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

Riccardo Maurelli^*, Giovanna Zambruno, Liliana Guerra^*, Claudia Abbruzzese^*, Goberdhan Dimri, Mara Gellini^*, Sergio Bondanza^* and Elena Dellambra^*,1

^* Laboratory of Tissue Engineering and Cutaneous Physiopathology and

Laboratory of Molecular and Cell Biology, I.D.I.–IRCCS, Istituto Dermopatico dell’Immacolata, Rome, Italy; and

Division of Cancer Biology, Department of Medicine, Evanston, Illionois, USA

¹Correspondence: Laboratory of Tissue Engineering and Cutaneous Physiopathology, IDI, Istituto Dermopatico dell’Immacolata, Via dei Castelli Romani, 83/85, Pomezia (Roma) 00040, Italy. E-Mail: e.dellambra{at}idi.it

SPECIFIC AIMS

Progressive increase of p16^INK4a expression has been shown to correlate with keratinocyte clonal evolution. However, it is not clear whether p16^INK4a accumulation is a triggering mechanism of human epidermal replicative senescence or a secondary event. Thus, the aim of our study was to investigate the role of p16^INK4a in human keratinocyte clonal evolution.

PRINCIPAL FINDINGS

1. p16^INK4a inactivation alone induces replicative senescence bypass in primary human keratinocyte
p16^INK4a was inactivated in primary human keratinocytes, using a defective retrovirus, carrying the full-length human cDNA of p16^INK4a (named FL), or a 100 bp fragment (named Exo1a), corresponding to exon 1 of INK4a/ARF locus, in antisense orientation. Primary keratinocytes transduced with an empty vector underwent senescence, whereas antisense p16^INK4a-transduced keratinocytes (both FL and Exo1a) bypassed replicative senescence and continued to divide at a rate comparable with young keratinocytes. p16^INK4a levels were undetectable already before bypass of senescence and p16^INK4a inactivation was maintained during subcultivation.

2. p16^INK4a down-regulation does not induces replicative senescence bypass in TA keratinocytes
Keratinocyte cultures approaching senescence, i.e., enriched in transient amplifying (TA) cells and depleted in stem cells, were transduced with the aforementioned retroviral vectors. Antisense p16^INK4a-transduced TA keratinocytes did not bypass replicative senescence, although antisense transcripts were highly expressed and p16^INK4a expression resulted down-regulated.

3. p16^INK4a inactivation impairs keratinocyte clonal evolution
Empty vector-transduced cells showed a progressive decrease of their colony-forming efficiency (CFE) during serial cultivation and a progressive increase of the percentage of paraclones. In contrast, antisense p16^INK4a-transduced keratinocytes displayed a progressive increase of CFE reaching values >60% (percentage of aborted colonies constantly <3%), which remained then constant during serial cultivation. Of note, this impairment of clonal evolution was observed in antisense p16^INK4a-transduced cells several passages before the onset of replicative senescence of empty-vector cells (Fig. 1 ). Bypass of senescence was accompanied by resumption of p63, a marker of high proliferative potential in epithelial cells. This finding suggests a recruitment of cells endowed with high proliferative potential in antisense p16^INK4a-transduced cultures.

View larger version (47K): [in this window] [in a new window]   Figure 1. Clonogenic potential of antisense p16INK4a transduced cells. A) CFE performed at selected cell passages indicated by numbers of cell doublings. B) CFE values of keratinocytes transduced with antisense p16INK4a (red bars) or with empty vector (blue bars) were plotted against cell passages. C) Aborted colony values (% of paraclones) of keratinocytes transduced with antisense p16INK4a (red line) or with empty vector (blue line) were plotted against cell passages.

4. Inactivation of p16^INK4a maintains transduced keratinocytes in the stem cell compartment
We examined the percentage of stem and TA cells in our transduced cultures. After bypass of senescence, cultures generated from antisense p16^INK4a-transduced cells were enriched in holoclones (considered the surface epithelial stem cells) and markedly depleted of paraclones (terminal TA cells).

