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The Journal of Immunology, 2005, 175: 4518-4527.
Copyright © 2005 by The American Association of Immunologists

Id3 Induces Growth Arrest and Caspase-2-Dependent Apoptosis in B Lymphocyte Progenitors1

Barbara L. Kee2

Department of Pathology, Committees on Immunology, Cancer Biology, and Developmental Biology, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The E-protein transcription factors E2A, HEB, and E2-2 play an essential role in the differentiation, proliferation, and survival of B lymphocyte progenitors (BLPs). In this study, we show that the E-protein antagonist Id3 induces apoptosis of both primary and transformed BLPs through a caspase-2-dependent mechanism that does not require p53 and is not inhibited by bcl-2. Id3 expressing B lineage cells show reduced expression of known E-protein target genes as well as multiple genes involved in cell proliferation. We hypothesize that Id3 induces activation of caspase-2 as a consequence of severe or "catastrophic" growth arrest. In support of this hypothesis, we show that chemical-induced growth arrest is sufficient to activate caspase-2 and induce apoptosis in BLPs. Our data suggest that E-proteins function in the control of differentiation and proliferation and that diminished E-protein activity results in apoptosis as a consequence of growth arrest.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Blymphopoiesis requires the coordinated activity of multiple transcriptional regulatory proteins, progressive rearrangement of the Ig H and L chain genes, and inputs from the extracellular microenvironment that are mediated predominantly through growth factors and cytokines. How these distinct activities are coordinated, integrated, and regulated is a major question in lymphocyte developmental biology. The earliest stages of B lymphocyte development are dependent on the activity of at least three transcriptional regulatory proteins E2A, early B cell factor (EBF),3 and Pax-5 that promote lineage-associated gene expression and rearrangement of the Ig loci (1). E2A and EBF are required for the emergence of pre-pro B cells from common lymphoid progenitors and, in their absence, no B lineage-associated genes are detected in mouse bone marrow (BM) or fetal liver (1, 2, 3, 4, 5). In contrast, B lymphocyte progenitors (BLPs) develop in the BM of Pax-5–/– mice, but are unable to progress beyond the pro-B lymphocyte stage owing to the absence of components of the pre-BCR signaling pathway (6, 7). Moreover, Pax5–/– pro-B lymphocytes are not fully committed to differentiation through the B lineage pathway (8).

E2A proteins are basic helix-loop-helix (HLH) transcription factors that are members of the E-protein family, which bind to the E-box sequence found in many developmentally regulated genes (9). In addition to E2A, the E-proteins E2-2 and HEB are expressed in B lineage cells but are present at low abundance and are not essential for B lymphopoiesis under normal conditions (10). Recent evidence indicates that E2A proteins are essential for B lymphocyte development because they raise the level of E-protein activity above a threshold required for the transcriptional induction of EBF in common lymphoid progenitors (11). EBF is required for B lymphopoiesis and promotes activation of most B lineage genes in synergy with E-proteins (12). The E-proteins are unique among the transcription factors controlling early B lymphocyte differentiation in that they have also been implicated in proliferation and survival (13). E2A is required for optimal expression of N-myc in response to IL-7R-derived signals and consequent expansion of early BLPs (11). Modulation of E-protein activity appears to have a physiological role in B lineage cells because the E-protein antagonist Id3 is a target of TGF-{beta} and is required for TGF-{beta}-induced growth arrest (14). Interestingly, in contrast to the slow growth phenotype observed in cells expressing only HEB and E2-2, ectopic expression of the E-protein antagonist Id3 results in apoptosis (13).

B lymphopoiesis occurs in the BM of adult mice in close association with stromal cells that provide factors essential for proliferation and survival such as c-kit ligand (KL), Flt3/Flk2 ligand, stromal cell-derived factor-1, and IL-7 (15, 16, 17, 18). IL-7 is the major cytokine driving expansion of committed pro-B lymphocytes and is correlated with the induction of c-myc and N-myc, transcription factors that induce proliferation and contribute to oncogenesis when expressed ectopically in B lineage cells (19, 20). However, the mechanisms that coordinate differentiation and IL-7-driven expansion of BLPs are just beginning to be explored.

Growth factor-dependent survival is mediated in large part by induction of anti-apoptotic members of the bcl2 family of proteins (21). These proteins act as protectors of mitochondrial integrity, and function to prevent the release of cytochrome c that occurs as a result of a decrease in mitochondrial membrane potential. Mitochondrial release of cytochrome c results in activation of caspase-9, an initiator caspase, which leads to activation of caspase-3, an effector caspase. Caspases are a family of cysteine proteases that are the major effectors of apoptosis and function in the destruction of proteins essential for cell viability such as lamins and inhibitors of DNase (22). The pathway of apoptosis initiated through disruption of the mitochondria is referred to as the intrinsic pathway because it is generally initiated by signals that are intrinsic to the cell. In contrast, extracellular proteins that bind to receptors of the TNF family, such as CD95/Fas, can directly initiate apoptosis through activation of receptor-associated caspase-8, which functions as an initiator caspase that can cleave and activate caspase-3 (23).

