Requirement for p53 in ionizing-radiation-inhibition of double-strand-break rejoining by human lymphoblasts.
Ionizing radiation (IR) triggers apoptosis, cell-cycle arrest, and DNA-repair induction in mammalian cells. These responses are mediated by proteins, including p53, which are activated or induced by IR. To determine the role of p53 in double-strand break (DSB) repair following irradiation of mammalian cells, we compared the abilities of unirradiated and irradiated TK6 human lymphoblast line and its derivatives TK6-E6-20C and TK6-E6-5E to repair restriction-enzyme-linearized shuttle pZ189 and the luciferase-reporter plasmid pGL3-control. TK6-E6-20C expresses wild-type p53 like the parental TK6 line, while TK6-E6-5E is p53 null. DSB-rejoining capacity was determined from the ratio of viable progenies arising from DSB-containing plasmids (linDNA) to the number of viable progenies from undamaged, supercoiled plasmids (scDNA). The ratio from the p53wt hosts was two- to three-fold higher than that from the p53null host, using either pZ189 or pGL3-control plasmid. After exposure of both hosts to 0.5 Gy gamma-radiation, DSB-rejoining capacity of p53null increased two-fold compared to unirradiated null controls, if transfection occurred immediately after irradiation. In contrast, the DSB-rejoining capacity of p53wt was unaffected by irradiation. If transfection was delayed for 2 h following irradiation, however, DSB-rejoining declined in both p53wt and p53null hosts. Irradiation also altered DSB-rejoining fidelity, measured from the mutation frequencies, among progenies of pZ189 linDNA. But, unlike rejoining capacity, changes in DSB-rejoining fidelity were similar in p53wt and p53null hosts. Changes in cell-cycle distribution in p53wt and p53null hosts were also similar following irradiation. These findings show that IR increases DSB-rejoining capacity in mammalian cells without functional p53, suggesting that p53 participates in suppressing DSB-rejoining following exposure of mammalian cells to IR.
Mallya
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Department of Oral Diagnosis, School of Dental Medicine, University of Connecticut Health Center, Farmington 06030-1605, USA.
Sikpi
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D002453:Cell Cycle / Q000528:radiation effects; D002460:Cell Line; D004247:DNA / Q000378:metabolism / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics / Q000528:radiation effects*; D004274:DNA, Recombinant / Q000378:metabolism / Q000528:radiation effects; D005720:Gamma Rays / Q000009:adverse effects*; D016158:Genes, p53; D006801:Humans; D008214:Lymphocytes / Q000528:radiation effects* / Q000648:ultrastructure; D016296:Mutagenesis; D010957:Plasmids / Q000528:radiation effects; D012343:RNA, Transfer / Q000235:genetics; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*
D004274:DNA, Recombinant; D016159:Tumor Suppressor Protein p53; D004247:DNA; D012343:RNA, Transfer