Journal of Cancer Research and Clinical Oncology

, Volume 139, Issue 9, pp 1591–1599

Generation and characterization of virus-free reprogrammed melanoma cells by the piggyBac transposon

Original Paper

Abstract

Purpose

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.

Keywords

piggyBac transposon Reprogramming Melanoma Stem cells Differentiation 

Supplementary material

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Supplementary material 1 (DOC 30 kb)
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Supplementary material 2 (TIFF 2,292 kb)
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Supplementary material 3 (TIFF 4,443 kb)
432_2013_1431_MOESM4_ESM.doc (44 kb)
Supplementary material 4 (DOC 43 kb)

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Department of Pathology, Institute of Basic Medical Sciences and School of Basic MedicineChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
  2. 2.Division of Structural Biology & Biochemistry, School of Biological SciencesNanyang Technological UniversitySingaporeSingapore

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