title: Molecular manipulation of keratin 8/18 intermediate filaments: modulators of FAS-mediated death signaling in human ovarian granulosa tumor cells.
abstract: BACKGROUND
  Granulosa cell tumors (GCT) are a rare ovarian neoplasm but prognosis is poor following recurrence. Keratin intermediate filaments expressed in these tumors are a diagnostic marker, yet paradoxically, may also constitute a target for therapeutic intervention. In the current study, we evaluated keratin 8/18 (K8/18) filament expression as a mechanism of resistance to apoptosis in GCT, specifically focusing on regulation of the cell surface death receptor, Fas (FAS).
  
  
  METHODS
  The GCT cell line, KGN, was transiently transfected with siRNA to KRT8 and KRT18 to reduce K8/18 filament expression. Expression of K8/18, FAS, and apoptotic proteins (PARP, cleaved PARP) were evaluated by fluorescence microscopy, flow cytometric analysis, and immunoblotting, respectively. The incidence of FAS-mediated apoptosis in KGN cells was measured by caspase 3/7 activity. All experiments were performed independently three to six times, using a fresh aliquot of KGN cells for each experiment. Quantitative data were analyzed by one- or two-way analysis of variance (ANOVA), followed by a Tukey's post-test for multiple comparisons; differences among means were considered statistically significant at P < 0.05.
  
  
  RESULTS
  Control cultures of KGN cells exhibited abundant K8/18 filament expression (~90 % of cells), and minimal expression of FAS (<25 % of cells). These cells were resistant to FAS-activating antibody (FasAb)-induced apoptosis, as determined by detection of cleaved PARP and measurement of caspase 3/7 activity. Conversely, siRNA-mediated knock-down of K8/18 filament expression enhanced FAS expression (> 70 % of cells) and facilitated FasAb-induced apoptosis, evident by increased caspase 3/7 activity (P < 0.05). Additional experiments revealed that inhibition of protein synthesis, but not MEK1/2 or PI3K signaling, also prompted FasAb-induced apoptosis.
  
  
  CONCLUSIONS
  The results demonstrated that K8/18 filaments provide resistance to apoptosis in GCT by impairing FAS expression. The abundance of keratin filaments in these cells and their role in apoptotic resistance provides a greater mechanistic understanding of ovarian tumorgenicity, specifically GCT, as well as a clinically-relevant target for potential therapeutic intervention.
authors.affiliation: Corning Life Sciences, Woburn, MA, 01801, USA. trisdalese@corning.com.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Trisdale
authors.affiliation: Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH, 03824, USA. nicoletteschwab5@gmail.com.
authors.forename: Nicolette M
authors.initials: NM
authors.lastname: Schwab
authors.affiliation: Veterans Affairs Medical Center and Olson Center for Women's Health, University of Nebraska Medical Center, Omaha, NE, 68198, USA. xhou@unmc.edu.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Hou
authors.affiliation: Veterans Affairs Medical Center and Olson Center for Women's Health, University of Nebraska Medical Center, Omaha, NE, 68198, USA. jsdavis@unmc.edu.
authors.forename: John S
authors.initials: JS
authors.lastname: Davis
authors.affiliation: Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH, 03824, USA. dave.townson@uvm.edu.|Current address: Department of Animal & Veterinary Sciences, University of Vermont, Burlington, VT, 05405, USA. dave.townson@uvm.edu.
authors.forename: David H
authors.initials: DH
authors.lastname: Townson
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D005260:Female; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006106:Granulosa Cell Tumor / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D053538:Keratin-18 / Q000235:genetics / Q000378:metabolism*; D053533:Keratin-8 / Q000235:genetics / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction; D019014:fas Receptor / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: C491901:FAS protein, human; C488421:KRT18 protein, human; D053538:Keratin-18; D053533:Keratin-8; D019014:fas Receptor