Role of Smac in human leukaemic cell apoptosis and proliferation

Article metrics

Abstract

Smac (or DIABLO) is a recently identified, novel proapoptotic molecule, which is released from mitochondria into the cytosol during apoptosis. Smac functions by eliminating the caspase-inhibitory properties of the inhibitors of apoptosis proteins (IAP), particularly XIAP. In this study, we stably transfected both full-length (FL) and mature (MT) Smac genes into the K562 and CEM leukaemic cell lines. Both FL and MT Smac transfectants increased the sensitivity of leukaemic cells to UV light-induced apoptosis and the activation of caspase-9 and caspase-3. Purified cytosol from the mature Smac transfectants, or the addition of human recombinant Smac protein or N-7 peptide into nontransfected cytosol, showed an increased sensitivity to cytochrome c-induced activation of caspase-3. The mature Smac enhanced the susceptibility of both K562 and CEM cells to TRAIL-induced apoptosis. Overexpression of the mature Smac protein also inhibited proliferation, as detected by reduced colony formation and Ki-67 expression in leukaemic cells. Cell cycle analysis revealed that Smac transfectants displayed significant G0/G1 arrest and reduction in 5-bromo-2′-deoxyuridine (BrdU) incorporation. Smac sensitized human acute myeloid leukaemia blasts to cytochrome c-induced activation of caspase-3. However, Smac failed to overcome Apaf-1-deficiency-mediated resistance to cytochrome c in primary leukaemic blasts. In summary, this study reveals that Smac/DIABLO exhibits a potential role in increasing apoptosis and suppressing proliferation in human leukaemic cells. Importantly, it also indicates that it is crucial to evaluate the levels of Apaf-1 and XIAP proteins in patient samples before using Smac peptide therapy in the treatment of human leukaemia.

Introduction

Resistance to apoptosis is the primary cause of treatment failure in patients with acute leukaemia. The mechanisms of resistance to apoptosis are either defects or blockages in apoptotic pathways. In general, there are two distinct apoptosis pathways, which have been defined as death receptor-mediated and mitochondrial-mediated apoptosis (Green and Reed, 1998). However, TNF family members, such as TNF-α, Fas (CD95) or TRAIL (TNF-related apoptosis-inducing ligand) can induce mitochondrial-independent and mitochondrial-dependent apoptosis, which depends on the type of cells (Scaffidi et al., 1998; Jia et al., 1999,2001a; Suliman et al., 2001; Deng et al., 2002). In the TRAIL-mediated apoptosis pathway, the activated upstream caspase-8 triggers the apoptotic process either by directly activating downstream caspase-3 or cleaving Bid to induce cytochrome c release from mitochondria (Jia et al., 2001a). In the mitochondrial pathway, death signals lead to changes in the mitochondrial outer membrane permeability and the subsequent release of proapoptotic factors. The released mitochondrial proteins include cytochrome c (Liu et al., 1996), apoptosis-inducing factor (AIF) (Susin et al., 1999), second mitochondria-derived activator of caspase (Smac/DIABLO) (Du et al., 2000; Verhagen et al., 2000), endonuclease G (Li et al., 2001) and OMI (Hegde et al., 2002). The released cytochrome c forms an essential part of the apoptosis complex, the ‘apoptosome’, which is composed of cytochrome c, Apaf-1 and procaspase-9. The formation of the ‘apoptosome’ induces autocatalytic processing of procaspase-9. The mature caspase-9 in turn activates its primary downstream target procaspase-3. Smac and/or OMI are also released from the mitochondria along with cytochrome c during apoptosis and function to promote caspase activation by eliminating the effect of inhibitor of apoptosis (IAP) family proteins (Du et al., 2000; Hegde et al., 2002).

The mechanisms of resistance to death receptor-mediated or mitochondria-independent apoptosis involve deficiencies in the death receptors, death effector proteins and caspase-8 or overexpression of IAPs (Chai et al., 2000; Fulda et al., 2001; Kabra et al., 2001; Partheniou et al., 2001; Zang et al., 2001). However, the resistance to mitochondria-dependent apoptosis is associated with the levels of Bcl-2 family proteins in the mitochondrial membrane, and levels of Apaf-1 and IAPs in the cytosol (Jia et al., 1999,2001b,2001c; Carson et al., 2002). Therefore, IAPs block both mitochondrial-dependent and mitochondrial-independent apoptotic pathways. The IAP protein family, which includes XIAP, c-IAP1 and c-IAP2, exerts its inhibitory role in apoptosis by binding to, and inhibiting active caspases, thus preventing amplification of the caspase cascade. Among IAPs, XIAP is the most potent and is characterized by the presence of three domains known as baculoviral IAP repeat (BIR) domains that are essential for inhibiting caspase activity (Deveraux et al., 1998). It has been reported that acute myelogenous leukaemia (AML) patients with lower levels of XIAP protein showed significantly longer survival and a tendency toward longer remission duration than those with higher levels of XIAP. However, high levels of XIAP protein in tumour cell lines were unexpectedly correlated with sensitivity to some anticancer drugs, particularly cytarabine and other nucleosides (Tamm et al., 2000). Therefore, the importance of XIAP in the context of other clinical prognostic factors is unknown.