Caspase-2 has recently been identified as a caspase that mediates apoptosis in response to cell stress such as mitotic catastrophe or DNA damage (24, 25). Caspase-2 has a long pro-domain typical of initiator caspases and has been shown to associate with RAIDD, an adaptor molecule implicated in TNFR signaling (26). Caspase-2, RAIDD, and PIDD associate in a high m.w. complex that functions as a death inducing complex called the "PIDDosome" (27). Induction of PIDD requires the transcription factor p53 suggesting that apoptosis induced by DNA damage may occur through activation of the PIDDosome (28). Indeed, caspase-2 has been shown to function upstream of the mitochondria to induce apoptosis in response to DNA damage (25). Thus, caspase-2 has been suggested to activate the intrinsic pathway of apoptosis in response to cell stress. However, caspase-2 cleaves substrates that may lead to cell death even in the absence of mitochondrial dysfunction, such as the Golgi protein golgin-160, suggesting that it may function directly in disruption of cellular functions (29).

We have shown previously that the E-protein antagonist Id3 induces apoptosis of BLPs (13, 14). Here, we demonstrate that ectopic expression of Id3 induces apoptosis of BLPs via a caspase-2-dependent mechanism that does not require p53 and cannot be inhibited by bcl2. Analysis of gene expression in primary and transformed B lineage cells suggests that activation of caspase-2 is likely the consequence of growth arrest induced by loss of multiple proteins involved in different aspects of proliferation. Our previous studies have shown that B-lineage cells expressing HEB and E2-2 in the absence of E2A fail to proliferate optimally in response to IL-7. We hypothesize that Id3 results in a more "catastrophic" growth arrest leading to induction of caspase-2 and apoptosis. Consistent with this hypothesis, we find that chemicals that induce growth arrest also lead to activation of caspase-2 and apoptosis. Our data demonstrate that E-protein activity directly links differentiation of B lineage cells to proliferation and survival, and further indicate that distinct thresholds of E-protein activity are required for activation of genes involved in survival, proliferation, and differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Mice were housed at the University of Chicago vivarium in accordance with the requirements of The University of Chicago Institutional Animal Care and Use Committee. C57B/6J and p53–/– mice were purchased from The Jackson Laboratory, and H2K-bcl2 transgenic mice were provided by Dr. J. Domen (Duke University, Durham, NC).

Cell culture

Primary BLP cultures were established after enrichment of B220+ cells on a magnetic column as described previously (11). Primary cells were cultured in OPTI-MEM supplemented with 10% FBS, 1x penicillin/streptomycin/glutamine, 50 µM 2-ME, a 1/100 dilution of IL-7 supernatant (from the J558-IL-7 producing line; a gift from Dr. F. Melchers, Basel Institute, Basel, Switzerland) and a 1/500 dilution of KL supernatant (from the CHO-MGF line; a gift from the Genetics Institute). Cell lines were cultured in RPMI 1640 supplemented with 10% FBS, 1x penicillin/streptomycin/glutamine (Invitrogen Life Technologies), 50 µM 2-ME. Caspase inhibitors, roscovitine, hydroxyurea, nocodazole, and 17{beta}-estradiol and 4-hydroxy-tamoxifen were purchased from Calbiochem.

Retroviral infection

The Id3, ER, ERId3, and E47ER retroviral vectors have been described elsewhere and were produced in Phoenix cells as described previously (14, 30). Cells were infected by spin-inoculation as described (31).

Caspase assays

Cell extracts were made at a concentration of 4 x 107 cells/ml by lysis in buffer 1 (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA containing protease inhibitors) for 15 min on ice. Debris was removed by centrifugation. Caspase activity was measured using 50 µl of extract in buffer 2 (10 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% CHAPS, and 10% sucrose) containing the relevant caspase substrate at a final concentration of 40 µM. The caspase substrates were z-VD-VAD-AFC (caspase-2), Ac-DEVD-AFC (caspase-3/7), z-VEID-AFC (caspase-6), Ac-LETD-AFC (caspase-8), and Ac-LEHD-AFC (caspase-9) and were purchased from Bachem Biosciences. The reaction was allowed to proceed with gentle agitation for 1 h at 37°C before determining fluorescence.

Apoptosis assay

Cells were stained with annexin V for 15 min before analysis as suggested by the manufacturer (BD Pharmingen). Dihydroethidium (DHE; Molecular Probes) was added to cell cultures at a concentration of 50 µM for 15 min before harvest and analysis. Cells were analyzed by flow cytometry using a FACSCalibur.

