Analysis of p53 binding to head-to-tail response element in cyclin B promoter using electrophoretic mobility shift assays.

<p><b>A</b>, MCF10A, MCF10A/OD, and MCF10A/Δp53 cell lines were incubated with 10 ng/ml SN38 for 24 hours (+SN) or were left untreated (–SN). Binding reactions were prepared by incubating nuclear extracts or recombinant p53 with a biotin-labeled probe corresponding to the head-to-tail response element in the cyclin B promoter, in the presence (+) or absence (−) of a 200-fold molar excess of specific DNA (unlabeled probe). All binding reactions were carried out in the presence of a 100-fold molar excess of unlabeled non-specific DNA. Complexes were separated on 4% native polyacrylamide gel electrophoresis, transferred to positively charged nylon membrane, and visualized using a streptavidin-horse radish peroxidase conjugate. <b>B</b>, A parallel gel was transferred to nitrocellulose membrane and immunoblotted with p53-specific antibodies.</p

Determination of p53 oligomerization status using glutaraldehyde cross-linking.

<p><b>A</b>, MCF10A, MCF10A/OD, and MCF10A/Δp53 and cells were incubated with 10 ng/ml SN38 (+SN38) for 24 hours or <b>B</b>, fresh media (untreated) for 24 hours. Cells were harvested in lysis buffer and incubated with the indicated concentrations of glutaraldehyde for 5 minutes. The cross-linked lysates were then analyzed by SDS-Page followed by immunoblotting with p53-specific antibodies. Cell lysates were loaded at 10,000 or 30,000 cells/lane as indicated.</p

Determination of cyclin B mRNA and protein levels.

<p>MCF10A, MCF10A/OD, and MCF10A/Δp53 cells were incubated with 0–30 ng/ml SN38 for 24 hours. RNA was purified from cell lysates and relative levels of cyclin B mRNA were analyzed using RT-PCR followed by agarose gel electrophoresis. Cell lysates were also analyzed for expression of cyclin B and actin protein using SDS-PAGE followed by immunoblotting with cyclin B- or actin-specific antibodies.</p

Comparison of head-to-head and head-to-tail p53 response elements.

<p>The consensus sequence of the standard p53 response element consists of two half-sites, each with quarter-sites oriented in a head-to-head manner (indicated by arrows). R =  purine, Y =  pyrimidine, W = A/T, and N = A/T/C/G. The p21 promoter contains a standard head-to-head p53 response element, while the MDR1 and cyclin B genes contain p53 response elements with head-to-tail orientations.</p

Analysis of p53 binding to head-to-tail response element using chromatin immunoprecipitation assays.

<p>MCF10A, MCF10A/OD, and MCF10A/Δp53 cells were incubated with either fresh media (untreated) or 10 ng/ml SN38 for 24 hours, followed by cross-linking in 1% formaldehyde. Chromatin was then purified, sonicated, and immunoprecipitated with anti-p53 antibody or negative IgG antibody. Unprecipitated chromatin was used as a positive control (input). DNA was isolated and amplified by PCR using primers flanking the head-to-tail response element in the cyclin B promoter or the RNA Polymerase II binding site from the GAPDH promoter. The amplified PCR fragments were analyzed by agarose gel electrophoresis.</p

Timecourse of cyclin B and p21 expression following SN38 treatment.

<p>MCF10A, MCF10A/OD, and MCF10A/Δp53 cells were treated with 30 ng/ml SN38. <b>A</b>, Cells were harvested at the indicated time points and lysates were analyzed using SDS-PAGE followed by immunoblotting with the indicated antibodies. Band intensity of scanned immunoblots was measured using Image J software and the measurements were normalized to background. <b>B</b>, p21 and <b>C</b>, cyclin B levels were plotted for MCF10A (black bars), MCF10A/OD (light gray bars), and MCF10A/Δp53 (dark gray bars).</p