INCORPORATION OF TRITIATED THYMIDINE STIMULATED BY UV RADIATION INTO HUMAN FIBROBLAST CULTURES

DNA repair synthesis was studied after UV irradiation in human fibroblasts cultured in vitro by measuring the UV-stimulated incorporation of [3H]thymidine into cells in which the semi-conservative DNA replication was inhibited by hydroxyurea. Experiments performed with 5 fibroblast lines derived from healthy donors showed a relatively fast initial process (that is completed within 1 h for 100 erg/mm2 and within 2 h for 500 erg/mm2) and a subsequent slower process, evident between 2 and 6 h after irradiation. The repair capacity of normal cells is expressed by the difference between the values of incorporation (in presence of hydroxyurea) of irradiated and control cells. The pattern of repair was similar in all 5 cell lines: repair capacity was positive and the amount of repair synthesis increased with incubation time after UV irradiation. Similar experiments were performed with fibroblasts derived from 5 patients with the classical xeroderma pigmentosum (XP) and from 1 patient with the De Sanctis-Cacchione syndrome. Normal and XP cells could be distinguished according to whether they displayed a positive or negative value of repair synthesis and/or according to the degree of the slope of the repair synthesis curve as a function of the incubation time after irradiation. The technique used in our experiments can demonstrate in a rapid and simple way a defect in the repair capacity in fibroblast cultures; the data are in good agreement with those obtained in the same XP cell lines by others who have measured unscheduled DNA synthesis in autoradiographs and repair replication after addition of BUdR [bromodeoxyuracil].