Smac is a novel mitochondria-derived proapoptotic protein. Its precursor contains a 55-residue mitochondrial targeting sequence at its N-terminal, which is cleaved in the mitochondria to generate the mature mitochondrial Smac (Srinivasula et al., 2000). The mature Smac together with cytochrome c can be released from mitochondria into cytosol in response to a variety of apoptotic signals. It has been reported that Smac is required for TRAIL-induced apoptosis in human melanoma and colon cancer cells (Zhang et al., 2001; Deng et al., 2002), and cytochrome c-induced apoptosis in human multiple myeloma and prostate cancer cells (Carson et al., 2002). Moreover, chemically synthesized Smac N-terminal peptides can effectively bind to the BIR3 domain of XIAP and therefore inhibit binding of caspase-9 to BIR3 in the reconstituted cytochrome c-induced caspase activation system (Chai et al., 2000; Liu et al., 2000; Srinivasula et al., 2001). Whether IAPs will represent a target for cancer therapy and the design of small molecules intended to bind to IAPs in a fashion similar to Smac are receiving considerable attention.

It was recently reported that Smac peptide enhances apoptosis induced by chemo- or immunotherapeutic agents in the leukaemic Jurkat cell line (Guo et al., 2002) and in malignant glioma cells in vivo (Fulda et al., 2002). In this study, we aim to evaluate whether Smac has an inhibitory effect on the proliferation of leukaemic cells and whether it can be widely used to enhance the effect of anticancer therapy in human leukaemia. We demonstrated that Smac induces cell cycle arrest in the G0/G1 phases in leukaemic cells and the sensitization effect of Smac on cytochrome c-dependent apoptosis in human leukaemia blasts relies on threshold amounts of Apaf-1 and XIAP.

Results

Smac gene transfection increases the susceptibility of leukaemic cell lines to TRAIL- or UV-induced apoptosis and activation of caspases

Leukaemic cell lines K562 and CEM were used in this study. The K562 cell line was remarkably resistant to mitochondrial-dependent apoptosis induced by UV light (Jia et al., 2001c), etoposide (Jia et al., 2001b) or daunorubicin (Liu et al., 2002) but sensitive to TRAIL-induced apoptosis, which was independent of the mitochondrial pathway. We tested whether Smac could sensitize leukaemic cells to TRAIL- or UV light-induced apoptosis. Full-length (FL) or mature (MT) Smac gene was transfected into K562 and CEM cell lines and stable transfectants were selected by G418 and limiting dilution. Increased Smac expression was detected in all the transfectants when compared with their parental cells (Figure 1a and b). We tested whether the mature Smac protein present in the cytosol can bind to XIAP in leukaemic cells. S-100 was extracted from K562 parental and its mature Smac-transfected K/MT cells. XIAP antibody was used to immunoprecipitate XIAP/Smac protein complex and detected by Western blotting using anti-Smac antibody. A strong protein band at 23 kDa was detected in the K/MT cell line (Figure 1c). This confirmed that part of XIAP protein in the mature Smac transfectant was bound to Smac protein.

Figure 1
figure1

Smac expression in the wild-type and transfected leukaemic cells. (a) Smac expression in wild-type (lane 1) and the FL Smac transfectants (lane 2). A volume of 300 μg protein was loaded in each lane. The monoclonal anti-Smac antibody was used at 1 : 500 dilution, incubated overnight at 4°C and room temperature for further 4 h before probing with HRP-anti-rat IgG. (b) Smac expression in wild-type (lane 1) and MT Smac transfectants (lane 2). (c) Immunoprecipitation of Smac with XIAP antibody. S-100 was extracted from K562 (lane 1) and K/MT (lane 2) cells. A volume of 8 μl (1 mg/ml) of XIAP antibody was used to pull down the XIAP/Smac complex from 500 μg protein containing S-100. Polyclonal anti-Smac antibody was used at 1 : 250 dilution to detect the binding of Smac to XIAP

The cytosolic mature Smac significantly sensitized both K562 and CEM cells to TRAIL-induced apoptosis, but the mitochondrial FL Smac only sensitized CEM cells to TRAIL-induced killing (Figure 2a, b). Neither FL nor MT Smac sensitized TRAIL-induced activation of caspase-8 and cleavage of Bid. Increased cleavage of procaspase-9 and procaspase-3 was detected in both FL and MT Smac-transfected K562 and CEM cells (Figure 2c, d). Both FL and MT Smac increased the sensitivity of K562 and CEM cells to UV light-induced apoptosis (Figure 3a, b) and cleavage of caspase-9 and caspase-3 (Figure 3c, d). These results indicated that the increased level of MT Smac confers increased sensitivity to both mitochondrial-dependent and mitochondrial-independent apoptosis. However, the greater levels of mitochondrial FL Smac only sensitized cells to mitochondrial-dependent apoptosis.

