Lack of correlation between early intracellular calcium ion rises and the onset of apoptosis in thymocytes.
Apoptosis, a well-recognized process of cell death, is usually defined by chromatin condensation, plasma membrane blebbing, reduction in cell volume, and in many cell types the cleavage of DNA into nucleosomal multiples, and finally the formation of apoptotic bodies. We have characterized the time of onset and the range of concentrations at which the toxins gliotoxin and thapsigargin induce apoptosis in thymocytes. We also looked for early changes in cytosolic calcium ion concentration ([Ca2+]i). Three methods were used to detect apoptosis: cellular morphology, DNA fragmentation and a flow cytometric method using ethidium bromide. Calcium fluxes were measured using both flow cytometry and bulk cell fluorimetry. Gliotoxin concentrations of 50 nmol/L to 10 mumol/L induced significant numbers of cells to become apoptotic in a dose dependent manner. At these concentrations there was no observable increase in [Ca2+]i as determined by flow cytometry or in bulk cells. However, when thymocytes were treated with gliotoxin at concentrations greater than 500 mumol/L, rises in [Ca2+]i were apparent, but these cells died by necrosis. Thapsigargin induced low levels of apoptosis in thymocytes; the maximum effect observable after a 10 nmol/L treatment. Thapsigargin is known to inhibit the Ca(2+)-ATPase in the endoplasmic reticulum thereby causing a sustained increase in [Ca2+]i in thymocytes. The rise in [Ca2+]i observed was quantitatively similar when thymocytes were treated with thapsigargin concentrations ranging between 10 and 100 nmol/L. These results led us to investigate the effect of dexamethasone on [Ca2+]i. In these experiments thymocytes showed no rises in [Ca2+]i above the control over 85 min following treatment with 10 mumol/L dexamethasone.
Beaver
J P
JP
John Curtin School of Medical Research, Australian National University, Canberra.
Waring
P
P
D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005912:Gliotoxin / Q000633:toxicity*; D051379:Mice; D013729:Terpenes / Q000633:toxicity*; D019284:Thapsigargin; D013950:Thymus Gland / Q000166:cytology*; D013997:Time Factors
D013729:Terpenes; D005912:Gliotoxin; D019284:Thapsigargin; D003907:Dexamethasone; D002118:Calcium