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.The replicative life span of normal human fibroblasts is limited by a senescence mechanism that responds to partial telomere shortening by triggering a p53-and p21 cip1 (p21)-dependent growth arrest (5,8,12). Expression of hTERT, the telomerase catalytic subunit, in presenescent fibroblasts and several other cell types, including retinal pigmented epithelial cells, vascular endothelial cells, and mesothelial cells, is sufficient to permanently evade this senescence mechanism and immortalize these cell types (7,17,78). Recent studies have indicated, however, that telomere shortening alone cannot completely explain the replicative life span limit and immortalization barrier exhibited by a diverse set of epithelial cell types.For keratinocytes (17, 31), mammary epithelial cells (11,21,31,56), bladder urothelial cells (51), and prostate epithelial cells (28, 57)...

Immortalisation of Normal Human Urothelial Cells Compromises Differentiation Capacity

“…Importantly, there was no emergence of a rare, rapidly-growing cell population that would have indicated clonal recovery. Accumulation of p16 has been associated with urothelial senescence in culture and is recognised as an essential block to neoplastic transformation [25]. In agreement with this and observations by Chapman et al [24], we saw a dramatic increase in p16 protein associated with the growth lag around the normal senescence point at passage 12 and growth rate recovery was accompanied by a reduction in p16 expression.…”

“…We now show that neither loss of p53 nor p16 function had any direct effect on differentiation, in agreement with the normal differentiation potential of keratinocytes with inactivated p16 function [25]. Less expected was the lack of any association between loss of p53 and differentiation, as changes in p53 have been associated with carcinoma in situ [5], thus implying that other changes may contribute to the dysplastic phenotype.…”

Does Notch play a tumor suppressor role across diverse squamous cell carcinomas?

“…Notch inhibition by DNMAML1 promotes invasive growth of transformed esophageal epithelial cells with EGFR overexpression and p53 dysfunction 68. Moreover, TP53 was frequently mutated in HNSCC cases 6, 7, 53, and the inactivation of p53 plays important role in HNSCC tumorigenesis 43, 69, 70. But it has not been clarified yet whether Notch signaling is closely involved in this.…”

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

“…When grown in co-culture with post-mitotic fibroblast feeder cells, human keratinocytes, exhibit a delay in passage dependent p16 INK4a (p16) expression and have an extended lifespan in culture (Ramirez et al, 2001;Rheinwald et al, 2002;Baek et al, 2003;Fu et al, 2003;Kang et al, 2003;Darbro et al, 2005). We have found that co-culture of keratinocytes with feeder cells delays the accumulation of p16 protein but does not prevent the eventual increase of p16 expression in late passage keratinocytes cultured with feeder cells .…”

“…Previous reports that have shown immortalization of cocultured human keratinocytes with TERT alone imply that telomere-dependent mechanisms are the sole barrier preventing continued proliferation in this culture system. However, the presence of inflection points in the growth curves of TERT immortalized keratinocytes co-cultured with feeders (Herbert et al, 2002;Rheinwald et al, 2002;Ramirez et al, 2003), combined with the observations that telomeres experience only limited shortening in co-cultured human keratinocytes (Kang et al, 2004), suggest that telomere-independent mechanisms may still be involved in limiting the replicative capacity of these cells. Thus, examination of p16 expression in TERT-transduced human keratinocytes co-cultured with feeder cells should provide vital information concerning not only the potential safety of telomerase-based cell therapies but also the mechanism of epithelial cell senescence in the co-culture environment.…”