Clin. exp. Immunol. (1992) 87, 465-471

Elevations in cytosolic free Ca2+ are not required to trigger apoptosis in human leukaemia cells S. V. LENNON, S. A. KILFEATHER*, M. B. HALLETTt, A. K. CAMPBELLt & T. G. COTTER Immunology Unit, Department of Biology, St Patrick's College, Maynooth, Co. Kildare, Republic of Ireland, *Department of Pharmacology, Royal College of Surgeons in Ireland, Dublin, Republic of Ireland, tDepartment of Surgery $Medical Biochemistry, University of Wales College of Medicine, Cardiff, UK

and

(Acceptedfor publication 6 November 1991)

SUMMARY Previous studies have indicated that Ca2+ is a trigger for apoptosis (programmed cell death) in thymocytes and related cell lines. Recently we have shown that levels of apoptosis in leukaemic cells are diminished in Ca2+-deficient conditions, indicating that Ca2+ may be important in the mechanism of apoptosis in these cells. In the present study we investigated the possibility that Ca2+ serves as a trigger for apoptosis in the human leukaemic cell line, HL-60. Using fura-2 to measure cytosolic free Ca2+ concentrations, [Ca2+], in cell suspensions, and by using ratio imaging of fura-2 in single cells, we did not observe an early significant increase in [Ca2+]i in HL-60 cells undergoing apoptosis. The latter stages of apoptosis were, however, accompanied by increasing [Ca2+]1; these increases were apparently a result of, rather than a cause of, apoptosis. Furthermore, apoptosis could be induced in HL-60 cells under conditions of vastly reduced [Ca2+]i achieved by loading these cells with fura-2 in the presence of EGTA. These results indicate that elevation of [Ca2+]j is not a prerequisite for apoptosis in HL-60 cells and that apoptosis can occur in these cells in the presence of low [Ca2+].

Keywords apoptosis programmed cell death calcium fura-2

INTRODUCTION Apoptosis or programmed cell death is the mode of cell death observed under physiological or mildly pathological conditions [1]. Apoptosis takes the form of a series of well-ordered stages [1,2], beginning with plasma membrane blebbing, and ending with the packaging of the cell's contents into vesicles termed apoptotic bodies which are subsequently phagocytosed by neighbouring cells in vivo [3,4] or undergo secondary necrosis in vitro [5]. The hallmarks of this important death process include nuclear fragmentation and endonuclease activation leading to fragmentation of the cell's nuclear DNA into oligonucleosomelength fragments [6]; processes which occur while membrane integrity is maintained. Examples of this important death process include death of tumour cells killed by cytotoxic T cells [7], receptor-mediated death in T cell hybridomas [8] and death of autoreactive thymocytes [9]. Possible roles of Ca2+ in apoptosis have been well documented. In thymocytes, a sustained increase in [Ca2+] occurs Correspondence: Dr T. G. Cotter, Immunology Unit, St Patrick's College, Maynooth, Co. Kildare, Republic of Ireland.

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immediately after addition of several apoptosis-inducing stimuli. Inhibition of this Ca2+ rise blocks DNA fragmentation and subsequent cell death [10-12]. Thus, there is considerable evidence for a role of Ca2+ in initiation of apoptosis in these cells. The importance of Ca2+ in apoptosis in other cell lines has also been demonstrated. For example, receptor-mediated apoptosis in T cell hybridomas [8] and pore-former-induced apoptosis in tumour target cells [13] are also dependent on extracellular Ca2 . Also, rat liver nuclei contain a Ca 2+dependent endonuclease which, when activated results in DNA fragmentation typical of apoptosis [14]. Recently we have shown that apoptosis is induced in a range of leukaemic cell lines by many different stimuli, when applied at experimentally determined low levels [15]. The levels of apoptosis were greatly diminished in Ca2+-deficient medium [15,16] and we have also demonstrated the presence of a Ca2+_ dependent, zinc-inhibitable endonuclease in HL-60 cells capable of inducing DNA fragmentation typical of apoptosis [17]. Furthermore HL-60 and other cell lines underwent apoptosis when cultured in zinc-deficient medium [18], and UV-induced apoptosis in HL-60 cells is zinc-inhibitable [16]. These findings

