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Clinical Cancer Research Vol. 6, 3756-3765, September 2000
© 2000 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Early Caspase Activation in Leukemic Cells Subject to Etoposide-induced G2-M Arrest: Evidence of Commitment to Apoptosis Rather Than Mitotic Cell Death1

Robert J. Sleiman and Bernard W. Stewart2

Children’s Cancer Institute Australia, Sydney Children’s Hospital, Sydney 2031 [R. J. S., B. W. S.], and School of Paediatrics, University of New South Wales, Sydney 2052 [B. W. S.], Australia


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
After exposure to cytotoxic drugs at relatively low concentration, many cell types undergo G2-M arrest and then either mitotic cell death or, in the case of hematopoietic cells, apoptosis. We have sought to examine this phenomenon in two lymphoblastoid cell lines. After continuous or short-term exposure to etoposide (final concentration, 0.5 µM), up to 80% of cells accumulated at G2-M by 24 h, and subsequently either underwent apoptosis or re-entered the cell cycle. In this and the other studies undertaken, the CEM and MOLT-4 lines behaved similarly. Progressive accumulation of cells at G2-M was accompanied by increasing levels of cyclin B1. Commitment to apoptosis was assessed by evidence of caspase activation using a number of different criteria. A decreased amount of Mr 32,000 procaspase-3 was evident 24–48 h after drug treatment. However, cleavage of caspase substrates poly(ADP-ribose) polymerase and lamin B indicated caspase activation occurring within 3–6 h of drug treatment. Protease activity in corresponding cell extracts increased progressively from 6 h or earlier to 24 h after the addition of etoposide to the medium. Such increase was consequent on drug treatment and not attributable to cells being at G2-M. Treatment with 1.5 mM caffeine abrogated etoposide-induced G2-M arrest, and in cells so treated, the etoposide-induced increase in protease activity was also abrogated. However, there was no impact of caffeine on cytotoxicity under these conditions. Although mitotic cell death is precipitated subsequent to prolonged G2-M arrest in many cell types, the present data suggest that commitment to apoptosis occurs in parallel to G2-M arrest in leukemic cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ionizing radiation and cytotoxic drugs, which induce DNA damage (either directly or indirectly), cause proliferating cells to undergo cycle arrest. In cells with functional p53, drug-induced arrest occurs at G1, and subsequent outcome is thought to depend on the adequacy of DNA repair (1) . Inadequate repair results in cell death (2) . After radiation- or drug-induced G1 arrest, cell death occurs by apoptosis, particularly in cells with functional p53 (3) . The cytotoxic drug etoposide targets topoisomerase II forming a cleavable complex to which, in turn, DNA damage, cell cycle arrest, and ultimately cell death is attributable (4) . Activation of caspase-3 (CPP32/prICE/Yama/apopain) occurs in response to cytotoxic drugs, including etoposide (5 , 6) , and is reckoned to mark an irreversible commit to apoptotic cell death (7) . Enzymatic activity of caspase-3 may be indicated by cleavage of PARP3 (8 , 9) .

The relationship between cell cycle arrest and apoptosis is best understood in the context of G1 arrest and the role of p53 and pRb, the latter being a caspase substrate (10 , 11) . However, drug- or radiation-induced death after G2-M arrest has been observed in a range of cell types specifically including cells with defective p53 (12, 13, 14, 15, 16, 17) . G2-M arrest in response to DNA damage is caused by inactivation of the cdc2 catalytic subunit (18 , 19) , an effect that may be mediated by cdc25 phosphatases (20 , 21) . In this context, apoptosis may occur (12 , 22) or the mode of cell death may be distinguished from apoptosis (13) . One mode of nonapoptotic cell death after G2-M arrest has been specifically described as "mitotic cell death" or "mitotic catastrophe" (16 , 23, 24, 25) . The phenomenon involves G2-M arrest followed by cell death associated with incomplete or defective mitosis, often indicated by formation of multinucleated cells. Cell death after G2-M arrest may be observed when drug concentrations lesser than those associated with G1 arrest are used (13 , 17 , 26 , 27) . G2-M arrest is thought to provide an opportunity for DNA repair, in the absence of which mitosis will be premature and result in a lethal outcome. This understanding is suggested by a range of studies reporting increased cell death if G2-M arrest is abrogated and increased survival (effectively equivalent to drug resistance) following prolongation of G2-M arrest (25 , 28, 29, 30) .

