How human self-renewal tissues co-ordinate proliferation with differentiation is unclear. Human epidermis undergoes continuous cell growth and differentiation and is permanently exposed to mutagenic hazard. Keratinocytes are thought to arrest cell growth and cell cycle prior to terminal differentiation. However, a growing body of evidence does not satisfy this model. For instance, it does not explain how skin maintains tissue structure in hyperproliferative benign lesions. We have developed and applied novel cell cycle techniques to human skin in situ and determined the dynamics of key cell cycle regulators of DNA replication or mitosis, such as cyclins E, A and B, or members of the anaphase promoting complex pathway: cdc14A, Ndc80/Hec1 and Aurora kinase B. The results show that actively cycling keratinocytes initiate terminal differentiation, arrest in mitosis, continue DNA replication in a special G2/M state, and become polyploid by mitotic slippage. They unambiguously demonstrate that cell cycle progression coexists with terminal differentiation, thus explaining how differentiating cells increase in size. Epidermal differentiating cells arrest in mitosis and a genotoxic-induced mitosis block rapidly pushes epidermal basal cells into differentiation and polyploidy. These observations unravel a novel mitosis-differentiation link that provides new insight into skin homeostasis and cancer. It might constitute a self-defence mechanism against oncogenic alterations such as Myc deregulation.

Human epidermis is continuously exposed to environmental mutagenic hazard and is the most frequent target of human cancer. How the epidermis coordinates proliferation with differentiation to maintain homeostasis, even in hyperproliferative conditions, is unclear. For instance, overactivation of the proto-oncogene MYC in keratinocytes stimulates differentiation. Here we explore the cell cycle regulation as proliferating human keratinocytes commit to terminal differentiation upon loss of anchorage or overactivation of MYC. The S-phase of the cell cycle is deregulated as mitotic regulators are inhibited in the onset of differentiation. Experimental inhibition of mitotic kinase cdk1 or kinases of the mitosis spindle checkpoint Aurora B or Pololike Kinase, triggered keratinocyte terminal differentiation. Furthermore, hyperactivation of the cell cycle by overexpressing the DNA replication regulator Cyclin E induced mitosis failure and differentiation. Inhibition of Cyclin E by shRNAs attenuated the induction of differentiation by MYC. In addition, we present evidence that Cyclin E induces DNA damage and the p53 pathway. The results provide novel clues for the mechanisms committing proliferative keratinocytes to differentiate, with implications for tissue homeostasis maintenance, HPV amplification and tumorigenesis.

Tumor suppressor p53 is a major cellular guardian of genome integrity, and its inactivation is the most frequent genetic alteration in cancer, rising up to 80% in squamous cell carcinoma (SCC). By adapting the small hairpin RNA (shRNA) technology, we inactivated endogenous p53 in primary epithelial cells from the epidermis of human skin. We show that either loss of endogenous p53 or overexpression of a temperature-sensitive dominant-negative conformation triggers a self-protective differentiation response, resulting in cell stratification and expulsion. These effects follow DNA damage and exit from mitosis without cell division. p53 preserves the proliferative potential of the stem cell compartment and limits the power of proto-oncogene MYC to drive cell cycle stress and differentiation. The results provide insight into the role of p53 in self-renewal homeostasis and help explain why p53 mutations do not initiate skin cancer but increase the likelihood that cancer cells will appear.