S. V. Lennon et al.

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suggest that activation of a Ca2+-dependent/zinc-inhibitable endonuclease present in these cells may be the trigger for apoptosis in HL-60 cells. In the present study, we investigated the possibility that increasing [Ca2+]i serves as a triggering signal for apoptosis in HL-60 cells. This was achieved using the fluorescent calcium indicator, fura-2, to observe changes in [Ca2+]i both in cell suspensions and in single cells. Surprisingly, we found that cells undergo blebbing and DNA fragmentation with no apparent early increase in [Ca2+],. Increases in [Ca2+], only occurred after DNA fragmentation and before secondary necrosis of these cells, i.e. in the final stages of apoptosis. Furthermore, apoptosis could be induced in cells following clamping of [Ca2+]- and removal of extracellular Ca2+. These results suggest that Ca2+ is not a trigger for apoptosis in HL-60 cells, and that apoptosis can proceed in these cells even in the presence of very low [Ca2+]. MATERIALS AND METHODS

Materials RPMI 1640 medium was obtained from GIBCO (Hertfordshire, U.K.). Fetal calf serum (FCS) was obtained from Flow Laboratories (Middlesex, UK). Hydrogen peroxide (H202), and phenol were obtained from BDH Chemicals (Poole, UK). Proteinase K was obtained from Boehringer Mannheim (Germany). Ethanol was obtained from Rathburn Chemicals (UK). Fura-2-AM was obtained from Sigma (Poole, UK) and from Molecular Probes (OR, USA). Calcium ionophore (A23187), Percoll and all other chemicals (except where otherwise stated) were obtained from Sigma. Cells and culture conditions HL-60, a promyelocytic leukaemia line [19], was cultured in RPMI 1640 medium supplemented with 10%/0 FCS. Cells were maintained at 37 C in a humidified 5% CO2 atmosphere and were passaged three times weekly. Exponentially growing cells were seeded atS x 1 05/ml for all experiments. When loading cells with fura-2-AM, either HBSS or Krebs medium was used. HBSS consisted of: 140 mm NaCl, 5 mm KCl, 0-3 mm Na2HPO4, 0 4 mM KH2PO4, I mM CaCl, 0 5 mM MgCI2, 05 mM MgSO4, 6 mm glucose and 25 mm HEPES, pH 7-2. Krebs medium consisted of 120 mm NaCl, 4-8 mm KCI, 1-2 mm KH2PO4, 1-2mM MgSO4, 1-3 mm CaCl2 and 25 mm HEPES, pH 7-2. For Ca2+deficient conditions, Ca2+ was replaced with 1 mm EGTA (ethyleneglycol-bis-[,B-amino-ethyl ether] NN'-tetra-acetic acid) in the above buffers.

Induction of apoptosis Apoptosis was induced by exposing exponentially growing cultures to either 100 pM H202, 5 5% ethanol or by UV irradiation. UV irradiation was carried out by exposing cells in polystyrene culture flasks (Nunc) from below to a 302 nm UV transilluminator source at a distance of 1 5 cm for 10 min and irradiated cells were subsequently incubated under standard culture conditions. Cell viability and morphology Cell viability was determined using the trypan blue exclusion test. Cell morphology was evaluated on Rapi Diff II stained cytocentrifuge samples. Apoptotic cells were identified by the

condensed and fragmented state of their nucleus [1,20]. Further verification of apoptosis in cultures was achieved by electrophoresis of DNA from cell cultures containing a high level of apoptosis or from purified populations of apoptotic cells, showing cleavage of the DNA into nucleosome size fragments. Apoptotic cells maintained the ability to exclude vital dyes as previously described [5,15,21].

