gram of apparently opposite functionality, characterized by the induction of the anti apoptotic IAP family member, cIAP2, a NF B target gene. cIAP2 e pression was significantly modulated at the mRNA and protein levels by retinoic acid in a cell conte t dependent manner. Using promoter mapping, promoter site directed inhibitor Gemcitabine mutagenesis, EMSAs and chro matin immunoprecipitation analysis we show that reti noic acid induces the recruitment of NF B proteins to NF B binding sites in the pro imal region of the cIAP2 promoter, thereby causing induction of cIAP2 e pres sion. In agreement with our data, the induction of NF B proteins binding and activity by retinoic acid has been reported in several cell systems such as neuroblas toma or leukemia cells.
Importantly, in addi tion to NF B proteins, the retinoid receptors, RAR and R R, are also recruited in vivo to the cIAP2 promoter upon retinoic acid treatment, despite the absence of bona fide RARE sites in this promoter by in silico analysis. Protein protein interaction between p50 p65 and R R that could contribute to stabilize the transcrip tional activation comple have been described. Despite the finding that mutation of an AP 1 motif decreases 9 cis RA inducibility, we could not detect in vivo recruitment of cJUN to the cIAP2 promoter in response to the retinoid. Although we cannot totally dis miss the possibility that cJUN takes part of the tran scriptional comple induced by retinoic acid, other AP 1 binding factors could be recruited to the promoter.
Importantly, although our data suggest that ligand bound RAR R R may be recruited directly to the tran scriptional activation comple we cannot discard that, in addition, retinoic acid induction of cIAP2 e pression proceeds via regulatory circuits, which are likely to involve retinoic acid target genes as well as cross talk with other signaling pathways. Thus, as reported for neuroblastoma Carfilzomib cells, retinoic acid could induce the activation in breast cancer cells of the phosphatidylinosi tol 3 Kinase Akt signaling pathway that finally results in NF B activation. Little is known about the anti apoptotic potential of retinoic acid. We provide evidence that in a cel lular conte t, present in T47D, ZR 75 1 and SK BR 3 cells, retinoic acid markedly upregulates cIAP2 e pres sion.
Retinoic acid CC 5013 significantly mitigates the apoptosis induced by chemotherapeutic agents in T47D and ZR 75 1 cells, while it is able to increase apoptosis by these compounds in H3396 cells where retinoic acid does not induce cIAP2 e pression. Many antiapoptotic proteins, such as Bcl 2, Mcl 1 and Bcl L, have been shown to inhibit chemotherapeutic agent induced apoptosis in diverse cell system models including hematopoietic and neuroblastoma cells. Additionally, it has been shown that the activation of genes encoding TRAF and IAP proteins by NF B serves to block apoptosis promoted by different insults including chemotherapy induced apoptosis in different cell types. In particu lar, overe pression of