Introduction

Didemnin B is a branched cyclic peptolide with anti-tumour, antiviral, and immunosuppressive effects.^{1, 2} Didemnin B has been shown to rapidly induce apoptosis in transformed human cells, particularly the human promyeloid HL-60 cells.³ It has been shown that this induction of apoptosis requires signalling through protein tyrosine phosphorylation and caspase activation.^{4, 5}

Rapamycin is a macrocyclic bacterial toxin, which is known for its ability to inhibit ribosomal translation activated through mitogenic stimulation. The mechanism by which this interference occurs has been reviewed.⁶ The major intracellular receptor for rapamycin is FK506-binding protein (FKBP)12, although rapamycin also binds to other members of the FKBP family.^{7, 8} In human cells, the FKBP12– rapamycin complex binds to mammalian target of rapamycin (mTOR), also known as rapamycin and FKBP12 target (RAFT)1⁹ and FKBP–rapamycin-associated protein (FRAP).¹⁰ There is evidence that rapamycin inhibits translation through formation of this complex.¹⁰ Rapamycin inhibits growth signalling through inhibition of p70 S6 kinase activation.¹¹ This inhibition of p70 S6 kinase requires binding of mTOR and rapamycin–FKBP12.¹²

In the present paper, we show that rapamycin inhibits the induction of apoptosis by didemnin B in HL-60 cells and investigate possible mechanisms of inhibition, in order to gain further insights into the mechanism of didemnin B-induced apoptosis.

Materials and methods

Materials

Didemnin B was kindly provided by the National Cancer Institute, Bethesda, MD, USA. Rapamycin and FK506 were gifts from Dr René Traber, Novartis Pharma Ltd, Basel, Switzerland. Roscovitine and olomoucine were kindly supplied by Dr Laurent Meijer from CNRS, Station Biologique, Roscoff cedex, France.

Cell culture

Human promyeloid HL-60 cells were maintained in logarithmic growth phase in RPMI-1640 medium (Life Technologies, Melbourne, Australia) supplemented with 10% (v/v) heat-inactivated foetal calf serum, 100 U/mL penicillin, 100 g/mL streptomycin, 1 mmol/L sodium pyruvate, 2 mmol/L L-glutamine and 5 mmol/L HEPES (pH 7.4) at 37°C in the presence of 5% CO₂ in a humidified incubator.

Determination of apoptotic morphology

The level of apoptosis in a cell culture (2–4 10⁵ cells/mL) at any given time was determined by centrifuging 100–200 L cells onto glass slides at 4000 g for 5 min using a cytospin (Shandon, Astmoor, England). Glass slides were precoated with 0.01% (w/v) poly-L-lysine. Cells were fixed in ethanol/acetic acid (3:1) for 5 min, then washed in dH₂O for 1 min and air-dried. Membrane blebbing could be observed in apoptotic cells using light microscopy with a Nikon microphot-FX fluorescence microscope. Alternatively, nuclei were stained with 1 g/mL 4´,6-diamidino-2-phenylindole.2HCl (DAPI; Sigma, Sydney, Australia), a DNA-binding fluorescent dye,¹³ for 5 min and the slides rinsed in dH₂O for 1 min and air-dried before mounting with anti-fade mounting medium (10 mg/mL p-phenylenediamine in 90% (v/v) glycerol, pH 9.0).¹⁴ Nuclear morphology was analysed under UV light (280 nm) using a Nikon microphot-FX fluorescence microscope. At least 400 cells were counted per slide to determine percentage apoptosis. Nuclei that were condensed were smaller and more brightly stained than normal nuclei. Those nuclei that were broken into small, spherical fragments or were condensed were counted as apoptotic.

Preparation of cytoplasts

Cytoplasts were prepared from HL-60 cells as described previously.¹⁵ A 12 mL Beckman ultracentrifuge tube was exposed to UV light overnight before adding 2 mL 25% (w/w) Ficoll, 2 mL 17% Ficoll, 0.5 mL 16% Ficoll, 0.5 mL 15% Ficoll and 2 mL 12.5% Ficoll in medium containing 10 g/mL cytochalasin B (Sigma). The gradient was equilibrated at 37°C in 5% CO₂ before use. HL-60 cells were adjusted to 2 10⁷ cells/mL in medium containing 12.5% (w/w) Ficoll and 10 g/mL cytochalasin B. Cells (3 mL) were then layered on top of the previously prepared discontinuous Ficoll gradient. An additional 2 mL medium containing 10 g/mL cytochalasin B was added on top of the cells and cells were centrifuged for 1 h at 80 000 g in a prewarmed SW41 rotor. The rotor was prewarmed by centrifugation for 3.5 h at 80 000 g in a Beckman L5-65 ultracentrifuge.