Cytosolic Ca2+ measurements

Cell suspensions. Cells were exposed to various apoptoticinducing stimuli (as described above). At various time intervals, 5 x 106 cells were taken from culture, washed once in culture medium, resuspended in1 ml of culture medium containing 2-5

and incubated for 20-30 min at 37 C in the dark. yMThefura-2-AM, cells were then washed three times in HBSS. Cells were then

106/ml

in this buffer and transferred to a resuspended at 2 x cuvette in a Perkin-Elmer (IOOOM) Fluorimeter with the cuvette holder maintained at 37 C. Cell viability in the cuvette was always >90%. Excitation and emission wavelengths were 336 nm and 536 nm respectively. values were calculated as Briefly, following recording of the previously described fluorescence corresponding to basal [Ca2+]j, Triton X-100 (final concentration 0-1% (v/v)) was added to lyse the cells and determine maximum fluorescence on release of dye into Ca2+ >1 mm. Minimum fluorescence was achieved by addition of EGTA (final concentration 10 mM) in 4M Tris. [Ca2+], was also measured in control cells for each time point. Changes in the autofluorescence of unloaded cells caused by the various additions were taken into account in the calculation of [Ca2 ], was determined using a value of 224 nm as the effective Kd

[Ca2+]j

[22].

[Ca2+],.

+ of the probe [22].

The ability of HL-60 cells to take up and hydrolyse Fura-2AM to fura-2 free acid was determined by manganese sensitivity of fluorescence observed after lysing fura-2-AM loaded cells. No compartmentalization of fura-2 was demonstrable, using digitonin to selectively permeabilize the plasma membrane [23]. Measurement of [Ca2+]I in single cells. Cells were loaded with 2-5-5 yM Fura-2-AM for 20-30 min at 37°C in the dark. The cells were then washed twice in Krebs medium, and resuspended in this buffer. The cells were then allowed to adhere to 0-1 mg/ml coverslips for 15 min and placed onto a poly-L-lysine-treated chamber and thermostatically controlled microperfusion 380 stage heater [25]. Fluorescence was excited at 350 nm and dual nm by coupling the output from a Spex Fluorolog wavelength fluorometer (Glen Spectra, UK) to a Zeiss photomicroscope III fitted with an Omega Optical dichroic mirror with 50% transmission at approximately 400 nm (Glen Spectra, UK). The emitted images were detected using an ISIS intensified CCD camera (Photonic Science, Tunbridge, UK) and ratio The images acquired using a Spex IM201 analysis system. accurate and subtractions set for were offsets background determination of 350/380 nm ratios. To minimize exposure to 340 nm light, images were acquired at 15 min intervals for throughout the experiments, the cells being unilluminated the remainder of the time. The intensity of the fura-2 signals was sufficiently high compared to the autofluorescence signal for calculation of ratio images to be uncontaminated by autofluorescence artefact. The Ca2+ concentration was calculated as described by Grynkiewicz et al. [22] from knowledge of the maximum and minimum ratio values, using a Kd of 224 nM. Whilst acquiring ratio images of the field of cells, either 100 gM

[24]

Apoptosis in human leukaemia cells

467

H202 or 5-5 % ethanol (both in Krebs medium containing 0-5 ,ug/ ml acridine orange) was added and the subsequent ratio images were recorded for analysis. Nuclear staining by acridine orange fluorescence was observed by excitation at 475 nm and nuclear images were recorded immediately after addition of stimulus and at various timepoints thereafter.

Table 1. Asynchronous increases in [Ca2+]i and apoptosis in HL-60 cells following exposure to ethanol or H202

Purification of apoptotic cells Apoptotic cells were isolated from normal cells as previously described [17]. Briefly, cells were washed twice in 0 01 M phosphate-buffered saline, pH 7 2 and resuspended in I ml of 1 06 g/ml Percoll solution. The cells were then layered onto a discontinuous Percoll gradient consisting of sequentiallylayered Percoll solutions with densities of 108 g/ml (I ml), 1075 g/ml (2 ml) and 107 g/ml (2 ml). Cells were then centrifuged in a swing-out rotor at 400 g for 30 min. The pellet thus formed contained a purified population of apoptotic cells, which were washed free of Percoll with PBS and resuspended in this buffer.

