A possible role of oxidative stress in the switch mechanism of the cell death mode from apoptosis to necrosis--studies on rho0 cells.

Apoptosis is induced not only during morphogenesis and embryogenesis but also under various pathological conditions, especially related to oxidative stress. Apoptotic cells are phagocytized by neighboring cells while necrotic cells cause local and general reactions sometimes lethal to our bodies. Data have been accumulated to demonstrate that the switch of the cell death mode from apoptosis to necrosis does occur. However, detailed mechanisms involved in the switch mechanism remain unsolved although decreases in the intracellular level of ATP and a burst in the cellular level of reactive oxygen species (ROS) have been proposed. Recently, we have shown that the population of apoptotic cells reaches maximum in human osteosarcoma 143B cells treated for 6h with menadione (MEN) while necrotic cells become predominant at 9h of the treatment. In the present study we have attempted to clarify the role of cellular ATP in the switch mechanism using rho(0) cells derived from human osteosarcoma rho+ cells. Results are summarized as follows: (1) Apoptotic and necrotic changes in rho(0) cells are much faster than rho+ cells after the treatment with MEN. (2) Cellular level of ATP in rho(0) cells remains essentially in the same level before and after the MEN-treatment while intracellular levels of superoxide continuously increase after the MEN-treatment. (3) rho+ cells treated with MEN in the presence of antimycin A plus oligomycin show similar changes to those of MEN-treated rho(0) cells. (4) MEN-induced increases in the cellular level of superoxide are distinctly suppressed by inhibitors of NADPH oxidase. These results suggest that the intracellular level of superoxide may be a key factor directly related to the switch mechanism from apoptosis to necrosis, and that decreases in cellular level of ATP accelerate both apoptotic and necrotic changes of the cells.

Role of mitochondria in the switch mechanism of the cell death mode from apoptosis to necrosis--studies on rho0 cells.

Detailed mechanisms of the switch of the cell death mode from apoptosis to necrosis remain to be solved, although the intracellular level of ATP and that of free radicals have been postulated to be the major factors involved in the mechanisms. In the present study menadione (MEN)-induced cell injury processes were studied using rho0 cells derived from human osteosarcoma 143B cells and parental rho+ cells co-treated with inhibitors of electron transfer chain of mitochondria or oligomycin, an inhibitor of ATP synthesis. Treatment of rho+ cells with 100 microM MEN induced apoptosis, which reached the maximum at 6 h, and was followed by an abrupt decrease thereafter, while necrotic cells (NC) increased continuously when they were judged by Annexin V and PI double staining. On the other hand, MEN induced apoptotic and necrotic changes much faster in rho0 cells compared to rho+ cells. The frequency to find apoptotic cells (AP) in the former cells was distinctly smaller than that to find NC judged by Annexin V and PI double staining. Electron microscopically, a major population of rho0 cells treated with MEN for 6 h consisted of intermediate cells, and a small number of AP co-existed. At 9 h of the treatment intermediate cells were exclusively seen, and AP were hardly detected. When parental rho+ cells were treated with MEN in the presence of oligomycin or oligomycin plus antimycin A both apoptotic and necrotic changes of the cells were distinctly accelerated. The intracellular level of superoxide in rho0 cells continuously increased after the MEN treatment, whereas that of ATP remained distinctly low before and after the MEN treatment compared to that in rho+ cells. These data suggest that the intracellular level of superoxide may be a key factor controlling the switch from apoptosis to necrosis.

Modification of physicochemical properties of actin filaments suppresses cell fragmentation in the execution phase of staurosporine-induced apoptotic processes.

Effects of jasplakinolide (JSP), a stabilizer of F-actin, and latrunculin A (LTA), a destabilizer of F-actin, on a series of events occurring in the execution phase of staurosporine (STS)-induced apoptotic processes were studied using human osteosarcoma 143B cells. Time-dependent apparent increases of the population of cells with collapsed membrane potential of mitochondria (Delta Psi(m)) caused by STS treatment were not due to actual decreases in the Delta Psi(m) per cell, but due to the fragmentation of cells resulting in decreases in the number of active mitochondria per cell. Decreases in the Delta Psi(m) in fragmented cells occurred late in the execution phase. Both JSP and LAT failed to prevent STS-induced release of cytochrome c from mitochondria followed by the activation of caspases 3 and 9, the cleavage of poly (ADP-ribose) polymerase (PARP) and apoptotic nuclear fragmentation. However, both drugs prevented STS-induced apoptotic cell fragmentation and decreases in the Delta Psi(m). These results indicate that physicochemical states of actin filaments play a certain role in the execution phase of STS-induced apoptotic processes.

Changes in physicochemical properties of microtubules lead to the formation of a single spherical structure of mitochondrial assembly enveloping nuclear chromatins.

Treatment of 143B cells with microtubule-active drugs (MADs) including taxol, nocodazole and colchicine induced distinct structural changes, such as rounding of the cells with perinuclear clustering of mitochondria, when the cells were treated for up to 10 h. When the incubation time with MADs was longer than 10 h, multinuclear cells appeared, and their population increased with time. In this study perinuclear clustering of mitochondria i.e. mitochondria encircling the aggregated chromatin of the nucleus that had lost the nuclear membrane was detected. This observation was distinct from that reported in the literature. Mitochondria were aligned in a few lines; the occurrence of mitochondria in even a single line is an extreme case, resulting in one plane of section for electron microscopy. Three-dimensional reconstructions of confocal microscopic images of mitochondria revealed that they were assembled as a spherical structure. The majority of the cells with perinuclear clustering of mitochondria remained intact for up to 24 h. Mitochondria were observed to be clustered around the nucleus in the orthodox configuration or in some cases they were moderately condensed, as observed electron microscopically. Annexin V and PI double staining of cells showed that more than 90% of cells were viable. In the case of treatment with taxol, membrane potential of mitochondria per cell was well maintained although it was moderately lowered in the case of treatment with nocodazole. Taking into consideration the previous data reported from our laboratory, the present results may assist in elucidation of the behaviour of mitochondria during the dividing processes of mammalian cells, which is yet to be clarified.

H2O2 activates ryanodine receptor but has little effect on recovery of releasable Ca2+ content after fatigue.

We studied whether hydrogen peroxide (H(2)O(2)) at