Bromodeoxyuridine/DNA analysis of replication in CHO cells after exposure to UV light.
The effects of ultraviolet light on cellular DNA replication were evaluated in an asynchronous Chinese hamster ovary cell population. BrdUrd incorporation was measured as a function of cell-cycle position, using an antibody against bromodeoxyuridine (BrdUrd) and dual parameter flow cytometric analysis. After exposure to UV light, there was an immediate reduction (approximately 50%) of BrdUrd incorporation in S phase cells, with most of the cells of the population being affected to a similar degree. At 5 h after UV, a population of cells with increased BrdUrd appeared as cells that were in G1 phase at the time of irradiation entered S phase with apparently increased rates of DNA synthesis. For 8 h after UV exposure, incorporation of BrdUrd by the original S phase cells remained constant, whereas a significant portion of original G1 cells possessed rates of BrdUrd incorporation surpassing even those of control cells. Maturation rates of DNA synthesized immediately before or after exposure to UV light, measured by alkaline elution, were similar. Therefore, DNA synthesis measured in the short pulse by anti-BrdUrd fluorescence after exposure to UV light was representative of genomic replication. Anti-BrdUrd measurements after DNA damage provide quantitative and qualitative information of cellular rates of DNA synthesis especially in instances where perturbation of cell-cycle progression is a dominant feature of the damage. In this study, striking differences of subsequent DNA synthesis rates between cells in G1 or S phase at the time of exposure were revealed.
Hoy
C A
CA
Department of Biological Sciences, Stanford University, CA 94305.
Carswell
C
C
Schimke
R T
RT
D000818:Animals; D000911:Antibodies, Monoclonal; D001973:Bromodeoxyuridine / Q000378:metabolism; D016466:CHO Cells; D002453:Cell Cycle / Q000528:radiation effects*; D002469:Cell Separation / Q000379:methods; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication / Q000528:radiation effects*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D016193:G1 Phase / Q000528:radiation effects; D011419:Propidium; D016196:S Phase / Q000528:radiation effects; D013997:Time Factors; D014466:Ultraviolet Rays
D000911:Antibodies, Monoclonal; D011419:Propidium; D004247:DNA; D001973:Bromodeoxyuridine; D016650:Fluorescein-5-isothiocyanate