APOPTOSIS OF A FIBROSARCOMA INDUCED BY PROTEIN-FREE CULTURE INVOLVES DNA CLEAVAGE TO LARGE FRAGMENTS BUT NOT INTERNUCLEOSOMAL FRAGMENTATION

A murine fibrosarcoma clone, Gc-4 SD, grows depending on fetal calf se rum. In MTT assay, protein-free cultivation resulted in a reduction of the viable cell number time-dependently. Electron-microscopic and flo w-cytometric analyses revealed that the reduction in growth was accomp anied by the appearance of apoptotic cells. However, no internucleosom al fragmentation was observed even after S1-nuclease treatment. On the other hand, pulse field gel electrophoresis revealed that cleavage of DNA into high-molecular-weight fragments estimated as 50 to 150 kilob ase pairs (kbp), with a peak of 100 kbp, was found in the serum-depriv ed cells. Large fragments disappeared from the DNA extracts when the s maller cells with high blue fluorescence with Hoechst 33342 were remov ed by now cytometry, suggesting direct correlation between the large D NA fragmentation and apoptosis. The addition of aurintricarboxylic aci d neither abolished the large DNA fragmentation nor inhibited the redu ction in the number of viable cells. Both cycloheximide and actinomyci n D enhanced the reduction in the number of viable cells as well as th e large DNA fragmentation. These results suggest that apoptosis of a f ibrosarcoma induced by protein-free culture involves a specific endoge nous endonuclease, which may be distinct from and independent of the A TA-sensitive endonuclease producing internucleosomal DNA fragmentation . (C) 1995 Wiley-Liss, Inc.