PDCD2, a protein whose expression is repressed by BCL6, induces apoptosis in human cells by activation of the caspase cascade
Abstract
We have previously reported that the human programmed cell death-2 gene (PDCD2), a target of BCL6 repression, is likely to be important in the pathogenesis of certain human lymphomas. We now demonstrate that transfection of a construct expressing PDCD2 induces apoptosis in human cell lines, that this occurs, at least in part, through activation of the caspase cascade, and, furthermore, that caspase inhibitors block this effect. Immunohistochemical studies in human benign lymphoid and lymphoma tissues support these findings. In addition, transfection of a VP16-BCL6 zinc fingers fusion protein, which competes with the binding of endogenous BCL6 in a Burkitt lymphoma cell line, increases PDCD2 protein expression and apoptosis, and knockdown of the PDCD2 protein in this cell line by PDCD2-specific small interfering RNA duplexes inhibits apoptosis. These studies indicate that one function of PDCD2 is to promote apoptosis in several human and mammalian cell lines and tissues, including lymphoma. Although the pathways involved in lymphomagenesis are likely to be multiple and complex, it is plausible that repression of PDCD2 expression by BCL6, which, in turn, leads to downregulation of apoptosis, is one mechanism involved in BCL6-associated lymphomatous transformation. The usefulness of increasing PDCD2 expression in the treatment of certain lymphomas merits further investigation.
Introduction
We have shown previously that the human programmed cell death-2 gene (PDCD2) is a target of BCL6 repression [1,2]. Human PDCD2 is an evolutionarily conserved gene that is expressed in many tissues, including lymphocytes. Because human BCL6 is a transcrip- tional repressor which is associated with lymphoma development, PDCD2 may be important in the pathogenesis of these tumors as well. Evidence supporting this concept includes the following: 1) PDCD2 was isolated from a human B cell lymphoma line (BJAB) by subtractive hybridization; during the course of these studies, we found that in cells in which BCL6 repressive effects were blocked, PDCD2 RNA was upregulated fivefold as compared with control cells in which BCL6 repression was not inhibited [1]; 2) in another human B cell lymphoma line (Daudi), ChIP assays indicated that BCL6 is bound to the region of the PDCD2 promoter containing the BCL6 consensus binding site [2]; 3) in a human B cell lymphoma line (BJAB), knockdown of BCL6 protein levels by siRNA increases PDCD2 protein expression [2]; 4) a reciprocal relationship between BCL6 and PDCD2 could be demonstrated in mouse lymphoid tissue and in human lymphomas, both B and T cell [2]; and 5) a Drosophila ortholog of PDCD2 (Zfrp8) functions in lymph gland development and controls cell proliferation [3].
The PDCD2 gene is highly homologous to a rat gene, Rp8 [4], which was identified initially as a cell death-associated mRNA in rat thymocytes [5]. Its protein appears to be a transcriptional repressor [6], but little is known about its function in human cells. Here we show that exogenous transfection of PDCD2 cDNA induces apoptosis in human cell lines, caspase inhibitors block this effect, and caspase 3/7 activities are significantly upregulated in PDCD2-transfected cells. Immunohistochemical studies in human benign lymphoid and lymphoma tissues support these observations, because the expression of BCL6 and cleaved caspase 3 is inversely correlated in lymphocytes and lymphoma cells (high BCL6 expression, little or no detectable caspase 3), whereas high levels of PDCD2 correlate with high levels of cleaved caspase 3.
Further, transfection of a VP16-BCL6 zinc fingers fusion construct, which competes with the binding of the endogenous wild-type BCL6 protein to its targets, including PDCD2 [1], in a B cell lymphoma line that expresses high levels of BCL6, leads to higher levels of PDCD2 protein expression and apoptosis. Knockdown of these elevated PDCD2 protein levels by PDCD2-specific siRNA duplexes inhibits apoptosis.
