Biochemical and morphological characterizations of DU-145 cell mortality in rabbit embryo-fetal fluid

Rabbit embryo-fetal fluid (EFF) contains regulatory factors of cell proliferation which increase the duration of the cell cycle, induce a quiescent status in some cells and lead up to cell death in others. The objective of this study was to demonstrate which of the two processes, namely necrosis or apoptosis, was responsible for the cell death. Inhibitors of protein synthesis, and nuclease and phospholipase A2 activities did not restore the viability of the cells treated with EFF. Using a combination of DNA labelling and extraction, it was possible to show that a large proportion of DNA was fragmented in the cells released in the supernatant while only a very small portion of DNA was fragmented in the monolayer cells. EFF did not induce fragmentation of DNA into nucleosome-sized subunits as analysed using polyacrylamide gel electrophoresis. Nevertheless, using cytofluorometric analysis, it was possible to demonstrate that 50% of the cells released in the supernatant contained a lower quantity of DNA per cell than in the control cells. This was also observed with EFF-treated monolayer cells but not in the control monolayer cells. The reduction of the DNA content per monolayer cell became significant at 48 h of treatment with EFF. Electron microscopic analysis did not reveal blebbing of the cells. However, depletion of glycogen, condensation of mitochondria and increasing number of lysosomes and residual bodies were observed upon treatment with EFF. From these experiments we conclude that the DU-145 cells treated with EFF do not die by apoptosis, but rather seem to die by necrosis.