Biochemical and morphological changes in the nuclear matrix prepared from apoptotic HL-60 cells: effect of different stabilizing procedures.
Apoptotic cell death is characterized by deep morphological changes that take place in the nucleus. It is unclear whether modifications also occur in the nuclear matrix, a mainly proteinaceous structure that conceivably acts as a nuclear framework. We have investigated whether biochemical and morphological alterations of the nuclear matrix prepared from apoptotic HL-60 cells were dependent on the manipulations to which isolated nuclei were subjected before DNase I digestion and 2 M NaCl extraction. Our results showed that the stabilizing procedures employed to preserve the inner fibrogranular network and nucleolar remnants of the matrix (i.e., a 37 degrees C incubation; exposure to sodium tetrathionate at 4 degrees C; exposure to sodium tetrathionate at 37 degrees C) had no effect on the protein recovery of apoptotic nuclear matrices, which was always approximately two- to fivefold less than in control matrices. Moreover, one- and two-dimensional gel analysis of nuclear matrix proteins showed that, in apoptotic samples, striking quantitative changes were present, as compared with controls. Once again, these changes were seen irrespective of the stabilizing procedures employed. Also, transmission electron microscope analysis showed similar morphological alterations in all types of apoptotic nuclear matrices. By contrast, the immunofluorescent distribution of the 240-kDa NuMA protein seen in apoptotic samples was more sensitive to the stabilizing treatments. Our results indicate that the biochemical and morphological changes of the apoptotic nuclear matrix are largely independent of the isolation protocols and strengthen the contention that destruction of the nuclear matrix network is one of the key events leading to apoptotic nuclear destruction.
Martelli
A M
AM
Dipartimento di Morfologia Umana Normale, Università di Trieste, Italy. martelli@univ.trieste.it
Bortul
R
R
Bareggi
R
R
Grill
V
V
Narducci
P
P
Zweyer
M
M
D017209:Apoptosis / Q000187:drug effects*; D015180:Electrophoresis, Gel, Two-Dimensional; D005455:Fluorescent Antibody Technique; D018922:HL-60 Cells; D006801:Humans; D008854:Microscopy, Electron; D015530:Nuclear Matrix / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure