Moreover, selleck chemicals llc rapidly high induction levels are achievable and the inducer, doxycycline , is well characterized. p53 overexpression does not promote cell cycle arrest and apoptosis in HeLa cells. We demonstrate that pro tein phosphatase 2A controls p53 functions and its inhibition activates p53, causing cell cycle arrest/ apoptosis in vitro and tumor growth inhibition in vivo. Interestingly, cyclin dependent kinase 5 regulates p53 phosphorylation essential for its activation. Taken together, we propose that non genotoxically overex pressed p53 can be activated by inhibiting its depho sphorylation in HPV positive cervical cancer cells. Inhibitors,Modulators,Libraries This strategy may be of therapeutic importance in p53 asso ciated gene therapy.

Materials Inhibitors,Modulators,Libraries and methods Chemicals and cell lines Antibodies against p53, GFP, pCdk5, Cdk5, p35, Bcl 2, cytochrome C, PARP, COX IV, b Tubulin, b Actin, HRP linked secondary antibodies, Control, p53 and PP2A siRNAs were purchased from Santa Cruz Biotechnology. Phospho specific p53 antibodies were purchased from Cell Signal ling Technology. FITC and rhodamine conjugated secondary antibodies were purchased from KPL. APO Direct TUNEL kit, Matrigel and Bax antibody was purchased from BD. Dox was purchased from Sigma. Hygromycin B and Tet system approved serum was purchased from Clontech. G418 and MTT were purchased from USB. Okadaic acid, mitotracker dye, DMEM, FBS and Lipofectamine2000 were Inhibitors,Modulators,Libraries pur chased from Invitrogen. Cdk2/5 inhibi tor, PFTa and U0126 were purchased from Calbiochem. Cdk5 siRNA was purchased from Dharmacon Inc. Development of cell lines is described in additional file 1.

Plasmids and transfection pC53 SN3 and pG13CAT were a kind gift from Dr. Bert Vogelstein, John Hopkins, Baltimore, MD. p53 fragment of pC53 SN3 was sub cloned in BamH1 site of pTRE and renamed as pTREp53. pG13CAT contains 13 repeats Inhibitors,Modulators,Libraries of p53 consensus binding site inserted in the 5 end to polyomavirus basal promoter linked to CAT reporter gene. Cells were Inhibitors,Modulators,Libraries co transfected with 2 ug of pG13CAT and 0. 5 ug of pEGFPC1 which serves as an internal control for transfection. Bcl 2 fragment from pRc/CMVBcl 2 was excised by HindIII and cloned into pTRE2 to obtain pTRE2Bcl 2. Cells were transfected with either 2. 0 ug or 0. 5 ug plasmid by Lipofectamine2000 transfection reagent as per manufacturers instructions.

Clonogenic survival assay Cells were treated with indicated concentrations of Dox, OA or Cdk2/5 inhibitor based on the experimental design http://www.selleckchem.com/products/Tubacin.html and incubated for 48 h. Cells were further grown for 21 days and thereafter colonies on the plate were stained with crystal voilet. Electrophoretic mobility shift assay To visualize the DNA binding activity of p53 in nuclear extracts of HTet23p53, HTet26p53, HTet43GFP and HeLa cells, EMSA was performed. After treatment with Dox, cells were harvested for the preparation of cyto plasmic and nuclear fractions by using nuclear extrac tion kit as per manufacturers instructions.