3 6

DNA isolation Cells, 106, were resuspended in 50 p1 of lysis buffer (10 mM EDTA, 50 mm Tris, pH 8-0, 0-5% sodium lauryl sarcosine, 0-5 mg/ml proteinase K) and incubated at 50 C for I h. RNAse A was then added to a final concentration of 0 5 mg/ml and incubation was continued at 50'C for 1 h. Samples were then extracted twice with equal volumes of phenol followed by two extractions with chloroform: isoamylalcohol (24:1). Samples were then pelleted at 13 000 g for 15 min in order to separate intact from fragmented chromatin. The supernatants were placed in separate tubes and precipitated overnight in two volumes of ice-cold ethanol at -70°C.

Electrophoresis of DNA Supernatant DNA was spun-down at 13 000 g for 15 min, airdried at room temperature for 5 min, and resuspended in 50 pi of TE buffer (0 01 M Tris, pH 8-0 containing 1 mm EDTA). Loading buffer (10 mm EDTA, 0-25% bromophenol blue, 50% glycerol) was then added at a 1:5 ratio. Samples were then heated to 65°C for 10 min in a water-bath and were then plunged into ice. Samples were then loaded onto a 1% agarose gel, and electrophoresis was carried out at 6 V per cm of gel in TBE buffer (2 mm EDTA, pH 8 0, 89 mm Tris, 89 mM boric acid). Molecular weight markers of 23 5, 9 6, 6-6, 4 3, 2-2, 22-1 and 0 5 kbp respectively were provided by an Hind III digest of A-DNA. After electrophoresis, DNA was visualized by soaking the gel in TBE containing 1 pg/ml ethidium bromide.

RESULTS Changes in [Ca 2+ in HL-60 cell cultures undergoing apoptosis Apoptosis was initiated in HL-60 cells by three different stimuli as described in Materials and Methods. At certain time intervals after addition of stimulus, cells were removed from culture, loaded with the acetoxymethyl ester of fura-2, and [Ca2+], measured (as described in Materials and Methods). [Ca2+], of control cells was also determined at each time point. Simultaneously, the percentage of apoptotic cells in both treated and control cultures was monitored on Rapi Diff II stained cytocentrifuge preparations. ,

% Apoptosis

[Ca2+], (nM)

Time

(hours) n Control Treated 5 5

15 2

17 50

P

500 cells) ± s.e.m. (b). Morphological features of apoptosis in HL-60 cells. Indicates nuclear fragmentation typical of apoptosis in cells treated with UV irradiation for 10 min and subsequently cultured under standard conditions for 3 h. 'N' indicates normal cells. (c). Electrophoresis of supernatant DNA extracted from purified populations of apoptotic cells. This indicates DNA fragmentation typical of apoptosis in HL-60 cells exposed to 10 min UV irradiation (lane 1), 100 pM H202 (lane 2), 5.50,, ethanol (lane 3). Lane 4 shows that no DNA fragmentation is associated with control cells. Lane 5 is a Hind III digest of .-DNA providing molecular size markers of 23-5, 9-6, 6t6, 4-3, 2 2, 2-1, and 0-5 kbp. 0

process. However, we have recently shown that A23187 can induce significant apoptosis in HL-60 cells cultured in Ca>+deficient medium [15]. It is apparent therefore that A23187 can also induce apoptosis by a mechanism independent of Ca>+ influx.

Imaging of Ca>+ in individual cells Due to the possibility of transient and asynchronous increases in [Ca2+], in individual cells undergoing apoptosis, effects which may have been undetectable in cell populations, the [Ca2+], distribution in individual cells was determined by ratio imaging of fura-2 signals.

