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Methylation of the p16INK4a promoter region in telomerase immortalized human keratinocytes co-cultured with feeder cells


Human keratinocytes grown in co-culture with fibroblast feeder cells have an extended in vitro lifespan and delayed accumulation of the tumor suppressor protein p16INK4a when compared to the same cells grown on tissue culture plastic alone. Previous studies have indicated that human keratinocytes can be immortalized by telomerase activity alone when grown in co-culture with feeder cells, suggesting that loss of the p16INK4a/Rb pathway is not required for immortalization. Using two independent human keratinocyte cell strains, we found that exogenous telomerase expression and co-culture with feeder cells results in efficient extension of lifespan without an apparent crisis. However, when these cells were transferred from the co-culture environment to plastic alone they experienced only a brief period of slowed growth before continuing to proliferate indefinitely. Examination of immortal cell lines demonstrated p16INK4a promoter methylation had occurred in both the absence and presence of feeder cells. Reintroduction of p16INK4a into immortal cell lines resulted in rapid growth arrest. Our results suggest that p16INK4a/Rb-induced telomere-independent senescence, although delayed in the presence of feeders, still provides a proliferation barrier to human keratinocytes in this culture system and that extended culture of telomerase-transduced keratinocytes on feeders can lead to the methylation of p16INK4a.

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We thank Robert Weinberg for providing the TERT-neo and TERT-hygro retroviral constructs, Denise Galloway for providing the pLXSH retroviral construct, and Francoise Gourronc for providing the p16-Bluescript vector, as well as Joseph Zabner for the use of his immunofluorescent microscope, Shiva Patil for assistance with cytogenetic analysis, and members of the Frederick Domann laboratory for help with bisulfite sequencing protocols. We are grateful to the other members of the Klingelhutz laboratory for helpful discussions. This work was supported by a grant to AJK from the National Institute on Aging (NIA), R01 AG18265 and a grant from the National Cancer Institute (NCI), R01 CA73612 to FED. Benjamin Darbro was supported by training grants from the National Heart, Lung, and Blood Institute (NHLBI), T32 HL07638, and the University of Iowa Medical Scientist Training Program (MSTP), T32 GM07337.

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Correspondence to A J Klingelhutz.

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Darbro, B., Lee, K., Nguyen, N. et al. Methylation of the p16INK4a promoter region in telomerase immortalized human keratinocytes co-cultured with feeder cells. Oncogene 25, 7421–7433 (2006).

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  • hTERT
  • CDKN2A
  • senescence
  • epigenetic
  • Rb
  • telomeres

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