Downregulation of Wip-1 phosphatase expression in MCF-7 breast cancer cells enhances doxorubicin-induced apoptosis through p53-mediated transcriptional activation of Bax.
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.
Kong
Weihong
W
Departments of Microbiology/Immunology, Kimmel Cancer Center, Thomas Jefferson University; Philadelphia, PA 19107, USA. weihong.kong@jefferson.edu
Jiang
Xiaoshan
X
Mercer
W Edward
WE
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing; D017930:Genes, Reporter; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D000071636:Protein Phosphatase 2C; D034622:RNA Interference; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000235:genetics*
D000903:Antibiotics, Antineoplastic; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D004317:Doxorubicin; C000607458:PPM1D protein, human; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C