Nuclear changes in necrotic HL-60 cells

Cell death in eukaryotes can occur by either apoptosis or necrosis. Apoptos is is characterized by well-defined nuclear changes which are thought to be the consequence of both proteolysis and DNA fragmentation. On the other ha nd, the nuclear modifications that occur during necrosis are largely less k nown. Here, we have investigated whether or not nuclear modifications occur during ethanol-induced necrotic cell death of HL-60 cells. By means oi imm unofluorescence staining, we demonstrate that the patterns given by antibod ies directed against some nuclear proteins (lamin B1, NuMA, topoisomerase l l alpha, SC-35, B23/nucleophosmin) changed in necrotic cells. The changes i n the spatial distribution of NuMA strongly resembled those described to oc cur during apoptosis. On the contrary, the fluorescent pattern characterist ic for other nuclear proteins (C23/nucleolin, UBF, fibrillarin, RNA polymer ase I) did not change during necrosis. By immunoblotting analysis, we obser ved that some nuclear proteins (SAF-A, SATB1, NuMA) were cleaved during nec rosis, and in the case of SATB1, the apoptotic signature fragment of 70 kDa was also present to the same extent in necrotic samples. Caspase inhibitor s did not prevent proteolytic cleavage of the aforementioned polypeptides d uring necrosis, while they were effective if apoptosis was induced. In cont rast, lamin B1 and topoisomerase ll alpha were uncleaved in necrotic cells, whereas they were proteolyzed during apoptosis. Transmission electron micr oscopy analysis revealed that slight morphological changes were present in the nuclear matrix fraction prepared from necrotic cells. However, these mo difications (mainly consisting of a rarefaction of the inner fibrogranular network) were not as striking as those we have previously described in apop totic HL-60 cells. Taken together, our results indicate that during necrosi s marked biochemical and morphological changes do occur at the nuclear leve l. These alterations are quite distinct from those known to take place duri ng apoptosis. Our results identify additional biochemical and morphological criteria that could be used to discriminate between the two types of cell death. (C) 2001 Wiley-Liss Inc.