Polyploidy and the mitosis path to epidermal cell fate

The cell cycle mechanisms coordinating proliferation with differentiation in epidermis remain intriguing and somewhat controversial. For decades it was assumed that differentiating keratinocytes underwent growth and cell cycle arrest in G0. This concept was supported by circumstantial data, namely the loss of proliferative capacity and the difficult detection of nucleotide incorporation in differentiating keratinocytes. However, older reports showing frequent mitotic figures in suprabasal layers of the skin and various other observations suggested otherwise (reviewed in [1]). We have obtained a large body of evidence for a role of mitotic checkpoints in squamous differentiation [2–5]. Our studies consistently showed that after a sustained block of mitosis keratinocytes undergo mitotic slippage or mitotic bypass, continue DNA replication and become tetraploid (4N) or polyploid (>4N) by a process known as endoreplication [1]. Whether the keratinocyte limiting factor is in G0/G1 or inG2/M is an important issue because it determines the way we understand homeostasis control and the targets that we should attack in disease. In addition, endoreplication provides a mechanism for the well known increase of cell size in differentiating keratinocytes. In a recent paper, Quek et al [6] report that two regulators of mitotic anaphase, CDC20 and CDH1, co-activators of the anaphase promoting complex (APC), influence differentiation in human keratinocytes. CDC20 is also a component of the mitotic checkpoint complex and participates in the spindle assembly checkpoint (SAC). This further supports a model in which mitosis is a limiting factor to keratinocyte cell fate. However, contrary to our findings, Quek et al state that differentiating keratinocytes stay inG0/G1with no signs of polyploidy. Their results are at variance with considerable data obtained by ourselves and others that are incompatible with a G0/G1 arrest in differentiating keratinocytes (refs in [1]). Here, we aim to reconcile these apparent contradictory data. In 2000 we reported that the mitotic inhibitory drug Nocodazole (Nz) rapidly induces keratinocyte differentiation and a striking increase in the proportion of polyploid cells [7]. We suggested that this was a normal process during keratinocyte differentiation. While Quek et al reproduce Nzinduced differentiation, they do not detect polyploidy upon the mitosis blockade. Unfortunately, the DNA content profiles are not shown. They further report lack of polyploidy in keratinocytes isolated from human skin. As explained elsewhere [1], it is technically difficult to obtain a significant proportion of differentiating (ergo polyploid) cells from the skin due to their strong attachments to each other. Harsh trypsin treatments break cells and nuclei. An intermediate treatment however allows a certain population of polyploid cells. This fraction increases along with differentiation [2]. It is unclear whether Quek et al had a significant proportion of differentiating cells in their samples. We also found a fraction of polyploid nuclei and chromosomal amplifications in situ [2]. Like us, Quek et al made use of the Rheinwald method [8]. In these conditions keratinocytes are cultured in the presence of “high calcium” concentration (1,2 mM), serum and a fibroblastic feeder layer which allow stratification. Quek et al do not find polyploid cells even in differentiating colonies (“paraclones”). We cannot reconcile this with our results and with the fact that binucleated and multinucleated keratinocytes or a large nucleus are very frequently observed in Rheinwald primary cultures and also (less easily) on skin sections in situ (Figure 1). Moreover, binucleated and multinucleated