Alterations in cytoskeletal and nuclear matrix-associated proteins during apoptosis

Evidence exists that apoptosis or programmed cell death plays an important role in tumor growth. Morphologically apoptosis is characterized by membrane blebbing and nuclear fragmentation in individual cells. In this study, we investigated changes in the nuclear organization in spontaneously occurring apoptotic cells in several cancer cell lines using several antibodies to nuclear matrix constituents. It appeared that nuclear matrix components remained detectable in cells undergoing spontaneous apoptosis. The same results were found when apoptosis was induced by cycloheximide in the non-small cell lung cancer cell line MR65. Using this induction method, the percentage of apoptotic cells in MR65 cells increased, allowing a more detailed and extensive examination of nuclear matrix alterations together with cytoskeletal changes. To study the expression of cytokeratins, type A- and B lamins, a nuclear matrix-associated 13 kDa U1RNP particle and the Ki67-antigen, immunocytochemistry in combination with confocal scanning laser microscopy was used. Apoptotic cells were identified based on nuclear morphology and the in situ nick translation assay. Whereas immunoreactivity against lamins and Ki67-Ag was rapidly lost during apoptosis, expression of the 13 kDa protein and, in early apoptotic stages, also cytokeratin expression, was observed to remain present. Dead cells lacked reactivity with all the antibodies tested. The persistence of nuclear matrix components is therefore a useful marker for the detection of apoptosis.