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Imaging, Diagnosis, Prognosis |
Authors' Affiliations: Departments of 1 Clinical Pathology and 2 Hematology, VU University Medical Center, Amsterdam, the Netherlands
Requests for reprints: Joost J. Oudejans, Department of Clinical Pathology, VU University Medical Center, P. O. Box 7057, 1007 MB Amsterdam, the Netherlands. Phone: 31-20-4444771; Fax: 31-20-4442964; E-mail: jj.oudejans{at}vumc.nl.
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Abstract |
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Experimental Design: Twenty-eight DLBCL patient samples were investigated for their expression levels of apoptosis-related genes using reverse transcription-multiplex ligation-dependent probe amplification analysis. Functional analysis of the intrinsic, caspase-9–mediated pathway was done using fluorescence-activated cell sorting analysis, Western blot analysis, and immunohistochemistry.
Results: Two DLBCL groups were identified: one with low expression levels of both proapoptotic and antiapoptotic genes and one group with high expression levels of these genes. DLBCL with high expression levels of proapoptotic and antiapoptotic genes frequently seemed to be refractory to clinical chemotherapy. Functional analysis in these latter DLBCL samples and DLBCL cell lines with comparable expression profiles revealed high levels of spontaneous caspase-9 activity without induction of apoptosis, indicating disruption of the apoptosis pathway downstream of caspase-9 activation. This disruption of the apoptosis pathway could be restored using a small-molecule XIAP antagonist.
Conclusions: We conclude that the intrinsic, caspase-9–mediated apoptosis pathway is constitutively activated in part of chemotherapy-refractory DLBCL with concomitant downstream inhibition of the convergence apoptosis pathway and that inhibition of XIAP might be an alternative therapy for chemotherapy-refractory DLBCL.
The intrinsic and extrinsic apoptosis pathways converge on the downstream effector caspases: caspase-3, caspase-6, and caspase-7. Activation of these caspases results in the execution of apoptosis (14). Effector caspases can be suppressed by antiapoptotic proteins called inhibitor of apoptosis proteins (IAP). These proteins contain one or more copies of the baculoviral repeat domain and, in some cases, a zinc ring-finger domain (15). The human genome encodes eight IAP family members: XIAP, cIAP1, cIAP2, NIAP, survivin, MLIAP, ILP2, and apollon. Overexpression of these proteins has been shown to result in resistance to agents that trigger the intrinsic and extrinsic apoptosis pathways (16, 17).
Using microarray expression analysis, we have recently shown that a subset of chemotherapy-refractory DLBCL is characterized by high expression levels of proapoptotic genes, particularly genes that are under transcriptional control of p53 and are involved in the intrinsic, caspase-9–mediated apoptosis pathway (18). These DLBCL samples were also characterized by high expression levels of apoptosis-inhibiting genes, providing an explanation of why expression of proapoptotic genes does not result in effective induction of apoptosis.
One of the limitations of microarray expression profiling of snap-frozen lymphoma tissues is that the tumor samples also contain tumor-infiltrating cells, including high numbers of nonneoplastic T lymphocytes. Infiltrating T cells express detectable levels of antiapoptotic genes, including Bcl-2 (19) and XIAP,3 and thus interfere with expression profiling of the lymphoma cells.
To obtain apoptosis expression profiles of only the neoplastic cell population, we determined expression levels of a previously validated panel of 36 apoptosis-related genes in isolated and purified neoplastic cells of DLBCL using a highly sensitive and quantitative reverse transcription-multiplex ligation-dependent probe amplification (RT-MLPA) analysis (20). Subsequently, we investigated whether the intrinsic apoptosis pathway is constitutively activated in DLBCL tumor cells that show high expression levels of proapoptotic genes.
Next, to understand the possible mechanisms that may underlie the differential chemotherapy sensitivity observed in the clinical samples, we investigated whether failure of chemotherapy to induce apoptosis is caused by downstream inhibition of the intrinsic apoptosis pathway in DLBCL cells and a panel of cell lines with apoptosis expression profiles that match with the lymphoma samples. Previously, we have shown that expression of XIAP is correlated with a poor clinical outcome in DLBCL (11). Schimmer et al. generated small antagonistic molecules that specifically interfere with the inhibitory function of XIAP (21–23). We used these antagonists to test whether neutralizing the effect of XIAP would result in restoring caspase-9–mediated apoptosis.