The human lymphoblastoid cell lines CEM and MOLT-4 both lack functional p53 (31 , 32) . In these and similar leukemia lines, cell death after cycle arrest at G2-M occurs in response to ionizing radiation or cytotoxic drugs (16 , 33) . We have studied such cell death induced by etoposide (34) . In the 24-h period after the addition of the drug to the medium (final concentration, 0.5 µM), the proportion of cells at G2-M progressively increases from 15 to 80% in the absence of cell death as determined by light microscopy and vital dye staining. In the period 24–48 h after addition of the drug, cell death occurs by apoptosis, there being no evidence of micronuclei or other indicators of mitotic catastrophe. Such apoptotic cell death may be related to the efficacy of etoposide in the treatment of acute lymphoblastic leukemia.

We have sought to elucidate the relationship between etoposide-induced arrest of cells at G2-M and caspase activation, caspase-3 activation being indicative of, if not absolutely required, for apoptosis (35) . Caspase-3 and its substrate caspase-6 are the major active caspases in a variety of leukemic lines specifically including CEM and MOLT-4 exposed to etoposide (36) . Moreover, caspase-6 has been considered to be the major laminase in cells undergoing apoptosis (37) , lamin B being subject to preferential degradation compared with lamins A and C (38) . Ectopic expression of an uncleavable mutant of lamin B caused a significant delay of E1A-induced p53-dependent apoptosis (39) .

On the basis of the apoptotic "timetable" for etoposide-induced death of CEM and MOLT-4 cells described earlier (34) and summarized above, caspase activation was anticipated at some time after drug treatment, and presumably in response to the prolonged cell cycle arrest (10) . We now report, however, that in etoposide-treated CEM and MOLT-4 cells, there was no quiescent period after which caspase activation was particularly marked. Rather, caspase activation occurred progressively after drug treatment, apparently in parallel with accumulation of cells at G2-M. To elucidate relevant mechanisms, we used synchronized cells to determine whether similar activation occurred as the proportion of cells at G2-M increased even in the absence of drug treatment. We examined whether abrogation of the G2-M checkpoint altered the pattern of caspase activation and subsequent cell death. We found that the relationship of caspase activation to cycle arrest in this system is indicative of apoptosis, and further distinguishes the death of lymphoblastoid cells after G2-M arrest from mitotic cell death, which occurs in G2-M-arrested carcinoma and other cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture Methods and Cellular Analysis.
Methods for maintenance of the CEM and MOLT-4 lines in culture, treatment with etoposide, and harvesting are described by Sleiman et al. (34) . The latter (34) also describes determination of cell cycle status by flow cytometry after staining with propidium iodide and quantitation of vital dye staining with trypan blue. For the purpose of synchronization, randomly dividing cultures of CEM and MOLT-4 cells were maintained to a density 3 x 105 cells/ml, at which point thymidine (final concentration, 2.5 mM) was added to the media. After 16 h, the cells were released by washing (three times) with prewarmed serum-free RPMI 1640, and suspending in fresh medium supplemented with 10% FCS together with a combination of thymidine and deoxycytidine (final concentration, 24 µM). At various times up to 9 h after this "release," cells were subject to isolation and analysis.

Semiquantitative RT-PCR.
Methods used for RNA isolation, reverse transcription, optimization of RT-PCR conditions, and analysis of relative band intensity have been published (34) . Primers used in the present study included caspase-3 (sense: 5'AGATGTCGATGCAGCAAACC-3'; antisense: 5'-CAGGTCCATTTGTTCCAAAA-3'; product length, 201 nucleotides); and ß2-microglobulin (sense: 5'ACCCCCACTGAAAAAGATGA-3'; antisense: 5'-ATCTTCAAACCTCCATGATG-3'; product length, 110 nucleotides).

Protein Isolation.
Proteins were analyzed by immunoblot in the cytosolic or nuclear fractions from the CEM and MOLT-4 cells. Cell fractionation of 5 x 107 cells was undertaken using the procedure described by Khachigian et al. (40) , with cytosolic protein being recovered from the supernatant generated after sedimentation of crude nuclei. The nuclear membrane fraction, used for the determination of lamin B, was that remaining after the final centrifugation to allow recovery of the nuclear extract (40) , and was suspended in the same buffer used for the nuclear extract. Preliminary studies indicated that nuclear extracts provided greatest insight in relation to cyclin B1 and PARP levels. Procaspase-3 was most readily detected in the cytosolic fraction, consistent with the findings by Krajewska et al. (41) .