Whilst ratio images were being acquired of a field of HL-60 cells (Fig. 3b), H202 was added at a concentration of 100 pM and subsequent ratio images of the cells observed. No significant changes in [Ca2+], occurred in HL-60 cells immediately after addition of stimulus, or during the formation of blebs on the cell surface (which occurred approximately 20-30 min after addition of stimulus). Simultaneous determinations of nuclear morphology were made using acridine orange fluorescence. Images recorded immediately after addition of the stimulus showed for the majority of cells a high intensity fluorescence from most of the cell body, indicative of a high nuclear: cytoplasmic size ratio

469

Apoptosis in human leukaemia cells 700 (a )

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2 600 r_

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.,, 500 u

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had previously been UV-irradiated. Again, similar results were observed as before, namely that nuclear condensation and fragmentation can proceed without any increase in [Ca>]. Hence, 3 h post UV-irradiation, when cytospin preparations of these cells showed that > 90% were undergoing apoptosis, the average [Ca2+], measured in individual cells was 40+8 nM (n= 10). However, towards the latter stages of apoptosis, namely 5 h post UV irradiation, when cells would be progressing towards secondary necrosis, [Ca2+], in cells was observed to increase (data not shown). This supports the data recorded for [Ca2+]i determined during apoptosis in cell suspensions, namely, that an increase in [Ca2+], could be a result, rather than a cause of, apoptosis.

*0 A23187 (nM)

a

Depletion of [Ca2 ]+ [Ca2] was depleted by loading the cells with fura-2 in the absence of extracellular Ca2+. This would be expected to severely deplete cells of their [Ca2+]j. This was verified by quantifying the [Ca2] in individual HL-60 cells loaded with fura-2 in this way. Under these conditions, HL-60 cells typically had [Ca2+]- of < 24 nm. However, such cells were still capable of undergoing apoptosis (Fig. 4).

a)

0

DISCUSSION

CL 0 CL

Cl

6 12 9 Time (h) Fig. 2. Dose response of HL-60 [Ca>+]i to the calcium ionophore A23 187 and induction of apoptosis by this agent. (a) Cells were treated with various concentrations of A23187 and their [Ca2+]i was determined approximately 2 min later. Results are expressed as triplicate determinations from a representative experiment (mean of triplicate + s.e.m.). (b) Time course of appearance of apoptotic cells in HL-60 cultures treated with various concentrations of ionophore (M). Results from a representative experiment are shown (mean of triplicate + s.e.m.). 0

3

typical of HL-60 cells (Fig. 3b). Images recorded at various timepoints after addition of stimulus indicated a progressive weakening of the intensity of acridine orange fluorescence, and reduction of the area of fluorescence indicative of nuclear condensation and fragmentation (Fig. 3d). Similar changes in acridine orange fluorescence were also observed in cells treated with ethanol or UV-irradiation. In control cells, no changes were observed in acridine orange fluorescence over the time scale of the experiment (data not shown). No significant changes in [Ca2+], was observed in HL-60 cells up to and including the time when nuclear condensation and fragmentation were apparent from acridine orange fluorescence (Fig. 3c). Hence, the HL-60 cells under investigation were able to undergo the main characteristics of apoptosis, namely nuclear condensation and fragmentation, without any significant increases in [Ca2+],. Similar results were also observed for ethanol-induced apoptosis (data not shown). Using the same experimental system as described above, ratio images of fura-2 fluorescence were obtained for cells which

