanox is not toxic for cells even at 125 uM in the conditions described above. Inhibition of translation by amlexanox was assayed by incorporating an L AHA modified amino acid in newly synthesized proteins. L AHA is then detected and measured using Click iT AHA new protein synthesis kit, Results of Figure 3B show that amlexanox has no signifi cant effect on the level of fluorescence, in contrast to cycloheximide which strongly reduces it, suggesting that the efficiency of translation is not altered by amlexanox. In addition, the phosphorylation status of eIF2 after amlexanox treatment was assessed since the induction of eIF2 phosphorylation as a function of inhibiting NMD has been demonstrated and that would impair translation process.