Cel The objective of the project is to measure different kinds of UVB-induced photoproducts and their repair in human skin. In time-course experiments the appearance of p53 mutations will be followed in the sample from which photoproduct levels are known. By comparing individuals with a risk of skin cancer the role of different types of photoproducts and their repair rates will be determined.Skin samples will be obtained either from healthy individuals or from patients undergoing skin biopsies for naevi, skin cancer or other skin diseases. These will be used for studies of adducts and mutations in the summer and winter time with consideration of sun exposure in the skin area. Biopsies distal from the area of lesion will be used for analysis, to avoid the presence of malignant tissue in the samples. Experimental irradiation of patients will be carried out on those suffering form psoriasis, atypical mole syndrome (AMS) and early onset basal cell cancer. Subjects will be irradiated with UVB-source on the buttock with a dose 2 x minimum erythema dose and sampled using a 3-5 mm punch biopsy at various intervals after irradiation. The biopsies will be immediately frozen in liquid nitrogen. The exact times of biopsy will be determined in preliminary experiments designed to follow the disappearance of the adducts and appearance of p53 mutations. UV-photoproducts will be analysed by a modified 32P-labelling technique, where DNA is digested to trinucleotides containing photoproducts and nucleosides with DNase I, snake venom phosphodiesterase and prostatic acid phosphatase. These enzymes are unable to cleave phosphodiester bonds between pydimidine dimers, and additionally leave an unmodified nucleotide to the 5'-end of the trinucleotide. This is important for good labelling T4 polynucleotide kinase. High performance liquid chromatography (HPLC) coupled with radioactivity detector as method of analysis has some advantages in comparison with TLC and it is being applied in the analysis of photoproducts. Detection of the cyclobutane dimers and 6-4 photoproducts is possible by HPLC with a on-line 32P-radioisotope detector. The experiments with DNA irradiated at 1000 J/m2 it is possible to identify most of the adducts. Of these two modifications TLC is slightly more sensitive but HPLC gives a better separation of the adducts. The slightly lower sensitivity of the HPLC method can be compensated by using more material in the analysis. In human skin explants adducts are detected at a dose 10 J/m2. The methods are currently optimised for detection of lesions at very low doses of UV-light, 1-5 J/m2. A novel allele specific PCR method will be used to detect CC to TT transitions. The method has been developed in two variations, mutant allele-specific PCR (AS-PCR) and mutant allele-specific ligase chain reaction (AS-LCR). The principle in both of these is that in the second PCR reaction one set of primers has a CC to TT mismatch either in the end to be extended by PCR or to be ligated. The codons selected for study are 245 and 247/248, which contain a CC sequence in the wild type and have been mutated in human skin tumors described in the literature. The technique has been used to detect mutations both in human sun-exposed skin and in cultured keratinocytes, exposed to 20 J/m2 or higher doses (Nakazawa et al. 1994). Thus as minimal erythema does are 50-100 j/m2 there should be no difficulties in detecting mutations in humans experimentally exposed to UVB. The cells with mutations will be studied with the in situ allele-specific PCR using radioactive or fluorescent primers to visualise the mutated cells in a skin biopsy. In removed malignant skin lesions it will be of interest to analyse how the mutated cells are located in respect to the cancerous lesion. Dziedzina nauki natural sciencesbiological sciencesgeneticsDNAnatural sciencesbiological sciencesgeneticsmutationnatural sciencesbiological sciencesgeneticsnucleotidesmedical and health sciencesclinical medicineoncologyskin cancerbasal cellsnatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes Program(-y) FP4-ENV 2C - Specific programme of research and technological development in the field of environment and climate, 1994-1998 Temat(-y) 01020202 - Alteration of processes as a result of UV-B radiation Zaproszenie do składania wniosków Data not available System finansowania CSC - Cost-sharing contracts Koordynator Karolinska Institute Wkład UE Brak danych Adres 141 57 Huddinge Szwecja Zobacz na mapie Koszt całkowity Brak danych Uczestnicy (2) Sortuj alfabetycznie Sortuj według wkładu UE Rozwiń wszystko Zwiń wszystko Centre International de Recherche sur le Cancer Francja Wkład UE Brak danych Adres 150 cours Albert Thomas 69372 Lyon Zobacz na mapie Koszt całkowity Brak danych Imperial Cancer Research Fund Zjednoczone Królestwo Wkład UE Brak danych Adres Newark Street E1 2AT London Zobacz na mapie Koszt całkowity Brak danych