title: Overexpression of Telomerase Protects Human and Murine Lung Epithelial Cells from Fas- and Bleomycin-Induced Apoptosis via FLIP Upregulation.
abstract: High doses of bleomycin administered to patients with lymphomas and other tumors lead to significant lung toxicity in general, and to apoptosis of epithelial cells, in particular. Apoptosis of alveolar epithelium is an important step in the pathogenesis of bleomycin-induced pulmonary fibrosis. The Fas-FasL pathway is one of the main apoptotic pathways involved. Telomerase is a ribonucleoprotein RNA-dependent DNA polymerase complex consisting of an RNA template and a catalytic protein, telomerase reverse transcriptase (TERT). Telomerase also possess extra-telomeric roles, including modulation of transcription of anti-apoptotic genes, differentiation signals, and more. We hypothesized that telomerase overexpression affects Fas-induced epithelial cell apoptosis by an extra-telomeric role such as regulation of anti-apoptotic genes, specifically FLICE-like inhibitory protein (FLIP). Telomerase in mouse (MLE) and human (A549) lung epithelial cell lines was upregulated by transient transfection using cDNA hTERT expression vector. Telomerase activity was detected using a real-time PCR-based system. Bleomycin, and bleomycin-induced Fas-mediated apoptosis following treatment with anti-Fas activating mAb or control IgG, were assessed by Annexin V staining, FACS analysis, and confocal microscopy; caspase cleavage by Western blot; FLIP or Fas molecule detection by Western blot and flow cytometry. hTERT transfection of lung epithelial cells resulted in a 100% increase in their telomerase activity. Fas-induced lung epithelial cell apoptosis was significantly reduced in hTERT-transfected cells compared to controls in all experiments. Lung epithelial cells with increased telomerase activity had higher levels of FLIP expression but membrane Fas expression was unchanged. Upregulation of hTERT+ in human lung epithelial cells and subsequent downregulation of FLIP by shFLIP-RNA annulled hTERT-mediated resistance to apoptosis. Telomerase-mediated FLIP overexpression may be a novel mechanism to confer protection from apoptosis in bleomycin-exposed human lung epithelial cells.
authors.affiliation: Laboratory for Lung Cellular & Molecular Biology, Institute of Pulmonary Medicine, Hadassah - Hebrew University Medical Center, Jerusalem, Israel.
authors.forename: Nissim
authors.initials: N
authors.lastname: Arish
authors.affiliation: Laboratory for Lung Cellular & Molecular Biology, Institute of Pulmonary Medicine, Hadassah - Hebrew University Medical Center, Jerusalem, Israel.
authors.forename: Pazit Y
authors.initials: PY
authors.lastname: Cohen
authors.affiliation: Laboratory for Lung Cellular & Molecular Biology, Institute of Pulmonary Medicine, Hadassah - Hebrew University Medical Center, Jerusalem, Israel.
authors.forename: Regina
authors.initials: R
authors.lastname: Golan-Gerstl
authors.affiliation: Laboratory for Lung Cellular & Molecular Biology, Institute of Pulmonary Medicine, Hadassah - Hebrew University Medical Center, Jerusalem, Israel; Department of Pulmonary and Critical Care Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, United States of America.
authors.forename: Zvi
authors.initials: Z
authors.lastname: Fridlender
authors.affiliation: Department of Emergency Medicine, Shaare Zedek Medical Center, Jerusalem, Israel.
authors.forename: Mark Richter
authors.initials: MR
authors.lastname: Dayan
authors.affiliation: Laboratory for Lung Cellular & Molecular Biology, Institute of Pulmonary Medicine, Hadassah - Hebrew University Medical Center, Jerusalem, Israel.
authors.forename: Philip
authors.initials: P
authors.lastname: Zisman
authors.affiliation: Laboratory for Lung Cellular & Molecular Biology, Institute of Pulmonary Medicine, Hadassah - Hebrew University Medical Center, Jerusalem, Israel; Department of Pathology, Boston University School of Medicine, Boston, MA, United States of America.
authors.forename: Raphael
authors.initials: R
authors.lastname: Breuer
authors.affiliation: Laboratory for Lung Cellular & Molecular Biology, Institute of Pulmonary Medicine, Hadassah - Hebrew University Medical Center, Jerusalem, Israel.
authors.forename: Shulamit B
authors.initials: SB
authors.lastname: Wallach-Dayan
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000009:adverse effects*; D017209:Apoptosis / Q000187:drug effects; D001761:Bleomycin / Q000009:adverse effects*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D051379:Mice; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D000903:Antibiotics, Antineoplastic; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D019014:fas Receptor; D001761:Bleomycin; D019098:Telomerase