Apoptosis of a fibrosarcoma induced by protein-free culture involves DNA cleavage to large fragments but not internucleosomal fragmentation

Abstract

A murine fibrosarcoma clone, Gc-4 SD, grows depending on fetal calf serum. In MTT assay, protein-free cultivation resulted in a reduction of the viable cell number time-dependently. Electron-microscopic and flow-cytometric analyses revealed that the reduction in growth was accompanied by the appearance of apoptotic cells. However, no internucleosomal fragmentation was observed even after SI-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 kilobase pairs (kbp), with a peak of 100 kbp, was found in the serum-deprived cells. Large fragments disappeared from the DNA extracts when the smaller cells with high blue fluorescence with Hoechst 33342 were removed by flow cytometry, suggesting direct correlation between the large DNA fragmentation and apoptosis. The addition of aurintricarboxylic acid neither abolished the large DNA fragmentation nor inhibited the reduction in the number of viable cells. Both cycloheximide and actinomycin D enhanced the reduction in the number of viable cells as well as the large DNA fragmentation. These results suggest that apoptosis of a fibrosarcoma induced by protein-free culture involves a specific endogenous endonuclease, which may be distinct from and independent of the ATA-sensitive endonuclease producing internucleosomal DNA fragmentation.