However, NRIF3 expression did not lead to apoptosis in a wide variety of other cell types. Apoptosis mediated by NRIF3 was documented by FACS analysis, binding of Annexin V, time lapse imaging, and TUNEL assay. This apoptotic activity was mapped to a short molarity calculator 30 amino acid region Inhibitors,Modulators,Libraries of NRIF3. We refer to this region as Death Domain 1 since it is necessary and sufficient to mediate apoptosis of breast cancer cells. DD1 does not interact with nuclear receptors, thus, this apoptotic effect of NRIF3 is inde pendent of its action as a nuclear receptor co activator. Change Inhibitors,Modulators,Libraries of Inhibitors,Modulators,Libraries Ser28 to Ala28 abrogates the ability of NRIF3 DD1 to mediate apoptosis suggesting that phosphorylation of Ser28 is important for this bio logical effect of NRIF3 DD1.
We cloned the intracellular target of NRIF3 DD1 and refer to this factor as DD1 Interacting Factor 1 which Inhibitors,Modulators,Libraries is a transcriptional repressor. Inhibitors,Modulators,Libraries Our studies indi cated that DIF 1 acts to selectively repress one or more pro apoptotic genes in breast cancer cells and this repression is reversed by the binding of NRIF3 DD1. The notion that DIF 1 represses pro apoptotic genes in breast cancer cells is further supported by the finding that knockdown of DIF 1 by siRNA leads to apoptosis of breast cancer cells but not of other cell types. including MCF 10A cells and C57MG cells, which are respectively immortalized normal human and mouse breast epithelial cell lines. Thus, DIF 1 acts as a death switch whose activity can be attenuated by the binding of NRIF3 DD1 leading to pro apoptotic gene expression in breast cancer cells.
Through microarray and expression studies, we identi fied FASTKD2 as the pro apoptotic target gene that is re pressed by DIF 1 in breast cancer cells but not other cell types. DIF 1 binds to the FASTKD2 gene in breast cancer cells but not to the FASTKD2 gene in other cell types. Knockdown of FASTKD2 by siRNA prevents NRIF3 DD1 mediated apoptosis http://www.selleckchem.com/products/Vandetanib.html in breast cancer cells while expression of FASTKD2 leads to apop tosis in all cell types. Our findings are consistent with a model where rapid and transient de repression of the FASTKD2 gene in breast cancer cells leads to apoptosis. Although, the NRIF3 DD1 DIF 1 pathway does not me diate apoptosis of a wide variety of non breast cancer cell lines, because of certain similarities and gene signatures between breast and prostate cancer we explored whether the NRIF3 DD1 FASTKD2 pathway mediates apoptosis of prostate cancer cell lines. We examined LNCaP cells which are androgen dependent as well as two other LNCaP cell lines which express high levels of androgen receptor but are andro gen independent with regard to growth. Interestingly, LNCaP AI and LNCaP abl are much more resistant to apoptosis than androgen dependent LNCaP AD cells.
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