A Two-Stage, p16^INK4A- and p53-Dependent Keratinocyte Senescence Mechanism That Limits Replicative Potential Independent of Telomere Status

Abstract

With increasing frequency during serial passage in culture, primary human keratinocytes express p16^INK4A (p16) and undergo senescence arrest. Keratinocytes engineered to express hTERT maintain long telomeres but typically are not immortalized unless, by mutation or other heritable event, they avoid or greatly reduce p16 expression. We have confirmed that keratinocytes undergo p16-related senescence during growth in culture, whether in the fibroblast feeder cell system or in the specialized K-sfm medium formulation, and that this mechanism can act as a barrier to immortalization following hTERT expression. We have characterized the p16-related arrest mechanism more precisely by interfering specifically with several regulators of cell cycle control. Epidermal, oral mucosal, corneal limbal, and conjunctival keratinocytes were transduced to express a p16-insensitive mutant cdk4 (cdk4^R24C), to abolish p16 control, and/or a dominant negative mutant p53 (p53DD), to abolish p53 function. Expression of either cdk4^R24C or p53DD alone had little effect on life span, but expression of both permitted cells to divide 25 to 43 population doublings (PD) beyond their normal limit. Keratinocytes from a p16^+/− individual transduced to express p53DD alone displayed a 31-PD life span extension associated with selective growth of variants that had lost the wild-type p16 allele. Cells in which both p53 and p16 were nonfunctional divided rapidly during their extended life span but experienced telomere erosion and ultimately ceased growth with very short telomeres. Expression of hTERT in these cells immortalized them. Keratinocytes engineered to express cdk4^R24C and hTERT but not p53DD did not exhibit an extended life span. Rare immortal variants exhibiting p53 pathway defects arose from them, however, indicating that the p53-dependent component of keratinocyte senescence is telomere independent. Mutational loss of p16 and p53 has been found to be a frequent early event in the development of squamous cell carcinoma. Our results suggest that such mutations endow keratinocytes with extended replicative potential which may serve to increase the probability of neoplastic progression.

View correction statement:

A Two-Stage, p16INK4A- and p53-Dependent Keratinocyte Senescence Mechanism That Limits Replicative Potential Independent of Telomere Status

We thank K. Münger for the p53DD retroviral vector, I. Gipson for providing ocular tissue specimens, D. Galloway for the HPV16 E6E7 retroviral vector, and J. Koh, C. Ngwu, E. Harlow, J. Bartek, F. Watt, and C. Loomis for providing antibodies. We thank James Rocco for helpful discussions.

This research was supported by grant R01-DE13178 from the NIDCR to J.G.R.; by grants from CIBAvision, Inc., and Inspire Pharmaceuticals, Inc., to J.G.R.; by Oral Cancer Program Project grant PO1-DE12467 from the NIDCR and by a Doris Duke Charitable Foundation Clinical Scientist Award to W.C.H.; and by a Howard Temin Award (CA94223) from the U.S. National Cancer Institute to W.C.H.

The research described in this report resulted from an ongoing collaboration between the laboratories of J.G.R. and W.C.H. and included key contributions from each of the authors. The order of the authors reflects their relative contributions to the project, without reference to senior or junior investigator status.