Downregulation of Wip-1 phosphatase expression in MCF-7 breast cancer cells enhances doxorubicin-induced apoptosis through p53-mediated transcriptional activation of Bax

• Published in: Cancer biology & therapy 2009-03, Vol.8 (6), p.555-563
• Main Authors: Kong, Weihong, Jiang, Xiaoshan, Mercer, W. Edward
• Format: Article
• Language: English
• Subjects: Antibiotics, Antineoplastic - pharmacology; Apoptosis - drug effects; Apoptosis - genetics; Base Sequence; bcl-2-Associated X Protein - genetics; Binding; Biology; Bioscience; Breast Neoplasms - genetics; Breast Neoplasms - metabolism; Calcium; Cancer; Cell; Cell Line, Tumor; Cycle; Doxorubicin - pharmacology; Female; Gene Expression Regulation, Neoplastic - drug effects; Gene Silencing; Genes, Reporter; Humans; Landes; Organogenesis; Phosphoprotein Phosphatases - genetics; Phosphorylation; Promoter Regions, Genetic; Protein Phosphatase 2C; Proteins; RNA Interference; Transcriptional Activation; Tumor Suppressor Protein p53 - metabolism
• ISSN: 1538-4047; 1555-8576
• DOI: 10.4161/cbt.8.6.7742

The human breast cancer cell line MCF-7 carries an amplified PPM1 D/Wip-1 gene and over expresses Wip-1 phosphatase protein. MCF-7 cells also harbor a wild type p53 gene. We established stable isogenic lines (MCF-Sp53 clones) which exhibit decreased levels of p53 protein. We show that although the PPM1 D gene is amplified in MCF-7 cells it is still expressed in a p53-dependent manner. Stable isogenic cell lines derived from MCF-7 cells (designated MCF-clones) were also established in which Wip-1 expression is significantly decreased by a plasmid-based PPM1D antisense RNA. Decreasing Wip-1 expression sensitized MCF-clones to doxorubicin-induced apoptosis. The enhanced apoptotic response was correlated with increased phosphorylation of N-terrninal p53-Ser15 and -Ser46 and increased expression of the pro-apoptotic Bax gene at both the mRNA and protein level. The enhanced apoptotic response was blocked by Bax-siRNA knock down suggesting that the increased response was a result of increased Bax protein expression. Moreover, reporter gene assays using the Waf-1 and Bax promoters to drive a luciferase gene revealed that luciferase activity driven by the Bax promoter was enhanced in MCF-clones while luciferase activity driven by the Waf-1 promoter was decreased relative to parental MCF-7 cells. The study reveals a novel molecular mechanism involving Wip-1 phosphatase, p53 phosphorylation and an enhanced apoptotic response mediated by transcriptional activation of the pro-apoptotic Bax gene. W. Edward Mercer Ph.D., who made great contributions to this paper, passed away on Thursday, October 30, 2008, after a brief illness. This paper is in memorial to his honorable attitude toward science and education.