The pellet was washed and stored at 220uC until used

ion of p21 expression and impairment of cell cycle progression and proliferation by silencing APE1 in pancreatic cell lines. We addressed this paradox by analyzing the role of APE1 in p21 expression in p53-expressing colon carcinoma HCT116 cells and its isogenic p53-null cells. We report here that APE1 functions as a constitutive co-repressor for p21 gene by its association with transcription factor AP4. However, APE1’s co-repressor function is overridden in the presence of p53 which after binding to APE1 activates the p21 gene. We have thus provided the first evidence for dual and opposite transcriptional co-regulatory roles of APE1 in controlling p21 expression which is dependent on p53 status. sc-2003) in the pre-cleared extracts of control and experimental cells. The immunoprecipitated proteins were resolved in SDSPAGE and identified by Western analysis with the indicated antibodies; HRP-conjugated mouse a-FLAG, mouse monoclonal a-p53 and goat aAP4. Depletion or overexpression of APE1 in experimental and control cells were detected by Western analysis of 4896 hours-post transfected cells with a-APE1 antibody. Luciferase Assay Cells were co-transfected with p21-promoter luciferase reporter construct and expression plasmid for APE1 or empty vector and luciferase activity in the extracts of 3648 hours-post transfected cells was measured in a luminometer using the luciferase assay kit. The luciferase activity was normalized with respect to total protein content of the lysates. RNA Isolation and Real Time RT-PCR Assay Total RNA 15276073 was isolated from cells with Qiagen RNeasy mini kit followed by DNase 1 treatment 8866946 and proceeded for cDNA synthesis using Superscript III 2883-98-9 first-strand synthesis kit. AP4 and p21 expression in the samples were analyzed by SYBR GREEN-based Real Time PCR using SYBR Premix Ex Taq and primers appropriate for p21, AP4 or HPRT1 expression. Data were represented as relative quantitation with respect to the reference samples set at 1 based on 22DDCT method. Materials and Methods Cell lines, Plasmids, siRNA and Transfection Human colorectal adenocarcinoma HCT116WT and HCT116p53null lines were kind gifts from Dr B. Vogelstein, Johns Hopkins University School of Medicine and were cultured in McCoy’s 5A medium supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin. Exponentially growing cells were treated with 10 mM etoposide and harvested after 5 hours. Wild Type APE1/N-terminal 42 amino acid deleted mutant APE1 expression plasmid in PCDNA3 backbone, PCMV 5.1 FLAG-tagged expression plasmid for WT APE1/N-terminal 33 amino acid deleted mutant APE1 were described elsewhere. p53 expression plasmid used in this study was kindly provided by Dr. A. L. Levine. siRNAs specific for APE1, AP4 mRNA and universal control siRNA were obtained from Sigma. Exponentially growing cells were transfected with Lipofectamine 2000 following manufacturer’s protocol and harvested for RNA isolation and RT-PCR, luciferase activity assay, chromatin immunoprecipitation assay, coimmunoprecipitation assay and Western analysis as required. Chromatin Immunoprecipitation Assay ChIP assay was performed after double crosslinking of cells with disuccinimidyl glutarate and formaldehyde, with Magna ChIP Protein A Magnetic beads using the following antibodies: a-APE1, a-AP4, a-p53 or control IgG as described previously. The immunoprecipitated purified DNA was then subjected to SYBR GREEN-based Real Time PCR with primers for p21 distal