Immunoblots.
Protein concentrations were estimated using BSA as a standard. Protein isolates (routinely 20 µg/lane) were electrophoresed through a 10% SDS-polyacrylamide gel at 100 V for 1.5 h and electrotransferred to nitrocellulose. Equal loading of lanes was confirmed by Coomassie blue staining of duplicate gels. After transfer, the membranes were blocked overnight at 4°C with 5% skim milk in TBST buffer [10 mM Tris-HCl buffer, 150 mM NaCl, 0.1% Tween-20 (pH 8.0)]. The membranes were then incubated with 0.5% skim milk in TBS [10 mM Tris-HCl buffer, 150 mM NaCl (pH 8.0)] containing 1:200 dilution of the antibodies to cyclin B1, caspase-3 (CPP32), or PARP (all purchased from Santa Cruz Biotechnology, Inc, Santa Cruz, CA) or lamin B (from Oncogene Research Products, Boston, MA), for a further 60 min at room temperature, washed twice with TBST, reblocked for 45 min, and then incubated with a 1:2000 dilution of the horseradish peroxidase-conjugated second antibody (Santa Cruz Biotechnology) in 0.5% skim milk with TBS buffer. Proteins were detected using Pierce SuperSignal Chemiluminescent Detection System in accordance with the manufacturer’s instructions (Pierce, Rockford IL) and analyzed using a GS-525 Molecular Imager System (Bio-Rad Laboratories).

Protease Activity in Vitro.
Protease activity primarily attributable to caspase-3 was determined on the basis of cleavage of pNA from the peptide substrate DEVD-pNA (ApoAlert, Clontech Labs) on the basis of absorbance at 405 nm.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Incubation of CEM cells in the presence of 0.5 µM etoposide resulted in progressive accumulation of up to 80% of cells at G2-M during the first 24 h, and then apoptotic cell death (as evidenced by less than G1 content) of most cells in the period up to 48 h (Fig. 1Citation , top section). Almost identical results were observed using MOLT-4 cells (data not shown). The data imply that cells at G2-M undergo cell death, and further evidence for this understanding was obtained by modifying the treatment protocol. The proportion of cells at G2-M (after 24 h) and the proportion undergoing apoptosis (after 48 h) were both less when exposure to the drug was restricted to a short period (a 3–12-h "pulse"), and the cells were returned to fresh media for the duration of the experiment. The impact of such drug treatment was barely discernible after a 3-h pulse, but was clearly evident using a 6- or 12-h pulse (Fig. 1Citation , middle and bottom sections). Under these latter conditions, there was little evidence of persistence of cells at G2-M. Rather, the surviving cells were at G1, suggesting that cells at G2-M either die or survive by progressing through mitosis.



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  		Fig. 1. Effect of etoposide (final concentration, 0.5 µM) on DNA content of CEM cells as determined by flow cytometric analysis after staining with propidium iodide. The profiles indicate cell cycle distribution (G1 to G2-M) as well as the appearance of apoptotic cells having less-than-G1 DNA content. Top row, the impact of continuous exposure to etoposide for periods from 3 to 48 h. In these and other studies, the vehicle control result was the same irrespective of whether such cells were isolated at zero time or after 48 h. Middle and bottom rows, the effect of "pulse" or short-term exposure to the drug, involving the addition of etoposide for 3, 6, or 12 h, and then resuspension in fresh media until analysis. The respective protocols are identified on each profile by the period in etoposide (before the slash) and the total time from the addition of etoposide until cells were isolated (after the slash). The individual profiles shown are typical of at least two analyses involving separate experiments.

