To gain insight into the transformation of epidermal cells into squamous carcinoma cells (SCC), we compared the response to ultraviolet B radiation (UVB) of normal human epidermal keratinocytes (NHEK) versus their transformed counterpart, SCC, using biological and molecular profiling. DNA microarray analyses (Affymetrix s , B12 000 genes) indicated that the major group of upregulated genes in keratinocytes fall into three categories: (i) antiapoptotic and cell survival factors, including chemokines of the CXC/CC subfamilies (e.g. IL-8, GRO-1, -2, -3, SCYA20), growth factors (e.g. HB-EGF, CTGF, INSL-4), and proinflammatory mediators (e.g. COX-2, S100A9), (ii) DNA repair-related genes (e.g. GADD45, ERCC, BTG-1, Histones), and (iii) ECM proteases (MMP-1, -10). The major downregulated genes are DNp63 and PUMILIO, two potential markers for the maintenance of keratinocyte stem cells. NHEK were found to be more resistant than SCC to UVB-induced apoptosis and this resistance was mainly because of the protection from cell death by secreted survival factors, since it can be transferred from NHEK to SCC cultures by the conditioned medium. Whereas the response of keratinocytes to UVB involved regulation of key checkpoint genes (p53, MDM2, p21 Cip1 , DNp63), as well as antiapoptotic and DNA repair-related genes -no or little regulation of these genes was observed in SCC. The effect of UVB on NHEK and SCC resulted in upregulation of 251 and 127 genes, respectively, and downregulation of 322 genes in NHEK and 117 genes in SCC. To further analyse these changes, we used a novel unsupervised coupled two-way clustering method that allowed the identification of groups of genes that clearly partitioned keratinocytes from SCC, including a group of genes whose constitutive expression levels were similar before UVB. This allowed the identification of discriminating genes not otherwise revealed by simple static comparison in the absence of UVB irradiation. The implication of the changes in gene profile in keratinocytes for epithelial cancer is discussed.

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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.

Crooked tail (Cd) mice bear a gain-of-function mutation in Lrp6, a co-receptor for canonical WNT signaling, and are a model of neural tube defects (NTDs), preventable with dietary folic acid (FA) supplementation. Whether the FA response reflects a direct influence of FA on LRP6 function was tested with prenatal supplementation in LRP6-deficient embryos. The enriched FA (10 ppm) diet reduced the occurrence of birth defects among all litters compared with the control (2 ppm FA) diet, but did so by increasing early lethality of Lrp6(-/-) embryos while actually increasing NTDs among nulls alive at embryonic days 10-13 (E10-13). Proliferation in cranial neural folds was reduced in homozygous Lrp6(-/-) mutants versus wild-type embryos at E10, and FA supplementation increased proliferation in wild-type but not mutant neuroepithelia. Canonical WNT activity was reduced in LRP6-deficient midbrain-hindbrain at E9.5, demonstrated in vivo by a TCF/LEF-reporter transgene. FA levels in media modulated the canonical WNT response in NIH3T3 cells, suggesting that although FA was required for optimal WNT signaling, even modest FA elevations attenuated LRP5/6-dependent canonical WNT responses. Gene expression analysis in embryos and adults showed striking interactions between targeted Lrp6 deficiency and FA supplementation, especially for mitochondrial function, folate and methionine metabolism, WNT signaling and cytoskeletal regulation that together implicate relevant signaling and metabolic pathways supporting cell proliferation, morphology and differentiation. We propose that FA supplementation rescues Lrp6(Cd/Cd) fetuses by normalizing hyperactive WNT activity, whereas in LRP6-deficient embryos, added FA further attenuates reduced WNT activity, thereby compromising development.