Materials and methods
Transient transfections, Western blot analysis, and flow cytometry
A FLAG-tagged construct (“pPDCD2-EGFP”) was prepared contain- ing full-length human PDCD2 cDNA subcloned in the pIRES-2 EGFP vector (Clontech, Mountain View, CA), which permits the gene of interest and the enhanced green fluorescent protein (EGFP) gene (designed for selection by flow cytometry) to be translated from a single bicistronic mRNA. Sequencing confirmed the intact coding region of PDCD2 and Western blotting confirmed expression of this construct. Western blotting was performed as described previously [2], except that incubation to detect FLAG-tagged pPDCD2-EGFP was with a monoclonal anti-FLAG affinity-purified antibody (Sigma, St. Louis, MO), and the alkaline phosphatase conjugate (Promega, Madison, WI) was anti-mouse IgG. Subsequently the membrane was re-incubated with affinity-isolated actin antibody produced in rabbit (catalog no. A2066, Sigma, St. Louis, MO), washed, and incubated with anti-rabbit IgG (Fc), alkaline phosphatase conjugate (Promega), and then washed again. Protein bands were detected with Western Blue® Stabilized Substrate for Alkaline Phosphatase (Promega). Human 293/293T cells grown under 5% CO2 in DMEM supplemented with 10% FCS were plated at 3 × 105 to 6 × 105 cells per 35 mm well and transiently transfected by calcium phosphate DNA co-precipitation with 0.9 µg of construct or an equimolar amount of vector; pBluescript II KS+ was added as carrier DNA to a total of 6 µg. K562 cells grown in RPMI medium 1640 supplemented with 10% FCS under 5% CO2 were transiently transfected by electroporation using a Gene Pulser transfection apparatus with capacitance extender (Bio-Rad Laborato- ries, Hercules, CA). Conditions were: 960 µF, 280 to 300 v, and 25 to 30 µg of the construct DNA (or an equimolar amount of the vector) for 10 × 106 cells in log phase in 0.5 ml of serum-free RPMI medium 1640. Adherent cell lines were harvested by gentle pipeting, without the use of trypsin, and care was taken to avoid scraping the cells. Cells were harvested over a 4-day period and labeled with PE-conjugated annexin V (to detect apoptosis) and LIVE/DEAD® Fixable Violet Dead Cell Stain (Invitrogen, Carlsbad, CA, for detection of membrane permeability). Flow cytometry was performed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA). Statistical analysis was performed using analysis of variance (ANOVA), which takes into account the differences between the two cell lines and variations across 4 days of data collection for each cell line in three independent studies.
The caspase 3 inhibitor z-DEVD-fmk (concentration range 25 to 55 µM, MBL, Naka-ku Nagoya, Japan) was added to the culture medium two hrs before transfection (calcium phosphate co-precip- itation) of 293T cells with pPDCD2-EGFP. For corresponding controls, the same volume of vehicle (dimethyl sulfoxide, DMSO) was used. Cells were harvested at 21 h. Statistical analysis was by Student’s t- test and included four independent studies in which apoptosis in the control (DMSO-treated) groups was at least 10% of the total cell population. The pan-caspase inhibitor z-VAD-fmk (Axxora, San Diego, CA) or the same volume of its vehicle (DMSO) was added to the culture medium 1 to 2 h before transfection of 293/293T cells with pPDCD2-EGFP by calcium phosphate co-precipitation. A total of 30 µM of the pan-caspase inhibitor was used, added in divided doses over a two-day period, and cells were harvested 47 h post transfection. Statistical analysis was by Student’s t-test to determine whether apoptosis in the control (DMSO-treated) group was significantly different from apoptosis in the z-VAD-fmk (inhibitor)-treated group, which was aribitrarily designated as 1, and included three indepen- dent studies in which at least 3400 GFP-positive cells were counted. A luminogenic caspase 3/7 substrate (Promega) was used to measure caspase 3 and 7 activities as per manufacturer’s guidelines in 293 cells transiently transfected by calcium phosphate co-precipita- tion with the pIRES-2 EGFP vector or pPDCD2-EGFP. A CMV-driven β-galactosidase expression construct was co-transfected as previously described [7]. Cells were harvested at 47 to 49 h in four independent studies. The data were normalized for transfection efficiency by using the values of β-galactosidase assays as described previously [7]. Statistical analysis was performed using Student’s t-test to deter- mine whether the mean relative luciferase activity in the PDCD2- transfected group was significantly different from the control (vector- transfected) group, which was designated as 1.
