title: Generation and characterization of virus-free reprogrammed melanoma cells by the piggyBac transposon.
abstract: OBJECTIVE
  Reprogramming of cancer cells to stem cell-like state provides a promising tool for the study of cancer pathogenesis and drug screening. However, most instances of direct reprogramming have been achieved by forced co-expression of defined transcription factors using viral vectors. Retroviral transduction as well as the ectopic expression of reprogramming factors may alter the differentiation potential of reprogrammed cancer cells or induce malignant transformation. Therefore, generation of reprogrammed cancer cells via virus-free reprogramming strategy needs to be studied.
  
  
  METHODS
  Melanoma cells were reprogrammed by co-expression of doxycycline-inducible Oct4, Sox2, Klf4, and c-Myc using the piggyBac (PB) transposon system. The expression level of genes was analyzed through RT-PCR, Western blot, and immunofluorescence. Epigenetic modification of genes was detected by bisulfite genomic sequencing. Post reprogrammed melanoma cells were generated through differentiation of reprogrammed melanoma cells. Sensitivity to chemotherapeutic agents and metastasis potential were investigated in post reprogrammed melanoma cells.
  
  
  RESULTS
  The virus-free reprogrammed melanoma cells were positive for stem cell markers including Oct4, Nanog, and SSEA-1, and the promoters of Nanog and Oct4 were demethylated. Moreover, reprogrammed melanoma cells gained differentiation potential and higher sensitivity to differentiation-inducing drugs. Post reprogrammed melanoma cells showed lower proliferation rate and metastatic potential compared with the parental cells.
  
  
  CONCLUSIONS
  Our results indicate that PB transposon-based method is applicable to generate virus-free reprogrammed melanoma cells. These cells can differentiate into other lineages with loss of malignant phenotypes, which may provide a more suitable source for molding of cancer pathogenesis.
authors.affiliation: Department of Pathology, Institute of Basic Medical Sciences and School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Yin
authors.forename: Yongna
authors.initials: Y
authors.lastname: Fan
authors.forename: Dingxin
authors.initials: D
authors.lastname: Qin
authors.forename: Xiaocui
authors.initials: X
authors.lastname: Xiaocui Bian
authors.forename: Xiaobao
authors.initials: X
authors.lastname: Bi
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D002454:Cell Differentiation; D049109:Cell Proliferation / Q000187:drug effects; D065150:Cellular Reprogramming; D004251:DNA Transposable Elements; D057890:Epigenomics; D005260:Female; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000008:administration & dosage; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D008546:Melanoma, Experimental / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009362:Neoplasm Metastasis; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology*; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014780:Viruses
chemical_list: D000970:Antineoplastic Agents; D015415:Biomarkers; D004251:DNA Transposable Elements; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489428:Myc protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D055748:SOXB1 Transcription Factors