5. Persistent inactivation of p16^INK4a is required to block clonal conversion
After bypass of senescence, antisense p16^INK4a-transduced cells were infected with a Cre-expressing adenovirus to ablate antisense expression. Ad-Cre infected cells showed senescence features. The transgene deletion was accompanied by re-expression of p16^INK4a, and by reduction of CFE.

6. Inactivation of p16^INK4a does not impair control mechanisms of keratinocyte growth
Antisense p16^INK4a-transduced keratinocytes were shown to 1) need growth factor and FCS; 2) grow only in anchorage-dependent manner; and 3) have a normal complement of 46 chromosomes.

7. Antisense p16^INK4a-transduced cells maintain their ability to reconstitute a differentiated human epidermis
Histological and immunohistochemical analysis of in vitro reconstituted skin equivalents showed that antisense p16^INK4a-transduced cells were able to stratify into basal and suprabasal layers, although signs of hyperproliferative activity were clearly evident.

8. p16^INK4a inactivation inhibits pRb and up-regulates p53 pathways
Inactivation of p16^INK4a inhibited the pRb pathway, maintaining the hyperphosphorylated state of pRb, and induced an up-regulation of the p14^ARF/p53/p21^Waf1 pathway.

9. p16^INK4a inactivation-induced bypass of senescence is accompanied by resumption of Bmi-1 and by maintenance of telomerase activity, hallmarks of tissue regenerative capacity
In antisense p16^INK4a-transduced keratinocytes, telomerase activity was resumed. This resumption was accompanied by elongation and stabilization of the telomeres. Luciferase assays showed that p16^INK4a levels are able to modulate telomerase promoter activity in normal human keratinocytes. Cell-type specific induction of telomerase by Bmi-1 has been reported. Interestingly, p16^INK4a inactivation-induced bypass of senescence was accompanied by resumption of Bmi-1 expression (Fig. 2 ). On the contrary, down-regulation of p16^INK4a in TA cells was unable to resume Bmi-1 and telomerase activity at levels comparable with young keratinocytes.

View larger version (80K): [in this window] [in a new window]   Figure 2. Telomerase activity and Bmi-1 expression. Telomeric repeat amplification protocol (TRAP; A) and TRF (B) assays were performed on cells transduced with empty vector (V lanes) and antisense p16INK4a-transduced cells (AS-Exo1a lanes) at different cell passages. C) Reporter telomerase promoter, p16INK4a, and lacZ control plasmids were cotransfected in primary keratinocytes. Promoter activities were calculated using the empty vector control (column 1) as reference. The ratio promoter:p16INK4a was 1:1 (column 2), 1:2 (column 3), and 1:3 (column 4). D) c-Myc, Mad1, and Bmi-1 expression was assessed by Western blot using cell extracts from empty vector (V-lanes) and Exo1a (AS-Exo1a-lanes) transduced cells at different cell passages. In A, B, and D, different cell passages were indicated by numbers of cell generations after infection.

10. Bmi-1 expression is required for maintaining the block of keratinocyte clonal evolution induced by p16^INK4a inactivation
Primary keratinocytes were infected with a defective retrovirus carrying a recombinant cDNA encoding the fusion protein p16^INK4a-enhanced green fluorescent protein. Keratinocytes immediately underwent senescence and were able to generate only paraclones. Up-regulation of p16^INK4a was accompanied by a decrease of telomerase expression and activity, and down-regulation of Bmi-1.

Inactivation of Bmi-1 in antisense p16^INK4a-transduced keratinocyte cultures resulted in increased percentage of Bmi-1 negative enlarged and flattened colonies. Importantly, in antisense p16^INK4a-antisense Bmi-1 transduced keratinocytes, down-regulation of Bmi-1 is followed by a strong increase of Mad1, a telomerase repressor, and a decrease of telomerase expression and activity in the absence of p16^INK4a up-regulation.