Figure 2
figure2

Effect of Smac on TRAIL-induced apoptosis and activation of caspases. The K562 cell line and its Smac transfectants, K/FL and K/MT (a and c), and the CEM cell line and its Smac transfectants, C/FL and C/MT (b and d), were treated with 500 ng/ml TRAIL for 2 and 4 h. TRAIL-induced apoptosis (a and b) was measured by flow cytometry. The significantly increased apoptosis (*P<0.01) in Smac transfectants was statistically analysed by t-test in comparison to the wild-type cells. Data shown are means ±s.d. from three independent experiments. (c and d) TRAIL-induced cleavage of caspase-8, caspase-9 and caspase-3, and Bid. Protein lysates 50–100, 500 μg protein were loaded in each lane. Monoclonal anti-caspase-8 and anti-caspase-9 and polyclonal anti-Bid antibodies were used at 1 : 1000 dilution. Polyclonal anti-caspase-3 antibody was used at 1 : 5000 dilution. Activated caspases, for example, cleavage bands, are indicated by ‘α’. Monoclonal anti-β-actin antibody (1 : 3000 dilution) was used as an equal loading control

Figure 3
figure3

Effect of Smac on UV light-induced apoptosis and activation of caspases. The K562 cell line and its Smac transfectants, K/FL and K/MT (a and c), and the CEM cell line and its Smac transfectants, C/FL and C/MT (b and d), were exposed with UV light for 2 min and further cultured for 4 h. UV light-induced apoptosis (a and b) was measured by flow cytometry. The significantly increased apoptosis (*P<0.01) in Smac transfectants was statistically analysed by t-test in comparison to the wild-type cells. Data shown are means±s.d. from three independent experiments. (c and d) UV light-induced cleavage of caspase-9 and caspase-3. The experimental conditions for Western blotting were as described in the legend to Figure 2

It has been reported that Smac acts by freeing caspase-9 from the XIAP/caspase-9 complex (Liu et al., 2000). We therefore tested whether the mature Smac transfectants obtain greater susceptibility to cytochrome c/dATP-induced activation of caspase-9 and caspase-3 in a cell-free system. S-100 was extracted from the wild-type K562 and CEM and their MT Smac transfectants. Cytochrome c and dATP was added into S-100 to initiate the activation of caspases. The MT Smac significantly increased the sensitivity of leukaemic cells to cytochrome c/dATP-induced activation of caspase-3 but not caspase-9 in both K562 and CEM cell lines (Figure 4a–d).

Figure 4
figure4

Cytochrome c-induced activation of caspase-9 and caspase-3. S-100 was extracted from control K562 (a and c), CEM (b and d) cell lines and the MT Smac transfectant K/MT and C/MT cell lines. Cytochrome c and dATP were added to 50 μg protein containing S-100 and the activation of caspase-9 (a and b) and caspase-3 (c and d) was measured at each time point. The significantly increased activation of caspase-3 (*P<0.01) was statistically analysed by ANOVA in comparison to the WT cells. The activity of caspase was expressed as μ M/h/mg protein. Data shown are means±s.d. from three independent experiments

Taken together, it was concluded that the cytosolic MT Smac binds to XIAP and acts a promoter of the activation of caspase-3. The increased levels of MT Smac sensitized both K562 and CEM cells to death receptor TRAIL-induced apoptosis. Both MT and FL Smac increased the sensitivity of leukaemic cells to mitochondrial-dependent apoptosis.

Smac transfectants reduce the proliferation rate in human leukaemic cell lines

We observed that the C/MT cell line had a remarkably reduced growth potential in the routine suspension culture. We were therefore interested in whether Smac had an antiproliferative effect on leukaemic cells. Wild-type K562 and CEM and their Smac transfectants were seeded in soft agar. Colony formation was assessed after 2 weeks. The MT Smac transfectants showed significantly reduced colony numbers in both K562 and CEM cell lines, but colony formation was normal in the FL Smac transfectants (Figure 5a). It was tested whether the reduced colony formation was associated with reduced Survivin expression in Smac-transfected leukaemic cells. Western blotting showed that decreased Survivin expression was seen only in the Smac-transfected K562 cells but not in the CEM cell line (Figure 5b). We could not conclude that the decreased proliferation is entirely because of the reduced levels of Survivin protein. The proliferative index was also measured by Ki-67 expression using flow cytometry. Both MT Smac transfectants, K/MT and C/MT, showed reduced Ki-67 expression from 76 to 62% in CEM cells and from 95 to 88% in K562 cells (Figure 6a, b). This indicates that Smac also inhibits cell cycling and therefore reduces proliferation of leukemic cells.

Figure 5
figure5

Inhibitory effect of Smac on the colony formation and Survivin expression. (a) Colony formation. Wild-type (WT) CEM and K562 cell lines; their full-length (FL) and mature (MT) Smac transfectants were seeded in 0.3% soft agar and cultured for 2 weeks. Numbers of colony per grid were counted under a phase-contrast microscope. Data shown are means±s.d. from three independent experiments. Significantly decreased colony formation (*P<0.01) in MT Smac-transfected cells was statistically analysed by t-test in comparison to their WT cell lines. (b) Survivin expression. Proteins were extracted from CEM (lane 1), C/FT (lane 2), C/MT (lane 3), K562 (lane 4), K/FL (lane 5) and K/MT (lane 6) and loaded as 100 μg/lane onto a 12% SDS–PAGE. The monoclonal anti-Survivin antibody was used at 1 : 200 dilution and β-actin (1 : 10 000 dilution) was used as a loading control

Figure 6
figure6

Inhibitory effect of Smac on Ki-67 expression. CEM (a) and K562 (b) cell lines and their Smac transfectants, both FL and MT, were permeabilized and stained with Ki-67 antibody (or control IgG) and PI. Expression of Ki-67 protein was assessed in the FL1-Height channel vs DNA content in the FL2-Area channel. Numbers indicated in each graph are percentage of Ki-67-positive cells. Data presented as mean±s.d. show representative results of one of three independently performed experiments

Cell cycle was further analysed by the BrdU incorporation verse DNA content in the resting CEM, C/FL and C/MT cell lines. The percentages of BrdU-positive cells were significantly reduced in both C/FL (25.9%) and C/MT (16.5%) cell lines compared with the CEM cell line (44.6%) (Figure 7a, b). DNA content analysis revealed that remarkable G0/G1 arrest in both C/FL (62.0%) and C/MT (73.8%) cells as compared with the wild-type CEM (54.87%) (Figure 7a, c). This result suggested that Smac has an effect in arresting cells in G0/G1.

