mice were fed for 10 days with PL (300 mg/kg) or vehicle then UV-irradiated, once. By 24 hours, UVinduced Cox-2 levels were increased in vehicle-fed and PL-fed mice, whereas by 48 and 72 hours, Cox-2 levels were four-to fivefold lower in PL-fed mice (P < 0.05). p53 expression/activity was increased in PL-fed versus vehicle-fed then UV-irradiated mice. UV-induced inflammation was decreased in PL-fed mice, as shown by ϳ60% decrease (P < 0.001) in neutrophil infiltration at 24 hours, and macrophages by ϳ50% (<0.02) at 24 and 48 hours. By 72 hours, 54 ؎ 5% cyclobutane pyrimidine dimers remained in vehiclefed versus 31 ؎ 5% in PL-fed skin (P < 0.003). The number of 8-hydroxy-2-deoxyguanosine-positive cells were decreased before UV irradiation by ϳ36% (P < 0.01), suggesting that PL reduces constitutive oxidative DNA damage. By 6 and 24 hours, the number of 8-hydroxy-2-deoxyguanosine-positive cells were ϳ59% (P < 0.01) and ϳ79% (P < 0.03) lower in PL-fed versus vehicle-fed mice. Finally, UV-induced mutations in PL-fed-mice were decreased by ϳ25% when assessed 2 weeks after the single UV exposure. These data demonstrate that PL extract supplementation affords the following photoprotective effects: p53 activation and reduction of acute inflammation via Cox-2 enzyme inhibition, increased cyclobutane pyrimidine dimer removal, and reduction of oxidative DNA damage.

We have shown that DNA oligonucleotides substantially homologous to the telomere 3-prime overhang sequence (T-oligos) increase DNA repair capacity (DRC) in cultured human cells and decrease UV-induced mutation rate and photocarcinogenesis in mouse skin. To investigate the protective effects of T-oligos in intact human skin, paired skin explants obtained from adult donors were treated with T-oligos or diluent alone for 24 h, then UVB- or sham-irradiated, and processed after 6, 24, 48, 72, and 96 h for histological analysis. After UV irradiation apoptotic epidermal cells were comparable in diluent- and T-oligo-treated skin. Proliferating (Ki67+) cells were sparse in sham-irradiated skin and for 24 h after UV in both diluent- and T-oligo-treated specimens. However, compared to diluent controls, at 48 and 72 h T-oligos significantly inhibited UV-induced rebound hyperproliferation. Maximum and comparable cyclobutane pyrimidine dimers (CPDs) were detected immediately after UV irradiation in diluent- and T-oligo-treated skin, but CPDs were strikingly reduced in T-oligo- vs. diluent-treated skin at 24, 48, and 72 h. Total and activated p53 protein was increased in T-oligo- vs. diluent-pretreated skin at the time of irradiation, and up to 3-fold increases persisted for 24 h post-UV. Over 5 days, UV irradiation and T-oligo comparably increased expression of melanogenic proteins and each increased epidermal melanin content 3- to 5-fold, with distinct nuclear capping in many keratinocytes. In combination, these findings predict that T-oligo treatment will increase melanogenesis, prolong epidermal arrest, and increase DNA repair rate after UV irradiation, thus decreasing the severity of acute and chronic photodamage in human skin. Moreover, the data document an inducible SOS-like response consisting of increased melanogenesis and increased DNA repair capacity in human skin following UV-induced damage that is also produced by T-oligos in the absence of initial damage.

Chronically irradiated murine skin and UV light-induced squamous cell carcinomas overexpress the inducible isoform of cyclooxygenase (COX-2), and COX-2 inhibition reduces photocarcinogenesis in mice. We have reported previously that DNA oligonucleotides substantially homologous to the telomere 3-overhang (T-oligos) induce DNA repair capacity and multiple other cancer prevention responses, in part through up-regulation and activation of p53. To determine whether T-oligos affect COX-2 expression, human newborn keratinocytes and fibroblasts were pretreated with T-oligos or diluent alone for 24 h, UVirradiated, and processed for Western blotting. In both cell types, T-oligos transcriptionally down-regulated base-line and UV light-induced COX-2 expression, coincident with p53 activation. In fibroblasts with wild type versus dominant negative p53 (p53 WT versus p53 DN ), T-oligos decreased constitutive expression of a COX-2 reporter plasmid by >50%. We then examined NFB, a known positive regulator of COX-2 transcription. In p53 WT but not in p53 DN fibroblasts and in human keratinocytes, T-oligos decreased readout of an NFB promoter-driven reporter plasmid and decreased NFB binding to DNA. After T-oligo treatment and subsequent UV irradiation, binding of the transcriptional co-activator protein p300 to NFB was decreased, whereas binding of p300 to p53 was increased. Human skin explants provided with T-oligos had markedly decreased COX-2 immunostaining both at base-line and post-UV light, coincident with increased p53 immunostaining. We conclude that T-oligos transcriptionally down-regulate COX-2 expression in human skin via activation and up-regulation of p53, at least in part by inhibiting NFB transcriptional activation. Decreased COX-2 expression may contribute to the observed ability of T-oligos to reduce photocarcinogenesis.Nonmelanoma skin cancer accounts for well over 1 million cases of human malignancy annually in the United States, and the incidence continues to rise (1-3). The major initiator and promoter of skin cancer is UVB radiation (4, 5). Among the contributing effects of UVB radiation on skin are the formation of cyclobutane-pyrimidine dimers and pyrimidine (6-4) photoproducts (6, 7), which lead to mutations in key regulatory genes (8), epidermal hyperplasia (9, 10) allowing for expansion of mutated clones (11), immunosuppression (12, 13), and inflammation (14, 15).One way inflammation in particular is thought to affect carcinogenesis is by promoting epidermal hyperplasia and proliferation through production of cytokines and various second messengers such as prostaglandin E 2 (16). The major enzyme responsible for the UVB-induced prostaglandin synthesis is cyclooxygenase-2 (COX-2), 5 the inducible isoform of the cyclooxygenase enzyme (17) that carries out the ratelimiting step of prostaglandin and thromboxane production (18 -20). COX-2 has been shown to be overexpressed in numerous human malignancies, including colon, lung, and breast cancers (21-24). In relation to skin cancer, UVB irradiation increas...

