Switch from p53 to MDM2 as differentiating human keratinocytes lose their proliferative potential and increase in cellular size

Abstract

p53 transcription factor is mutated in most skin cell carcinomas and in more than 50% of all human malignancies. One of its transcriptional targets is MDM2, which in turn down-regulates p53. The role of the p53/MDM2 regulatory loop upon genotoxic stress is well documented, but less is known about its role in normal tissue homeostasis. We have explored this pathway during the di erent transitions of the human epidermal di erentiation programme and after isolating stem cells, transit amplifying cells or di erentiating cells from epidermis. Maximum expression of p53 was found in proliferating keratinocytes. A striking and transient induction of MDM2 and a down-modulation of p53 characterized the transition from proliferation to di erentiation in primary human keratinocytes. These changes were delayed in late di erentiating carcinoma cells, and were clearly di erent in suspended primary ®broblasts. Interestingly, these changes correlated with an increase in cell size, at the time of irreversible commitment to di erentiation. Induction of MDM2 was also associated with suppression of proliferation in normal, or hyperproliferative, psoriatic epidermis. Moreover, both proteins were induced as keratinocytes were driven to leave the stem cell compartment by c-Myc activation. Overall, our results show a critical regulation of the p53/MDM2 pathway at the epidermal transition from proliferation to di erentiation.

Cyclin E drives human keratinocyte growth into differentiation

“…Induction of p21 during keratinocyte differentiation has been reported previously. 16,17,23,41 In our experiments, p21 was transiently induced in suspended keratinocytes and bound Cyclin A/cdk1 and Cyclin B/cdk1 at the time of the commitment to differentiation (6 h), whereas the binding to cdk2 peaked later on. Interestingly, p21 is required for the downregulation of Cyclin A in mouse keratinocytes.…”

p53 deficiency exacerbates pleiotropic mitotic defects, changes in nuclearity and polyploidy in transdifferentiating pancreatic acinar cells

“…Thus, p21 may provide a means of evading apoptosis, during the transition between differentiated states, as shown in several systems (Poluha et al, 1996;Wang and Walsh, 1996;Lawlor and Rotwein, 2000). Moreover, keratinocyte differentiation is accompanied by induction of mdm2 and concomitant downmodulation of p53 activity and, interestingly, is coincident with increased cell size and ploidy (Dazard et al, 2000). However, in the mammary gland, overexpression of mdm2 generated polyploid cells irrespective of p53 status, suggesting additional p53-independent roles for mdm2 in the regulation of DNA synthesis (Lundgren et al, 1997).…”

Genome-wide comparison of human keratinocyte and squamous cell carcinoma responses to UVB irradiation: implications for skin and epithelial cancer

“…Total cell lysates, protein resolution by PAGE, transfer on nitrocellulose membrane (Bio-Rad), and immunoblotting were performed as previously described (Dazard et al, 2000). Detection was performed using the Western blotting-enhanced chemiluminescence substrate (ECL þ plus, Amersham Pharmacia Biotech, Buckinghamshire, England) following the supplier's protocol.…”

“…Fixation and staining were performed as previously described (Dazard et al, 2000). All cells were resuspended and filtered through a 70 mm mesh prior to analysis by flow cytometry on a Becton Dickinson FACScan s .…”

“…Establishment of primary cultures and cell maintenance was performed as previously described (Dazard et al, 2000). Keratinocytes were freshly isolated from neonatal foreskin specimens derived from a normal human Caucasian.…”