PUMA Regulates BITC-Induced Apoptosis Figure 4. Overexpression of Bcl-xL and Bcl-2 confers protection against BITC-induced apoptosis in MCF-7 cells. (A) Western blotting for Bcl-xL and Bcl-2 using lysates from MCF-7 cells treated with DMSO (control) or BITC (2.5 or 5 mM) for the indicated time periods. Number above band indicates change in level compared to the corresponding DMSO-treated control. (B) Western blotting for Bcl-xL using lysates from MCF-7 cells transiently transfected with empty vector or Bcl-xL plasmid and treated for 24 h with DMSO or 5 mM BITC. Number above band represents change in level relative to empty vector transfected cells treated with DMSO (first lane). (C) Quantitation of histone-associated DNA fragment release into the cytosol in MCF-7 cells transiently transfected with empty vector or vector encoding for Bcl-xL and treated for 24 h with DMSO or 5 mM BITC. (D) Western blotting for Bcl-2 using lysates from MCF-7 cells transiently transfected with empty vector or Bcl-2 plasmid and treated for 24 h with DMSO or 5 mM BITC. Number above band represents change in level relative to empty vector transfected cells treated with DMSO (first lane). (E) Quantitation of histone-associated DNA fragment release into the cytosol in MCF-7 cells transiently transfected with empty vector or vector encoding for Bcl-2 and treated for 24 h with DMSO or 5 mM BITC. Data in C, E are expressed as enrichment relative to corresponding DMSO-treated control (mean 6 SD, n = 3). Significantly different (P,0.05) acompared with corresponding DMSO-treated control, and bbetween empty vector transfected cells and Bcl-xL or Bcl-2 overexpressing cells by one-way ANOVA followed by Bonferroni’s multiple comparison test. The experiments were repeated twice and data from one representative experiment are shown. doi:10.1371/journal.pone.0032267.g004 (b) BITC-induced apoptosis is partially but significantly attenuated by RNA interference of PUMA, (c) the PUMA knockout variant of HCT-116 is significantly more resistant to BITC-induced apoptosis compared with wild-type HCT-116 cells, and (d) BITC adminis- tration increases levels of PUMA protein in MDA-MB-231 tumor xenografts in vivo. Because BITC-mediated induction of PUMA is discernible in cultured and xenografted breast cancer cells, this protein represents a viable biomarker of BITC response. Role of p53 in regulation of PUMA expression is well established [20]. PUMA is expressed at a low level in normal tissues but it is highly sensitive to induction in response to a wide variety of stresses [20,28]. In stressed cells (e.g., DNA damage), p53 is recruited to the two p53-responsive elements in the PUMA promoter [29]. Binding of p53 to the PUMA promoter alters acetylation of histones H3 and H4 leading to opening of the chromatin structure and hence transcriptional activation [30]. The results of the present study indicate that BITC treatment causes both p53-dependent and p53-independent induction of PUMA as this effect is observed in both MCF-7 and MDA-MB-231 cells. However, as expected the MCF-7 cell line is relatively more PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e32267