Differentiation-dependent p53 regulation of nucleotide excision repair in keratinocytes

The role of the tumor suppressor p53 in repair of ultraviolet light (UV)-induced DNA damage was evaluated using a host-cell reactivation (HCR) assay. HCR determines a cell's ability to repair UV-damaged DNA through reactivation of a transfected CAT reporter plasmid. Most UV damage is removed through nucleotide excision repair (NER). Primary murine keratinocytes isolated from p53-deficient and wild-type p53 mice were used in the HCR assay. The NER was reduced in p53(-/-) keratinocytes as compared with p53(+/+) keratinocytes. The reduced DNA repair in p53(-/-) mice was confirmed with a radioimmunoassay comparing cyclobutane dimers (CPDs) and (6-4) photoproducts in p53(+/+) and p53(-/-) keratinocytes after the cells were exposed to UV irradiation. Our results demonstrate that wild- type p53 plays a significant role in regulating NER. Furthermore, as there is evidence that p53 protein levels decrease after keratinocytes become differentiated, we sought to determine whether p53 plays a role in NER in differentiated keratinocytes. Differentiation of the keratinocytes by increasing the Ca²⁺ concentration in the culture media resulted in a marked reduction in NER equally in both p53(+/+) and p53(-/-) groups. This finding suggests that reduced DNA repair after differentiation is p53 independent. A similar reduction in HCR was confirmed in differentiated human keratinocytes. These data, taken together, indicate that p53 or p53-regulated proteins enhance NER in basal undifferentiated keratinocytes but not in differentiated cells. As non-melanoma skin cancers originate from the basal keratinocytes, our findings suggest that loss of p53 may contribute to the pathogenesis of this common skin cancer.