BIOCHEMICAL AND MORPHOLOGICAL CHARACTERIZATIONS OF DU-145 CELL MORTALITY IN RABBIT EMBRYO-FETAL FLUID

Rabbit embryo-fetal fluid (EFF) contains regulatory factors of cell pr oliferation which increase the duration of the cell cycle, induce a qu iescent 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. Inh ibitors of protein synthesis, and nuclease and phospholipase A2 activi ties did not restore the viability of the cells treated with EFF. Usin g a combination of DNA labelling and extraction, it was possible to sh ow that a large proportion of DNA was fragmented in the cells released in the supernatant while only a very small portion of DNA was fragmen ted in the monolayer cells. EFF did not induce fragmentation of DNA in to nucleosome-sized subunits as analysed using polyacrylamide gel elec trophoresis. Nevertheless, using cytofluorometric analysis, it was pos sible 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 th e control monolayer cells. The reduction of the DNA content per monola yer cell became significant at 48 h of treatment with EFF. Electron mi croscopic analysis did not reveal blebbing of the cells. However, depl etion 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 wit h EFF do not die by apoptosis, but rather seem to die by necrosis.