DNA-dependent protein kinase and cellular response to ionizing radiation

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The overall goal of the present project has been to elucidate a role of DNA-dependent protein kinase (DNA-PK) in the process of cellular response to ionizing radiation. Toward this goal, the purposes of the present project were (1) to purify and characterize DNA-PK from human leukemic MOLT-4 cells, which would show quick cell death process after irradiation, (2) to analyze the change in the activity in irradiated cells, (3) to examine the activity in various cell lines, and (4) to look into the correlation between the activity and cellular radiosensitization by heat treatment.1) Purified DNA-PK from MOLT-4 cells consisted of 3 subunits : p70, p86, and p470. p70 and p86 were reacted to anti-Ku antigen ; p470 was responsible for the phosphorylation activity. Double stranded DNA was shown to activate the enzyme in dose dependent manner, but single stranded DNA was not effective.2) Purified DNA-PK lost its activity when incubated at 44ﾟC.When Ku subunits were heated and and then mixed with p470, the inactivation was same as when the whole enzyme was heated ; the inactivation of Ku was responsible for the inactivation of the enzyme.3) In 10 Gy-irradiated MOLT-4, the activity was constant up to 1hr after irradiation. The decrease in DNA-PK activity was observed 2 hr after irradiation or later. By Western analysis using anti-p470 antibody the fragmentation of p470 was observed from 6 hr after irradiation.4) DNA-PK activities were higher in human cells than in the cells of rodent origin. In rodent cells, when heated at 44ﾟC,the activity was decreased quickly, while the activity was stable in human cells at least up to 3 hr. Heat sensitization of cells to ionizing radiation was, as revealed as colony forming ability, larger in rodent cells than in human cells.