Abstract
Many reports have documented apoptotic death in different cell types within hours of exposure to cytotoxic drugs; lower drug concentrations may cause cell cycle arrest at G2/M and subsequent death, which has been distinguished from 'classic' apoptosis. We have analysed etoposide-induced cell death in two lymphoblastoid T-cell lines, CCRF-CEM and MOLT-4, specifically in relation to DNA cleavage as indicated by pulse-field gel and conventional electrophoresis. High (5 microM) concentration etoposide causes 50-kb cleavage of DNA that occurs at the same time as apoptotic morphology and internucleosomal cleavage. At lower concentrations (0.5-0.05 microM), sequential change may be discerned with altered gene expression being similar to that at high dose, but preceding cell cycle arrest and 50-kb cleavage. These last changes, in turn, clearly precede internucleosomal fragmentation of DNA, vital dye staining and morphological evidence cell death. The pattern of higher order fragmentation constitutes a sensitive indicator of commitment to cell death in these cells. Morphological evidence of cell death is associated with internucleosomal fragmentation in one of the lines, but the pattern of 50-kb DNA cleavage provides the clearest evidence of commonality in death processes occurring at low and high drug concentration.
Highlights
We have previously examined the response of CEM and MOLT-4 cells to etoposide in terms of internucleosomal fragmentation of DNA, cell cycle distribution and morphological change (Catchpoole and Stewart, 1993; 1995)
There was a uniformity in response of both the CEM and the MOLT-4 populations: exposure to etoposide caused immediate apoptotic cell death as determined by morphological criteria
The response was independent of drug concentration: if this were reduced from 100,UM to 5 m,M apoptosis was still evident within
Summary
MOLT-4 and CEM cell lines were maintained in RPMI-1640 medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum (Biosciences Pty, Australia) at 370C. Cancer Research Campaign 1998 growth were treated with etoposide (Sigma Chemicals, St Louis, MO, USA) in dimethyl sulphoxide (DMSO, final concentration, 0.05%), the vehicle alone being added to control cultures. Cell numbers were determined by phase-contrast light microscopy using a haemocytometer. Procedures for treatment and harvesting have been described (Catchpoole and Stewart, 1993)
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