Doctor of Philosophy
Faculty of Science
Walter, Marc, Membrane alterations during apoptosis: a study into the possible involvement of SREBP-2, Doctor of Philosophy thesis, Faculty of Science, University of Wollongong, 2001. http://ro.uow.edu.au/theses/1868
A major aim of this research was to examine the biological relevance during apoptosis of the caspase-mediated cleavage of sterol-regulatory-element-binding-protein-2 (SREBP-2), a transcription factor that is a key regulator of cholesterol synthesis. The cleavage of SREBP-2 was investigated in the human HL60, U937, and Jurkat cell lines exposed to the apoptosis inducers etoposide, TNFa/cycloheximide, and staurosporine. The literature indicates that these cell lines and inducers have been used before in a variety of studies concerned with different aspects of apoptosis. However, the mode of cell death induced is dosage dependent and can vary with different conditions between different laboratories. A variety of methods and assays were used to establish apoptosis as the mode of cell death induced in the current studies. Flow cytometric analyses measured the exposure of phosphatidylserine and changes in cell size and membrane permeability which are characteristic of apoptosis. Immuno-blot assays detected the proteolytic processing of caspase-3, while fluorogenic substrates were used to measure caspase-3-like and caspase- 8-like activities in lysates prepared from cells undergoing apoptosis.
A polyclonal antibody generated against recombinant SREBP-2 was used to measure the cleavage of SREBP-2 in HL60, U937, and Jurkat cells undergoing apoptosis. Immunoblot-analyses showed that SREBP-2 is cleaved during apoptosis in all three cell lines regardless of the inducer. W h e n the proteolytic activation of SREBP-2 by site-1- and site-2-protease was inhibited by 25-hydroxycholesterol in etoposide-induced H L 6 0 cells, SREBP-2 cleavage was only detected in cell lysates 2 h after the induction of apoptosis. Whe n the caspase inhibitor Z-VAD-fmk was added, no SREBP-2 cleavage was detected suggesting that, in this system, cleavage of SREBP-2 during apoptosis is caspasemediated and occurs 2 h after the induction of apoptosis.
SREBP-2 functions as a key regulator of cholesterol synthesis, so that its cleavage during apoptosis should have an impact on cholesterol levels. Analyses of cholesterol levels of whole cells undergoing apoptosis showed a drop in cholesterol content in the first hour after the induction of apoptosis in all three cell lines, regardless of the inducer. However, after further time, etoposide-induced cells showed a constant increase in cholesterol content beginning 2 h after the induction of apoptosis and coinciding with the cleavage SREBP-2 by caspases. In contrast, cells induced to undergo apoptosis with staurosporine or TNFa/cycIoheximide had constantly declining cholesterol levels, despite SREBP-2 cleavage. These data suggested that either the cholesterol levels (and therefore SREBP-cleavage) are not important for the apoptotic program or that, in those cells that experience continuously declining cholesterol levels during apoptosis, another mechanism is employed to compensate for low cholesterol levels.
The main function of cholesterol in a cell is that of a spacer molecule within the plasma membrane, regulating phospholipid packing and, indirectly, membrane permeability. Flow cytometric analyses of the membrane permeabilities of HL60, U937, and Jurkat cells during apoptosis showed a limited increase in membrane permeability to an intermediate level (i.e. above control cells but less than dead cells) as early as 1 h etoposide-induced HL60 cells and 4 h after induction of apoptosis in the other cell systems. Only when the cells were dead, were high membrane permeabilities detected. This was 6 h after the induction of apoptosis with etoposide in HL60 cells and 4 h in other cell systems. It was also noted that cells with continuously declining cholesterol levels reduced in size more rapidly than those with transiently increasing cholesterol levels. In order to test the hypothesis that apoptotic cells with continuously declining cholesterol levels maintain a low membrane permeability as a result of cell shrinkage, phospholipid packing of cells undergoing apoptosis was analyzed.
Phospholipid packing of the cell membrane was analyzed by flow cytometry using the probe merocyanine 540. In order to confirm that cholesterol regulates the phospholipid packing in plasma membranes, normal cells were either depleted or loaded with cholesterol using cyclodextrins and analyzed for changes in the phospholipid packing.
Cholesterol-depleted cells showed decreased phospholipid packing and increased membrane permeability while cholesterol-loaded cells showed increased phospholipid packing and low membrane permeability. These data confirmed that cholesterol influences phospholipid packing of the plasma membrane and thereby indirectly affects the permeability. However, cells undergoing apoptosis showed an increase in phospholipid packing in the first 3 h after the induction of apoptosis regardless of the cholesterol The phospholipid packing only decreased when the cells were dead. It was also noted that in the first 3 h after the induction of apoptosis, all cells tested reduced in size. shrinkage was more prominent in those cell systems with continuously decreasing cholesterol levels than those with increasing cholesterol levels. These data suggested to compensate for decreasing cholesterol levels, cells undergoing apoptosis might undergo rapid cell shrinkage to increase the phospholipid packing of the plasma membrane and maintain a low membrane permeability.