Cells were recovered from each interface using a plastic transfer pipette and 10 mL medium (prewarmed to 37°C) was added, to wash. Cells were centrifuged for 5 min at 200 g followed by a second wash in 10 mL medium (prewarmed to 37°C). After centrifugation for 5 min at 200 g, cells were resuspended in 5 mL medium and aliquots were centrifuged onto glass slides at 4000 g for 5 min using a cytospin (Shandon) and stained with 1 g/mL DAPI as described above in order to check for enucleation. Cells recovered from the 12.5% interface were found to contain greater than 99% enucleated cells (cytoplasts). Cytoplast concentration was determined by trypan blue exclusion, and cytoplasts were then diluted to 4 105 cytoplasts/mL. Didemnin B and staurosporine (Sigma) were added 1–2 h after recovery.

Results

Inhibition of didemnin B-induced apoptosis by rapamycin

To determine the effect of rapamycin on didemnin B-induced apoptosis, HL-60 cells were pretreated with 0, 1 or 10 mol/L rapamycin for 1 h before treatment with 1 mol/L didemnin B. Apoptotic morphology of HL-60 cells was determined by staining with DAPI, as described in Materials and Methods. The percentage apoptosis induced by didemnin B after pretreatment with rapamycin is plotted versus time, where 0 h indicates the initiation of didemnin B treatment (Figure 1). Rapamycin is shown to inhibit the induction of apoptosis by didemnin B in a concentration-dependent manner.

Figure 1.

Inhibition of didemnin B-induced apoptosis by rapamycin in HL-60 cells. The percentage apoptosis induced by 1 mol/L didemnin B after 1 h pretreatment with rapamycin is plotted versus time, where 0 h is the time of didemnin B addition. The concentrations of rapamycin used were 0 (, ), 1 (, ), and 10 mol/L (, ). Open symbols represent controls and closed symbols represent didemnin B-treated samples. Data are the mean SD of three experiments.

Full figure and legend (8K)

Non-requirement for FKBP12–rapamycin complex in inhibition of didemnin B-induced apoptosis by rapamycin

To determine whether rapamycin inhibits apoptosis through its ability to bind FKBP12 and subsequently inhibits p70 S6 kinase activation, the effects of competition for binding of FKBP12 with FK506 were investigated using a protocol previously established in our laboratory.¹⁶ HL-60 cells were pretreated with 10 mol/L FK506 for 1 h before treatment with 1 mol/L rapamycin for 15 min prior to the addition of 1 mol/L didemnin B. The percentage apoptosis induced by didemnin B in the presence of rapamycin, FK506 or rapa-mycin and FK506 is plotted versus time of treatment with 1 mol/L didemnin B (Figure 2). While FK506 alone has a slightly inhibitory effect on the induction of apoptosis by didemnin B, the pretreatment with FK506 does not inhibit rapamycin- mediated inhibition of didemnin B-induced apoptosis in HL-60 cells. Therefore, an excess of FK506 does not prevent the effect of rapamycin by inhibiting its binding to an FKBP, suggesting that an FKBP12–rapamycin complex is not involved in the inhibition of didemnin B-induced apoptosis by rapamycin.

Figure 2.

Inhibition of didemnin B-induced apoptosis by rapamycin in the presence of FK506 in HL-60 cells. The percentage apoptosis induced by 1 mol/L didemnin B after 1 h 15 min pretreatment with 10 mol/L FK506 (), 15 min pretreatment with 1 mol/L rapamycin () or 1 h pretreatment with 10 mol/L FK506 followed by 15 min pretreatment with 1 mol/L rapamycin () is plotted versus time, where 0 h is the time of didemnin B addition. Control cells () were untreated or treated with 1 mol/L didemnin B alone (). The data are the mean SD of two experiments.