 
In human melanoma cells, {gamma}-irradiation causes G2-M arrest and inhibits cyclin B1 synthesis (42) , and after ionizing irradiation of HeLa cells, the level of cyclin B1 protein is considered to be the rate-limiting factor accounting for G2 delay (43) . In contrast, however, cyclin B1 accumulates in HeLa cells arrested by UV light (44) or etoposide (21 , 45) . In our studies, cyclin B1 was readily detectable in preparations of nuclear protein. After the addition of 0.5 µM etoposide to the medium, the amount of nuclear cyclin B1 progressively increased in the 3–24-h period (Fig. 2)Citation . In corresponding cytoplasmic preparations, the protein was not detected except after 12 h, when trace amounts were apparent and after 24 h, when a clear band was evident. Accumulation of cyclin B1 in the cytoplasm suggests a difference in intracellular transport between lymphoblastoid and HeLa cells accumulating cyclin B1 after similar treatment (45) .



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  		Fig. 2. Immunoblot analysis of cyclin B1 (nuclear and cytoplasmic) and cytoplasmic procaspase-3 and actin (loading control) isolated from CEM and MOLT-4 (M4) cells incubated in the presence of 0.5 µM etoposide for 3, 6, 12, 24, or 48 h, and the respective vehicle control (VC) cells as labeled on the figure. Protein isolation procedures and antibodies used are described in the "Materials and Methods" section. Right, in this and other immunoblots, molecular weights from appropriate standards are indicated. Results are typical of those from at least two separate series of experiments.

 
Caspase family members are located in the cytosol as zymogens, with cleavage of procaspase-3 (Mr 32,000) to generate Mr 17,000 and Mr 12,000 fragments being considered indicative of commitment to apoptosis (7) . In cytosolic preparations from both CEM and MOLT-4, the procaspase-3 band was either markedly decreased (CEM) or lost (MOLT-4) during the 24–48 h after the addition of the drug (Fig. 2)Citation . In common with several studies using a range of other cell types (6 , 46 , 47) , the Mr 17,000 and Mr 12,000 fragments were not detected. Apart from its loss during apoptosis, other changes in the staining intensity of the procaspase-3 band were evident. By comparison to control preparations, an increased amount of procaspase-3 was evident in cytosolic extracts from CEM cells after 6–12 h, and to a lesser extent in MOLT-4 cells after 3-h exposure to etoposide (Fig. 2)Citation . The data suggested increased synthesis, and we sought evidence for change in gene expression using semiquantitative RT-PCR. These studies indicated an increased amount (up to 2.9 increase in band intensity) of the corresponding mRNA 3–12 h after the addition of etoposide to CEM cells, with no evidence of change (ratios, 1.1–1.3) in corresponding MOLT-4 preparations (data not shown).

Further insight regarding caspase-3 activation was obtained by reference to known substrates: PARP, which is directly cleaved by capase-3, and lamin B, a substrate for capase-6, which is itself cleaved by caspase-3 (48) . In the case of PARP, preparations of nuclear protein isolated from CEM and MOLT-4 cells treated with 0.5 µM etoposide revealed early change. In both lines, loss of the Mr 120,000 band, and accumulation of the Mr 85,000 fragment was clearly evident 3 h after the addition of the drug. In the case of CEM cells, there was partial loss of the Mr 120,000 band over the course of the first 24 h after the addition of the drug, whereas the MOLT-4 preparations indicated complete loss of this band by 6 h (Fig. 3Citation , top). Cleavage of lamin B was evident up to 18 h before morphological evidence of cell death. In both CEM and MOLT-4 preparations, the appearance of the Mr 45,000 fragment derived from lamin B was the most sensitive indicator of lamin B cleavage (Fig. 3Citation , bottom).



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  		Fig. 3. Progressive degradation, as indicated by immunoblot analysis, of caspase substrates PARP and lamin B in CEM and MOLT-4 (M4) cells incubated in the presence of 0.5 µM etoposide for 3, 6, 12, 24, or 48 h by comparison with the result obtained from vehicle control (VC) cells. After appropriate incubation, protein was isolated from nuclear and membrane fractions, respectively, as described in the "Materials and Methods" section.

 
Although loss of procaspase-3 is not evident until 24–48 h after the addition of the drug (Fig. 2)Citation , immunoblot analysis of PARP and lamin B suggested the possibility of caspase-3 activation at earlier times (Fig. 3)Citation . To gain additional understanding of the pattern of caspase activation, protease activity was assayed in cell extracts using an appropriate synthetic peptide. This activity increased in response to etoposide treatment, the effect being evident within 6 h. There was progressive increase in peptidase activity exhibited by extracts of each cell line prepared from 6 to 24 h after exposure to etoposide (Fig. 4)Citation . Such activity was reduced by >80% in the presence of a specific inhibitor of caspase-3 (data not shown). By 24 h, protease activity in extracts from cultures to which etoposide had been added was >10-fold that detected in control cell preparations. The result is suggestive of caspase-3 activation, or activation of some other caspase, occurring in parallel with accumulation of cells at G2-M rather than being subsequent to such accumulation.



