title: Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines.
abstract: LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies.
authors.affiliation: Department of Radiotherapy-Oncology, Democritus University of Thrace Medical School, University General Hospital of Alexandroupolis, Alexandroupolis, Greece.
authors.forename: Michael I
authors.initials: MI
authors.lastname: Koukourakis
authors.affiliation: Department of Radiotherapy-Oncology, Democritus University of Thrace Medical School, University General Hospital of Alexandroupolis, Alexandroupolis, Greece.
authors.forename: Dimitra
authors.initials: D
authors.lastname: Kalamida
authors.affiliation: Department of Pathology, Democritus University of Thrace Medical School, University General Hospital of Alexandroupolis, Alexandroupolis, Greece.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Giatromanolaki
authors.affiliation: Department of Radiotherapy-Oncology, Democritus University of Thrace Medical School, University General Hospital of Alexandroupolis, Alexandroupolis, Greece; Cancer Research United Kingdom Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, Headington, Oxford, OX3 9DU, United Kingdom.
authors.forename: Christos E
authors.initials: CE
authors.lastname: Zois
authors.affiliation: Department of Pathology, Democritus University of Thrace Medical School, University General Hospital of Alexandroupolis, Alexandroupolis, Greece.
authors.forename: Efthimios
authors.initials: E
authors.lastname: Sivridis
authors.affiliation: Department of Radiotherapy-Oncology, Democritus University of Thrace Medical School, University General Hospital of Alexandroupolis, Alexandroupolis, Greece.
authors.forename: Stamatia
authors.initials: S
authors.lastname: Pouliliou
authors.affiliation: Department of Radiotherapy-Oncology, Democritus University of Thrace Medical School, University General Hospital of Alexandroupolis, Alexandroupolis, Greece.
authors.forename: Achilleas
authors.initials: A
authors.lastname: Mitrakas
authors.affiliation: Nuffield Department of Cellular Sciences, John Raddcliffe Hospital, Headington, Oxford, OX3 9DU, United Kingdom.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Gatter
authors.affiliation: Cancer Research United Kingdom Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, Headington, Oxford, OX3 9DU, United Kingdom.
authors.forename: Adrian L
authors.initials: AL
authors.lastname: Harris
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000906:Antibodies / Q000276:immunology; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D000071186:Beclin-1; D045744:Cell Line, Tumor; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000235:genetics; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D007559:Ivermectin / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009369:Neoplasms / Q000473:pathology*; D034622:RNA Interference; D034741:RNA, Small Interfering
chemical_list: D000906:Antibodies; D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D005231:Fatty Acids, Unsaturated; C477012:MAP1LC3A protein, human; C502070:MAP1LC3B protein, human; C502071:MAP1LC3C protein, human; D018942:Macrolides; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; C057620:bafilomycin A; D007559:Ivermectin; C038753:leptomycin B