Figure 7
figure7

BrdU incorporation and propidium iodide staining. (a) BrdU incorporation is shown as log fluorescence using the FL1-Height channel (upper panel) and relative DNA content is measured in FL2-A as PI staining (upper and lower panels). BrdU-positive cells were gated after excluding negative cells that were not treated with BrdU but were probed with antibodies. BrdU-positive cells represent S phase cells actively synthesizing DNA. (b) Percentages of BrdU-positive cells were calculated from dot plots. (c) Percentages of G0/G1 cells were calculated from the histograms of DNA content (lower panel of a). Data shown are the mean±s.d. from at least two independent experiments. Significant difference was statistically analysed by t-test. *P<0.01, **P<0.0001

Purified Smac protein and N-7 peptide enhance cytochrome c-induced activation of caspase-3

We tested whether recombinant mature Smac protein or Smac N-7 peptide could enhance cytochrome c-induced activation of caspase-3. Smac protein (2.5 μ M) or N-7 peptide (500 μ M) was incubated with S-100 for 30 min on ice prior to the addition of cytochrome c and dATP. Both Smac protein and Smac N-7 peptide significantly increased the activation of caspase-3 in K562 and CEM cell lines (Figure 8a, b), which was consistent with transfected Smac. N-7 peptide, either purchased from Calbiochem or synthesized by Alta Bioscience (Birmingham, UK), produced a similar result. This suggests that both purified Smac protein and synthesized N-7 Smac peptide can increase the sensitivity of leukaemic cell lines to cytochrome c-induced activation of caspase.

Figure 8
figure8

Effect of recombinant Smac protein or N-7 Smac peptide on cytochrome c-induced activation of caspase-3 in CEM (a) and K562 (b) cell lines. Smac protein (2.5 μ M) or N-7 peptide (500 μ M) was added to 50 μg protein-containing S-100 (extracted from CEM cell line) and incubated on ice for 30 min. Cytochrome c/dATP were then added to initiate the reaction. Caspase activity was defined as micromolar AFC release per milligram protein per hour (μ M/h/mg protein). Data shown are means±s.d. from three independent experiments. Significantly increased activity of caspase-3 (P<0.01) in Smac protein pretreated S-100 was analysed by the t-test

Smac promotes cytochrome c-induced activation of caspase-3 in human leukaemic blasts which express both Apaf-1 and XIAP

The potential role of Smac in cytochrome c-dependent apoptosis was evaluated in leukaemia blasts from previously untreated patients with AML (eight patients) and ALL (four patients). The FAB type and percentage of blasts of samples are listed in the Table 1. S-100 was extracted from isolated blasts and preincubated with Smac N-7 peptide (500 μ M). The Smac-enhanced sensitivity of leukaemic blasts to cytochrome c-induced activation of caspase-3 was seen only in the sample AML-7, which expressed significant amounts of Apaf-1 and XIAP (Figure 9a–c). Smac had no effect on cells lacking Apaf-1 (AML-1, -2, -4, -6, -8, and ALL-1, -2, -3 and -4) or XIAP (AML-2, -4 and -5). As Apaf-1 and XIAP are not expressed in all leukaemic cells, this indicates that the effect of Smac on cytochrome c-dependent apoptosis is limited to certain types of cells and is dependent on the levels of Apaf-1 and XIAP in leukaemic cells.

Table 1 FAB types and percentages of blasts of leukaemic samples
Figure 9
figure9

Effect of N-7 Smac peptide on cytochrome c-induced activation of caspase-3 (a) and the association with expression of XIAP and Apaf-1 (b and c). (a) Caspase-3 activation. N-7 Smac peptide (500 μ M) was added to 50 μg protein-containing S-100 (extracted from leukaemia blasts) and incubated on ice for 30 min. Cytochrome c/dATP were then added to initiate the reaction. Caspase activity was defined as micromolar AFC release per milligram protein per hour (μ M/h/mg protein). (b) XIAP and Apaf-1 expression. A volume of 50 μg protein was loaded in each lane. Monoclonal antibody for XIAP (Transduction Laboratory) was used at 1 : 200 dilution and polyclonal Apaf-1 antibody (Alexis) was used at 1 : 1000 dilution. (c) The ratios of XIAP/actin and Apaf-1/actin of each sample were the intensities of individual bands normalized with respect to actin band intensities in the same lane. ‘P’ indicates Smac protein, ‘N’ indicates N7 peptide, ‘Cyt.c’ means cytochrome c

Discussion

It has been previously shown that the Apaf-1 deficiency confers resistance to cytochrome c-dependent apoptosis in both human leukaemic cell lines and human AML blasts (Jia et al., 2001b). We aimed to study whether Smac could enhance the sensitivity of leukaemic cells to cytochrome c-dependent and -independent apoptosis; and whether Smac has an inhibitory effect on the proliferation of leukaemic cells. We found that enforced overexpression of Smac protein sensitized leukaemic cells to both TRAIL- and UV light-induced apoptosis. Interestingly, the mature Smac transfectants showed a significantly reduced proliferation rate and G0/G1 arrest.