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  		Fig. 4. Protease activity mediated by extracts of CEM (top) and MOLT-4 (bottom) cells incubated for the period indicated in the presence of 0.5 µM etoposide (•). The effect of 1.5 mM caffeine added to the cultures at the same time as etoposide is indicated ({circ}). Protease activity is expressed as multiples of the mean determined in vehicle control preparations, which were 93 and 232 µmol of DEVD-pNA cleaved/106 cells for CEM and MOLT-4 cells, respectively, each point being the result of at least two assays, with the SE indicated.

 
The responsiveness, to etoposide treatment, of protease activity assayed in vitro suggested that this parameter be used to examine the relationships between cell cycle arrest and caspase activation. In the first instance, we sought to determine whether, in the absence of drug treatment, accumulation of cells at G2-M would result in such an increase in protease activity. This hypothesis was tested using CEM and MOLT-4 cells, which were synchronized using a thymidine block. The results obtained using the two lymphoblastoid lines were virtually identical. After "release" from the thymidine block, flow cytometric analysis established a progressive increase in the proportion of cells at G2-M. The relevant proportion increased from <5% at the time of release to 60–70% by 6–9 h (Fig. 5)Citation . In extracts prepared from such cells, protease activity was consistently twice that in extracts from randomly dividing cells, such change being presumably a consequence of the synchronization process. However, there was no pattern of variation in protease activity in relation to cell cycle progression (Fig. 5)Citation . Accordingly, increased activity in the drug-treated preparations is attributable to the cytotoxic agent rather than cell cycle arrest per se.



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  		Fig. 5. Variation in protease activity mediated by extracts of CEM (top) and MOLT-4 (bottom) cells at various times up to 9 h after "release" from thymidine block as described in the "Materials and Methods" section. The efficacy of this procedure as a means of synchronization is indicated by change in the proportion of cells at G2-M (•, left axis). Protease activity determined using the corresponding cell extracts ({circ}, right axis) is expressed in arbitrary units relative to activity exhibited in extracts from randomly dividing cells (see legend to Fig. 4Citation ).

 
Because increased protease activity is not attributable to an increased proportion of cells at G2-M, but is clearly a consequence of drug treatment (Fig. 4)Citation , we explored whether abrogation of etoposide-induced G2-M arrest would perturb this pattern. Consistent with observations made using other systems (see Refs. 28 , 30 , and 49 and references therein), in the presence of 1.5 mM caffeine, accumulation of cells at G2-M after the addition of 0.5 µM etoposide was abrogated. Assayed after 24 h, the proportion of cells at G2-M was reduced from ~80% (as a result of etoposide treatment) to a level only marginally different from the control by the addition of caffeine (Fig. 6)Citation . Caffeine itself had no impact on cell cycle distribution (Table 1)Citation . Moreover, a number of indicators, including cell viability as determined by trypan blue staining (data not shown) and the proportion of cells exhibiting less-than-G1 DNA content by flow cytometry (Table 1)Citation , established that exposure to 1.5 mM caffeine was not cytotoxic.



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  		Fig. 6. Abrogation of etoposide-induced arrest at G2-M of CEM (top) and MOLT-4 cells (bottom) by caffeine. After incubation in the presence of 0.5 µM etoposide for the periods indicated, flow cytometric analysis (see Fig. 1Citation ) was undertaken. The proportion of cells at G2-M in cultures treated with etoposide (•) is contrasted with that in cultures treated identically except for the addition of caffeine (1.5 mM final concentration) with etoposide ({circ}). All points are the results of at least two determinations with the SE shown.

 

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  		Table 1 Perturbation by caffeine of the cell cycle distribution exhibited by CEM and MOLT-4 cells after 24-h continuous exposure to 0.5 µM etoposide

 
The addition of caffeine had a marked effect on protease activity: the progressive increase observed after etoposide treatment did not occur. A similar effect was observed in both CEM and MOLT-4 cells, with protease activity increasing only 2- to 4-fold by comparison with a 15-fold increase otherwise caused by etoposide (Fig. 4)Citation . Appropriate control experiments indicated that exposure of cells to 1.5 mM caffeine had no effect on the low level of protease activity in otherwise untreated cells.