Gene array analysis

GFP+ cells were sorted from ER- and ERId3-expressing cells 6 h after addition of 1 µM 17{beta}-estradiol (32 h postinfection). RNA was extracted using TRIzol reagent, and the integrity of the RNA was assessed by Northern blot analysis. The target preparation protocol was as described in the Affymetrix GeneChip Expression Analysis Manual with minor modifications. Briefly, 10 µg of total RNA was used to synthesize double-stranded cDNA using the Superscript Choice System (Invitrogen Life Technologies). First-strand cDNA synthesis was primed with a T7-(dT24) oligonucleotide. From the phase-log gel-purified cDNA, biotin-labeled antisense cRNA was synthesized using BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics). After precipitation with 4 M lithium chloride, 20 µg of cRNA was fragmented in fragmentation buffer (40 mM Tris-acetate (pH 8.1), 100 mM KOAc, 30 mM MgOAc) for 35 min at 94°C, and then 10 µg of fragmented cRNA was hybridized to Affymetrix mouse U74v2 chips for 16 h at 45°C and 60 rpm in an Affymetrix Hybridization Oven 640. The arrays were washed and stained with streptavidin PE in Affymetrix Fluidics Station 450 using Affymetrix GeneChip protocol and then scanned using an Affymetrix GeneChip Scanner 3000. The data were analyzed using Affymetrix Microarray Suite 5.0 and Microsoft Excel.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Id3 induces apoptosis in BLPs

We have shown previously that ectopic expression of Id3 in BLPs leads to the induction of apoptosis indicating that E-proteins are required for survival (13). To study the mechanism by which Id3 promotes BLP cell death, we established an assay in which a retrovirus producing Id3, or an estradiol inducible from of Id3 (ERId3), and enhanced GFP is used to antagonize E-protein activity (32). Within 24 h after addition of 17{beta}-estradiol to primary BLPs infected with the ERId3-producing retrovirus, a subset of GFP+ cells bound annexin V, a marker of apoptosis (Fig. 1A). In contrast, BLPs infected with an ER-producing retrovirus did not bind annexin V after addition of 17{beta}-estradiol. In addition, GFP+ cells were rapidly lost from ERId3 virus-infected, but not ER virus-infected, cultures suggesting a specific loss of ERId3-expressing cells (Fig. 1B). Interestingly, at any time point after infection, only a subset of GFP+ cells (10–30%) bound annexin V, suggesting that the induction of apoptosis may occur asynchronously in these cells (Fig. 1C). However, by 96 h after infection, most GFP+ cells had disappeared from ERId3-expressing cultures and the cells stained with trypan blue, indicating that the ERId3-expressing cells had died.



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  		FIGURE 1. Induction of apoptosis by Id3 in BLPs. A, B220+ cells from mouse BM were expanded in KL plus IL-7 for 4 days before infection with retrovirus producing ER, or ERId3, and GFP. Twenty-four hours postinfection, the cells were treated with 17{beta}-estradiol (1 µM). Forty-eight hours postinfection, the cells were stained with annexin V and examined by flow cytometry. B, The percentage of cells expressing GFP, and therefore ER ({blacksquare}) or ERId3 ({square}), was determined by flow cytometry 0, 8, 36, and 72 h after addition of 17{beta}-estradiol. C, The percentage of GFP+ ER-expressing (gray bars) or ERId3-expressing (black bars) cells binding annexin V was determined 0, 8, and 36 h after addition of 17{beta}-estradiol. D, 70Z/3 pre-B cells were infected with retrovirus producing ER or ERId3 plus GFP in addition to retrovirus producing E47ER plus hCD25 or hCD25 only, and the percent of hCD25+ cells expressing GFP was examined 0, 24, and 48 h after addition of 17{beta}-estradiol and tamoxifen (1 µM). The percentage of hCD25+ cells expressing GFP is shown for populations infected with hCD25 plus ER-GFP ({square}), hCD25 plus ERId3 ({blacksquare}), E47ER-hCD25 plus ER-GFP ({circ}), and E47ER plus ERId3-GFP (•).

 
To determine whether Id3-induced cell death was mediated through inhibition of E-proteins, we infected a pre-B cell line, which undergoes apoptosis in response to Id3 (see below), with both ERId3 and a tamoxifen-inducible form of the E-protein E47 (E47ER) (30). The E47ER retrovirus produces both E47ER and hCD25 from a single mRNA interrupted by an internal ribosomal entry site. Therefore, cells infected with both the ERId3- and E47ER-producing retroviruses can be identified by coexpression of GFP and hCD25. Remarkably, E47ER rescued the survival of ERId3-expressing cells as evidenced by the maintenance of GFP+ cells when both ERId3 and E47ER are expressed in the same cells (Fig. 1D). In contrast, ERId3-expressing cells coinfected with a retrovirus producing hCD25 only showed a rapid decline in GFP+ cells, consistent with the induction of apoptosis (Fig. 1D). Therefore, Id3-induced apoptosis can be prevented by ectopic expression of the E-protein E47. In addition, retroviral mediated expression of Id2 leads to the death of BLPs (data not shown). This finding is consistent with the hypothesis that Id3-induced apoptosis is mediated through inhibition of E-proteins, because the only common feature between Id3 and Id2 is the HLH domain that can interact with E-proteins.

