Efficient immortalization of primary human cells by p16INK4a-specific short hairpin RNA or Bmi-1, combined with introduction of hTERT

Cancer Sci. 2007 Feb;98(2):147-54. doi: 10.1111/j.1349-7006.2006.00373.x.

Abstract

Activation of telomerase is sufficient for immortalization of some types of human cells but additional factors may also be essential. It has been proposed that stress imposed by inadequate culture conditions induces senescence due to accumulation of p16(INK4a). Here, we present evidence that many human cell types undergo senescence by activation of the p16(INK4a)/Rb pathway, and that introduction of Bmi-1 can inhibit p16(INK4a) expression and extend the life span of human epithelial cells derived from skin, mammary gland and lung. Introduction of p16(INK4a)-specific short hairpin RNA, as well as Bmi-1, suppressed p16(INK4a) expression in human mammary epithelial cells without promoter methylation, and extended their life span. Subsequent introduction of hTERT, the telomerase catalytic subunit, into cells with low p16(INK4a) levels resulted in efficient immortalization of three cell types without crisis or growth arrest. The majority of the human mammary epithelial cells thus immortalized showed almost normal ploidy as judged by G-banding and spectral karyotyping analysis. Our data suggest that inhibition of p16(INK4a) and introduction of hTERT can immortalize many human cell types with little chromosomal instability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalysis
  • Cell Proliferation
  • Cells, Cultured
  • Cellular Senescence
  • Cyclin-Dependent Kinase Inhibitor p16 / genetics*
  • Cyclin-Dependent Kinase Inhibitor p16 / metabolism*
  • DNA Methylation
  • Down-Regulation
  • Enzyme Activation
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism
  • Humans
  • Karyotyping
  • Keratinocytes / metabolism
  • Nuclear Proteins / genetics*
  • Nuclear Proteins / metabolism*
  • Polycomb Repressive Complex 1
  • Promoter Regions, Genetic / genetics
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins / metabolism*
  • RNA Interference
  • Repressor Proteins / genetics*
  • Repressor Proteins / metabolism*
  • Telomerase / genetics
  • Telomerase / metabolism*
  • Up-Regulation

Substances

  • BMI1 protein, human
  • Cyclin-Dependent Kinase Inhibitor p16
  • Nuclear Proteins
  • Proto-Oncogene Proteins
  • Repressor Proteins
  • Polycomb Repressive Complex 1
  • Telomerase