title: Characterization of a novel PTEN mutation in MDA-MB-453 breast carcinoma cell line.
abstract: BACKGROUND
  Cowden Syndrome (CS) patients with germ line point mutations in the PTEN gene are at high risk for developing breast cancer. It is believed that cells harboring these mutant PTEN 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.
  
  
  METHODS
  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 PTEN 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 Pten(-/-) mouse embryo fibroblasts (MEFS). Histidine (His)-tagged PTEN fusion protein was generated in Sf9 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.
  
  
  RESULTS
  We found a mutation in the PTEN 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 Pten(-/-) MEFS.
  
  
  CONCLUSIONS
  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.
authors: Department of Oncological Sciences, The Mount Sinai School of Medicine, New York, NY 10029, USA.
authors: Gobind
authors: G
authors: Singh
authors: Leticia
authors: L
authors: Odriozola
authors: Hong
authors: H
authors: Guan
authors: Colin R
authors: CR
authors: Kennedy
authors: Andrew M
authors: AM
authors: Chan
mesh_terms: D000328:Adult; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D002277:Carcinoma / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis; D005260:Female; D006223:Hamartoma Syndrome, Multiple / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009154:Mutation; D051059:PTEN Phosphohydrolase / Q000235:genetics* / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D003062:Codon; D025521:Tumor Suppressor Proteins; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human