Id3 induces death in transformed B lineage cells representing different stages of differentiation

We also tested the ability of Id3 to inhibit the survival of transformed B lineage cells. As was the case with primary BLPs, all of the pro-B and pre-B lymphocyte cell lines that we tested showed evidence of cell death after activation of ERId3 (Fig. 2). In these cell lines, apoptosis was measured as a loss of GFP+ cells from the culture and an increase in reactivity with DHE, an indicator of reactive oxygen species, which is associated with mitochondrial dysfunction in lymphocytes (33). Apoptosis in the 70Z/3 and 38B9 cell lines was also detected using an Ab that recognizes the activated form of caspase-3 and CaspaTag, a fluorescent pan-caspase substrate (data not shown). The immature B lymphocyte cell line Wehi 231 and a hybridoma cell line FS1 also became DHE+ within 12 h after addition of 17{beta}-estradiol to ERId3, but not ER, virus-infected cultures, and the frequency of GFP+ cells declined (Fig. 2). In addition, primary LPS-activated splenic B lymphocytes were induced to bind DHE after activation of ERId3 (Fig. 2B). Therefore, primary and transformed cells representing multiple stages of B lymphocyte development are susceptible to Id3-induced apoptosis indicating that they require E-protein activity for their survival.



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  		FIGURE 2. Transformed cell lines representing multiple stages of B cell differentiation are susceptible to Id3-induced apoptosis. A, Cell lines were infected with ER-GFP (closed symbols)- or ERId3-GFP (open symbols)-producing retrovirus, and the percentage of cells expressing GFP was determined by flow cytometry 0, 12, and 24 h after addition of 17{beta}-estradiol. 70Z/3 = pre-B ({diamond}, {diamondsuit}), 38B9 = pro-B ({triangleup}, {blacktriangleup}), Wehi231 = immature B ({circ}, •), FS1 = activated mature B/hybridoma ({square}, {blacksquare}). B, Cell lines and LPS blasts were infected with ER-GFP (grey bars) or ERId3-GFP (black bars), and the percentage of GFP+ cells showing increased staining with DHE was determined 12 h after addition of 17{beta}-estradiol.

 
Id3-induced apoptosis is not inhibited by Bcl-2 and does not require p53

E-proteins have been implicated in the expression of the receptor for IL-7, an essential cytokine for pro-B and large pre-B lymphocyte survival (34, 35). However, we found that the GFP+ and GFP– cells from both Id3 and GFP virus-infected cultures expressed similar levels of IL-7R{alpha} on the cell surface 36 h after infection even though >20% of the Id3-expressing cells bound annexin V (data not shown). However, we note that IL-7R{alpha} expression is reduced on the 38B9 pro-B and 70Z/3 pre-B cell lines 24 h after addition of 17{beta}-estradiol to ERId3-expressing cells although IL-7 is not required for the survival of these transformed cell lines. Therefore, we conclude that loss of IL-7R{alpha} expression is not the cause of cell death after expression of Id3 in BLPs.

To gain further insight into the mechanism of Id3-induced apoptosis we tested the ability of Id3 to induce apoptosis in BLPs isolated from H2K-bcl2 transgenic mice (36). Bcl-2-expressing cells are generally resistant to apoptosis initiated through the intrinsic pathway (37). Remarkably, H2K-bcl2 transgenic BLPs expressing Id3 bound annexin V within 36 h after infection and the percentage of GFP+ cells declined 8-fold by 96 h postinfection (Fig. 3, A and B). In comparison with Id3-expressing wild-type (WT) BLPs, the percentage of GFP+ cells binding annexin V was slightly decreased (12 vs 19%); however, cell death was not inhibited. Therefore, expression of Bcl-2 in BLPs is not sufficient to rescue these cells from Id3-induced apoptosis.



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  		FIGURE 3. Id3-induced apoptosis is not inhibited by Bcl2 and does not require p53. A, B220+ cells isolated from BM of H2K-bcl2 transgenic or WT mice were expanded in KL plus IL-7 for 4 days before infection with retrovirus producing GFP only or Id3 plus GFP. Thirty-six hours postinfection, the cells were stained with annexin V and examined by flow cytometry. B, Ninety-six hours postinfection, the percentage of WT (gray bars) or H2K-bcl2 transgenic (black bars) BLPs expressing GFP in populations infected with GFP- or Id3-GFP-producing virus was determined by flow cytometry. C, B220+ cells isolated from BM of p53–/– and p53+/+ mice were expanded in KL plus IL-7 for 4 days before infection with retrovirus producing GFP or Id3 plus GFP. Seventy-two hours postinfection, the cells were stained with annexin V and examined by flow cytometry. D, Ninety-six hours postinfection, the percentage of p53+/+ (gray bars) and p53–/– (black bars) BLPs expressing GFP in populations infected with GFP- or Id3-GFP-producing virus was determined by flow cytometry.

