f the super Nutlin-3a Mdm2 inhibitor repressor of NF B activation, I Ba SR, abrogates protection of etoposide induced apoptosis by 9 cis RA To e plore whether NF B activation may play a role in protection of etoposide induced apoptosis by 9 cis RA, we generated T47D cells stably overe pressing a consti tutively active form of I Ba, I Ba SR. This mutated version of I Ba contains serine to alanine mutations at residues 32 and 36, which confer resistance to signal induced phosphorylation and subsequent pro teasome mediated degradation. Thus, NF B dimers remain bound to I Ba SR in the cytosol, and their translocation to the nucleus and the subse quent transcriptional regulation of their target genes is impaired. The cDNA coding for I Ba SR was inserted into de pcDNA3. 1 vector and after transfection in T47D cells, a pool of neomycin resistant cells was isolated.

To determine the effectiveness of the super repressor in the stable cell line, we assessed by western blotting I Ba resistance to TNFa induced degradation. For this purpose, whole cell e tracts were prepared from T47D I BaSR cells and control cells i. e. a pool of neomycin resistant cells iso lated after transfection of the pcDNA3. 1 vector. As e pected, I Ba was degraded after 15 min treatment with TNFa in T47D vector cells, while degradation of I Ba was not affected by TNFa treat ment in the case of T47D I BaSR cells. Furthermore, the e pression of I Ba SR had no effect on proliferation rate of the stable cell line in the absence of treatment. Therefore, T47D I BaSR cells are a good tool to determine the involvement of the NF B pathway in the protection of apoptosis by 9 cis RA.

As a further control of the efficiency of NF B inacti vation, we evaluated both the basal and 9 cis RA induced level of the NF B dependent cIAP2 mRNA and protein in the I Ba mutant cell line by real time PCR and western blot, and found that cIAP2 Dacomitinib levels were specifically down regulated when compared to control cells. To evaluate the impact of I BaSR overe pression in 9 cis RA protection against etoposide mediated apoptosis, we compared by Western blotting, as a measurement of cell death, the level of activation of caspase 3 between T47D vector cells and T47D I BaSR cells.

While the level of cleaved caspase 3 was induced by etoposide in control cells and strongly abrogated when cells were pretreated with 9 cis RA, overe pression of the I Ba mutant did not affect notably caspase 3 activation by etoposide, but restored very significantly the activation of cleaved cas pase 3 by fairly etoposide in the presence of 9 cis RA. We also compared the apoptosis induced by etoposide in the presence or absence of 9 cis RA pretreatment in T47D vector and T47D I BaSR cells by propidium iodide staining and FACS analysis. As seen in Fig. 7D, while pretreatment with 9 cis RA inhibits etoposide mediated cell death in T47D vector cells, apoptosis was enhanced in T47D I BaSR cells treated with 9 cis RA alone, and these cells were equally sensitive to etoposide in the p