Thapsigargin induces mitochondrial dysfunction and apoptosis in the mastocytoma P815 cell line and in mouse thymocytes.
Thapsigargin is a plant-derived inhibitor of the endoplasmic reticulum Ca(2+)-ATPase.Treatment with thapsigargin leads to a rapid, large and prolonged increase in the intracellular calcium ion concentration ([Ca(2+)](i)). Previously thapsigargin has been shown to inhibit proliferation and induce apoptosis. Here we report the results of thapsigargin treatment in thymocytes harvested from 10-day-old mice and in the P815 mastocytoma cell line. In thapsigargin-treated cells we observed enlarged mitochondria with disrupted cristae structure. These mitochondria closely resembled those observed after the induction of phase transition. To determine if the mitochondria were functioning normally the cells were stained with rhodamine 123 (R123) and analysed with flow cytometry. After thapsigargin treatment the R123 staining decreased, indicative of a loss of mitochondrial membrane potential. Furthermore intracellular ATP concentrations were also found to be reduced in cells treated with thapsigargin. Taken together these results indicate an increase in the [Ca(2+)](i) caused by thapsigargin treatment results in dysfunctional mitochondria and reduced ATP. We propose that this decrease in the concentration of ATP provokes the onset of thapsigargin-induced apoptosis. To investigate the effect of thapsigargin treatment on the cell cycle, rapidly cycling P815 cells were sorted into populations enriched for either G(0)/G(1) or S/G(2)/M phases, and these populations were then treated with thapsigargin. Thapsigargin treatment induced a cell cycle block before S phase. We propose that the block in the cell cycle induced by thapsigargin was a result of the decreased intracellular ATP concentration interfering with the energy requiring processes of DNA replication. The block could also be related to the high intracellular calcium ion concentration that would interfere with the subtle calcium transients involved in the cell's preparations for replication and mitosis. Apoptosis occurred to an equal extent in both populations of cells.
Beaver
J P
JP
John Curtin School of Medical Research, Australian National University, Canberra, Australia.
Waring
P
P