Caffeine is known to abrogate G2-M arrest in mammalian cells, and in some irradiated or drug-treated populations, it has been shown to increase lethality of the respective drug or radiation treatment (28 , 30) . However, in our experiments, no altered cytotoxicity was evident as a result of the addition of caffeine. Thus, after 48 h in the presence of 0.5 µM etoposide, the proportion of trypan blue-positive CEM or MOLT-4 cells was not affected by caffeine (final concentration, 1.5 mM, added at the same time as etoposide). Likewise, when determined after shorter incubation times, the proportion of nonviable cells was not affected by the addition of caffeine (Fig. 7)Citation .



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  		Fig. 7. Loss of viability after continuous exposure to 0.5 µM etoposide, as determined by trypan blue staining, in CEM (top) and MOLT-4 (bottom) cells. Closed bars, the effect of the drug; open bars, results obtained in the presence of caffeine as described in the legend to Fig. 6Citation . Quantitation of trypan blue staining was undertaken as previously described (34) .

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Death of Lymphoblastoid Cells after G2-M Arrest.
Pathways to cell death in CEM or MOLT-4 cultures exposed to etoposide are markedly concentration-dependent. Drug concentrations of >=5 µM cause G1 arrest within 6 h, and at the same time, apoptotic morphology and vital dye staining are observed (50) . Such etoposide concentrations equate with those used in most studies of drug-induced apoptosis and provide most present understanding of the relationship between cell cycle arrest, caspase activation, and apoptosis (11 , 51) . In response to <=0.5 µM, but otherwise cytotoxic concentrations of etoposide, cell cycle arrest, in this case at G2-M, precedes cell death rather than being coincident with it. After 24-h exposure to 0.5 µM etoposide, <20% of G2-M arrested cells are dead, although the entire population will die in the ensuring 48 h (34) . These results are similar to, but not identical with, dose-dependent differences described for etoposide and certain other drugs in different cell types (26 , 27 , 52) . Apoptotic morphology is evident in the etoposide-treated lymphoblastoid cells irrespective of the concentration used. This is not the case in HeLa and some other cell types when failure of feedback control of G2-M transition results in mitotic catastrophic cell death in response to drugs or radiation (25 , 53 , 54) . A possible rationale for such difference in the mode of cell death otherwise associated with G2-M arrest involves consideration of cell type, and specifically the perception that apoptotic cell death is readily evoked in hematopoietic cells (55) . Indeed, most of the observations we have recorded here are compatible with a prompt, rather than a delayed or default, commitment to apoptosis in the lymphoblastoid populations studied.

Using short-term ("pulse") exposure to etoposide, we initially sought to dissociate G2-M arrest from cell death by identifying a treatment time that resulted in G2-M arrest, but no appreciable cell death. We could not achieve this, apparently because of a tight relationship between G2-M arrest and cell death. Preliminary studies using continuous exposure protocols indicated that progressive reduction in drug concentration, although resulting in diminution of G2-M arrest, was paralleled by reduced cytotoxicity. We therefore examined pulse exposure to etoposide using a fixed drug concentration (0.5 µM) that is unequivocally cytotoxic after continuous exposure. Again, G2-M arrest could not be dissociated from cell death. Thus, pulse exposure for 3 h did not cause appreciable G2-M arrest or cell death (as indicated by less-than-G1 DNA content), whereas exposure to etoposide for >=6 h caused both G2-M arrest and cell death in some, but not all cells (Fig. 1)Citation . The G1 fraction evident 48 h after a 6- or 12-h "pulse" exposure to etoposide most likely comprises cells that were able to undergo mitosis having recovered from G2-M arrest. Thus, cells that complete mitosis survive, whereas those subject to sustained arrest die: a principle that has been proposed to explain drug-induced cell death (10) .

