Identification of p53 and Its Isoforms in Human Breast Carcinoma Cells
Figure 6
(a) Western blot analysis of p53 expressed in the nuclear fraction of human breast cancer cells revealed a wide spectrum of isoforms (49, 48, 47, 45, 36, 35, 34, 29, 28, and 24 kDa). These isoforms were not present in nontransformed cells (control cases, lines 1 and 2). Only the Δ133p53β (35 kDa) and ~36 kDa isoforms are present at higher levels in the invasive breast carcinomas (lines 4, 5, and 7) compared to noninvasive cases (lines 3 and 6). The analysis suggested coexpression of mutant forms of p53 and/or wild-type p53 with p53 isoforms indicated. Expression of multiple p53 isoforms varied between phenotypically distinct subset of cases. In normal breast tissues, expression of p53 is either very low or not measurable. Equal amounts of nuclear proteins (50 μg/lane) from extracts of normal and breast cancer tissues were separated on 12% SDS-PAGE and transferred to nitrocellulose membrane. Incubation with CM-1 rabbit polyclonal antibody (diluted 1 : 1000) was followed by incubation with biotinylated swine anti-rabbit Ig serum (diluted 1 : 1000) and strept ABComplex-HRP conjugated as described in Section 2. HRP was detected with DAB as chromogen. Bottom panel shows actin loading control. All Western immunoblotting experiments were performed at least twice with similar results. Lines 1 and 2—normal breast tissue. Lines 4, 5, and 7—invasive ductal breast carcinomas. Lines 3 and 6—noninvasive breast carcinomas. Molecular mass markers are presented on the left (MW). The position of the p53 is indicated by the arrow. The arrows denote the position of the p53 isoforms. (b) Relative abundance of p53 and its isoforms in the nuclear fraction of breast cancer cells. The levels of p53 and its isoforms in each sample were quantified using transmission scanning and analyzed by Gel-Pro Analyzer 3.1. Significant increase of the p53 and its isoforms (35 and 36 kDa) is evident in nuclear fraction of breast cancer cells.