An increase in phospholipid packing combined with cell shrinkage during apoptosis could have implications for processes other than maintenance of membrane permeability. It was speculated that these changes in the plasma membrane during apoptosis might affect endocytosis. Therefore, another aim of this thesis was to investigate the involvement the process of endocytosis in apoptosis. Blebbing of the plasma membrane is a major characteristic of cells undergoing apoptosis but the cause of this behaviour is unknown. Rapidly endocytosing cells display a ruffling or blebbing membrane similar to the blebbing seen during apoptosis. It has been hypothesized that endocytosis could account for plasma membrane blebbing during apoptosis. In order to test this hypothesis, etoposide-induced HL60 cells and TNFα/cycloheximide-induced U937 cells were analyzed for endocytosis by flow cytometry and confocal microscopy using the dye FM1- 43. The flow cytometric analyses showed a sudden increase in endocytosis of FM1-43 stained plasma membrane 2 h after the induction of apoptosis in HL60 cells with etoposide. After 2 h, endocytosis declined to a lower level but remained at a level greater than in normal cells. TNFα/cycloheximide-induced U937 cells showed very little endocytosis 2 hours after the induction of apoptosis and this later declined to almost zero. The flow cytometric analyses were confirmed by confocal microscopy. Several areas with accumulated FM1-43 were identified within etoposide-induced HL60 cells indicating endocytosis, while TNFα/cycloheximide-induced U937 cells showed no such areas. Cholesterol is a necesαary component for endocytosis as it induces membrane curvature which is necessary for the formation of endocytic vesicles. Cholesterol-depleted cells not capable of endocytosis. Here it was shown that etoposide-induced HL60 cells (which increase cholesterol levels at an early stage in apoptosis) endocytose during apoptosis TNFα/cycloheximide-induced U937 cells do not, probably because of declining cholesterol levels in the plasma membrane which prevents the formation of endocytic vesicles. However, light microscopy showed that etoposide-induced HL60 cells as well as TNFα/ cycloheximide-induced U937 cells have blebbing plasma membranes 3 h after the induction of apoptosis. Thus, at least in TNFα/cycloheximide-induced U937 cells, endocytosis is not necessary for apoptotic membrane blebbing.
The changes in intracellular cholesterol levels during apoptosis might also affect the cholesterol levels of mitochondrial membranes. In many cell systems, mitochondria release apoptogenic factors into the cytosol to trigger apoptotic execution. The mechanism that causes the release of apoptogenic factors is unknown. However, it was shown by others that changes in the cholesterol content of mitochondrial membranes alters their permeability. It was hypothesized that changes in cholesterol content of mitochondria during apoptosis might trigger the release of apoptogenic factors. As a first step towards testing this hypothesis, changes in cholesterol content of the outer membrane of mitochondria isolated from etoposide-induced Jurkat cells were analyzed by flow cytometry using the cholesterol-binding dye filipin. However, the cholesterol content Jurkat cell mitochondria was too low to be detected by filipin. Coincident with these studies, the feasibility of using filipin to measure changes in cell surface cholesterol content was demonstrated.
Another aim of this research was to detect novel caspase and non-caspase substrates cleaved during apoptosis. Alterations in the proteome of apoptotic cells versus normal cells were analyzed by high-resolution 2 dimensional polyacrylamide-gel-electrophoresis (2D-PAGE). Cell lysates from normal and etoposide-induced HL60 cells were divided into cytosolic, plasma membrane and nuclear membrane proteins fractions. Visual comparison of silver-stained 2D-gels of cytosolic proteins isolated from normal and apoptotic cells detected nine new protein spots in the electrophoretogram of proteins apoptotic cells. Since silver-stained proteins cannot be analyzed by conventional sequence analysis, the 2D-PAGE protocol was optimized to allow the use of dyes less sensitve than silver-stain but compatible with conventional N-terminal sequence analysis. Subsequent 2D-PAGE using a collodial Coomassie Brilliant Blue G250 dye detected two proteins with molecular masses of 20 and 25 kDa and isoelectric points of 6.0 and 5.8, respectively. These protein spots were transferred onto PVDF membranes by either western blotting or adsorption from gel eluates and their N-terminal amino acid sequence analyzed by a commercial service. However, the protein content transferred onto PVDF membranes by either method was too small to identify the proteins. Due to a lack in availability of more sophisticated equipment at the time of the study and time limitations, the study was not taken further. Nevertheless, this work demonstrated the usefulness high-resolution 2D-PAGE in detecting apoptosis-specific peptides in cells undergoing apoptosis.