An ultrastructural and immunohistochemical study of PC12 cells during apoptosis induced by serum deprivation with special reference to autophagy and lysosomal cathepsins

In addition to the caspase family of proteinases, cathepsin D, a lysosomal aspartic proteinase, has been suggested to act as a proapoptotic mediator i n mammalian cells. To further understand the roles of cathepsins B and D in apoptosis of the cells, we examined the precise alteration processes of ul trastructures and immunoreactivity for these enzymes in PC12 cells cultured under serum deprivation. Laser scanning microscopy showed immunoreactivity for cathepsins B and D to be finely distributed in the cytoplasm of PC12 cells at the onset of cultu re under serum deprivation. At 3 h after the onset of culture, the immunore activity for cathepsin B slightly decreased in the cells, while immunodepos its for cathepsin D in the cells became more intense and larger in size tha n those at 0 h. Positive staining for TUNEL in nuclei of the cells appeared at 6 h, though fewer in number. Corresponding to the increase in the numbe r of TUNEL-positive cells at 12 h and 24 h, the immunoreactivity for cathep sin B was drastically diminished in the cells, whereas that for cathepsin D was significantly augmented, especially in TUNEL-positive cells. Electron microscopically, autophagic vacuoles/autolysosomes appeared in the cytoplas m of the cells 3 h after the onset of culture. A distinct nuclear change sh owing relatively condensed chromatin first appeared in the peripheral part of the nuclei at 6 h, The number of PC12 cells having nuclei with chromatin condensation increased especially at 24 h, while these cells showed shrink age of both their cytoplasm and nuclei. Dense bodies and autophagic vacuole s with limiting membranes were seen in these cells, These results showing the occurrence of autophagy and imbalance of protein amounts between cathepsins B and D during apoptosis may argue for our hypot hesis that these enzymes are, in part, involved in the cell death cascade f or PC12 cells following serum deprivation.