3664

The p53 homolog p63 exists as multiple isotypes of 2 subclasses, TA and ΔN. ΔNp63 isotypes lack the amino-terminal TA domain that is homologous to p53, and can block both p53 and TAp63 mediated transcription. Two ΔNp63 isotypes are expressed in primary mouse keratinocyte cultures. The full length form, ΔNp63α, predominates in proliferating cells and is downregulated with 0.12mM Ca2+ induced differentiation, while a shorter ΔNp63 form lacking the α-terminus persists in differentiating cultures. Overexpression of ΔNp63α has been described in human squamous cell carcinomas. Consistent with these reports, the p63 expression pattern in chemically induced mouse skin tumors that was observed with a pan-p63 antibody was also seen using either ΔN or α specific antibodies. To identify functional differences between ΔNp63 isoforms and the role of the carboxyl-terminus, we overexpressed ΔNp63α or ΔNp63p40, a shortened form that truncates immediately after the oligomerization domain, in cultured keratinocytes. Overexpression of either ΔNp63 isoform blocks differentiation associated growth arrest. As shown previously, ΔNp63α overexpression blocks Ca2+ induced keratinocyte differentiation. This effect requires the α-carboxyl terminus, as it is not observed with overexpressed ΔNp63p40. Overexpressed TAp63α does not mimic the effects of overexpressed ΔNp63α. These data suggest that while the ΔN domain of ΔNp63 is sufficient to block growth arrest, cooperation between the ΔN and α domains is required to block differentiation. To gain mechanistic insight into the functional differences between ΔN isotypes, keratinocytes overexpressing ΔNp63α, ΔNp63p40 or β-gal were cultured in medium containing 0.12mM Ca2+ and lysates were analyzed for altered transcription factor binding activity using Panomics Array I. Several ΔNp63 regulated transcription factors were identified, including NFkB, which was upregulated by ΔNp63p40 relative to β-gal and ΔNp63α. Western blotting confirmed that ΔNp63p40 enhances nuclear NFkB p65 levels under both proliferating and differentiating conditions. A smaller enhancement by ΔNp63p40 was seen with NFkB p50. No enhancement was seen in the ΔNp63α cultures or β-gal control cultures under either proliferating or differentiating conditions. Ongoing studies are aimed at dissecting the role of ΔNp63p40 directed upregulation of NFkB in keratinocytes and it’s impact on normal and neoplastic conditions.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]