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Abstract
Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16INK4a cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16INK4a-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT+ keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16INK4a expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16INK4a-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems.
ACKNOWLEDGMENTS
We thank J. Benwood, D. Long-Woodward, and K. O'Toole for technical assistance and S. Verselis for information about the mutant p53 allele in our Li-Fraumeni cell line. We thank D. Galloway for the HPV16 E7 retroviral vector, J. Koh, S. C, Ngwu, and E. Harlow for p16INK4a antibodies, and T.-T. Sun, C. A. Loomis, and F. M. Watt for keratin and involucrin antibodies.
This research was supported by Oral Cancer Program Project grant PO1 DE12467 from the NIDCR, Skin Disease Research Center grant P30 AR42689 from the NIAMS, and a research grant from Organogenesis, Inc. (J.G.R.). In addition, portions of this work were supported by a Daniel K. Ludwig and American Cancer Society Professorship (R.A.W.), a Culpeper Biomedical Initiative Pilot Grant (W.C.H. and R.A.W.), a Damon-Runyon/Walter Winchell Cancer Research Fund award, a Howard Hughes Medical Institute postdoctoral fellowship, and a Herman and Margaret Sokol postdoctoral fellowship (W.C.H.), and a Starr Foundation and American Cancer Society Clinical Research Professorship (F.P.L.).