Full figure and legend (7K)

Effects of wortmannin and PD098059 on didemnin B-induced apoptosis

Because the lack of effect of FK506 on rapamycin-mediated inhibition of didemnin B-induced apoptosis suggests that rapamycin does not act through inhibition of p70 S6 kinase activation in this instance, possible involvement of the mitogen-activated protein (MAP) kinase pathway was further investigated using specific inhibitors. HL-60 cells were pretreated, before didemnin B treatment, for 1 h with different concentrations of wortmannin (Sigma), a phosphatidylinositol 3-kinase inhibitor,¹⁷ or PD098059 (2´-amino-3´-methoxyflavone; Calbiochem, San Diego, CA, USA), a MAP kinase kinase (MEK) inhibitor that prevents the activation of MAP kinase.¹⁸ The percentage apoptosis induced by 3 h treatment with 1 mol/L didemnin B, as determined by DAPI staining, is plotted versus the concentration of wortmannin or PD098059 (Figure 3). Wortmannin and PD098059 do not inhibit the induction of apoptosis by didemnin B, indicating that phosphatidylinositol 3-kinase and MEK are not involved in didemnin B-induced apoptosis.

Figure 3.

Effects of wortmannin and PD098059 on didemnin B-induced apoptosis in HL-60 cells. HL-60 cells were pretreated for 1 h with different concentrations of (a) wortmannin or (b) PD098059, followed by a 3 h treatment with 1 mol/L didemnin B () or no further treatment (). The data are the mean SD of two experiments.

Full figure and legend (10K)

Effects of cyclin-dependent kinase inhibitors on didemnin B-induced apoptosis

Rapamycin is known to inhibit cyclin-dependent kinase activation, although whether this is through inhibition of p70 S6 kinase activation or not is still uncertain.^{19, 20} We, therefore, investigated the possible role of cyclin-dependent kinases in didemnin B-induced apoptosis. The cyclin-dependent kinase inhibitors roscovitine and olomoucine^{21, 22} were used to determine whether the inhibition of didemnin B-induced apoptosis by rapamycin may occur through inhibition of cyclin-dependent kinase activity.

HL-60 cells were pretreated with different concentrations of roscovitine or olomoucine for 1 h before didemnin B treatment and determination of apoptosis by DAPI staining, as described in Materials and Methods. Percentage apoptosis after 3 h with 1 mol/L didemnin B is plotted versus concentration of roscovitine or olomoucine (Figure 4). Roscovitine and olomoucine both induce apoptosis in HL-60 cells in a concentration-dependent manner; however, they do not inhibit the induction of apoptosis by didemnin B. Due to its variable effect on these cells, a concentration of 1 mmol/L olomoucine is too high to draw conclusions from its effect (Figure 4b). These results suggest that rapamycin does not inhibit didemnin B-induced apoptosis through inhibition of cyclin-dependent kinases.

Figure 4.

Apoptosis induced by didemnin B in HL-60 cells in the presence of cyclin-dependent kinase inhibitors. HL-60 cells were pretreated for 1 h with different concentrations of (a) roscovitine or (b) olomoucine followed by a further 3 h treatment with 1 mol/L didemnin B () or no further treatment (). The data are the mean SD of two (a) or four (b) experiments.

Full figure and legend (13K)

Effect of didemnin B on apoptosis in cytoplasts

Because the effect of rapamycin on didemnin B-induced apoptosis cannot be accounted for by its known biochemical functions and its effect is not lessened by pretreatment with an excess of FK506, the possible involvement of FKBP25 was investigated. Because FKBP25 has a higher binding affinity for rapamycin than FK506,²³ it is the most likely FKBP yet identified that may be involved in inhibition of didemnin B-induced apoptosis by rapamycin.

The cellular localization of FKBP25 has been described to be nuclear,^{24, 25} so we prepared enucleated HL-60 cells (cytoplasts) to analyse whether didemnin B depends on nuclei to induce apoptosis. Cytoplasts were prepared from HL-60 cells, as described in Materials and Methods, and incubated with 1 mol/L didemnin B for 3 h or 5 mol/L staurosporine for 4 h. The appearance of apoptotic morphology was determined using phase-contrast microscopy as described in Materials and Methods. The percentage apoptosis (as determined by membrane blebbing) induced by didemnin B and staurosporine in HL-60 cells and cytoplasts is compared in Figure 5. The morphology of HL-60 cells and cytoplasts after treatment with 1 mol/L didemnin B for 3 h or 5 mol/L staurosporine for 4 h is shown in Figure 6. Didemnin B does not induce apoptotic morphology in HL-60 cytoplasts, although staurosporine does, suggesting that a nucleus is required for didemnin B-induced apoptosis, unlike some other mechanisms of induction of apoptosis.

Figure 5.

