title: Generation and characterization of epidermolysis bullosa simplex cell lines: scratch assays show faster migration with disruptive keratin mutations.
abstract: BACKGROUND
  Epidermolysis bullosa simplex (EBS) is an inherited skin fragility disorder caused by mutations in keratin intermediate filament proteins. While discoveries of these mutations have increased understanding of the role of keratins and other intermediate filaments in epithelial tissues, progress towards the development of therapy for these disorders is much slower.
  
  
  OBJECTIVE
  Cell culture model systems that display these structural defects are needed for analysis of the cellular consequences of the mutations and to enable possible therapeutic strategies to be developed. Our aim was to generate immortalized cell lines as such model systems for the study of EBS.
  
  
  METHODS
  We generated a series of stable cell lines expressing EBS-associated keratin mutations, by immortalizing keratinocytes from EBS-affected skin biopsies with either simian virus 40 (SV40) T antigen or human papillomavirus 16 (HPV16) E6/E7, and assessed their keratin expression (by immunofluorescence), proliferation rates and migratory behaviour (in outgrowth and scratch wound assays).
  
  
  RESULTS
  Clonal immortalized keratinocyte cell lines KEB-1, KEB-2, KEB-3 (using SV40 T antigen) and KEB-4, KEB-7 and NEB-1 (using HPV16 E6/E7) were established. These include two lines from a single individual with Weber-Cockayne EBS (i.e. KEB-3 and KEB-4, mutation K14 V270M), and three cell lines from a second family, two from siblings carrying the same mutation (KEB-1, KEB-2 lines from Dowling-Meara EBS, mutation K5 E475G) and one from an unaffected relative (NEB-1). The sixth cell line (KEB-7), with a previously unreported severe mutation (K14 R125P), was the only one to show keratin aggregates in resting conditions. Despite variations in the immortalization procedure, there was no significant difference between cell lines in keratin expression, outgrowth capabilities or response to transient heat shock. However, cell migration, as measured by speed of scratch wound closure, was significantly faster in cells with severe EBS mutations.
  
  
  CONCLUSIONS
  These cell lines provide useful culture systems in which to assess aspects of EBS-induced cell changes. The faster migration after scratch wounding of the EBS keratinocytes may be a consequence of the known upregulation of stress-activated kinase pathways in these cells.
authors.affiliation: Cancer Research UK Cell Structure Research Group, Dundee University School of Life Sciences, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, U.K.
authors.forename: S M
authors.initials: SM
authors.lastname: Morley
authors.forename: M
authors.initials: M
authors.lastname: D'Alessandro
authors.forename: C
authors.initials: C
authors.lastname: Sexton
authors.forename: E L
authors.initials: EL
authors.lastname: Rugg
authors.forename: H
authors.initials: H
authors.lastname: Navsaria
authors.forename: C S
authors.initials: CS
authors.lastname: Shemanko
authors.forename: M
authors.initials: M
authors.lastname: Huber
authors.forename: D
authors.initials: D
authors.lastname: Hohl
authors.forename: A I
authors.initials: AI
authors.lastname: Heagerty
authors.forename: I M
authors.initials: IM
authors.lastname: Leigh
authors.forename: E B
authors.initials: EB
authors.lastname: Lane
mesh_terms: D002455:Cell Division / Q000235:genetics; D002460:Cell Line / Q000378:metabolism* / Q000473:pathology; D002465:Cell Movement / Q000235:genetics; D002472:Cell Transformation, Viral; D002675:Child, Preschool; D004252:DNA Mutational Analysis / Q000379:methods; D016110:Epidermolysis Bullosa Simplex / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D006358:Hot Temperature; D006801:Humans; D007382:Intermediate Filaments / Q000235:genetics; D015603:Keratinocytes / Q000473:pathology; D007633:Keratins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D027383:Papillomaviridae; D013539:Simian virus 40; D014945:Wound Healing / Q000235:genetics*
chemical_list: D007633:Keratins