Changes in c-myc expression and the kinetics of dexamethasone-induced programmed cell death (apoptosis) in human lymphoid leukaemia cells.
The kinetics of dexamethasone-induced death of CCRF CEM clone C7A human lymphoblastic leukaemia cells was determined with respect to changes in the expression of the c-myc protein. Cell death was characterised as being by apoptosis: cells with an intact plasma membrane had condensed chromatin and were characterised as having approximately 300 kbp fragments when DNA integrity was analysed by pulsed-field electrophoresis. Onset of apoptosis required a minimum of 36 h exposure to 5 microM dexamethasone; before this time no apoptotic cells were observed. This 36 h incubation period appeared to be necessary to prime the cells for subsequent death by apoptosis. In the continued presence of dexamethasone the percentage of apoptotic cells increased to 60% apoptotic cells by 54 h. Investigation of changes in c-myc protein showed that it was undetectable after 12 h of incubation with dexamethasone, although cells were not committed to die at this time. Cells were treated with dexamethasone for 54 h and for various pulsed periods with a non-toxic concentration of cycloheximide (200 nM). When cycloheximide was present during the first 36 h priming period of dexamethasone treatment, there was an immediate loss of c-myc protein and apoptosis at 54 h was completely inhibited. In contrast, there was no inhibition of apoptosis when dexamethasone-treated cells were incubated with an 18 h pulse of cycloheximide added after 36 h. Cells exposed to dexamethasone for 36 h ('primed') were given various periods of dexamethasone-free incubation before readdition of dexamethasone for a further 18 h. The longer the cells were free of drug after priming, the less susceptible they became to apoptosis, suggesting a slow decay of their 'memory' of the initial 36 h period of exposure. Cycloheximide inhibited the decay of this memory. Removal of dexamethasone after a 36 h exposure was characterised by a subsequent 24 h suppression of c-myc protein expression. Despite this, 90% of cells became refractory to apoptosis before the reappearance of c-myc protein. The evidence does not support the hypothesis that changes in c-myc expression are required for the engagement of apoptosis of CEM cells.
Wood
A C
AC
Cancer Research Campaign Molecular and Cellular Pharmacology Group, School of Biological Sciences, University of Manchester, UK.
Waters
C M
CM
Garner
A
A
Hickman
J A
JA
D017209:Apoptosis / Q000187:drug effects*; D003513:Cycloheximide / Q000494:pharmacology; D004273:DNA, Neoplasm / Q000032:analysis; D003907:Dexamethasone / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D016521:Electrophoresis, Gel, Pulsed-Field; D016193:G1 Phase / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D007945:Leukemia, Lymphoid / Q000235:genetics / Q000378:metabolism; D015458:Leukemia, T-Cell / Q000235:genetics / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects*
D004273:DNA, Neoplasm; D016271:Proto-Oncogene Proteins c-myc; D003907:Dexamethasone; D003513:Cycloheximide