Cells were incubated in DNA labeling solution for 2 h at 37 C and analyzed by FACS Calibur. PI stains total DNA and FITC conjugated Wortmannin solubility dUTP stains apoptotic cells. Statistical analysis Statistical comparisons are made using students paired t test using SPSS10. 0 and P value 0. 05 was considered Inhibitors,Modulators,Libraries significant. Results Development and screening of HeLaTet On p53 inducible cell system Seven out of 24 p53 transfected clones and nine out of 12 GFP transfected clones exhibited induction in the presence of Dox. Two clones HeLaTet On p53 23 S and HeLaTet On p53 26 S along with HeLaTet On BIEGFP 43 with low leaky and high regulatory expres sion were selected for further studies. Growth properties of clones for 6 days were similar to parental HeLa cells. Also, protein concentration did not alter between the clones and the parental cells.

Dox upto 2000 ng/ml was non Inhibitors,Modulators,Libraries toxic. Inhibitors,Modulators,Libraries Tight regulation of p53 expression was confirmed by addition of 100 and 1000 ng/ml of Dox. p53 expression was induced Inhibitors,Modulators,Libraries in response to Dox in a dose dependent manner. Also, GFP protein expression was tightly regulated. As E6 downregulation induces cell death, E6 mRNA levels in p53 and GFP expressing clones as well as in parental HeLa cells was detected by RT PCR. No alteration in e6 expression fol lowing treatment with Dox was observed. p53 localization and nuclear retention is essential for execution of its transcriptional and tumor suppressor activities. However, in cancer cells wild type p53 is sequestered in cytoplasm by various molecules which prevent its functioning.

p53 induced in response to Dox in a dose dependent manner is predominantly loca lized in the nucleus. No alteration in p53 protein expression was detected in Dox treated HTet43GFP and parental HeLa cells. In HTet43GFP cells GFP protein expression is Inhibitors,Modulators,Libraries tightly regulated by Dox. p53 overexpression does not cause cell cycle arrest or growth inhibition in HeLa cells even though it possesses DNA binding activity PI staining for the cell cycle analysis depicted no altera tion in cell cycle phases in p53 overexpressing cells as compared to HTet43GFP or HeLa cells. Long term consequence of p53 overexpression was investigated by clonogenic survival assay. Almost equal numbered and sized colonies were formed by p53 over expressing HTet23p53 and HTet26p53 cells.

As Dox induced p53 was localized in the nucleus, its in vitro DNA binding activity by electrophoretic mobi lity our site shift assay and in vivo transcriptional activ ity by chloramphenicol acetyl transferase reporter gene was evaluated. Increased binding of p53 to its con sensus sequence in HTet23p53 and HTet26p53 but not in HTet43GFP and HeLa cells after Dox addition was detected. Also, there was increase in CAT activity in p53 overexpressing HTet23p53 and HTet26p53 cells and no increase was detected in HTet43GFP and HeLa cells. Specificity of CAT activity was confirmed by PFTa treatment.