Resistance to apoptosis can be caused by either overexpression of the antiapoptotic proteins (Bcl-2, Bcl-XL or IAPs) or constitutive deficiency in the proapoptotic proteins (Bax, Bak or Apaf-1). The chronic myeloid leukaemia cell line, K562, is highly resistant to chemotherapeutic drugs, such as etoposide or daunorubicin, as well as UV light-induced apoptosis (Jia et al., 2001b,2001c; Liu et al., 2002). However, the constitutive deficiency of proapoptotic proteins, such as p53 or Apaf-1, did not affect the sensitivity of K562 cells to TRAIL-induced apoptosis (Jia et al., 2001a). Stable MT Smac transfectants expressed the cytosolic MT Smac protein that bound to XIAP. The MT Smac increased the sensitivity of both K562 and CEM cells to TRAIL-induced apoptosis downstream of mitochondria. However, the FL mitochondrial Smac only sensitized CEM cells to TRAIL-induced apoptosis. As expected, both FL and MT Smac increased the sensitivity of K562 and CEM cells to UV light-induced apoptosis and cytochrome c-induced activation of caspase-3. Smac did not enhance cytochrome c-induced activation of caspase-9. This is in agreement with other reports that Smac promotes apoptosis by removing XIAP from processed caspase-9 but not procaspase-9 (Ekert et al., 2001). However, Smac sensitized leukaemic cells to UV light-induced activation of caspase-9, probably because of the feedback activation by the activated caspase-3. TRAIL-induced activation of caspase-9 is not solely because of cytochrome c. It also relies on the activation of caspase-8. The caspase-8-initiated activation of caspase-3 may have feedback effect on the caspase-9. Thus, we have shown that Smac can enhance mitochondria-dependent and -independent apoptosis in leukaemic cell lines expressing relatively low levels of Apaf-1.

Importantly, we found that cytosolic Smac can inhibit proliferation, as shown by decreased colony formation and Ki-67 expression. Cell cycle analysis revealed that both cytosolic and mitochondrial Smac arrests leukaemic cells in G0/G1 and reduces BrdU incorporation. This indicates that Smac blocks G0/G1 to S transition. Smac, per se, does not induce apoptotic cell death. Leukaemic cells, which overexpressed Smac, survived after selection by long-term, high-dose treatment with G418. However, their growth rate was remarkably reduced in both culture medium and in soft agar. The G0/G1 arrest, reduced Ki-67 expression and BrdU incorporation in Smac transfectants indicate that the Smac gene inhibits cell cycling. It has been reported that AML patients with low levels of XIAP enjoyed a significantly longer survival and tended to have longer median remission durations (Tamm et al., 2000). Overexpression of another IAP family protein Survivin, which contains a single BIR domain, was associated with a higher number of proliferating cells in some cancers (Altieri, 2001). Like other IAPs, Smac can also bind to Survivin via its BIR domain (Du et al., 2000). The inhibitory effect of Smac on proliferation may be because of eliminating Survivin and XIAP.

The ability of Smac to sensitize cells to apoptotic stimuli offers new therapeutic opportunities in cancer and this is an area of active investigation. The limitations of gene therapy for solid tumour and leukaemic cells means that it would be almost impossible to inject genes directly into all these cells. Therefore, the use of peptides designed to include the functional domain of the parent protein may be an easier way to facilitate tumour killing by chemotherapy. The short sequences derived from the N-terminus of Smac can interact with XIAP, c-IAP1 and c-IAP2 and promote the activation of caspase-9, caspase-3 and caspase-7 (Chai et al., 2000; Liu et al., 2000; Srinivasula et al., 2000). In this study, it was revealed that Smac N-7 peptide, like Smac transfectants and purified Smac protein, sensitized leukaemic cell lines to cytochrome c-induced activation of caspase-3. Using cell-permeable peptides, another group also confirmed that both Smac-4 and Smac-7 peptides potentiated epothilone B or TRAIL-induced apoptosis in the human leukaemic Jurkat cell line (Guo et al., 2002).

We have previously reported a deficiency of Apaf-1 protein in human AML blasts. Apaf-1 deficiency is one mechanism by which leukaemic blasts become resistant to cytochrome c-induced activation of caspase-3 and cytochrome c-dependent apoptosis (Jia et al., 2001c). We tested whether eliminating the inhibitory effect of XIAP by Smac could overcome Apaf-1 deficiency-associated resistance to apoptosis in primary leukaemic blasts. Smac was unable to sensitize blasts lacking either Apaf-1 or XIAP. Unlike leukaemic cell lines, the proapoptotic protein Apaf-1 and antiapoptotic protein XIAP are not expressed in all primary leukaemic blasts. In all four cases of ALL tested, the blasts expressed XIAP but not Apaf-1. Smac could not overcome XIAP-mediated resistance to cytochrome c-dependent apoptosis in the absence of Apaf-1.

In summary, we suggest that Smac can sensitize leukaemic cells to both cytochrome c-dependent and -independent apoptosis. Smac also functions to induce G0/G1 arrest and therefore to inhibit the proliferation rate. However, the ability of Smac to augment cytochrome c-dependent apoptosis in human leukaemic cells requires both Apaf-1 and XIAP. We therefore propose that it is crucial to evaluate the levels of Apaf-1 and XIAP proteins in patient samples before using Smac peptide therapy in the treatment of human leukaemia.