Caspase-3 Activation in G2-M-arrested CEM and MOLT-4 Lines.
Schimke et al. (10) reported that death in a number of cell types occurs subsequent to cycle arrest, this outcome being characterized as the "ultimate repair." Applied to the present observations, such understanding implies that apoptosis is "triggered" when cells remain arrested at G2-M for a critical period. Activation of caspase-3 marks irreversible commitment to apoptosis and is therefore described as an "effector" caspase (56) . Using the present experimental system, therefore, examination was made of progressive cycle arrest and commitment to apoptosis. The results of immunoblot analysis initially indicated a difference in time course, with cyclin B1 accumulation being apparent from 3–6 h after the addition of etoposide to the media, whereas loss of Mr 32,000 procaspase-3 was not evident until the 24–48-h period (Fig. 2)Citation . However, two data sets mitigate against the notion of delayed caspase activation. The first set involves caspase substrates. In both CEM and MOLT-4 cells, cleavage of the caspase-3 substrate PARP is evident within 3 h of drug treatment, and at least in the case of MOLT-4 cells, it is lost by 6 h (Fig. 3Citation , top). Lamin B cleavage, most immediately attributable to caspase-6, that is, an effect downstream of caspase-3 activation, is also evident in both cell types within 6 h after the addition of etoposide (Fig. 3Citation , bottom). Measurement of protease activity in vitro also indicates short-term activation (Fig. 4)Citation . Moreover, the progressive increase in activity provides no basis for a two-stage process involving accumulation of cells at G2-M, and then caspase activation occurring at some critical time later. The result was unexpected. We thought that caspase activation would occur after a period of delay, such delay being described in many systems, and perceived as a period during which repair is attempted; failure of repair results in a commitment to cell death (16 , 23, 24, 25) .

Although caspase-3 activation has been comprehensively monitored during drug-induced cell death within 4–6 h (5 , 36 , 47 , 57) , there are few studies on caspase activation associated with G2-M arrest. A report by Martins et al. (22) describes how caspase activation was delayed after a 1-h pulse exposure to 17 µM etoposide, which, in a comparable line not expressing bcr-abl, caused apoptosis in <6 h. Their studies reveal that the consequences delayed caspase activation, which otherwise occurred immediately after G2-M arrest. Granted apparent persistence of procaspase-3 in our studies (Fig. 2)Citation , it is possible that protease activity evident in vitro activity is attributable to other caspases. Otherwise, persistence of the Mr 32,000 procaspase-3 band after etoposide treatment suggests synthesis of procaspase-3 coincident with activation. Accumulation of procaspase-3 has been noted in the 2 h after exposure of HL-60 cells to etoposide (47) , after which the band was gradually lost. Likewise, synthesis of PARP, before its later degradation, has been observed during slow (8–10 days) spontaneous apoptosis in a human osteosarcoma line (58) . These various findings, together with our data, suggest a complex system in which caspase-3 regulation may not be wholly explained in terms of cleavage of procaspase-3 alone.

A Relationship between G2-M Arrest and Caspase Activation.
In their recent report on etoposide-induced caspase activation, Martins et al. (22) note "the poorly understood biochemical events between generation of topoisomerase II-mediated DNA damage and the execution phase of programmed cell death." The relationship between topoisomerase-II-mediated DNA damage and G2-M cell cycle arrest has been extensively studied (59) and, specifically in relation to ionizing radiation, abrogation of G2-M arrest may result in increased cell death (30 , 60) . Lock et al. (28) reported that caffeine potentiates etoposide-induced death of HeLa cells, this being correlated with specific tyrosine dephosphorylation and activation of cdc2 kinase, and hence, mitotic progression from G2. Likewise, the effect of 7-hydroxystaurosporine in mediating cell death in response to drugs (29 , 49) is consistent with this agent’s impact on G2-M arrest. Granted such definition of the relationship between G2-M arrest and death, the experiments described here were focused on a narrower relationship: that between G2-M arrest and caspase activation.

The addition of caffeine together with etoposide abrogates the G2-M arrest otherwise observed in CEM and MOLT-4 cells exposed to the cytotoxic agent (Table 1)Citation . Such treatment with caffeine is found to affect the increase in protease activity evident in response to etoposide (Fig. 4)Citation . At least in the lymphoblastoid cells studied here, it appears that etoposide treatment causes both progressive G2-M cell cycle arrest and progressive caspase activation. The impact of caffeine suggests that these two events may be related. Relevant data generated in other systems have been principally concerned with arrest at G1 (61) . Thus Wang et al. (62) have demonstrated that dysregulation of the cyclin-dependent kinase inhibitor p21 occurs early in 1-ß-D-arabinofuranosylcytosine-induced apoptosis and is associated with activation of the apoptotic protease cascade. In the context of Fas-mediated apoptosis, it is postulated that proteases may cleave cell cycle regulatory proteins (63) . These scant, but disparate observations suggest that cell cycle arrest and caspase activation may progressively affect each other, rather than one event being characterized as the cause of the other.

