title: Macromolecular crowding effect is critical for maintaining SIRT1's nuclear localization in cancer cells.
abstract: SIRT1 is a principle class III histone deacetylase which exhibits versatile functions in stress response, development, and pathological processes including cancer. Although SIRT1 deacetylates a wide range of nuclear and cytoplasmic proteins, its subcellular localization in cancer cells has been controversial. In this study, we uncovered the inconsistent reports about SIRT1 subcellular localization is partially due to different analysis approaches. While immunofluorescence and live cell imaging reveal a predominant nuclear localization of SIRT1, conventional cell fractionation often results in a severe leaking of SIRT1 into the cytoplasm. Such a leakage is mainly caused by loss of cytoplasmic macromolecular crowding effect as well as hypotonic dwelling during the isolation of the nuclei. We also developed an improved cell fractionation procedure which maintains SIRT1 in its original subcellular localization. Analyzing a variety of human cancer cell lines using this approach and other methods demonstrate that SIRT1 predominantly localizes to the nucleus in cancer cells.
authors.affiliation: a Department of Tumor Biology , H. Lee Moffitt Cancer Center and Research Institute , Tampa , FL , USA.
authors.forename: Lidong
authors.initials: L
authors.lastname: Sun
authors.affiliation: a Department of Tumor Biology , H. Lee Moffitt Cancer Center and Research Institute , Tampa , FL , USA.
authors.forename: Jia
authors.initials: J
authors.lastname: Fang
mesh_terms: D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D005591:Chemical Fractionation; D004072:Digitonin; D005362:Ficoll; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology; D046911:Macromolecular Substances / Q000378:metabolism*; D009369:Neoplasms / Q000378:metabolism*; D056564:Sirtuin 1 / Q000378:metabolism*; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
chemical_list: D007038:Hypotonic Solutions; D046911:Macromolecular Substances; D005362:Ficoll; D056564:Sirtuin 1; D004072:Digitonin
keywords: SIRT1; cancer; histone deacetylases; nuclear localization; protein fractionation