Location of Repository

Characterization of a novel PTEN mutation in MDA-MB-453 breast carcinoma cell line

By Singh Gobind, Odriozola Leticia, Guan Hong, Kennedy Colin R and Chan Andrew M

Abstract

<p>Abstract</p> <p>Background</p> <p>Cowden Syndrome (CS) patients with germ line point mutations in the <it>PTEN </it>gene are at high risk for developing breast cancer. It is believed that cells harboring these mutant <it>PTEN </it>alleles are predisposed to malignant conversion. This article will characterize the biochemical and biological properties of a mutant PTEN protein found in a commonly used metastatic breast cancer cell line.</p> <p>Methods</p> <p>The expression of PTEN in human breast carcinoma cell lines was evaluated by Western blotting analysis. Cell line MDA-MB-453 was selected for further analysis. Mutation analysis of the <it>PTEN </it>gene was carried out using DNA isolated from MDA-MB-453. Site-directed mutagenesis was used to generate a PTEN E307K mutant cDNA and ectopic expressed in PC3, U87MG, MCF7 and <it>Pten</it><sup>-/- </sup>mouse embryo fibroblasts (MEFS). Histidine (His)-tagged PTEN fusion protein was generated in <it>Sf9 </it>baculovirus expression system. Lipid phosphatase and ubiquitination assays were carried out to characterize the biochemical properties of PTEN E307K mutant. The intracellular localization of PTEN E307K was determined by subcellular fractionation experiments. The ability of PTEN E307K to alter cell growth, migration and apoptosis was analyzed in multiple PTEN-null cell lines.</p> <p>Results</p> <p>We found a mutation in the <it>PTEN </it>gene at codon 307 in MDA-MB-453 cell line. The glutamate (E) to lysine (K) substitution rendered the mutant protein to migrate with a faster mobility on SDS-PAGE gels. Biochemically, the PTEN E307K mutant displayed similar lipid phosphatase and growth suppressing activities when compared to wild-type (WT) protein. However, the PTEN E307K mutant was present at higher levels in the membrane fraction and suppressed Akt activation to a greater extent than the WT protein. Additionally, the PTEN E307K mutant was polyubiquitinated to a greater extent by NEDD4-1 and displayed reduced nuclear localization. Finally, the PTEN E307K mutant failed to confer chemosensitivity to cisplatinum when re-expressed in <it>Pten</it><sup>-/- </sup>MEFS.</p> <p>Conclusions</p> <p>Mutation at codon 307 in PTEN C2 loop alters its subcellular distribution with greater membrane localization while being excluded from the cell nucleus. This mutation may predispose breast epithelial cells to malignant transformation. Also, tumor cells harboring this mutation may be less susceptible to the cytotoxic effects of chemotherapeutics.</p

Topics: Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282, Internal medicine, RC31-1245, Medicine, R, DOAJ:Oncology, DOAJ:Medicine (General), DOAJ:Health Sciences
Publisher: BioMed Central
Year: 2011
DOI identifier: 10.1186/1471-2407-11-490
OAI identifier: oai:doaj.org/article:1bebb19e39024a6ebd97f2df7d086b66
Journal:
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • https://doaj.org/toc/1471-2407 (external link)
  • http://www.biomedcentral.com/1... (external link)
  • https://doaj.org/article/1bebb... (external link)
  • Suggested articles


    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.