As shown in the Figure 8H, we found http://www.selleckchem.com/products/Gefitinib.html TGFb to specifically induce binding of both Smad3 and p/CAF to DNA. Furthermore, we found that gene silencing of p/CAF and p21, using siR NAs, prevented Smad3 binding to the SBE, suggesting that both p21 and p/CAF are required for Smad3 DNA binding and Smad3 mediated transcriptional activity. Having shown that p21 is indeed required for Smad3 mediated transcriptional activity, we then assessed the effect of knocking down p/CAF on Smad3 transcriptional activity using the CAGA12 luc reporter construct. As shown in Figure 8J, the results clearly indicate that p/CAF is required for TGFb induced Smad3 transcriptional activity. Collectively, these data indicate that p21 and p/CAF reg ulate TGFb transcriptional activity by controlling Smad3 occupancy on its DNA binding elements.

TGFb induces a complex formation between Smad3, p21 and p/CAF, further leading to Smad3 acetylation by p/CAF. Further more, both p21 and p/CAF are required for Smad3 DNA binding and Smad3 mediated transcriptional activity, highlighting a novel mechanism by which the p21/p/CAF/ Smad3 complex contribute to the activation of TGFb tar get gene transcription. High expression of p/CAF/p21/p Smad3 is associated with lymph node positivity Lymph node involvement is an important prognostic indicator in clinical breast cancer outcomes. High expression of TGFb1 is correlated with a high incidence of lymph node metastasis.

To examine the associa tion among active TGFb/Smad signaling, p21 and p/ CAF with lymph node metastasis, we performed immu nohistochemistry to measure the expression levels of serine 423/425 phosphorylated Smad3, p21 and p/CAF in tissue microarray containing 50 invasive ductal breast tumors, 25 of which are lymph node posi tive. The immunoreactivity for pSmad3, p21 and p/CAF protein expression in tumor cells was graded and described in Methods. We considered a score of 0 to 2 as a low phosphorylation/expression level and a score of 3 to 4 as a high phosphorylation/expression level. As shown in Figure 9A, B, lymph node negative patients showed low levels of pSmad3, p21 and p/CAF expres sions, whereas a significant enrichment of high pSmad3, p21 and p/CAF was observed in patients with positive lymph nodes. To distinguish between tumor cells and stroma, we used a cytokeratin antibody, a cell marker specific to neoplastic cells of epithelial origin.

As shown in Figure S9, p21 is specifically expressed at a higher level in breast tumor cells but not in stroma cells. We also examined the relationships between pSmad3 levels and p/CAF Carfilzomib protein expression with p21 protein levels, using the Pearsons correlation test. As shown in Figure 9C, our results clearly indicate that high levels of phos phorylated Smad3 and p/CAF expression significantly correlate with high p21 protein expression.