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Molecular and Cellular Biology, July 2002, p. 5157-5172, Vol. 22, No. 14
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.14.5157-5172.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

James G. Rheinwald,1,2* William C. Hahn,3,4,5 Matthew R. Ramsey,1,2 Jenny Y. Wu,1,2 Zongyou Guo,1,2 Hensin Tsao,6 Michele De Luca,7 Caterina Catricalà,8 and Kathleen M. O'Toole1,2

Department of Adult Oncology, Dana-Farber Cancer Institute,3 Department of Medicine,4 Department of Dermatology,1 Harvard Skin Disease Research Center,Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115,2 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142,5 Department of Dermatology and The Wellman Laboratories of Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114,6 Laboratory of Tissue Engineering, Istituto Dermatopatico dell'Immacolata,7 San Gallicano Hospital, Rome, Italy8

Received 6 November 2001/ Returned for modification 7 December 2001/ Accepted 12 April 2002

With increasing frequency during serial passage in culture, primary human keratinocytes express p16INK4A (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 (cdk4R24C), to abolish p16 control, and/or a dominant negative mutant p53 (p53DD), to abolish p53 function. Expression of either cdk4R24C 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 cdk4R24C 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.

* Corresponding author. Mailing address: Harvard Institutes of Medicine, Room 664, 77 Ave. Louis Pasteur, Boston, MA 02115. Phone: (617) 525-5553. Fax: (617) 525-5571. E-mail: JRheinwald{at}rics.bwh.harvard.edu.

{dagger} This paper is dedicated to Howard Green and I. C. (Gunny) Gunsalus, who encouraged, inspired, and supported J.G.R. as a student to embark upon a career as research scientist 30 years ago.

Molecular and Cellular Biology, July 2002, p. 5157-5172, Vol. 22, No. 14
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.14.5157-5172.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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