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Biochemical Characterization of the Protein Activity Responsible for High Molecular Weight DNA Fragmentation during Drug-induced Apoptosis¹

Department of Clinical Investigation, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Cleavage of cellular DNA into high molecular weight (predominantly 50 kb) fragments is an early event during apoptosis. We previously reported that this fragmentation was a Ca²⁺-independent process during apoptosis, which was induced by anticancer agents in human leukemia cells. The present study demonstrated that a high molecular weight DNA fragmentation activity (HDFA) was induced in the drug-treated cells and, upon fusion of the drug-treated cells with untreated target cells prelabeled with [¹⁴C]thymidine, caused fragmentation of the labeled DNA in the target cells. Furthermore, extracts of the drug-treated cells caused high molecular weight DNA fragmentation in nuclei isolated from untreated cells. Biochemical characterization of HDFA revealed the following properties: HDFA was proteinaceous in nature, as evidenced by its inactivation by heating or by digestion with proteinase K; HDFA required Mg²⁺ for optimal activity but was inhibited by Zn²⁺ and K⁺; HDFA was active in vitro at pH 6.0–8.0 and was inactive under more acidic conditions (pH < 6.0); addition of ATP (0.5–2 mM) substantially potentiated HDFA activity in isolated nuclei; and HDFA was not inhibited by actin (an inhibitor of DNase I) but was inhibited by the extracts from K562 cells, which were resistant to drug-induced apoptosis. The specific inhibitor of cysteine proteases (interleukin 1ß-converting enzyme protease family) blocked the generation of drug-induced high molecular weight DNA fragmentation in whole cells, whereas in isolated nuclei, the cysteine protease inhibitors did not prevent the cleavage of chromatin by exogenous HDFA. These results suggest that, once HDFA is activated during apoptosis, it does not require the presence of cysteine proteases for its endonucleolytic activity and that the cysteine proteases may be involved in the apoptotic process upstream of the activation of HDFA in whole cells.

¹ This work was supported in part by Grants CA28596, CA32839, and CA55164 from the National Cancer Institute, the Department of Health and Human Services, and by Grant DHP-1 from the American Cancer Society.

² To whom requests for reprints should be addressed, at Department of Clinical Investigation, Box 71, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030. Phone: (713) 792-7742; Fax: (713) 794-4316.

Received 2/ 3/97. Accepted 6/18/97.

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