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Materials and Methods |
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Lymphoma and tonsil cell suspensions were isolated from tissue biopsies by a combination of mechanical and enzymatic dissociation and frozen until further testing (25). Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll density gradient centrifugation and frozen until further testing. Cells were thawed 1 h before experimental testing and cultured in Iscove's modified Dulbecco's medium (BioWhittaker) supplemented with 10% FCS and 1% penicillin and streptomycin at 37°C with 5% CO2 in a humidified atmosphere. Nonneoplastic T cells were removed by using super paramagnetic dynabeads coated with anti-CD3 antibodies (Dynal). The purity of the resulting tumor cell fractions was determined by morphologic examination of the tumor cells in CD3-stained (clone CBC.37, DakoCytomation) and CD20-stained (clone L26, DakoCytomation) cytospin preparations. This procedure resulted in <5% contaminating, nonneoplastic CD20-negative cells in all cases tested. GCBs were fluorescence-activated cell sorting (FACS) sorted from tonsil B-cell suspensions using antibodies against the cell surface markers CD38 (clone HB7, BD Biosciences) and IgD (DakoCytomation; ref. 26).
The SUDHL4, SUDHL5, and HT DLBCL cell lines and the Nalm6 (originally obtained from the German Collection of Microorganisms and Cell Cultures) and CEM leukemia cell lines (originally obtained from the American Type Culture Collection) used in this study were cultured in RPMI 1640 (BioWhittaker) containing 10% FCS and 100 IU penicillin/100 µg/mL streptomycin.
RT-MLPA analysis. Total RNA of isolated lymphoma cells and cell lines was prepared using RNABee solution (Tel-Test, Inc.) according to the manufacturer's recommendations.4 RT-MLPA was done on total RNA as described previously (20).
Data were analyzed using Genotype and GeneScan software (Applied Biosystems). The β-glucuronidase housekeeping gene was used as internal reference to minimize possible effects of unequal amounts of mRNA. The ratios were subsequently divided by the mean expression ratios for that particular gene for optimal comparison of the samples in all patients. To identify clusters of correlated genes, hierarchical cluster analysis using TIGR software5 was used (27). Cluster analysis was done by average linkage analysis using Euclidian distance.
Assessment of caspase-dependent cell death. Induction of apoptosis in lymphoma cells and cell lines was determined as described previously and done in triplicate (25). Etoposide (Sigma) was used to assess the functional status of the intrinsic, caspase-9–mediated pathway. Briefly, cell lines were treated with 500 nmol/L etoposide for 4, 7, 16, and 24 h. Cell lines were preincubated with 25 µmol/L of the caspase-8 inhibitor LETD-fmk or the caspase-9 inhibitor LEHD-fmk (Alexis Biochemicals) 1 h before treatment. XIAP was inhibited using small-molecule XIAP antagonists. Lymphoma cells and cell lines were incubated with 10, 25, and 50 µmol/L of XIAP antagonist 1396-12 or the inactive control 1396-28 for 4 or 24 h (21–23).
Apoptosis was measured using a standard number of fluorescent beads (Fluorospheres, Becton Dickinson) in combination with 7-aminoactinomycin (ViaProbe, BD PharMingen) to determine the number of viable (7-aminoactinomycin negative) cells in each individual experiment. Fluorescence was detected by the FACSCalibur flow cytometer and analyzed using CellQuest software (both Becton Dickinson).
Detection of caspase-9 and caspase-3/7 activity. Caspase-9 activity was determined using a luminescent assay (Caspase-Glo 9 assay, Promega Benelux). Cells were lysed and incubated with the LEHD-containing substrate for 25 min at 37°C. Subsequently, luminescence was measured using a microplate luminometer (Victor2, Wallac, Perkin-Elmer). In addition, caspase-9 activity was measured in each individual cell using FACS analysis according to the manufacturer's instructions [CaspaTag, caspase-9 (LEHD) activity kit]. Briefly, cells (106 per mL) were incubated with 30x working dilution FAM-LEHD-fmk for 1 h at 37°C under 5% CO2. Cells were washed and fluorescence was measured on a FACSCalibur flow cytometer. The caspase-9 activity was calculated as follows: (mean fluorescence intensity of FAM-LEHD-fmk–treated cells) / (mean fluorescence intensity of unstained cells).