CONCLUSIONS AND SIGNIFICANCE

In the present study, we demonstrate that direct and persistent inactivation of p16^INK4a in primary human keratinocytes is per se sufficient to allow bypass of replicative senescence by impairing keratinocyte clonal evolution and maintaining primary human keratinocytes in the stem cell compartment. p16^INK4a inactivation is accompanied by maintenance of Bmi-1 expression and telomerase activity, hallmarks of tissue regenerative capacity. In our transduced cells, sustained telomerase activity in turn results in telomere maintenance and immortalization of cultures. We also show that inactivation of Bmi-1 in antisense p16^INK4a-transduced keratinocyte colonies is accompanied by senescence features. Importantly, down-regulation of Bmi-1 is followed by a strong increase of Mad1 and the subsequent decrease of telomerase expression and activity in the absence of p16^INK4a up-regulation. These data demonstrate that Bmi-1 expression is required for maintaining the block of keratinocyte clonal evolution induced by p16^INK4a inactivation.

It has been reported that hTERT-mediated keratinocyte immortalization is accompanied by p16^INK4a expression. Our data indicate that in those cells up-regulation of p16^INK4a reflects attempts of feedback compensation but does not induce senescence because hTERT is constitutively active. We show that, as expected, inactivation of p16^INK4a in primary human keratinocytes inhibits the pRb pathway maintaining the hyperphosphorylated state of pRB. It has been reported that inactivation of pRb pathway, by ectopic expression of Cdk4, a p16^INK4a-insensitive mutant Cdk4^R24C, or human papilloma virus E7 oncoprotein, in epithelial cells can extend the life span but does not lead to keratinocyte immortalization. However, keratinocyte transduction results in p16^INK4a overexpression and telomere shortening. On the basis of our findings, we speculate that in those cells, despite inhibition of the pRb pathway, up-regulation of p16^INK4a does not allow Bmi-1 expression and telomerase activity resumption and subsequent cell immortalization.

Our data also demonstrate that p16^INK4a inactivation impairs keratinocyte clonal evolution in the presence of p14^ARF and p53 expression, identifying p16^INK4a as a major regulator of human keratinocyte clonal evolution. In addition, we show that p16^INK4a expression directly affects clonal evolution, modulates telomerase promoter activity, and down-regulates telomerase and Bmi-1 expressions and telomerase activity.

We demonstrate that Bmi-1 expression and telomerase activity decrease during clonal evolution of primary human keratinocytes. Immunocytochemistry results suggest that irreversible growth arrest and terminal differentiation are associated with decrease of Bmi-1 expression and cytoplasmic distribution. As reported, the ability of Bmi-1 to mediate cellular transformation in rodent fibroblasts correlates with its nuclear localization. Interestingly, antisense p16^INK4a-transduced immortalized keratinocytes, in which we observe a block of clonal evolution, display a predominantly nuclear Bmi-1 expression, whereas inactivation of Bmi-1 in these cells induces senescence features. These data support the involvement of Bmi-1 in the maintenance of stemness also in human keratinocytes.

Antisense-p16^INK4a transduction in cultures approaching senescence does not affect clonal evolution, and the onset of senescence is anyway triggered. This finding suggests an irreversible role of p16^INK4a accumulation in inducing clonal conversion. In keeping with data obtained in senescent fibroblasts, we hypothesize that irreversible epigenetic changes have already occurred in transient amplifying keratinocytes, in which p16^INK4a is highly expressed, and that down-regulation of p16^INK4a is no more able to affect the proliferative capacity of cells already committed to terminal differentiation. Thus, antisense-p16^INK4a transduction allows keratinocytes to bypass replicative senescence only in primary cultures bearing holoclones.

Altogether, our findings indicate that the tumor suppressor gene p16^INK4a regulates keratinocyte clonal evolution and that inactivation of p16^INK4a in epidermal stem cells is necessary for maintaining stemness in vitro.

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic diagram shows that inactivation of p16INK4a in early passage primary human keratinocyte cultures (bearing holoclones) is per se sufficient to induce cellular immortalization by impairing keratinocyte clonal evolution and maintaining cells in stem cell compartment. Inactivation of p16INK4a in late-passage primary human keratinocyte cultures (depleted in holoclones) does not affect clonal evolution, and onset of senescence is anyway triggered. Similar findings are obtained when overexpressing p16INK4a in early passage primary human keratinocytes.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4480fje

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This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-4480fjev2 20/9/1516    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Maurelli, R. Articles by Dellambra, E. Search for Related Content PubMed PubMed Citation Articles by Maurelli, R. Articles by Dellambra, E.