We performed RT PCR analysis under similar growth conditions, to analyze the status of p53 regulated genes p21, Bax, and GADD45. As is seen in Fig. 1E, no significant alteration in the expression pattern of these genes was detected in MCF7As3 and MCF 7As6 clones when comparing to the expression in adult MCF 7 in addition to control MCF 7H cells. These genes may be applying p53 independent paths for their expression. Since both As3 and As6 clones were usually related, for chk inhibitor investigations and further studies, MCF 7As3 and MCF 7As6 were pooled together and termed as MCF 7As53 cell line. The antisense p53 expressing MCF 7As53 cells, parental MCF7 cells, and resistant clone MCF 7H were compared and further characterized for other p53 related proteins in addition to for breast carcinoma particular marker substances. ER plays an important role in MCF 7 cells and breast cancer development are ER positive breast cancer model. As illustrated in Fig. 2A, no huge difference in ER expression levels was detected in the three cell lines and the degree of ER expression was identical. Aside from ER position MCF 7As53 cells showed normal FP degrees, which is a well known carcinoembryonic antigen expressed in breast carcinoma. Plastid Bax, a common p53 managed protein, was also not altered very significantly. No differences were found in the appearance of Mdm2 oncoprotein, the main element upstream regulator of p53, which targets it to proteasome mediated degradation and checks its transactivation properties. Mdm2 is amplified or overexpressed in several human cancers, including ovarian cancer, breast cancer, osteosarcoma, and lymphoma. Still another critical molecule is p73, which is really a p53 family protein with structural and functional homology and shares similarities with the tumor suppressor gene with respect to activation of transcription from p53 open marketers, along with directly or indirectly affecting either p53 activity or expression levels. In comparison to those in adult cells the constant state p73 protein levels in the MCF 7As53 cell line were similar. These results imply MCF7As53 showed no major variability at molecular level apart from the p53 expression. The house keeping proteins such as T tubulin and W actin were employed as internal controls for protein loading as well as for comparing changes in the protein Dizocilpine selleck expression pattern in the cells. In some experiments comparative profile of compounds were gathered from various duplicate gels. We conducted CAT reporter assay, further to verify that indeed p53 downregulation also leads to decline in p53 dependent transactivation activity. MCF 7 and MCF 7As53 cells were separately transfected with either pG13 CAT or pWWPCAT constructs as described in Materials and methods.