Materials and methods

Antibodies and reagents

The following antibodies were used in this study: anti-caspase-8 (12F5), anti-Smac (10G7), anti-Smac polyclonal and anti-Apaf-1 polyclonal antibodies (Alexis Biochemicals, Lausen, Switzerland); anti-XIAP (2F1), anti-caspase-9 (5B4) and anti-caspase-3 polyclonal antibodies (Stressgen-Bioquote, York, UK); anti-actin (AC-74), anti-Flag M2 and anti-BrdU (Clone BU-33) antibodies; FITC-conjugated anti-mouse IgG (Sigma, Dorset, UK); anti-Bid polyclonal antibody (R&D Systems, Minneapolis, USA); anti-Ki-67 (B56) antibody (BD PharMingen, San Diego, CA, USA); and anti-XIAP antibody (BD Transduction Laboratory, San Diego, CA, USA). Anti-Survivin antibody (D-8), HRP-conjugated anti-mouse, anti-rabbit and anti-rat IgGs are from Santa Cruz, Inc. (Santa Cruz, CA, USA). E. coli DH5α strain, DMRIE-C reagent, serum-free OPTI-MEM medium and Geneticin (G418) sulphate were purchased from Gibco BRL (West Sussex, UK). Plasmid DNA Mini-Prep Kit was from QIAGEN (Valencia, CA, USA). TRAIL was obtained from Biomol-Affiniti (Exeter, UK). 5-bromo-2′-deoxyuridine (BrdU), propidium iodide (PI), RNase A, bovine heart cytochrome c, dATP, Gum agar, culture medium, RPMI-1640 and Eagle-MEM, FCS and all other chemicals were purchased from Sigma. Fluorescent substrates, Ac-Leu-Glu-His-Asp-AFC (Ac-LEHD-AFC) for caspase-9 and Ac-Asp-Glu-Val-Asp-AFC (Ac-DEVD-AFC) for caspase-3 and Smac N-7 peptide (H-AVPIAQK-OH) were obtained from Calbiochem (Nottingham, UK). Purified MT Smac protein (the sequence corresponding to amino acids 56–239 of human Smac) was from R&D Systems. Dynalbeads (M-450) were from Dynal Biotech Ltd (Wirral, UK).

Leukaemic cell lines, leukaemia blasts and gene transfection

The human myeloid leukaemic K562 cell line and T-lymphoblastic cell line CEM were used in this study. The cell lines were cultured in RPMI-1640 medium as described previously (Jia et al., 2001c). Human AML and acute lymphoid leukaemia (ALL) blasts were obtained from untreated patients with AML or ALL at presentation and were separated over a Ficoll–Hypaque gradient.

Flag-C-tagged FL Smac pCDNA3 and Flag-C-tagged MT Smac (without the mitochondrial targeting sequence) plasmids (Srinivasula et al., 2000) were grown in E. coli DH5α strain and were purified using QIAGEN Plasmid DNA Mini-Prep Kit. A volume of 6 μl of plasmid DNA was transfected into 2 × 106 leukaemic cells using DMRIE-C reagent in serum-free OPTI-MEM medium. After culturing at 37°C for 5 h, cell culture conditions were recovered by the addition of 15% foetal calf serum (FCS) containing RPMI-1640 medium. For stable transfection of Smac genes in the leukaemic cell lines, transfectants were selected in the presence of 1.0–1.2 mg/ml Geneticin (G418) sulphate for 1 month and then further selected by single-cell cloning in 96-well plates. K/FL (clone F4) and C/FL (clone D3) transfectants, which overexpress the FL Smac gene, and K/MT (clone D5) and C/MT (clone C1), which overexpress the MT Smac gene were determined by a dot blot using the monoclonal anti-Flag-M2 antibody and confirmed by Smac overexpression by Western blotting.

Assessment of apoptosis by flow cytometry

To induce apoptosis in intact cells, leukaemic cells (5 × 105/ml) were treated with 500 ng/ml TRAIL (4) or exposed to UV irradiation (120 mJ/cm2) (Chromato-UV-E Transiluminator, Model TM-20) for 2 min and further cultured for up to 4 h (Jia et al., 2001b). Cells were permeabilized with 70% ethanol and stained with 50 μg/ml PI. PI fluorescence of nuclei was measured by flow cytometry (FACScan, Becton Dickinson, Oxford, UK) (Jia et al., 2001c).

Cytochrome c-induced activation of caspases in a cell-free system

Cells were suspended in 0.5 ml of Buffer A (250 mM sucrose, 10 mM HEPES–KOH, pH 7.4, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 20 μ M cytochalasin B) and incubated for 20 min on ice. Cells were then broken with a glass Dounce homogenizer (Jencons-PRL, Leighton Buzzard, UK). Cell homogenates were centrifuged at 16 000 g for 40 min at 4°C. Purified cytosol (S-100 fraction) was obtained by passing through a 0.22-μm Ultrafilter (Sigma) at 16 000 g for 20 min. Cell-free reactions were set up in a 25 μl reaction volumes. S-100 (50 μg) in Buffer A was incubated with or without bovine heart cytochrome c (50 ng)/dATP (10 nM) at 30°C up to 1 h. The reaction mixture was diluted to 95 μl with buffer A. The caspase-initiated cleavage of fluorigenic substrates was initiated by the addition of 5 μl of 400 μ M (final concentration was 20 μ M) fluorescent substrates, Ac-LEHD-AFC, for caspase-9 or Ac-DEVD-AFC, for caspase-3. After incubation at 30°C for 15 min, the reaction was stopped by the addition of 50 μl of 1% sodium acetate trihydrate in 175 mM acetic acid. The fluorescence at 400/505 nm was measured with a TD-700 fluorimeter (Turner Designs, Sunnyvale, CA, USA). Measurements were calibrated against a standard linear regression curve of AFC. Caspase activity was defined as micromolar AFC release per milligram protein per hour (μ M/h/mg protein) (Jia et al., 2001b).