Additionally, to be able to track cells by flow cytometry, we subcloned the BCL6 zinc fingers fused at the 5-prime end to VP16 (VP16-BCL6ZF) [1] in the pIRES-2 EGFP vector (described above). The VP16-BCL6 zinc fingers construct we described previously [1] was modified to include the BCL6 stop codon [8]. We then studied apoptosis in BJAB cells (Epstein-Barr virus-negative Burkitt lympho- ma cell line) which were transiently transfected by electroporation. Transfection of 111 to 150 nM siRNAs by electroporation and Western blot analysis were performed as described previously [2]. Conditions for transient transfection of DNA plasmids (35 µg of the pIRES-2 EGFP vector or VP16-BCL6ZF subcloned in this vector) were: 960 µF, 240 v for 10 × 106 cells in 0.5 ml of serum-free RPMI medium 1640 using a Gene Pulser transfection apparatus (Bio-Rad Laboratories, Hercules, CA) with capacitance extender. For the studies involving siRNAs and plasmids, the VP16-BCL6 zinc fingers construct described above was electroporated 48 to 65 h after pre-treatment of cells with human siRNA molecules specific for PDCD2 (human PDCD2 siGENOME SMARTpool, Dharmacon, LaFayette, CO) or siCONTROL nontargeting siRNA 1 (Dharmacon). Scanning densitometry was used to normalize the relative difference in protein expression on Western blots based on the level of intensity of β-actin.
Immunohistochemisty
Immunohistochemistry was performed on formalin or B5-fixed paraffin-embedded tissue sections using the following antibodies: rabbit monoclonal anti-cleaved caspase 3 (Asp 175) (5A1E; Cell Signaling Technology®, Danvers, MA) at dilution 1:50, mouse monoclonal anti-human BCL6 (Clone PG-B6p, DAKO, Carpinteria, CA) at dilution 1:20, and rabbit polyclonal anti-PDCD2 [1] at dilution 1:100. Antigen retrieval was performed in Bond™ Epitope Retrieval Solution 2 for 20 min (Leica Microsystems, Newcastle Upon Tyne, UK). Immunostaining was done on the automated Bond™ system (Leica Biosystems, Melbourne, Australia) using Bond™ Polymer Refine Detection or Bond™ Polymer AP Red detection according to a modified manufacturer’s protocol (25 min incubation with primary antibody, 15 min post-primary step, 25 min polymer). For dual staining, sections were first stained for cleaved caspase 3 using Bond™ Polymer Refine Detection followed by staining for BCL6 or PDCD2 using Bond™ Polymer AP Red detection. Anti-PDCD2 was applied twice (2 × 25 min) for dual staining for cleaved caspase 3 and PDCD2. Mayer’s hematoxylin (DAKO) was used as a counterstain. The appropriate positive and negative controls were performed in each case.
Results
Direct transfection of PDCD2 induces apoptosis in human cell lines
We used two mammalian cell lines, 293T (readily transfectable adherent cell line from human embryonic kidney) and K562 (human erythroleukemia suspension cell line) in transient transfection studies of PDCD2. We chose these cell lines because of high efficiency of transfection (in the case of 293T) and relatively lower (though still detectable) endogenous expression levels of PDCD2 (in the case of K562 cells, our unpublished data) as well as little or no expression of BCL6 [9]. Western blotting confirmed PDCD2 expression after transient transfection (Fig. 1A). Transfection efficiency ranged from 16% to 53% in the 293T cells and from 3% to 8% in K562 cells. When the construct expressing the full-length PDCD2 cDNA (pPDCD2-EGFP) or the pIRES-2 EGFP empty vector were transiently transfected in the two cell lines, apoptosis events were significantly higher in the GFP- positive PDCD2-transfected cells than in the vector (control)- transfected GFP-positive cells (PDCD2-transfected vs. vector-trans- fected, P = 0.016, Figs. 1B and C).
PDCD2 promotes activation of caspase 3
Studies performed in pPDCD2-EGFP-transfected 293 and 293T cells to evaluate the effects of a caspase 3 inhibitor (z-DEVD-fmk) and a pan-caspase inhibitor (z-VAD-fmk) on apoptosis revealed that these drugs induced a significant downregulation of apoptosis (fold differences in apoptosis, DMSO vs. z-DEVD-fmk, P =0.01, four independent studies, Figs. 2A and B; fold differences in apoptosis, DMSO vs. z-VAD-fmk, P = 0.003, three independent studies, Fig. 2C) as compared with control cells treated with DMSO alone. Additionally, caspase 3/7 luminescent assays, which were normalized for trans- fection efficiency by using the values of β-galactosidase assays (see Materials and methods), showed significantly higher caspase activity in PDCD2-transfected cells as compared with vector-transfected controls (P = 0.029, four independent studies, Fig. 2D).
