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Radiation-induced Apoptosis of Ewing's Sarcoma Cells: DNA Fragmentation and Proteolysis of Poly(ADP-ribose) Polymerase¹

Department of Radiation Medicine, Division of Radiation Research, Georgetown University Medical Center, Washington, DC 20007

Ewing's sarcoma (ES) cells express high levels of poly(ADP-ribose) polymerase (PADPRP) and are responsive to killing by ionizing radiation. We have determined that ionizing radiation induced a pronounced but reversible G₂-M phase cell cycle arrest that was maximum by 24 h after exposure. Following the release from this block, floating cells began to appear. These floating cells were shown to be apoptotic by flow cytometric and DNA fragmentation analyses. We found that apoptosis is a significant component of radiation-induced death in ES cells and that this is accomplished in conjunction with proteolytic cleavage of PADPRP. Two fragments of M_r 25,000 and M_r 29,000 containing the PADPRP DNA-binding domain were identified in floating (apoptotic) cells, whereas only the full-length M_r 116,000 native protein was detected in adherent cells that retained DNA intact. These data are consistent with PADPRP cleavage being an early step in the apoptotic cascade of biochemical events in ES cells after ionizing radiation exposure.

¹ This work was supported by Grant CA 58986 from the NIH.

² To whom requests for reprints should be addressed, at Department of Radiation Medicine, Georgetown University Medical Center, The Research Building, E-207, 3970 Reservoir Road, Washington, DC 20007.

Received 7/ 6/95. Accepted 8/18/95.

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