Dependence of lethality induced by a direct DNA perturbation of synchronized human diploid fibroblasts on different periods of the DNA synthetic period (S Phase)

Abstract

The cytotoxic effect of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of cultured human diploid fibroblasts was examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-hour periods during the S phase with 5-bromodeoxyuridine (0.1-10 microM), followed by irradiation with near-UV (5-10 min). The 5-bromodeoxyuridine-plus-irradiation treatment was cytotoxic, while treatment with 5-bromodeoxyuridine alone or irradiation alone was not cytotoxic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 hours of S phase, whereas scarce lethality was observed in late S phase. The extent of substitution of 5-bromodeoxyuridine for thymidine in newly synthesized DNA was similar in every period of the S phase. Furthermore, no specific period during S phase was significantly more sensitive to treatment with respect to DNA damage, as determined by an induction of unscheduled DNA synthesis. These results suggest that a certain region or regions in the DNA of human diploid fibroblasts, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by 5-bromodeoxyuridine treatment plus near-UV irradiation for cell survival.

Temporal order of replication of genes responsible for hypoxanthine phosphoribosyl transferase and Na+/K+ ATPase in chemically transformed human fibroblasts

“…This method provided cells with over 90% synchrony, although approximately 20% of the cells were noncycling. Similar level of synchrony was obtained in human diploid fibroblasts by this method (Tsutsui et al, 1984). Use of HU blockage to obtain synchronization did not induce any chromosome aberrations.…”

“…Treatment of synchronized human diploid fibroblasts with BrdU during S phase and subsequent irradiation with near-UV light resulted in single-strand breaks in cellular DNA, detectable by alkaline sucrose gradient analysis and resulted in DNA damage, as determined by an induction of unscheduled DNA synthesis (UDS) (Tsutsui et al, 1984). BrdU or irradiation alone did not cause measurable DNA strand breakage and DNA damage.…”

“…The major photoproduct is uracil, which is formed by the dissociation of bromouracil in the radical formation, and these radicals may extract a n electron from the furanose backbone. This ultimately leads to a break in the DNA, particularly when it is subjected to alkaline conditions Hutchinson, 1973;Roufa, 1976;Tsutsui et al, 19791, as well as leads to DNA damage, as determined by an induction of unscheduled DNA synthesis (Tsutsui et al, 1984).…”

“…We also reported that a direct perturbation of DNA by combined BrdU and near-UV irradiation treatment is sufficient to initiate neoplastic transformation of Syrian hamster embryo cells in culture , and that a certain region or regions in the DNA of Syrian hamster embryo cells may be the more sensitive targets to the perturbation of DNA for the initiation of transformation (Tsutsui et al, 1979). Furthermore, we demonstrated by use of this method that a certain region or regions in DNA of human diploid fibroblasts may be more sensitive to the DNA perturbation for cell survival (Tsutsui et al, 1984). In this paper, we have examined the cytotoxicity of BrdU-plusirradiation treatment to a neoplastically transformed cell line derived from human diploid fibroblasts to study the temporal order of replication of genes involved in cell survival as well as hy oxanthine phosphoribosyl transferase (HPRT) and Na /K+ ATPase.…”