Immunohistochemical studies in human benign lymphoid and lymphoma tissues
Staining for BCL6 and cleaved caspase 3 in human lymphoid germinal centers and lymphomas, both B and T cell types as analyzed previously [2], revealed a negative correlation between BCL6 and caspase 3 expression in both normal and malignant lymphocytes [high BCL6, absent cleaved caspase 3 (Fig. 3, 1B and 2B); absent BCL6, high level of cleaved caspase 3 (Fig. 3, 3B)]. The cleaved caspase 3 and PDCD2 proteins could both be detected within the cytoplasm of the same cells (Fig. 3, 3C, lower panel).
Inhibition of BCL6 binding by transfection of the BCL6 zinc fingers in a human B cell lymphoma line (BJAB) increases PDCD2 protein expression and apoptosis which are inhibited by selective knockdown of PDCD2
We previously showed that transfection of a BCL6 zinc fingers cDNA construct, which we fused to the activating domain of VP16 (VP16-BCL6ZF), competes very effectively with the endogenous BCL6 protein in BJAB cells, leading to inhibition of BCL6 repressive effects and, in turn, resulting in upregulation of PDCD2 RNA [1]. Further, knockdown of endogenous BCL6 protein levels by specific siRNA in these cells also increased PDCD2 protein expression [2].
We now show that transient transfection of the BCL6 zinc fingers increases PDCD2 protein expression in BJAB cells and leads to an increase in apoptosis. The upregulation of PDCD2 protein in BJAB cells transfected with the zinc fingers as compared with the vector control was detected by Western blotting (Fig. 4A) and induced an increase in apoptosis (right lower quadrant in the flow diagram, arrow in Fig. 4B). Pretreatment of BJAB cells with human siRNA molecules specific for PDCD2 (P) followed by transfection of the BCL6 zinc fingers (F) led to downregulation of the PDCD2 protein levels (P/F) as compared with cells pretreated with siCONTROL nontargeting siRNA 1 (C) followed by transfection of the BCL6 zinc fingers (C/F) (Fig. 4C). Two independent studies showed similar results. After transfection of the siRNA-treated cells with the BCL6 zinc fingers, we followed apoptotic events in PDCD2 siRNA-pretreated cells as compared with cells pretreated with siCONTROL nontargeting siRNA 1. The effects of the VP16-BCL6 zinc fingers on apoptosis, as measured by annexin V binding in the GFP-positive cells, are inhibited in the PDCD2 siRNA- treated group (Fig. 4D, right panels, right lower quadrants) as com- pared with the controls (Fig. 4D, left panels, right lower quadrants). The increased percentage of live cells in the PDCD2 siRNA-treated groups (flow diagrams, Fig. 4D, lower left quadrants in the panels on the right as compared with the lower left quadrants in the flow diagrams of the control groups on the left) likely reflects down- regulation of apoptosis by the PDCD2-specific siRNA duplexes. The increased number of cells noted in the upper left quadrant in the cells pretreated with the control siRNA at 43 h may represent increased apoptosis in this group. Because other BCL6 targets (e.g., p53, a strong regulator of apoptosis) [10] are very likely also upregulated in our dominant negative cell system, it is to be expected that pro-apoptotic effects induced by the BCL6 fingers will be reversed only partially by a selective reduction in the PDCD2 protein levels.
The graph in Fig. 4E reflects data pooled from two independent experiments of cells harvested at 43 and 50 h. In this graph, apoptotic events in cells pretreated with control siRNA, then transfected with the BCL6 fingers (C/F), are normalized to 1; apoptotic events are reduced in cells (P/F) pretreated with PDCD2 siRNA, then transfected with the fingers (mean±SD= 0.74 ± 0.087). Thus, we were able to demonstrate that the effects of the BCL6 zinc fingers on apoptosis are indeed due, at least in part, to PDCD2.
Discussion
We and others identified the BCL6 gene through its involvement in chromosomal rearrangements that accompany lymphomas in humans, especially those of the diffuse large B cell type [8,11,12]. In other lymphomas, mutations occur 5′ to the BCL6 coding region [13]. Studies in mouse models have confirmed the oncogenic role of deregulated BCL6 expression in lymphoma pathogenesis [14,15]. The protein encoded by BCL6 is known to be a strong transcriptional repressor that is needed for the formation of lymph node germinal centers [16,17] and is expressed at high levels in human lymph node germinal center B cells, most cortical thymocytes, and some human B and T cell lymphomas [18]. The C-terminal zinc finger region of the BCL6 protein binds DNA in a sequence-specific manner [19,20], and transcriptional repression by BCL6 is conveyed through an N-terminal POZ domain and a second, more centrally located domain [7,21–23]. Numerous targets of the BCL6 repressive effects have been identified, including genes implicated in B cell activation and differentiation, inflammation, cell cycle, DNA repair, chromatin formation, transcrip- tional regulation, and protein stability, both in normal and malignant B cells [24,25]. It has been shown that BCL6 binds to the promoters of about 3000 genes and that in diffuse large B cell lymphomas there is a gain of many target genes as well as a loss of binding to targets that BCL6 binds in normal B cells [25]. However, even though a large number of promoters is bound by BCL6 physically, apparently only a fraction of these (∼ 1200) undergoes transcriptional repression in germinal center B cells [26]. Additionally, BCL6 can repress a number of oncogenes and interact with various corepressors in order to repress genes involved in a variety of different biologic pathways [25]. These data indicate that the networks encompassing the genes that cooperate with BCL6 and the transcriptional effects of BCL6, both under normal conditions and when BCL6 expression is deregulated, are very complex [25,26].
