title: Suppressive effects of phosphorylated ascorbate on ultraviolet B radiation-induced DNA damage and differential expression of the wild-type and mutated p53 tumor-suppressor gene in keratinocytes.
abstract: Ultraviolet B radiation (UVB)-induced DNA damage such as cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PP) in mouse epidermal keratinocytes (Pam212) was suppressed by pre-irradiation administration of two autooxidation-resistant vitamin C derivatives, ascorbic acid (Asc)-2-O-phosphate (Asc2P) and dehydro-Asc, but not by Asc or Asc-2-O-α-glucoside (Asc2G). According to Western blot analysis of the UVB-irradiated keratinocytes, expression of the tumor suppressor gene p53 was immediately enhanced from low pre-irradiation levels to the nearly maximum level at 2 h post-irradiation. In contrast, the p53-antagonizing oncogene mdm2 was scarcely expressed at 0-1 h post-irradiation, but was drastically enhanced at 2-3 h post-irradiation, eventually paralleling p53 expression. This suggests that the period for the actual effective function of p53 proteins may be restricted to the initial 0-3 h after UVB irradiation. p53 expression reached the maximum at 3 h post-irradiation and remained unchanged until at least 6 h, reaching its maximum at UVB doses of 20-100 mJ/cm2 - nearly equal to the minimum erythema dose (MED) or to the doubled score for human skin. However, at 200-500 mJ/cm2, p53 expression was markedly suppressed. This suggests a risk for potential photocarcinogenesis at doses greater than double the MED. UVB-induced p53 expression was notably suppressed by Asc2P or dehydro-Asc, but not by Asc. Nuclear expression of the wild-type p53 gene (wt-p53) was markedly promoted at 6 h post-irradiation, and significantly suppressed by Asc, Asc2G or dehydro-Asc, as well as by Asc2P. In contrast, mutation-type p53 (mt-p53), which is known to interfere with wt-p53 function, was not markedly expressed in the nuclei and was explicitly suppressed by Asc2P but not by Asc, dehydro-Asc or Asc2G, which was not cytoprotective against UVB. Thus, compared to non-cytoprotective Asc or the other derivatives, Asc2P suppressed UVB-induced CPD and 6-4PP formation, resulting in both cytoprotective effects and the preferential suppression of mt-p53 over wt-p53.
authors.affiliation: Laboratory of Cell-Death Control Biotechnology, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Hiroshima 727-0023, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Sugimoto
authors.forename: Yasukazu
authors.initials: Y
authors.lastname: Saitoh
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Ichihashi
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Miwa