Western blotting

Proteins were subjected to standard SDS–PAGE at 20–40 mA/gel and transferred onto PVDF membrane (Sigma) at 100 V for 1 h. PVDF membrane was blocked with 1% nonfat milk in PBST for 1 h and probed for various proteins using monoclonal or polyclonal antibodies (as described individually in the figure legends). Bound antibodies were detected using appropriate HRP-conjugated secondary antibodies, followed by detection using SuperSignal ECL. The density of each band was analysed using an AlphalmagerTM 2000 Densitometer (Alpha Innotech Corp. San Jose, CA, USA).

Immunoprecipitation using Dynabeads

To detect MT Smac binding to XIAP, S-100 was extracted from K562 and K/MT cells. The anti-mouse IgG Dynabeads (M-450) were washed three times with Buffer A and incubated with the monoclonal anti-XIAP antibody (2F1) at a ratio of 50 μl of beads to 8 μl antibody for 3 h at 4°C on a rotor. Coated beads were washed and then added into 500 μg protein containing S-100. Pulling down the XIAP/Smac immune complex was performed at 4°C overnight on a rotor. The beads were then washed three times with Buffer A and resuspended in gel-loading solution containing SDS. After boiling for 3 min, isolated proteins were separated by a 12% SDS–PAGE and detected by a polyclonal anti-Smac antibody.

Soft agar assay for colony formation

Gum agar (2%) was melted in a microwave and cooled to 50–60°C in a water bath. Eagle-MEM medium and 20% FCS were prewarmed to 40°C in a water bath. Agar was mixed with medium and FCS to give 0.5% agar and 10% FCS. A volume of 2 ml of 0.5% agar was added to each 35 mm Petri dish and allowed to set. The top agar was prepared with 2% agar, Eagle-MEM medium and FCS to give 0.3% agar and 10% FCS. A volume of 1.8 ml of 0.3% agar was mixed with 0.2 ml of cell suspension (containing 2 × 104 cells) by vortexing the contents vigorously until the cells were evenly suspended. The cell-containing mixture was plated in a 2 ml volume on the top agar. The dish was overlaid with 1 ml of Eagle-MEM medium containing supplements. Cells were incubated for 2 weeks at 37°C in 5% CO2 before counting colonies.

Detection of cycling cells

The Ki-67 protein is exclusively expressed during the cell division cycle in the G1, S and G2 phases as well as mitosis and therefore it has been used to detect the percentages of cycling cells (Gerlach et al., 1998). Ki-67 expression in the wild-type and Smac transfectants was performed using a washless staining method for both Ki-67 and DNA. Briefly, 105 cells were suspended in 50 μl ice-cold freezing buffer (250 mM sucrose, 5% DMSO, 40 mM Na citrate, pH 7.6) and frozen at −70°C for 30 min. After thawing, 200 μl of lysis-DNA staining solution (0.5% v/v NP-40, 20 μg/ml PI, 0.2 mg/ml RNase A and 0.5 mM EDTA in PBS, pH 7.2) was added to the tube with treated cells and incubated on ice for 15 min. The diluted (1 : 10) monoclonal anti-Ki-67 antibody (25 μl) or the equivalent amount of isotype control antibody (DAKO) was added and incubated for 15 min on ice. A volume of 25 μl of FITC-conjugated anti-mouse antibody (Sigma) was added and further incubated for at least 15 min prior to flow cytometry. The dual-parameter analysis of Ki-67 vs DNA content was analysed by an FACScan flow cytomter. Expression of Ki-67 was calculated by determining fluorescence emission at the FL1-H channel.

BrdU incorporation

CEM, C/FL or C/MT cells in 106/ml were incubated with 1 μ M BrdU for 20 min at 37°C, fixed with 70% ice-cold ethanol and then stored at 4°C overnight. After washing twice with PBS, cells were resuspended in 5 ml of 0.04% pepsin in 0.1 N HCl and incubated for 30 min on a rotor at 37°C. Digested nuclei were collected by centrifugation at 1200 g for 10 min and were then further treated by adding 3 ml of 2 N HCl for 20 min at 37°C. The nuclear suspension was neutralized by the addition of 6 ml of 0.1 M sodium borate. After washes with PBSTB (0.5% Tween-20 and 0.5% BSA in PBS), nuclei were probed with anti-BrdU monoclonal antibody (1 : 500 dilution) overnight at 4°C. Nuclei were washed twice with PBSTB and stained with FITC-conjugated anti-mouse IgG (1 : 100 dilution) for 1 h at 4°C. After washing by PBSTB, nuclear DNA was stained with 10 μg/ml PI for 1 h at 4°C in the dark. Both BrdU and DNA contents were analysed by flow cytometry at FL1-Hight channel vs FL2-Aera channel (Terry and White, 2001).