Using the BCL6 zinc fingers, which readily bind DNA but lack repressive effects, to compete with high levels of endogenous BCL6 in a Burkitt lymphoma cell line, we previously identified the PDCD2 gene as a target of BCL6 repression [1]. Human PDCD2 encompasses an open reading frame encoding 344 amino acids and is an evo- lutionarily conserved gene that is expressed in many organs [4]. It contains a zinc finger MYND domain [27] and interacts with N-CoR/ mSin3A complexes (as does BCL6), which repress transcription by recruiting histone deacetylase [6,28]. Human PDCD2 is situated on chromosome 6q27, a region implicated in translocations and deletions in leukemias and various kinds of lymphomas [4,29]. Although we identified PDCD2 as a BCL6 target in a lymphoma cell line [1], our further studies indicate that the inverse relationship between PDCD2 and BCL6 is present not only in human lymphomas, both B cell and T cell types [2], but in nonmalignant lymphoid tissues as well, e.g., human tonsil [1] and Peyer’s patches of mouse intestine [2].
PDCD2 is highly homologous to a rat gene (Rp8) that was initially identified in association with programmed cell death in immature thymocytes [5], and, although some subsequent reports showed data consistent with these observations [30], some others gave conflicting
results concerning the association of Rp8 with apoptosis [31,32]. The studies we report here indicate that PDCD2 plays a role in the induction of apoptosis in human cells, and that at least one mech- anism by which this occurs is activation of the caspase cascade. We demonstrate that transfection of a construct expressing PDCD2 leads to apoptosis in two human cell lines, that caspase inhibitors block this effect, and that caspase activity is enhanced in PDCD2- transfected cells as compared with controls. Immunohistochemical studies in human benign lymphoid and lymphoma tissues support these findings. Inhibition of endogenous BCL6 binding in a human B lymphoma cell line induces increased levels of PDCD2 protein and
apoptosis; knockdown of the PDCD2 protein by PDCD2-specific siRNA molecules leads to inhibition of apoptosis.
The protein encoded by the Drosophila ortholog of PDCD2 (Zfrp8) has been reported to function in lymph gland development and to control cell proliferation [3]. It is possible that PDCD2 may play more than one role in the regulation of cell survival as, for example, has been demonstrated in the case of p53, which has been shown to induce apoptosis as well as cellular senescence [33], and BCL2 (inhibition of apoptosis, induction of cell cycle arrest, and accelerated senescence) [34]. Kusam et al. [35] reported recently that BCL6- transformed B cell lines show strong decreases in the expression of several genes, including PDCD2, and that PDCD2 repression is impor- tant for the transformation of primary B cells.
From studies of B cell chronic lymphocytic leukemia and multiple myeloma, which are characterized by a slow, progressive accumula- tion of malignant cells [36] and manifest resistance to a number of drug therapies [37], it is evident that even a small reduction in apoptosis may have profound effects in vivo over a period of time. Further, it has been shown that the expression level of a protein marginally exceeding the endogenous level is sufficient to trigger apoptosis [38]. Thus, it is plausible that even small perturbations in the regulation of PDCD2 function (e.g., through the repressive effects that BCL6 exerts on this target) may translate into deregulation of apoptosis events, which are likely to be an important factor in the pathogenesis of lymphoma. In summary, taken together, our observations confirm and extend the evidence that a function of PDCD2 in mammalian and human cells is promotion of apoptosis.
Although the events that lead to lymphomatous transformation evolve over a period of time and are due to multiple genetic events [39], further study of PDCD2 appears to BI-3802 be merited in the devel- opment of novel lymphoma therapies.