nducibil ity, http://www.selleckchem.com/products/BAY-73-4506.html similar to that found with 200 cIAP2 Luc construct, which contains an additional NF B site. However, the induction by 9 cis RA of the 247 cIAP2 Luc construct, which contains three NF B sites and two AP1 sites, was significantly higher, suggesting a potential role of these elements in the stimu lation by the retinoid. No significant variation was seen when 9 cis RA induced transcriptional activity between 247 cIAP2 Luc and longer cIAP2 promoter constructs was compared. Because no obvious RAREs could be found in the 247 bp retinoic acid responsive sequence, we systematically mutated each putative cis acting ele ment in the background of 247 cIAP2 Luc to test if one of these response elements could mediate this response.

Site directed mutagenesis of these response elements showed the critical importance of two NF B binding sites at positions 210 and 147 and one potential AP1 binding site at position 220, partially overlapping with the NF B binding site 1, and highlighted the contribution of the AP1 binding site at position 233 and the IRF E site at position 130 to retinoid induced promoter activity. To further reinforce these data, SK BR 3 cells were transiently co transfected with the 247 cIAP2 reporter gene and either an e pression vector coding for a domi nant negative mutant of I Ba, I Ba SR or an e pression vector coding for a dominant negative of c JUN, to test whether 9 cis RA inducibility was impaired.

E pression of the dominant negative mutant of I Ba totally blocked retinoid inducibility of the cIAP2 promoter, GSK-3 whereas e pression of TAM 67 only partially suppressed retinoid induced cIAP2 pro moter activity, thus confirming the critical role of NF B in the induction of cIAP2 e pression by 9 cis RA. Together, these data clearly demonstrate that NF B is critically involved in mediating the retinoic acid dependent transcriptional activation of the cIAP2 promoter and that potentially other factors, particularly c JUN and IRFs, contribute to the overall response. Since mutations of NF B binding sites resulted in a major decrease of 9 cis RA inducibility, we tested these sites in electrophoretic mobility shift assays. EMSAs with e tracts of T47D cells demonstrated that 9 cis RA induces the binding of a protein comple to the cIAP2 NF B1 and NF B3 sites. Incubation with antibodies against p65 inhibited binding, revealing the presence of this NF B family member in these comple es.

Therefore, we conclude that 9 cis RA induces the for mation of p65 containing comple es at the NF B bind ing sites of the cIAP2 promoter. To gain a deeper insight into the molecular mechan Crizotinib NSCLC isms underlying 9 cis RA induction of cIAP2 transcription, we performed chromatin immunoprecipi tation assays to assess the in vivo recruitment of p65, RAR, R Ra and c JUN to the cIAP2 promoter in untreated and 9 cis RA treated T47D cells. ChIP assays revealed that 9 cis RA induced acetylation of histone H3 at the cIAP2 promoter, a hallmark of transcriptional activation. In