Caffeine may potentiate the cytotoxic effects of a variety of DNA-damaging agents, such as ionizing radiation, alkylating compounds, and cisplatinum analogues (64) . Caffeine may abrogate etoposide-induced G2-M arrest and increase the incidence of mitotic cell death (28) , although there appear to be caffeine-sensitive and -insensitive pathways operative in G2-M-arrested HeLa cells (65) . Sensitization of irradiation-induced cell killing by caffeine is greater in fibroblasts or carcinoma cells lacking p53 (30 , 66 , 67) . p53 null cells are sensitized to UV in the presence of caffeine, but this does not involve G2-M delay (68) .

As distinct from mitotic cell death observed in HeLa and other populations, hematopoietic cells exhibit G2-M arrest and apoptosis after irradiation or treatment with cytotoxic drugs (33 , 34) . {gamma}-Irradiation induces death of CEM cells by apoptosis after G2-M arrest (69) . In common with our observations (Fig. 1)Citation , G2-M arrest after irradiation appears to be reversible. Caffeine potentiates cytotoxicity in lymphoma cells concomitantly exposed to cisplatin, specifically in the context of affecting G2-M arrest (70) . However, in CEM and MOLT-4 cells, we found that although caffeine abrogated G2-M arrest in response to etoposide, there was no demonstrable effect on cell death. Thus, an impact of caffeine treatment on the cytotoxicity of etoposide, as observed during mitotic catastrophe (28) , was not evident in either line (Fig. 7)Citation . Cell death via mitotic catastrophe is critically dependent on cell type and specifically occurs in HeLa cells. In these populations, enhanced radiosensitivity in the presence of caffeine is attributable to apoptosis occurring ahead of mitotic cell death (71) . In hematopoietic cells on the other hand, caffeine may enhanced drug-induced apoptosis after G2-M arrest (70) , but depending on the hematopoietic cell line and the inducing agent, caffeine may have either a neutral or even a protective effect (72, 73, 74) .

In summary, the conspicuous feature of apoptotic cell death after G2-M arrest in the hematopoietic cells reported here is the prompt occurrence of caspase activation. This early activation is incompatible with the notion of cell death being precipitated by mitotic catastrophe after inadequate repair, as might be anticipated from studies of mitotic cell death in many cell types. A central tenet of the recent review by Brown and Wouters (75) of factors that determine drug-induced cell death is that distinction should be made between nonhematopoietic and hematopoietic cells. The latter are specifically "programmed" to undergo drug-induced apoptosis (76) , and, unlike other cell types, perhaps p53 status is not a prime determinant of responsiveness (69) . Caspase activation in parallel with, rather than following, G2-M arrest may be a manifestation of this susceptibility. Yet even within subtypes of leukemia cells, distinctly different patterns of drug-induced cell death have been recognized with, for example, HL-60 cells being distinguished from MOLT-4 (77) . In pediatric disease, etoposide seems to have made a greater impact in the treatment of acute lymphoblastic leukemia than it has in acute myelogenous leukemia (78) , which may be related to the mode of cell death induced in the respective malignant populations. The identification of specific biochemical criteria for different pathways of drug-induced cell death, of which the pattern of caspase activation may be one, will facilitate the demonstration of these pathways in patient material, and consequently their relevance to successful chemotherapy.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a grant from the National Health and Medical Research Council (Australia). Back

2 To whom requests for reprints should be addressed, at Cancer Control Program, South Eastern Sydney Public Health Unit, Locked Bag 88, Randwick 2031 NSW, Australia. Phone: 61-2-9382-8249; Fax: 61-2-9382-8334; E-mail: Stewartb{at}sesahs.nsw.gov.au Back

3 The abbreviations used are: PARP, poly(ADP-ribose) polymerase; pNA, p-nitroanilide; RT-PCR, reverse transcription-PCR. Back

Received 1/25/00; revised 5/22/00; accepted 5/22/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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