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.
UV-induced DNA damage gives rise to mutations and skin cancer. We have previously reported that treatment of skin cells in vitro with thymidine dinucleotide (pTT) activates p53 and increases the ability of cells to repair subsequent UV-induced DNA damage by enhancing endogenous DNA repair capacity. Here we show that topical pTT pretreatment enhances the rate of DNA photoproduct removal, decreases UV-induced mutations, and reduces photocarcinogenesis in UV-irradiated hairless WT repair-proficient and Xpc ؉/؊ heterozygous partially repair-deficient mice, both transgenic for the lacZ͞pUR288 mutation-indicator gene. These data support the existence of inducible mammalian DNA damage responses that increase DNA repair capacity after DNA damage and hence reduce the impact of future exposures to environmental carcinogens. The ability of topically applied pTT to induce protective physiologic responses that normally result from DNA damage suggests a previously undescribed means of reducing skin cancer in high-risk individuals.
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...
The bleeding time (BT) is widely used in clinical medicine as a screening test of platelet function, although its deficiencies in such a role are well recognized. The Platelet Function Analyzer (PFA)-100 measures the ability of platelets activated in a high-shear environment to occlude an aperture in a membrane treated with collagen and epinephrine (CEPI) or collagen and ADP (CADP). The time taken for flow across the membrane to stop (closure time) is recorded. This study compared the PFA-100 with the BT as a screening test of platelet dysfunction in 113 hospital inpatients. The PFA-100 test was performed initially using the CEPI cartridge; CADP tests were performed on those with abnormal (> 163 s) CEPI closure times. Whole blood platelet aggregation studies and chart review were performed on patients in whom the BT and PFA-100 results did not agree.Abnormal bleeding times and PFA-100 results were obtained in 20.4% and 35.4% of patients, respectively. The results of BT and PFA-100 agreed in 74.3% of patients. Of the 29 patients in whom the BT and PFA-100 results were discordant, whole blood platelet aggregation studies supported the PFA-100 result in 25 (86.2%). The PFA-100 was more sensitive to aspirin-induced platelet dysfunction and was more rapidly and cheaply performed than the BT. Since the PFA-100 test reflects platelet function better than the BT, we conclude that this test could replace the BT as a first-line screening test for platelet dysfunction in clinical practice.
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