title: Translational up-regulation and high-level protein expression from plasmid vectors by mTOR activation via different pathways in PC3 and 293T cells.
abstract: BACKGROUND
  Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines.
  
  
  RESULTS
  In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), β-galactosidase (β-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3.
  
  
  CONCLUSIONS
  Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium.
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
authors.forename: Prashanthi
authors.initials: P
authors.lastname: Karyala
authors.forename: Nima D
authors.initials: ND
authors.lastname: Namsa
authors.forename: Durga Rao
authors.initials: DR
authors.lastname: Chilakalapudi
mesh_terms: D020001:1-Butanol / Q000737:chemistry; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010712:Phosphatidic Acids / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D015854:Up-Regulation; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D010712:Phosphatidic Acids; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D020001:1-Butanol; C546842:MTOR protein, human; D020797:Receptor, Platelet-Derived Growth Factor beta; D058570:TOR Serine-Threonine Kinases; D001616:beta-Galactosidase