 
We also tested the requirement for p53 in Id3-induced apoptosis. p53 initiates apoptosis through the intrinsic pathway in response to genotoxic stress such as DNA damage (38). Infection of p53–/– BLPs with Id3 retrovirus resulted in the appearance of annexin V+ cells at levels similar to those observed in p53+/+ cells (26 vs 19% of GFP+ cells) (Fig. 3C). In addition, GFP+ cells declined 20-fold over the course of 96 h in culture (Fig. 3D). In contrast, the GFP-producing retrovirus did not induce annexin V binding in either p53–/– or p53+/+ BLPs, and the percentage of cells expressing GFP remained constant in these cultures. Taken together, our data indicate that Id3-induced apoptosis occurs in the absence of p53 and is not inhibited by Bcl-2.

Id3 promotes activation of caspases-2, -3, and -9

We have shown previously that Id3 induces apoptosis through a caspase-dependent mechanism (14). Therefore, we determined which caspases become activated by Id3 in B lineage cells using a panel of fluorogenic caspase substrates. We found that caspase-2, -3/7, and -9, but not -6 or -8, were activated within 8 h after addition of 17{beta}-estradiol to ERId3, but not ER, virus-infected cells (Fig. 4A). This was the case in both primary BLPs and transformed B lineage cell lines (data not shown). The absence of caspase-8 activation indicates that Id3 does not induce apoptosis through activation of death receptors such as Fas/CD95 or the TNFR. Caspase-3 and -9 can be activated through the intrinsic pathway of apoptosis; however, the inability of bcl2 to rescue Id3-induced apoptosis suggests that this pathway is not essential. Therefore, caspase-2 was of interest, particularly given its ability to function as an initiator caspase upstream of the mitochondria during stress-induced apoptosis (25). Interestingly, we found that caspase-2, -3, and -9 were activated in both H2K-bcl2 transgenic and p53–/– BLPs infected with ERId3-, but not ER-producing, virus, consistent with the susceptibility of these cells to Id3-induced apoptosis (Fig. 4B). Therefore, caspase-2 may play an essential role in apoptosis in response to inhibition of E-protein activity in B lineage cells.



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  		FIGURE 4. Induction of caspases-2, -3, and -9 by Id3. A, 70Z/3 pre-B cells were infected with ER- or ERId3-producing retrovirus. Twenty-four hours after infection, the cells were treated with 17{beta}-estradiol (1 µM) for 8 h. The cells were harvested, and whole cell extracts were incubated with each of the indicated caspase substrates for 1 h before the level of fluorescence in each reaction was assayed. Each extract was measured in triplicate. The percentage of cells expressing GFP in the ER and ERId3 virus-infected cultures was 69 and 65%, respectively. Identical results were obtained using primary IL-7-dependent BLPs. B, Relative fluorescence after assay with each of the indicated caspase substrates was determine for extracts prepared from KL plus IL-7 cultures of H2K-bcl2 transgenic or p53–/– BLPs infected with ER (black bars)- or ERId3 (gray bars)-producing virus. Each extract was measured in triplicate. Infection efficiency for ER and ERId3 in H2K-bcl2 transgenic BLPS was 60 and 58%, and in p53–/– BLPs was 75% and 70%, respectively. The H2K-bcl2 transgenic BLPs and p53–/– BLPs were assayed in independent experiments.  

 
Caspase-2 is required for Id3-induced apoptosis

To determine whether caspase-2 is required for Id3-induced apoptosis we tested the ability of the caspase-2 inhibitor VD-VAD-fmk to interfere with ERId3-induced binding of annexin V in BLPs. ERId3-expressing BLPs were treated with VD-VAD-fmk for 30 min before addition of 17{beta}-estradiol, and the cells were examined 8 h later for their ability to bind annexin V. Remarkably, VD-VAD-fmk was as efficient as z-VAD-fmk, a pan-caspase inhibitor, at antagonizing Id3-induced binding of annexin V (Fig. 5). In contrast, the caspase-3 inhibitor z-DEVD-fmk was not able to prevent Id3-induced annexin V binding, although a partial inhibition was observed consistently (Fig. 5). We were unable to assess the ability of the caspase-9 inhibitor LEVD-CHO to inhibit Id3-induced annexin V binding because it caused significant toxicity when added to BLPs even in the absence of Id3. Therefore, we conclude that activation of caspase-2, but not caspase-3, is essential for the induction of annexin V binding by Id3 in BLPs. Our data also suggest that caspase-3 may play a significant but nonessential role in this apoptotic pathway.



