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

Y OHSAWA, K ISAHARA, S KANAMORI… - Archives of histology …, 1998 - jstage.jst.go.jp
Y OHSAWA, K ISAHARA, S KANAMORI, M SHIBATA, S KAMETAKA, T GOTOW…
Archives of histology and cytology, 1998jstage.jst.go.jp
In addition to the caspase family of proteinases, cathepsin D, a lysosomal aspartic
proteinase, has been suggested to act as a proapoptotic mediator in mammalian cells. To
further understand the roles of cathepsins B and D in apoptosis of the cells, we examined
the precise alteration processes of ultrastructures 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 …
Summary
In addition to the caspase family of proteinases, cathepsin D, a lysosomal aspartic proteinase, has been suggested to act as a proapoptotic mediator in mammalian cells. To further understand the roles of cathepsins B and D in apoptosis of the cells, we examined the precise alteration processes of ultrastructures 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 culture under serum deprivation. At 3h after the onset of culture, the immunoreactivity for cathepsin B slightly decreased in the cells, while immunodeposits for cathepsin D in the cells became more intense and larger in size than those at 0h. Positive staining for TUNEL in nuclei of the cells appeared at 6h, though fewer in number. Corresponding to the increase in the number of TUNEL-positive cells at 12h and 24h, the immunoreactivity for cathepsin 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 cytoplasm of the cells 3h after the onset of culture. A distinct nuclear change showing relatively condensed chromatin first appeared in the peripheral part of the nuclei at 6h. The number of PC12 cellshaving nuclei with chromatin condensation increased especially at 24h, while these cells showed shrinkage of both their cytoplasm and nuclei. Dense bodies and autophagic vacuoles 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 ourhypothesis that these enzymes are, in part, involved in the cell death cascade for PC12 cells following serum deprivation.
Lysosomes, as an acidic compartment with limiting membranes, are ubiquitous in all animal cells and play an important role in the maintenance of the cellular metabolic turnover by degrading unneeded intra-and extracellular materials into biological monomers which are then reutilized by the cells (UCHIYAMA et al., 1994). Cathepsins B and D are representative cysteine and aspartic proteinases in lysosomes of mammalian cells. They are involved in the Golgi-derived vesicles which fuse with autophagic/heterophagic vacuoles, forming auto/heterolysosomes. Wehave previously shown that autophagy actively occurs in the apoptotic processes of CAl pyramidal neurons in the gerbilhippocampus after brief ischemia and of effete epithelial cells at the villous tip of the small intestine (NITATORI et al., 1995; SHIBAHARA et al., 1995). The role of autophagy, frequently occurring in such dying cells, is believed to protect the cells from death (CLARKS, 1990). Apoptosis, a major type of active cell death, is accompanied by characteristic morphological alterations consisting of the shrinkage of cytoplasm, nuclear chromatin condensation, fragmentation of cells into small pieces, andheterophagocytosis by neighboring cells (KERB et al., 1972). Ithas been suggested that calpain, serine proteinases, granzymes and the ubiquitin-proteasome pathway of protein degradation play a role in the apoptotic process (SOLARY et al., 1998). Degradation of substrates by these proteinases may result in the release of apoptotic factors from mitochondria which activates the caspase family of proteinases, or the direct activation of the proteinases (SOLARY et al., 1998). In addition to these proteinases, wehave recently demonstrated the existence of a cell death pathway
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