References

  1. Altieri DC . (2001). Trends Mol. Med., 7, 542–547.

  2. Carson JP, Behnam M, Sutton JN, Du C, Wang X, Hunt DF, Weber MJ and Kulik G . (2002). Cancer Res., 62, 18–23.

  3. Chai J, Du C, Wu JW, Kyin S, Wang X and Shi Y . (2000). Nature, 406, 855–862.

  4. Deng Y, Lin Y and Wu X . (2002). Genes Dev., 16, 33–45.

  5. Deveraux QL, Roy N, Stennicke HR, Van Arsdale T, Zhou Q, Srinivasula SM, Alnemri ES, Salvesen GS and Reed JC . (1998). EMBO J., 17, 2215–2223.

  6. Du C, Fang M, Li Y, Li L and Wang X . (2000). Cell, 102, 33–42.

  7. Ekert PG, Silke J, Hawkins CJ, Verhagen AM and Vaux DL . (2001). J. Cell Biol., 152, 483–490.

  8. Fulda S, Kufer MU, Meyer E, van Valen F, Dockhorn-Dworniczak B and Debatin KM . (2001). Oncogene, 20, 5865–5877.

  9. Fulda S, Wick W, Weller M and Debatin KM . (2002). Nat. Med., 8, 808–815.

  10. Gerlach C, Kubbutat M, Schwab V, Key G, Flad HD and Gerdes J . (1998). Lab. Invest., 78, 129–130.

  11. Green DR and Reed JC . (1998). Science, 281, 1309–1312.

  12. Guo F, Nimmanapalli R, Paranawithana S, Wittman S, Griffin D, Bali P, O'Bryan E, Fumero C, Wang HG and Bhalla K . (2002). Blood, 99, 3419–3426.

  13. Hegde R, Srinivasula SM, Zhang Z, Wassell R, Mukattash R, Cilenti L, DuBois G, Lazebnik Y, Fernandes-Alnemri T and Alnemri ES . (2002). J. Biol. Chem., 277, 432–438.

  14. Jia L, Macey MG, Yin Y, Newland AC and Kelsey SM . (1999). Blood, 93, 2353–2359.

  15. Jia L, Patwari Y, Kelsey SM and Newland AC . (2001a). Biochem. Biophys. Res. Commun., 283, 1037–1045.

  16. Jia L, Patwari Y, Srinivasula SM, Newland AC, Alnemri ES and Kelsey SM . (2001b). Oncogene, 20, 4817–4826.

  17. Jia L, Srinivasula SM, Liu FT, Newland AC, Alnemri ES and Kelsey SM . (2001c). Blood, 98, 414–421.

  18. Kabra NH, Kang C, Hsing LC, Zhang J and Winoto A . (2001). Proc. Natl. Acad. Sci. USA, 98, 6307–6312.

  19. Liu FT, Kelsey SM, Newland AC and Jia L . (2002). Br. J. Haematol., 117, 333–342.

  20. Liu X, Kim CN, Yang J, Jemmerson R and Wang X . (1996). Cell, 86, 147–157.

  21. Li LY, Luo X and Wang X . (2001). Nature, 412, 95–99.

  22. Liu Z, Sun C, Olejniczak ET, Meadows RP, Betz SF, Oost T, Herrmann J, Wu JC and Fesik SW . (2000). Nature, 408, 1004–1008.

  23. Partheniou F, Kelsey SM, Srinivasula SM, Newland AC, Alnemri ES and Jia L . (2001). Biochem. Biophys. Res. Commun., 287, 181–189.

  24. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH and Peter ME . (1998). EMBO J., 17, 1675–1687.

  25. Srinivasula SM, Datta P, Fan XJ, Fernandes-Alnemri T, Huang Z and Alnemri ES . (2000). J. Biol. Chem., 275, 36152–36157.

  26. Srinivasula SM, Hegde R, Saleh A, Datta P, Shiozaki E, Chai J, Lee RA, Fernandes-Alnemri T, Shi Y and Alnemri ES . (2001). Nature, 410, 112–116.

  27. Suliman A, Lam A, Datta R and Srivastava RK . (2001). Oncogene, 20, 2122–2133.

  28. Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Costantini P,, Loeffler M and Kroemer G . (1999). Nature, 397, 441–446.

  29. Tamm I, Kornblau SM, Segall H, Krajewski S, Welsh K, Kitada S, Scudiero DA, Tudor G, Qui YH and Reed JC . (2000). Clin. Cancer Res., 6, 1796–1803.

  30. Terry NHA and White RA (2001). Methods Cell. Biol., 63, 355–374.

  31. Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE, Moritz RL, Simpson RJ and Vaux DL . (2000). Cell, 102, 43–53.

  32. Zang DY, Goodwin RG, Loken MR, Bryant E and Deeg HJ . (2001). Blood, 98, 3058–3065.

  33. Zhang XD, Zhang XY, Gray CP, Nguyen T and Hersey P . (2001). Cancer Res., 61, 7339–7348.

Download references

Acknowledgements

We acknowledge the Cancer Research UK Medical Oncology Unit for collection and storage of peripheral blood and bone marrow samples. This work was supported by grants from by the Leukaemia Research Fund (9946) to SMK and LJ and Research Advisor Committee of the Royal London Hospital (RAC389) to LJ.

Author information

Correspondence to Li Jia.

Rights and permissions

Reprints and Permissions

About this article

Keywords

  • apoptosis
  • Apaf-1
  • caspases
  • IAPs
  • Smac

Further reading