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  		FIGURE 5. Inhibition of caspase-2 prevents Id3-induced annexin V binding. B220+ cells were isolated from BM of WT mice and expanded in KL plus IL-7 for 4 days before infection with ERId3 virus. The cells were treated with vehicle (DMSO) or caspase inhibitors (50 µM) for 30 min before addition of 17{beta}-estradiol (1 µM) or vehicle (ethanol). The cells were stained with annexin V and analyzed by flow cytometry 8 h after addition of 17{beta}-estradiol. The percentage of GFP+ cells binding annexin V is presented. One representative experiment of three is shown. z-VAD, pan-caspase inhibitor; z-VD-VAD, caspase-2 inhibitor; DEVD, caspase-3 inhibitor. One of three representative experiments is shown.

 
Id3 inhibits expression of genes associated with B lineage differentiation and proliferation

To gain insight into how Id3 promotes activation of caspase-2, we examined gene expression in primary BLPs and the 70Z/3 pre-B cell line expressing ERId3 or ER 6 h after addition of 17{beta}-estradiol. We chose the 6-h time point because this is the earliest time after addition of 17{beta}-estradiol at which an increase in annexin V binding can be detected consistently on BLPs and caspase activation can be detected in both cell types (data not shown). We reasoned that if ERId3 is able to initiate apoptosis within this time then the essential E-protein target genes must be altered within this time. However, using this time point requires that the target genes have a half-life <6 h, necessitating that many E-protein target genes may not be detected. In addition, we only considered genes that were altered in both primary BLPs and 70Z/3 cells based on the assumption that there is a common E-protein target gene(s) in these cells that is antagonized by Id3 to induce cell death. Given these assumptions, we looked for genes that were decreased or increased by at least 2-fold in both cell types 6 h after induction of ERId3 as compared with ER (Table I).


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  		Table I. Genes altered by ER-Id3 as compared with ER in both 70Z/3 and BLPs

 
The majority of genes were expressed at equivalent levels in ERId3 and ER virus-infected cells (data not shown). However, we identified 19 genes that were decreased in ERId3-expressing cells as compared with ER-expressing cells (Table I). Among the genes decreased in both cell types are previously reported E-protein targets including IL-7R{alpha}, Ig{kappa}, the surrogate L chain proteins {lambda}5 and VpreB, and B lymphoid kinase (35, 39, 40). Genes with protein products that function in B lymphocyte development, but have not been implicated as E-protein targets, were also decreased, such as the chemokine receptor CXCR4 (lcr-4) and CD72 (16, 41). Notably, multiple genes required for cell proliferation were decreased in both cell types including c-myc, nucleostemin, peter pan, and adenylosuccinate synthetase (42, 43). In addition, we have previously shown that N-myc mRNA is decreased by ERId3 in both cell types (11). Five genes were increased by expression of ERId3 in both BLPs and 70Z/3 cells (Table I). One of these proteins, Tob, has been implicated as a negative regulator of mature T lymphocyte expansion (44). A combination of Northern blot analysis, RT-PCR, and flow cytometry confirmed the differential expression of many of the genes identified on the gene array in BLPs or 70Z/3 cells including IL7R{alpha}, {lambda}5, CXCR4, c-myc, nucleostemin, peter pan, and Tob (data not shown). Therefore, genes associated with lymphocyte differentiation and proliferation are inhibited by ectopic expression of Id3 in B lineage cells. However, surprisingly, none of the differentially expressed genes had a clear function in lymphocyte survival.

An analysis of genes that were inhibited by ERId3 but not ER in BLPs that were either not inhibited by >2-fold or not expressed in 70Z/3 cells revealed a role for Id3 in suppression of numerous previously identified E2A target genes. These genes include the recombinase activating genes Rag-1 and Rag-2, TdT, Ig{kappa}, and EBF (data not shown). In addition, cyclin D2 and cyclin D3 were found to be decreased by Id3 in primary BLPs but were altered by <2-fold in 70Z/3 cells including cyclin D2 and cyclin D3 (gene array data may be viewed at <http://madam.bsd.uchicago.edu:8080/>). Taken together, our data indicate that, within 6 h after activation of ERId3, E-protein-dependent genes are altered in expression as are many genes involved in lymphocyte differentiation or proliferation.

