Error bars show standard deviation

Error bars show standard deviation. response to drugs, correlating with reduced p53 apoptotic transactivation. The drug-induced apoptotic cell death, reduced by high glucose, was finally restored by the phosphatase inhibitor calyculin A. Conclusions These data indicate that high glucose specifically inhibited Ser46 phosphorylation thus reducing p53 apoptotic activity. These results uncover a new mechanism of p53 inactivation providing an interesting novel molecular link between metabolic diseases such as diabetes or obesity and tumor progression and resistance to therapies. gene with Ser46A mutation (non phosphorylatable Ser46), reduced p53 apoptotic transactivation [17], strengthening the apoptotic role for this p53 posttranslational modification. Hyperglicaemia is a pathophysiological condition characterized by high blood glucose concentration that has been shown to predispose to cancer development and progression [18]. Hyperglicaemia is often a consequence of a Western lifestyle that is associated with metabolic syndrome and type-2 diabetes or obesity. Epidemiological evidence suggests that patients with diabetes mellitus are at significantly higher risk of developing many types of cancers [19]. Foods with high glycemic load are most closely correlated with higher recurrence of colon cancer [20]. Moreover, hyperglicaemia may inhibit tumor response to therapies conferring resistance to chemotherapy-induced cell death [21-24]. Glucose metabolism has been shown to reduce p53-dependent transcription of apoptotic Puma gene, although the molecular mechanism of such inactivation was not elucidated [25]. Therefore, in this study we sought to investigate whether high glucose (HG) culture condition might target p53Ser46 in cancer cells and have an impact on p53-induced drug response. Materials and methods Cell culture and reagents In this study human lung cancer H1299 (p53 null), colon cancer RKO and NS-1643 HCT116 (carrying wild-type p53), HCT116-p53-/-, lung cancer A549 and ovarian cancer 2008 cells (carrying wild-type p53), were used. Cells were routinely cultured in DMEM (Life Technology-Invitrogen) containing 1?g/L D-glucose, supplemented with 10% heat-inactivated fetal bovine serum (FBS) plus glutamine and antibiotics. NS-1643 For high glucose (HG) treatment, cells were transferred to DMEM containing 4.5?g/L D-glucose (Life Technology-Invitrogen), as previously reported [22,23], supplemented with 2% FBS for 24?h before adding chemotherapeutic drugs Adriamycin (ADR) or cisplatin (CDDP) to the culture media respectively at 2?g/ml and 5?g/ml for additional NS-1643 16?h (for ChIP assay) or 24?h (for all the other experiments). Phosphatase inhibitor calyculin A [26] (Sigma) was added at 1 nM along with drugs. Viability and tunel assays For viability assay, subconfluent cells were plated in duplicate in 60?mm Petri dishes and 24?h later transferred to HG medium or DMEM with 1?g/L D-glucose, both containing 2% FBS. The day after, cells were treated with ADR or CDDP for 24?hours. Both floating and adherent Ctsl cells were collected and cell viability was determined by Trypan blue exclusion by direct counting with a haemocytometer, as reported [27]. Tunel assays were essentially performed as described [28]. Briefly, 4×104 cells were spun on a slide by cytocentrifugation and subsequently fixed in 4% paraformaldheyde for 30?min at room temperature. After rinsing with PBS, the samples were permeabilized in a solution of 0.01% Triton X-100 in sodium citrate for 2?min. Samples, washed with PBS, were then incubated in the TUNEL reaction mix for 1?h at 37C according to the manufactures instructions (Roche, Germany). Cells were counter-stained with Hoechst 33342 before analysis with a fluorescent microscope (Zeiss). Chromatin-immunoprecipitation (ChIP) assay ChIP assay was carried out essentially as previously described [29]. Protein complexes were cross-linked to DNA in living cells by adding formaldehyde directly to the cell culture medium at 1% final concentration. Chromatin extracts containing DNA fragments with an average size of 500?bp were incubated overnight at 4C with milk shaking using polyclonal anti-p53 antibody (FL393, Santa Cruz Biotechnology). Before use, protein G (Pierce) was blocked with 1?g/L sheared herring sperm DNA and 1?g/L BSA for 3?h at 4C and NS-1643 then incubated with chromatin and antibodies for 2?h at 4C. PCR was performed with HOT-MASTER Taq (Eppendorf) using 2?L of immuniprecipitated DNA and promoter-specific primers. Immunoprecipitation with non-specific immunoglobulins (IgG; Santa Cruz Biotechnology) was performed as negative controls. The amount of precipitated chromatin measured in each PCR was normalized with the.