title: Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance.
abstract: BACKGROUND
  Subsets of cells with stem-like properties have been previously isolated from human epithelial cancers and their resistance to apoptosis-inducing stimuli has been related to carcinoma recurrence and treatment failure. The aim of this study was to investigate the mechanisms of resistance to apoptosis-inducing agents of cells with stem-like properties in both normal and malignant human epithelia.
  
  
  METHODS
  Cells isolated from fresh human head and neck carcinomas (n = 11), cell lines derived from head and neck, prostate and breast human carcinomas (n = 7), and from normal human oral mucosa (n = 5), were exposed to various apoptosis-inducing stimuli (UV, Tumour Necrosis Factor, Cisplatin, Etoposide, and Neocarzinostatin). Flow cytometry for CD44 and epithelial-specific antigen (ESA) expression, colony morphology, tumour sphere formation and rapid adherence assays were used to identify the subset of cells with stem-like properties. Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed (Western Blot, qPCR). The role of G2-checkpoint regulators Chk1 and Chk2 was investigated by use of debromohymenialdisine (DBH) and siRNA.
  
  
  RESULTS
  In both cancer biopsies and carcinoma cell lines a subset of CD44(high) cells showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the percentage of cells in G2-phase and the rate of apoptosis was found. Pulse-chase with iododeoxyuridine (IdU) demonstrated that CD44(high) carcinoma cells spent longer time in G2, even in un-treated controls. These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis. Low passage cultures of normal keratinocytes were also found to contain a subset of CD44(high) cells showing increased clonogenicity, and a similar pattern of G2-block associated with apoptotic resistance.
  
  
  CONCLUSIONS
  These data indicate that both normal and malignant human epithelial cells with stem-like properties show greater resistance to apoptosis associated with extended G2 cell cycle phase, and that this property is not a consequence of neoplastic transformation. Targeting G2 checkpoint proteins releases these cells from the G2-block and makes them more prone to apoptosis, implying an opportunity for improved therapeutic approaches.
authors.affiliation: 1Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, 4 Newark Street, Whitechapel, London E1 2AT, UK.
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Harper
authors.forename: Daniela Elena
authors.initials: DE
authors.lastname: Costea
authors.forename: Luke
authors.initials: L
authors.lastname: Gammon
authors.forename: Bilal
authors.initials: B
authors.lastname: Fazil
authors.forename: Adrian
authors.initials: A
authors.lastname: Biddle
authors.forename: Ian C
authors.initials: IC
authors.lastname: Mackenzie
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D002448:Cell Adhesion / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D005047:Etoposide / Q000494:pharmacology; D016195:G2 Phase / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D015603:Keratinocytes / Q000187:drug effects / Q000473:pathology; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology* / Q000528:radiation effects; D011836:Radiation Tolerance; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014466:Ultraviolet Rays; D009353:Zinostatin / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; C497483:CD44 protein, human; D018960:Hyaluronan Receptors; D014409:Tumor Necrosis Factor-alpha; D005047:Etoposide; D009353:Zinostatin; D002945:Cisplatin