Id3 induces growth arrest in B lineage cells

The results of our gene array analysis led us to hypothesize that caspase-2 may be activated in response to a severe growth arrest induced by Id3 through alterations in expression of genes involved in many aspects of proliferation including ribosome biosynthesis (nucleostemin), purine synthesis (adenylosuccinate synthetase), and cell cycle regulation (N-myc and c-myc). Indeed, activation of ERId3 in 70Z/3 pre-B cells led to the rapid accumulation of cells in the G1-phase (65 vs 43% at 24 h) and a loss of cells from the S-phase (13 vs 50% at 24 h) of the cell cycle when compared with ER-expressing cells (Fig. 6). Similar results were observed in primary BLPs and were also observed when BLPs were treated with a TAT-Id3 fusion protein (14). Therefore, Id3 inhibits expression of multiple genes involved in varied aspects of B lymphocyte proliferation and induces growth arrest in B lineage cells.



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  		FIGURE 6. Id3 induces growth arrest in B lineage cells. 70Z/3 pre-B lymphocytes were infected with ER or ERId3 retrovirus. Twenty-four hours postinfection, GFP+ cells were isolated by sorting on the flow cytometer and placed in culture for 8 h. 17{beta}-Estradiol was added to independent wells for 0, 11, 24, or 36 h. BrdU was added to the cultures for the final 15 min. The cells were harvested and stained with anti-BrdU and propidium iodide. The percentage of cells in each phase of the cell cycle is indicated.

 
Growth arrest induces apoptosis and activates caspase-2 in B lineage cells

Our analysis of gene expression combined with the observation that Id3 induces growth arrest lead us to hypothesize that growth arrest may place a stress on the cell leading to activation of caspase-2. To test the ability of cell cycle arrest to promote caspase activation, various chemicals known to cause cell cycle arrest were examined for their ability to induce cell cycle arrest and activate caspase-2, -3/7, and -9. These chemicals included roscovitine, which prevents activation of CDKs resulting in a G1-phase arrest; nocodazole, which inhibits microtubules resulting in a G2/M-phase arrest;, and hydroxyurea, which prevents DNA replication resulting in a G1-phase arrest (Fig. 7A). Analysis of extracts obtained from cells treated with roscovitine, nocodazole, or hydroxyurea for 8 h demonstrated that each of these chemicals induces an increase in caspase-2, -3/7, and -9 activity (Fig. 7B). In addition, an increase in annexin V binding was observed in BLPs treated with roscovitine (10.5%), nocodazole (6%), or hydroxyurea (9.7%) as compared with untreated cells (2.2%), indicating that growth arrest can lead to apoptosis in primary cells (Fig. 7C). Moreover, the majority of primary BLPs and transformed B lineage cell lines died within 24 h after treatment with each of these chemicals as determined by trypan blue exclusion (data not shown). These data are consistent with the hypothesis that severe growth arrest leads to activation of caspase-2 and apoptosis in B lineage cells and that Id3 induces apoptosis by inhibiting expression of E-protein-dependent genes that are required for proper cell cycle progression.



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  		FIGURE 7. Cell cycle arrest induces apoptosis in BLPs. A, 70Z/3 pre-B cells were cultured in the presence of DMSO, roscovitine (25 µM), nocodazole (200 ng/ml), or hydroxyurea (1 mM) for 8 h, and BrdU was added for the final 15 min of culture. The cells were harvested and stained with anti-BrdU Ab and propidium iodide before analysis by flow cytometry. B, Extracts prepared from 70Z/3 pre-B cells cultured with DMSO, roscovitine, nocodazole, or hydroxyurea for 8 h were tested for their ability to hydrolyze fluorogenic substrates for caspase-2, caspase-3, and caspase-9. Each assay was performed in triplicate. C, B220+ BM cells were expanded for 4 days in KL plus IL-7 and infected with ER virus to allow detection of GFP+ cells, as a marker of cell integrity. Twenty-four hours postinfection, the cells were treated with vehicle (DMSO), roscovitine, nocodazole, or hydroxyurea, and the cells were stained with annexin V 8 h later. The percentage of GFP+ cells staining with annexin V is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We show in this study that Id3 induces apoptosis in B lineage cells through a pathway dependent on activation of caspase-2. Our gene expression data lead us to suggest that the most likely mechanism for caspase-2 activation is growth arrest induced by inhibition of multiple genes involved in proliferation including c-myc, N-myc, nucleostemin, peter pan, and adenylosuccinate synthetase. Previous studies from our lab and others have defined a role for E-proteins in B lymphocyte differentiation through control of a transcriptional hierarchy leading to expression of genes involved in lymphocyte differentiation (45). The data presented in this study help to define a second major role for E-proteins in maintaining homeostasis in B lineage cells by preventing catastrophic growth arrest and apoptosis.