The concept of Ca2+ playing a pivotal role in apoptotic cell death was provided mainly from work conducted on thymocytes [10-12] and related cell lines [8] where a sustained Ca2+ influx occurs following induction of apoptosis by different stimuli. Inhibition of Ca2+ elevation in these cells blocks DNA fragmentation and cell death. Ca>2+ influx may be enhanced by a cytosolic factor which is newly synthesized after treatment of these cells with an apoptosis-inducing stimulus [10,11]. Hence, by blocking protein or RNA synthesis, the synthesis of this factor and the increase in [Ca2+]i and subsequent cell death are blocked. There is evidence, however, that Ca2+ increases are not a universal requirement for apoptosis in all cells. A recent report has indicated that apoptosis induced by glucocorticoid and novobiocin in human CEM-C7 cells involves activation of a constitutive Ca2 + -independent endonuclease [26]. However, other work, using 45Ca2+ to measure Ca2+ uptake in these cells undergoing glucocorticoid-induced apoptosis could not entirely rule out the involvement of Ca2+ in the initiation of apoptosis or release of Ca2+ from internal stores during apoptosis [27]. In the present investigation, we have shown that apoptosis can be initiated in HL-60 cells without any increase in [Ca2+],. Furthermore, apoptosis could still be induced in the HL-60 cells in conditions of exceedingly low [Ca2+] (< 24 nM) achieved by using fura-2 to buffer [Ca2+]i and EGTA to deplete the extracellular Ca>. In cell suspensions, a gradual increase in [Ca2 + ], was observed during the development of apoptosis in HL-60 cells. However, A23 187 at a concentration which was found to mimic this increase in [Ca2+]- did not induce apoptosis in these cells, indicating that this Ca2+ increase alone was not a signal for initiation of apoptosis, but possibly a secondary event. Determination of [Ca2+], in individual cells by using ratio measurements of fura-2 indicated that an increase in [Ca2+], occurred only after nuclear fragmentation had occurred. This latter increase in [Ca>]2 is probably indicative of a progressive breakdown in

470

S. V. Lennon et al.

tt

*so

Fig. 3. Imaging of [Ca2+]i and nuclear shape in individual HL-60 cells before and after exposure to an apoptotic-inducing stimulus. Certain cells are nuflbered (1-3) in each plate to facilitate interpretation. (a) [Ca2+ ] in untreated HL-60 cells. (b) Nuclear shape of HL-60 cells recorded immediately after addition of stimulus. Indicates normal cells, with a high nuclear: cytoplasmic ratio. (c) [Ca2 + ]i in HL-60 cells 3 h after addition of 100 gM H202. Note no significant change in [Ca2+]j relative to (a). (d) Nuclear shape in HL-60 cells 3 h after addition of H202. Note a reduction in intensity ofacridine orange fluorescence and a decrease in the area offluorescence, indicative of nuclear condensation and fragmentation. Calcium colours: Black < 24 nm [Ca 2+11. Purple 24-74 nm [Ca2+],. =

=

2 kbp -

23-5

-96

66 -4.3 -

22

-21

O

Fig. 4. DNA fragmentation in cells with low [Ca2+],. Indicates DNA fragmentation typical of apoptosis in HL-60 cells which were loaded with fura-2 in the absence of extracellular Ca2 exposed to u.v. irradiation for 10 min and then cultured under normal conditions for 3 h (lane 1). Lane 2 is a HindIII digest of A-DNA providing molecular size markers of 23-5, 9 6, 6 6, 4-3, 2-2, 21 and 05 kbp. ,

,

Ca2+ homeostasis prior to secondary necrosis in these cells. Due to the heterogeneity of the apoptotic response, cells advance towards secondary necrosis at different rates and an overall increase in [Ca2+]i within the cell population develops over time as cells accumulate at the final stages of apoptosis. This could account for the slow increase in cell suspension [Ca2+]j observed during apoptosis. It is apparent, therefore, that any increase in [Ca2+]i in HL-60 cells undergoing apoptosis is a consequence rather than a cause of apoptosis. Previously we have identified an endonuclease in HL-60 cell nuclei which could be activated with millimolar concentrations of Ca2+ to effect DNA fragmentation of the type observed during apoptosis of these cells [17]. Similar endonucleases have been identified in thymocytes [28] and in a T cell clone [29], and activation of these endonucleases is thought to be a primary step in the process of apoptosis. In the present investigation, HL-60 cells which had been loaded with fura-2 in the absence of extracellular Ca2+ typically had [Ca2+], of

Elevations in cytosolic free Ca2+ are not required to trigger apoptosis in human leukaemia cells.

Previous studies have indicated that Ca2+ is a trigger for apoptosis (programmed cell death) in thymocytes and related cell lines. Recently we have sh...
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