Inactivation of p16^INK4a(inhibitor of cyclin‐dependent kinase 4A) immortalizes primary human keratinocytes by maintaining cells in the stem cell compartment

Abstract

Replicative senescence of human keratinocytes is determined by a progressive decline of clonogenic and dividing cells, and its timing is controlled by clonal evolution (i.e., the transition from stem cells to transient amplifying and postmitotic cells). Progressive increase of p16INK4a (inhibitor of cyclin-dependent kinase 4A) expression has been shown to correlate with keratinocyte clonal evolution. Thus, the aim of our study is to understand whether p16INK4a accumulation is a triggering mechanism of epidermal clonal evolution or a secondary event. We show that inactivation of p16INK4a, by an antisense strategy, allows primary human keratinocytes to escape replicative senescence. Specifically, p16INK4a inactivation alone blocks clonal evolution and maintains keratinocytes in the stem cell compartment. Antisense excision is followed by keratinocyte senescence, confirming that persistent p16INK4a inactivation is required for maintenance of clonal evolution block. Immortalization is accompanied by resumption of B-Cell Specific Moloney murine leukemia virus site 1 (Bmi-1) expression and telomerase activity, hallmarks of tissue regenerative capacity. In turn, Bmi-1 expression is necessary to maintain the impairment of clonal evolution induced by p16INK4a inactivation. Finally, p16INK4a down-regulation in transient amplifying keratinocytes does not affect clonal evolution, and cells undergo senescence. Thus, p16INK4a inactivation appears to selectively prevent clonal conversion in cells endowed with a high proliferative potential. These data indicate that p16INK4a regulates keratinocyte clonal evolution and that inactivation of p16INK4a in epidermal stem cells is necessary for maintaining stemness.

Function of JunB in Transient Amplifying Cell Senescence and Progression of Human Prostate Cancer

“…Cellular senescence dependent on p16 may be induced in response to telomere erosion and/or by another as yet unidentified stimulus. Maurelli et al (42) report that, in primary human keratinocytes, senescence bypass is accomplished only when p16 is down-regulated in those cells endowed with a high proliferative potential. Moreover, elevated intracellular level of reactive oxygen species and protein kinase Cy activation has been found to maintain senescent human cell cycle arrest, resulting in blocking against immortalization, even after p16/pRb signals are inactivated (20).…”

Inducible re-expression of p16 in an orthotopic mouse model of pancreatic cancer inhibits lymphangiogenesis and lymphatic metastasis

“…p16 INK4a -deficient mice present with increased incidence of spontaneous and carcinogen-induced cancers (Kim and Sharpless, 2006). More recently p16 INK4a was associated with the clonal evolution of epidermal cells such that inactivation of p16 maintained epidermal cells in the stem cell compartment (Maurelli et al, 2006). In extension of these findings, knockout models have unveiled an age-dependent, tissue-specific function of p16 in limiting stem cell renewal (Kim and Sharpless, 2006).…”

Oncogenic Ras: A double-edged sword for human epidermal stem and transient amplifying cells

“…4,7,11 It is undetectable in holoclones and well-expressed in meroclones and paraclones. 8 Bmi-1 is a component of Polycomb Repressive Complex 1 (PRC1) that regulate the balance of SC dormancy and activation by modifying chromatin and suppressing gene expression.…”

“…8 What is more, inactivation of p16 INK4a in human epidermal SCs is necessary for maintaining their stemness and this process might be regulated by Bmi-1 expression. 7 Bmi-1 modulates p16 INK4a levels and delays clonal conversion even in primary keratinocytes from elderly donors and from young patients suffering from premature aged syndromes. 8 In addition, Cbx4, a PRC1 associated protein, maintains SCs in an undifferentiated state by regulating Polycomb Group (PcG) proteins (Ezh2, Dnmt1 and Bmi-1) and preventing the active SC state.…”