Inactivation of p16INK4a (inhibitor of cyclin-dependent kinase 4A) immortalizes primary human keratinocytes by maintaining cells in the stem cell compartment.
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.
Maurelli
Riccardo
R
Laboratory of Tissue Engineering and Cutaneous Physiopathology, I.D.I.-IRCCS, Istituto Dermopatico dell'Immacolata, Via dei Castelli Romani, 83/85, Pomezia (Roma), 00040 Italy. e.dellambra@idi.it
Zambruno
Giovanna
G
Guerra
Liliana
L
Abbruzzese
Claudia
C
Dimri
Goberdhan
G
Gellini
Mara
M
Bondanza
Sergio
S
Dellambra
Elena
E
D016475:3T3 Cells; D000818:Animals; D001402:B-Lymphocytes / Q000502:physiology / Q000821:virology; D016922:Cellular Senescence / Q000502:physiology; D003001:Cloning, Molecular; D003114:Colony-Forming Units Assay; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000037:antagonists & inhibitors* / Q000235:genetics; D017931:DNA Primers; D000078404:Epidermal Cells; D004817:Epidermis / Q000502:physiology; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008979:Moloney murine leukemia virus; D016133:Polymerase Chain Reaction; D012038:Regeneration; D013234:Stem Cells / Q000502:physiology*; D014162:Transfection
D019941:Cyclin-Dependent Kinase Inhibitor p16; D017931:DNA Primers