Effects of didemnin B and staurosporine on apoptosis in cytoplasts. Cytoplasts were prepared from HL-60 cells as described previously and HL-60 cells were treated with 10 g/mL cytochalasin B for the equivalent time as a control. Cytoplasts () and control cells () were untreated (sample 1) or treated with 1 mol/L didemnin B for 3 h (sample 2) or 5 mol/L staurosporine for 4 h (sample 3). Percentage apoptosis was determined by examining cell morphology as described previously. The data are the mean SD of two experiments.

Full figure and legend (7K)

Figure 6.

Effects of didemnin B and staurosporine on the morphology of HL-60 cells and cytoplasts. Cytoplasts were prepared from HL-60 cells as described previously and HL-60 cells were treated with 10 g/mL cytochalasin B for the equivalent time as a control. Control cells (a–c) and cytoplasts (d–f) were treated with 1 mol/L didemnin B for 3 h (b,e), 5 mol/L staurosporine for 4 h (c,f) or untreated (a,d). Cellular morphology was viewed under a light microscope as described previously.

Full figure and legend (330K)

Discussion

The inhibition of didemnin B-induced apoptosis by rapamycin suggests at a first glance the possible involvement of the MAP kinase pathway in didemnin B-induced apoptosis; however, further investigation has shown that this involvement is unlikely. Because FK506 does not compete with the effects of rapamycin in didemnin B-induced apoptosis, it is unlikely that p70 S6 kinase activation is involved, because an excess of FK506 would be expected to prevent rapamycin-mediated inhibition. We have previously shown in the laboratory that 10 mol/L rapamycin can almost completely compete for binding to FKBP12 with 1 mol/L FK506 in various cell lines.¹⁶ Our results, therefore, strongly suggest a mechanism of action other than binding to FKBP12 for rapamycin in didemnin B-induced apoptosis.

Activation of phosphatidylinositol 3-kinase can lead to activation of p70 S6 kinase.^{26, 27} Like rapamycin, wortmannin can inhibit the activation of p70 S6 kinase.²⁸ It has also been shown that p70 S6 kinase can be activated by a wortmannin-insensitive pathway involving Raf and MEK, which can be inhibited by PD098059.²⁹ The lack of inhibition of didemnin B-induced apoptosis by wortmannin and PD098059 further supports the conclusion that rapamycin does not inhibit didemnin B-induced apoptosis through inhibition of p70 S6 kinase and the MAP kinase pathway. A similar situation has been described recently for the up-regulation of insulin-like growth factor binding protein-5 in C2 myoblasts, which can be inhibited by rapamycin but not by wortmannin or LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran- 4-one).³⁰

Although rapamycin has also been shown to inhibit cyclin-dependent kinase activation, this function is not likely to be responsible for inhibition of didemnin B-induced apoptosis, because the cyclin-dependent kinase inhibitors roscovitine and olomoucine do not inhibit didemnin B-induced apoptosis.

The evidence presented here shows that inhibition of p70 S6 kinase and cyclin-dependent kinase is not likely to be involved in the inhibition of didemnin B-induced apoptosis by rapamycin and therefore some other mechanism is likely to be involved. This mechanism probably involves a binding protein that has greater affinity for rapamycin than FK506. All of the identified FKBP, except FKBP25, have similar binding affinities for rapamycin and FK506.^{31, 32, 33, 34} However, it has been shown that FKBP25 has a much greater affinity for rapamycin than for FK506,²³ suggesting that it is a likely candidate to mediate the inhibitory effect of rapamycin in didemnin B-induced apoptosis, because FK506 was unable to compete with rapamycin.

It has previously been shown that cytoplasts can undergo morphological changes associated with apoptosis, indicating that a nucleus is not required for the initiation of apoptosis in all cases.³⁵ The fact that HL-60 cytoplasts do not undergo morphological characteristics of apoptosis when treated with didemnin B, although they are capable of these changes when treated with staurosporine, suggests that the nucleus is required for didemnin B-induced apoptosis. This characteristic of didemnin B-induced apoptosis further supports the possibility that FKBP25 is involved, because FKBP25 has been shown to be localized to the nucleus.^{24, 25} This evidence for a possible role of FKBP25 in didemnin B-induced apoptosis suggests that the theoretical involvement of casein kinase II and nucleolin, which have been shown to associate with FKBP25,²⁴ should be further investigated along with the involvement of FKBP25.

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Acknowledgements

We thank Dr Laurent Meijer for providing roscovitine and olomoucine for these experiments. We also appreciate the gift of didemnin B from the National Cancer Institute, Drug Synthesis and Chemistry Branch.