A reduction in cell volume is a fundamental feature of apoptosis. This thesis has characterised changes in cell volume, together with nuclear changes, occurring in several models of apoptosis including, murine L1210 B-cells treated with mechlorethamine and human CEM-C7 A lymphoblastoid cells treated with dexamethasone, etoposide or subjected to serum deprivation. In the etoposide and glucocorticoid apoptotic models, cell volume was measured by electronic cell sizing and flow cytometry and two distinct phases were observed. In the dexamethasone model, the first phase of cell shrinkage began 12 hours after the addition of dexamethasone (5 ILM) and progressed until 36 hours when chromatin condensation was detected in intact cells. Removal of dexamethasone before 36 hours (the pre-commitment period), resulted in reversal of the volume decrease and prevented the appearance of nuclear changes. In the etoposide model, cells initially swelled, within the first 8 hours, and then rapidly lost volume at the later time points. Multiparameter flow cytometry indicated that cell shrinkage and chromatin condensation were occurring simultaneously in individual cells. The presence of the anti-apoptotic protein Bcl-2 abrogated chromatin condensation without affecting cell shrinkage or cytosolic acidification. Partial inhibition of protein synthesis also delayed chromatin condensation without affecting cell shrinkage. Possible mechanisms behind the first and second phase of cell shrinkage, in the dexamethasone model, were investigated. In the first phase, there was a net loss of potassium but no change in cellular buoyant density or potassium efflux. There were no significant differences in the rates of volume recovery after either hypertonic or hypotonic stimuli. These observations favour a mechanism of cell shrinkage involving loss of the entire cytoplasmic contents, possibly following proteolysis, rather than loss of only osmolytes and water. The second phase of volume loss was coincident with chromatin condensation and was associated with cellular fragmentation and a reduction in cellular density. In conclusion, the exact relationship of apoptotic cell volume changes to chromatin condensation appears to vary from cell model to cell model with cell volume loss being mediated by multiple mechanisms which are both dependent and independent of cellular fragmentation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:488152 |
| Date | January 1996 |
| Creators | Benson, Roderick Simon Patrick |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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