Caspase-3/7 activity was determined using a fluorescent assay (Roche) according to the manufacturer's instructions. Cells were lysed and incubated with the DEVD-rhodamine 110 substrate for 1 h at 37°C. The amount of free rhodamine was measured with a 492-nm excitation filter and a 535-nm emission filter using a microplate fluorescence reader (TECAN Spectrafluor). Caspase activity was determined as caspase activity levels of treated samples minus caspase activity levels of untreated samples. For cell lines and lymphoma samples, determination of caspase activity levels was done in triplicate.
Measurement of 
m. Mitochondrial membrane depolarization was detected using the fluorescent probe tetramethylrhodamine ethyl ester perchlorate (Invitrogen) that accumulates in mitochondria. Briefly, cells (106 per mL) were incubated with 25 nmol/L tetramethylrhodamine ethyl ester perchlorate at 37°C for 10 min in the dark and analyzed using FACS analysis. Mitochondrial membrane depolarization was observed as a shift to the left in the emission spectra. was determined as the % unstained cells in etoposide-treated samples minus the % unstained cells in untreated samples.
To further investigate whether the mitochondrial potential is continuously decreased in active caspase-9–positive cells, we used the uncoupler SF6847 (tyrphostin A9, Sigma-Aldrich), which dissipates the mitochondrial membrane potential. Cells were incubated with 10 µmol/L SF6847 for 15 min.
Western blot analysis. Total protein extracts were prepared from untreated cell lines using lysis buffer [50 mmol/L Tris-HCl (pH 8.0), 0.5% NP40, 5 mmol/L EDTA] containing protease inhibitors. Antibodies against the following proteins were used: Bax (1:1,000; Dako), Bak (1:1,000; Cell Signaling Technology), AIF (1:1,000; Cell Signaling Technology), Puma (1:1,000; Cell Signaling Technology), MCL1 (1:1,000; Dako), and XIAP (clone 2F1, 1 µg/mL; MBL). Proteins were visualized with the enhanced chemiluminescence technique (Amersham Pharmacia Biotech). Cellular β-actin (AB-1 kit, 6.25 ng/mL; Oncogene Research Products) was used as a loading control for each Western blot sample.
Immunohistochemistry. Expression of AIF, Bim, MCL1, and XIAP was investigated at the protein level in three high apoptosis expression profile cases and three low apoptosis expression profile cases as determined by RT-MLPA analysis. Sections (3 µm thick) from paraffin-embedded biopsy specimens of DLBCL were stained with the following antibodies: Bim (NeoMarkers, Lab Vision Corp.), AIF, MCL1, and XIAP. The antibodies were incubated overnight. For detection of AIF, Bim, MCL1, and XIAP, a standard highly sensitive EnVision horseradish peroxidase system (DakoCytomation) was used. Antibodies were visualized with the chromogen 3,3'-diaminobenzidine.
Statistical analysis. The Mann-Whitney U test was used to compare means between groups. Qualitative variables were analyzed using the Pearson 
2 test of the Fisher's exact test. Analyses were done using Statistical Package for the Social Sciences software (version 11.5; SPSS, Inc.). P values of <0.05 were considered significant.
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Results |
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High expression levels of proapoptotic genes correspond with high levels of spontaneous caspase-9 activity in DLBCL samples. To investigate whether high expression levels of proapoptotic genes reflect constitutive activation of the intrinsic apoptosis pathway, we compared levels of spontaneous caspase-9 activity in available isolated lymphoma cells of 11 cases characterized by high expression levels of proapoptotic and antiapoptotic genes with 7 cases with low expression levels of these genes. DLBCL samples with high expression levels of proapoptotic genes showed significantly higher levels of caspase-9 activity compared with the lymphoma cells with relative low levels of proapoptotic genes (P = 0.008; see Fig. 1D). The peripheral blood B cells and GCBs showed low levels of caspase-9 activity similar to the DLBCL cells with low expression levels of proapoptotic genes.
Constitutive caspase-9 activation without caspase-3 activation and apoptosis in cell lines with high expression levels of proapoptotic genes. A more detailed functional analysis of the intrinsic apoptosis pathway was done in cell lines with apoptosis profiles comparable with both groups of DLBCL. Cluster analysis of RT-MLPA–detected gene expression values was done identical to that for the DLBCL samples. All the three available DLBCL cell lines (HT, SUDHL4, and SUDHL5) showed high expression levels of proapoptotic and most antiapoptotic genes, whereas the two leukemia cell lines (Nalm6 and CEM) showed low expression levels of proapoptotic and most antiapoptotic genes (Fig. 1B). Western blot analysis confirmed the relatively high expression of the proapoptotic genes Bax, Bak, and AIF at the protein level in HT, SUDHL4, and SUDHL5 cell lines (Fig. 2A ).
