Co-Regulation of p16INK4a and Migratory Genes in Culture Conditions that Lead to Premature Senescence in Human Keratinocytes

Abstract

Cellular stasis, also known as telomere-independent senescence, prevents many epithelial cells from becoming immortalized by telomerase alone. As human keratinocytes age in culture, protein levels of the tumor suppressor p16INK4a continue to increase, resulting in growth arrest independent of telomere length. Differences in culture conditions have been shown to modulate both p16INK4a expression and replicative capacity of human keratinocytes; however, the mechanism of p16INK4a induction under these conditions is unknown. Using multiple primary keratinocyte cell strains, we verified a delay in p16INK4a induction and an extended lifespan of human keratinocytes when grown in co-culture with post-mitotic fibroblast feeder cells as compared with keratinocytes grown on tissue culture plastic alone. Evaluation of gene expression levels in the two culture conditions by microarray analysis, and subsequent validation, demonstrated that keratinocytes cultured on plastic alone had significantly increased expression of many genes involved in keratinocyte migration and reduced expression levels of genes involved in keratinocyte differentiation. Higher levels of p16INK4a expression were present in cells that also displayed increased amounts of autophosphorylated focal adhesion kinase and urokinase plaminogen activator receptor (uPAR), both markers of keratinocyte migration. Furthermore, when tyrosine phosphorylation or urokinase-type plasminogen activator (uPA)/uPAR function was inhibited, both keratinocyte migration and p16INK4a expression were reduced. Our results indicate that keratinocytes cultured in the absence of feeder cells exhibit a migratory phenotype and suggest that p16INK4a is selectively induced under these conditions by a mechanism involving tyrosine kinase activity and the urokinase plasminogen activation system.

Senescence-Associated Oxidative DNA Damage Promotes the Generation of Neoplastic Cells

“…S4B and C). Senescent cells, including keratinocytes, have also been described as irreversibly arrested through induction of cyclin-dependent kinase inhibitors p16 and p21 (20)(21)(22). A quantitative reverse transcription-PCR analysis showed, as expected, that both cyclin-dependent kinase inhibitor mRNAs increased at the first plateau but decreased again in post-senescent-emerging cells (Supplementary Fig.…”

Epigenetic alterations of the SERPINE1 gene in oral squamous cell carcinomas and normal oral mucosa

“…In vitro studies of wound healing indicated that PAI-1 modulates the complex process of injury resolution through control of focalized plasmin-mediated matrix remodeling and cell migration. Certain ''senescence-associated'' genes (i.e., CDKN2A and Tp53) may actually function in wound repair and early malignant program by inhibiting proliferation but also promoting migration (Chan et al, 2001;Ploplis et al, 2004;Darbro et al, 2005;Kortlever et al, 2006;Natarajan et al, 2006). Indeed, keratinocytes at the leading edge during wound re-epithelialization are less mitotically active than cells more displaced from the motile front (Dabelsteen and Mackenzie, 1976) and have been shown to express relatively high levels of PAI-1 by other studies (Garlick and Taichman, 1994;Jensen and Lavker, 1996;Providence and Higgins, 2004;Qi et al, 2008;Shetty et al, 2008a,b).…”

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

“…This increase in p16 expression eventually leads to growth arrest by telomere-independent mechanisms. There is a growing body of evidence that suggests induction of epithelial cell migration is associated with upregulation of p16 expression ( Darbro et al, 2005). Previously, we have shown that human keratinocytes cultured in the absence of feeder cells acquire a phenotype consistent with a migratory response and that this migratory stimulus may be responsible for inducing p16-mediated, telomere-independent senescence in this culture condition .…”

“…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 .…”

“…Total RNA was collected using TriReagent (Molecular Research Center, Cincinnati, OH, USA) and purified according to the manufacturer's instructions. Total protein was collected using WE16 lysis buffer, as previously described (Foster and Galloway, 1996) Immunoblot analysis Immunoblot analyses of cellular protein levels were performed as previously described (Foster et al, 1994;Darbro et al, 2005). The following antibodies were used in this study: p16 (Pharmingen, San Diego, CA, USA: G175-405) and Actin (Santa Cruz Biotechnology Inc.: I-19).…”