Drug-induced death of leukaemic cells after G2/M arrest: higher order DNA fragmentation as an indicator of mechanism

Abstract

Summary 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 G/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,M) 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 ,UM), 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.

Morphological and molecular evidence of differentiation during etoposide-induced apoptosis in human lymphoblastoid cells

“…21 We have previously used semi-quantitative reverse transcriptase (RT) ± PCR to demonstrate increased expression of purine nucleoside phosphorylase in PMAtreated CEM cells, and recorded an even more marked increase in etoposide. 16 Similar RT ± PCR analysis indicated a marked increase in the amount of terminal deoxynucleotidyl transferase mRNA within 3 h of treatment with 5 mM etoposide, and the effect was evident in preparations isolated up to 24 h after drug treatment (Figure 1). However, when the drug concentration was decreased to 0.5 mM, no increased expression was evident during the first 6 h, and by 12 h relative band intensity suggested a decrease in expression of up to 50%.…”

“…Finally, although both etoposide and PMA induce CD3 expression, the apoptotic outcome is markedly different. Etoposide causes apoptosis in all (50 ± 0.5 mM) or 20% (0.05 mM) of CEM cells, 16 while PMA treatment did not cause cell death as indicated by ultrastructural and annexin V studies described later in this Results section. Figure 1 RNA isolated from control and etoposide-treated CEM preparations was reverse transcribed and subject to semiquantitative RT ± PCR analysis as previously described.…”

“…20 Such fragmentation is evident in lymphoblastoid and other cell populations shortly after exposure to etoposide at cytotoxic concentrations; 22,23 this result being previously recorded during studies in our laboratory. 16 Further analysis has now been undertaken comparing the effect of PMA with etoposide in CEM cells. As expected on the basis of previous studies, 16 a band corresponding to 50 kilobase was evident following pulsed-field gel electrophoresis of DNA isolated from cells exposed to etoposide (0.5 mM) for 6, 12, 24 or 72 h (data not shown).…”

“…Figure 1 RNA isolated from control and etoposide-treated CEM preparations was reverse transcribed and subject to semiquantitative RT ± PCR analysis as previously described. 16 After densitometric analysis of the PCR bands, the ratio between the respective target gene and the housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase) was calculated from at least two independent experiments and the results shown are the mean standard error for each determination. …”

“…14 We have studied induction of apoptosis in lymphoblastoid leukaemic cells by etoposide. 15,16 In respect of all parameters studied, no differences could be discerned between CEM and MOLT-4 cells and study of etoposideinduced caspase activation did not reveal differences (RJ Sleiman and BW Stewart, unpublished results). Accordingly, the present study utilized CEM cells on the basis that this line typifies the response of lymphoblastoid cell populations.…”