title: Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance.
abstract: BACKGROUND
  Cancer-initiating cells (CICs) are proposed to be responsible for the generation of metastasis and resistance to therapy. Accumulating evidences indicates CICs are found among different human cancers and cell lines derived from them. Few studies address the characteristics of CICs in cervical cancer. We identify biological features of CICs from four of the best-know human cell lines from uterine cervix tumors. (HeLa, SiHa, Ca Ski, C-4 I).
  
  
  METHODS
  Cells were cultured as spheres under stem-cell conditions. Flow cytometry was used to detect expression of CD34, CD49f and CD133 antigens and Hoechst 33342 staining to identify side population (SP). Magnetic and fluorescence-activated cell sorting was applied to enrich and purify populations used to evaluate tumorigenicity in nude mice. cDNA microarray analysis and in vitro radioresistance assay were carried out under standard conditions.
  
  
  RESULTS
  CICs, enriched as spheroids, were capable to generate reproducible tumor phenotypes in nu-nu mice and serial propagation. Injection of 1 × 10³ dissociated spheroid cells induced tumors in the majority of animals, whereas injection of 1 × 10⁵ monolayer cells remained nontumorigenic. Sphere-derived CICs expressed CD49f surface marker. Gene profiling analysis of HeLa and SiHa spheroid cells showed up-regulation of CICs markers characteristic of the female reproductive system. Importantly, epithelial to mesenchymal (EMT) transition-associated markers were found highly expressed in spheroid cells. More importantly, gene expression analysis indicated that genes required for radioresistance were also up-regulated, including components of the double-strand break (DSB) DNA repair machinery and the metabolism of reactive oxygen species (ROS). Dose-dependent radiation assay indicated indeed that CICs-enriched populations exhibit an increased resistance to ionizing radiation (IR).
  
  
  CONCLUSIONS
  We characterized a self-renewing subpopulation of CICs found among four well known human cancer-derived cell lines (HeLa, SiHa, Ca Ski and C-4 I) and found that they express characteristic markers of stem cell, EMT and radioresistance. The fact that CICs demonstrated a higher degree of resistance to radiation than differentiated cells suggests that specific detection and targeting of CICs could be highly valuable for the therapy of tumors from the uterine cervix.
authors.affiliation: Programa de Doctorado en Ciencias Bioquímicas, Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, Mexico.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: López
authors.forename: Adela
authors.initials: A
authors.lastname: Poitevin
authors.forename: Veverly
authors.initials: V
authors.lastname: Mendoza-Martínez
authors.forename: Carlos
authors.initials: C
authors.lastname: Pérez-Plasencia
authors.forename: Alejandro
authors.initials: A
authors.lastname: García-Carrancá
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001562:Benzimidazoles / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D045744:Cell Line, Tumor / Q000473:pathology; D002471:Cell Transformation, Neoplastic; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics / Q000502:physiology; D005260:Female; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D046228:Microarray Analysis; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000528:radiation effects*; D011836:Radiation Tolerance; D018874:Spheroids, Cellular / Q000473:pathology; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology* / Q000532:radiotherapy; D023041:Xenograft Model Antitumor Assays
chemical_list: D015703:Antigens, CD; D001562:Benzimidazoles; D014408:Biomarkers, Tumor; C017807:bisbenzimide ethoxide trihydrochloride