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As active caspase-8 is also capable of activating caspase-9 via truncation of Bid, we investigated whether active caspase-8 was responsible for the activation of caspase-9 in the cell lines with high levels of caspase-9 activity. High levels of caspase-9 activity in HT, SUDHL4, and SUDHL5 cells could not be blocked by simultaneous incubation with the caspase-8 inhibitor LETD-fmk (Fig. 2B).
Etoposide induces caspase-9 activation and apoptosis only in cell lines without constitutive caspase-9 activation. Cell lines were treated with etoposide to test the functionality of the intrinsic, caspase-9–mediated apoptosis pathway. After induction with etoposide, a strong increase in caspase-9 and caspase-3/7 activity and apoptosis in Nalm6 and CEM cells was found (Figs. 2C and 3A
). Simultaneous incubation with the specific caspase-9 inhibitor LEHD-fmk showed that etoposide induced caspase-3/7 activation, and apoptosis in these cell lines was dependent on activation of caspase-9 (Fig. 3A, left). Furthermore, we observed mitochondrial depolarization of these cells after incubation with etoposide (Fig. 3B and C). In contrast, depolarization of the mitochondrial membrane already occurred in untreated DLBCL cell lines with continuous caspase-9 activity. In these cell lines, incubation with the uncoupler SF6847 hardly resulted in an additional loss of mitochondrial potential (m). Etoposide failed to further increase caspase-9 activity and to induce caspase-3/7 activation (Figs. 2D and 3A, right). Moreover, no additional mitochondrial depolarization was observed in SUDHL4, SUDHL5, and HT (Fig. 3B and C). In SUDHL5 cells, a little increase in caspase-9 and caspase-3/7 activity was observed after induction with etoposide (Fig. 3A, right).
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Discussion |
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We further investigated this using functional analysis in isolated lymphoma cells of DLBCL samples and lymphoma cell lines (SUDHL4, SUDHL5, and HT). We could show that high expression levels of proapoptotic genes are indeed associated with high levels of spontaneous caspase-9 activity and loss of mitochondrial membrane potential. Apparently, in DLBCL cells with a high apoptosis profile, the intrinsic apoptosis pathway is already triggered and part of the mitochondrial membrane potential is gone. These results might implicate that the normal oxidative phosphorylation is (partly) disrupted in cells with constitutive caspase-9 activation. However, the role of mitochondrial dysfunction in tumor cells is controversial. Recent articles show that mitochondrial outer membrane permeabilization with cytochrome c release might be compatible with the preservation of mitochondrial integrity and function (29, 30). However, it has also been shown that tumor cells frequently depend on glycolysis even under aerobic conditions, suggesting a disruption of oxidative phosphorylation (31). Our studies do not allow drawing definitive conclusions on this issue. Thus, whether oxidative phosphorylation is indeed disrupted in cells with constitutive caspase-9 activity will be subject to further studies.
Cases with high levels of spontaneous caspase-9 activity did not show concomitant caspase-3/7 activation and apoptosis. Consistent with our previous microarray data (18), DLBCL cases with a high apoptosis profile were clinically refractory to chemotherapy, which was further substantiated by our observation that etoposide failed to induce additional caspase-9 activation, loss of mitochondrial membrane potential, and apoptosis in cell lines with high levels of proapoptotic genes. Apparently, failure of chemotherapy to induce apoptosis in chemotherapy-refractory DLBCL is caused by inhibition of the intrinsic pathway downstream of caspase-9 activation.
Downstream inhibition of the apoptosis pathways can be caused by expression of the members of the IAP family, including XIAP. DLBCL samples with high expression levels of proapoptotic genes also showed high levels of antiapoptotic genes, including most members of the IAP family, which explains the inhibition of the intrinsic apoptosis pathway in these cases. This notion was supported by our previous observation that expression of XIAP in neoplastic cells of primary nodal DLBCL is associated with a poor clinical outcome (11). Inhibition of the inhibitory effect of XIAP resulted in a rapid increase in caspase-3/7 activity and apoptosis without an increase in caspase-9 activity, indicating that the XIAP antagonist mediates its effect downstream from caspase-9 activation in the apoptotic pathways, consistent with data by Carter et al. (21) and Wang et al. (23). However, the effect of the XIAP antagonist was completely blocked by the caspase-9 inhibitor, indicating that XIAP antagonist-induced apoptosis is caspase-9 dependent in these DLBCL cells and that caspase-9 activation itself is not caused by the XIAP inhibitor but results from an intrinsic cellular drive for constitutive caspase-9 activation.