To document and quantify inducible photoprotective effects in human skin, explant cultures were treated once with thymidine dinucleotide (pTT) or diluent alone or UV-irradiated. Both pTT and UV increased the melanogenic protein levels on days 1-5 and comparably increased melanocyte dendricity and epidermal melanin content. Explants treated with pTT or UV but not with diluent alone showed initial inhibition of epidermal proliferation followed by mild reactive hyperplasia; melanocyte proliferation was minimal. To determine whether pTT and UV provide comparable protection against subsequent UV-induced DNA damage, explants were pTT- or diluent-treated or UV-irradiated. All explants were then irradiated with the same UV dose 72 hours later. Compared to diluent alone, pTT or UV pretreatment decreased the number of epidermal cells positive for cyclobutane pyrimidine dimers (CPDs) 50% immediately post-irradiation. In pTT- and UV- versus diluent-pretreated explants, the rate of CPD removal was also more rapid, approximately 80 vs 45% of the initial burden within 72 hours. These data confirm and quantify comparable SOS-like responses in human skin after pTT or UV irradiation, attributable to both increased epidermal melanin and increased DNA repair rate, in the case of pTT in the absence of initial damage.

Treatment with thymidine dinucleotide (pTT) has well documented DNA-protective effects and reduces development of squamous cell carcinoma in UV-irradiated mice. The preventive effect of pTT on basal cell carcinoma (BCC) was evaluated in UV-irradiated Ptch-1 ؉/؊ mice, a model of the human disease Gorlin syndrome. Topical pTT treatment significantly reduced the number and size (P < 0.001) of BCCs in murine skin after 7 months of chronic irradiation. Skin biopsies collected 24 hours after the final UV exposure showed that pTT reduced the number of nuclei positive for cyclobutane pyrimidine dimers by 40% (P < 0.0002) and for 8-hydroxy-2-deoxyguanosine by 61% (P < 0.01 compared with vehicle control). Immunostaining with an antibody specific for mutated p53 revealed 63% fewer positive patches in BCCs of pTTtreated mice compared with controls (P < 0.01), and the number of Ki-67-positive cells was decreased by 56% (P < 0.01) in pTT-treated tumor-free epidermis and by 76% (P < 0.001) in BCC tumor nests (P < 0.001). Terminal dUTP nick-end labeling staining revealed a 213% increase (P < 0.04) in the number of apoptotic cells in BCCs of pTT-treated mice. Cox-2 immunostaining was decreased by 80% in tumor-free epidermis of pTT-treated mice compared with controls (P < 0.01). We conclude that topical pTT treatment during a prolonged period of intermittent UV exposure decreases the number and size of UV-induced BCCs through several anti-cancer mechanisms. 1 Like squamous cell carcinomas (SCCs), BCCs are associated with fair skin and chronic sunlight exposure, 2,3 but the relationship is more complex and studies to date have failed to show that sun protection reduces development of BCCs, in contrast to SCCs.1 Moreover, BCCs and SCCs differ in their responsiveness to certain chemopreventive agents. For example, BCC development is inhibited strongly by topical retinoids but not by oral nonsteroidal anti-inflammatory drugs (NSAIDs) or green tea.4,5 By contrast, murine SCC photocarcinogenesis is inhibited poorly by topical retinoids and quite well by oral NSAIDs or green tea. 4,6 These differing effects of UV radiation and chemopreventive agents on BCC versus SCC, in combination with the large socioeconomic burden of BCCs, 7 stimulated us to test thymidine dinucleotide (pTT), an agent already demonstrated to prevent SCCs in UV-irradiated mice 8 for its ability to reduce BCC development.Studies during the past decade have tied BCC tumorigenesis quite firmly to activated hedgehog signaling, a Research involving processing tissue for evaluation of microscopic BCCs, routine histology, immunofluorostaining image, and statistical analyses, and manuscript preparation were performed at the