Taken together, these data indicate that resistance to chemotherapy-induced apoptosis in DLBCL may be caused by downstream inhibition of the intrinsic apoptosis pathway and not by failure of chemotherapy to activate the intrinsic apoptosis pathway. Furthermore, our study implies that neutralizing the apoptosis-inhibiting function of XIAP may be important in the treatment of chemotherapy-refractory DLBCL.
Previous genome-wide microarray expression profiling studies showed that DLBCL can be categorized in two major subtypes: one resembling GCBs and the other resembling in vitro ABCs (32–34). Nuclear factor-
B–mediated up-regulation of antiapoptotic genes probably explains the poor outcome in patients with ABC-like DLBCL (35, 36). However, in our study, DLBCLs with high levels of proapoptotic and antiapoptotic genes were of both the GCB- and ABC-like phenotypes, indicating that downstream disruption of the apoptosis pathway is not restricted to only ABC-like DLBCL.
What may cause constitutive activation of the intrinsic apoptosis pathway? In nonneoplastic lymphoid cells, expression of the proapoptotic BH3-only proteins (Puma, Noxa, Bad, Bim, Bik, BMF, and Bid) can be induced by a variety of cellular signals, including cytokine withdrawal, antigen receptor cross-linking, glucocorticoid exposure, and DNA damage (37). Expression of these proapoptotic genes is mostly induced in a p53-dependent manner (38). In addition, several mitogenic oncogenes, including activated E2F1, also induce apoptosis in a p53-dependent manner as a "fail-safe" program, which, when intact, will prevent tumor development (38–40). Which of the above mechanisms causes the constitutive activation of the intrinsic apoptosis pathway remains uncertain. In our previous microarray experiments, high expression levels of proapoptotic genes correlated with high expression levels of E2F1, suggesting that high expression levels of proapoptotic genes might be explained by constitutive expression of E2F1 (38, 39).
We conclude that constitutive activation of the intrinsic apoptosis pathway with concomitant inhibition downstream of caspase-9 activation plays a central role in chemotherapy resistance in DLBCL. Moreover, our results suggest that inhibition of XIAP function might be an alternative therapy for chemotherapy-refractory DLBCL and that profiling of apoptosis genes in DLBCL cells may allow for prospective identification of patients that are expected to benefit from therapy with the small-molecule XIAP antagonist.
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Acknowledgments |
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Footnotes |
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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.
3 Cillessen et al., unpublished data. 
Received 12/ 7/06; revised 8/15/07; accepted 9/10/07.
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m loss, caspase-9 activity, and apoptosis in Jurkat cells. Exp Cell Res 2000;258:223–35.[CrossRef][Medline]B activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells. J Exp Med 2001;194:1861–74.B activation to suppress p53-independent apoptosis induced by oncogenic Ras. Science 1997;278:1812–5.This article has been cited by other articles:
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  S. A. G. M. Cillessen, J. C. Reed, K. Welsh, C. Pinilla, R. Houghten, E. Hooijberg, J. Deurhof, K. C. M. Castricum, P. Kortman, C. J. Hess, et al. Small-molecule XIAP antagonist restores caspase-9 mediated apoptosis in XIAP-positive diffuse large B-cell lymphoma cells Blood, January 1, 2008; 111(1): 369 - 375. [Abstract] [Full Text] [PDF]
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), CEM (–), SUDHL4 (
), SUDHL5 (
), and HT (
). Levels of caspase-9 and caspase-3/7 activity and the percentage of apoptotic cells were determined in triplicate. Cell lines with a low apoptosis profile were also induced with 500 nmol/L etoposide in the presence of 25 µmol/L caspase-9 inhibitor and caspase-3 activity was determined. Etoposide-induced apoptosis was entirely dependent on caspase-9 activation. 
, negative; —, etoposide; 
, 25 µmol/L XIAP antagonist + 25 µmol/L caspase-9 inhibitor.