The conclusion that apoptosis is occurring through a caspase-2-dependent pathway is based, in part, on cleavage of VD-VAD-AFC by cellular extracts of Id3-infected cells and by the ability of VD-VAD-fmk to inhibit Id3-induced exposure of phosphadidylserine (PS) on the cell surface. VD-VAD is a high-affinity substrate for caspase-2; however, it can be cleaved at high concentrations by caspases-3 and -7 (46). Therefore, it is important that DEVD-fmk, a selective inhibitor of caspase-3/7, was much less efficient than VD-VAD-fmk at inhibiting Id3-induced PS exposure. The inability of bcl2 to rescue Id3-induced apoptosis further suggests that Id3-induces an apoptotic pathway distinct from the classical intrinsic pathway. In addition, we were able to detect very faint bands of a size consistent with the cleaved products of caspase-2 and -3 by Western blot analysis suggesting that caspase-2 and -3 are cleaved in these cells (data not shown). The difficulty in detecting these cleaved products is likely due to their highly labile nature and the heterogeneity of cells undergoing apoptosis in response to Id3. In general, only 10–30% of Id3-expressing cells show PS on the cell surface at any given point in time, and only a subset of these would be expected to possess detectable cleaved caspases making detection of cleaved products difficult. We hypothesize that this heterogeneity in apoptosis may reflect the asynchronous nature of cell cycle progression in the population of cells under analysis. Taken together with our observation that BLPs from H2K-bcl2 transgenic mice are sensitive to Id3-induced apoptosis, our data indicate that caspase-2, rather than caspase-3, is the initiator of this pathway to cell death.

Caspase-2 is activated in response to cellular stress induced by DNA damage and growth arrest; however, it is not known how these stress responses are coupled to activation of caspase-2 (24, 25). Caspase-2 can be activated by aggregation in a protein complex including PIDD and RAIDD and may not require proteolytic processing (27). PIDD is induced by p53; however, Id3-induced apoptosis is p53 independent, suggesting that PIDD may not be essential for caspase-2 activation in these cells. Moreover, neither PIDD nor RAIDD expression were induced by Id3 in BLPs (data not shown). Two mRNAs have been identified that code for a long (L) and a short (S) form of caspase-2 that can activate or inhibit apoptosis, respectively (47). Therefore, loss of caspase-2S or increased expression of caspase-2L could lead to apoptosis. However, caspase-2S mRNA was not detected in BLPs and the level of caspase-2L mRNA did not change after expression of Id3 (data not shown). Therefore, the mechanism coupling Id3-induced growth arrest to caspase-2 activation remains to be determined.

Caspase-2-deficient mice do not show an obvious defect in B lymphocyte development, although mature B lymphocytes are resistant to granzyme B and perforin-induced apoptosis (48). However, the lack of a phenotype in these mice does not exclude an important role for caspase-2 in homeostasis of B lineage cells. It may be that the requirement for caspase-2 is only manifest under conditions of stress, for example, in cells that lose E-protein activity through either a loss in E-protein gene expression or through induction of Id proteins (for example, after exposure to TGF-{beta}) (14). Alternatively, another caspase such as caspase-9 may replace the function of caspase-2 in these mice as is observed in the nervous system (49). Our data suggest that a severe or catastrophic growth arrest may be required for induction of caspase-2 in BLPs. This possibility is suggested by the fact that BLPs with a low abundance of E-protein activity, such as we reported in E2A–/–EBF+ BLPs, proliferate poorly in response to stromal cells plus IL-7, but do not undergo obvious apoptosis (11). However, like WT BLPs, the E2A–/–EBF+ BLPs undergo apoptosis when E-protein activity is further reduced by introduction of Id3. This finding suggests that a more severe or different form of growth arrest may occur when E-protein levels fall below a critical threshold. This is consistent with our finding that c-myc, nucleostemin, peter pan, cyclin D2, and cyclin D3 mRNA levels only decline when E-protein activity falls below this critical level because these genes are expressed in E2A–/–EBF+ BLPs at levels similar to WT BLPs (11). Our data also suggest that BLPs have significant latitude to modulate E-protein activity and decrease proliferation without inducing apoptosis. Only under conditions where E-protein activity is inhibited beyond this threshold would caspase-2 become activated and kill the cell.

We conclude that inhibition of E-protein activity is likely to lead to apoptosis through induction of a catastrophic growth arrest. This conclusion is based on the observed decrease in multiple genes whose protein products are involved in proliferation, evidence of growth arrest, and the ability of forced cell cycle arrest (using roscovitine, nocodazole, or hydroxyurea) to induce caspase-2 activity and PS exposure on BLPs. Interestingly, the E-protein-dependent genes identified in our gene array screen participate in proliferation in distinct ways. For example, c-myc and N-myc are basic HLH transcription factors that likely activate genes required for cell cycle progression, although the essential targets have yet to be identified. In contrast, adenylosuccinate synthetase is required for purine synthesis, nucleostemin is thought to play a role in ribosome production and the functions of peter pan have not been well described (42, 43, 50). Notably, neither N-myc nor cyclin D2 expression can overcome Id3-induced apoptosis consistent with the hypothesis that multiple E-protein-dependent genes are involved in this catastrophic event (data not shown). Taken together, our data indicate that caspase-2 is activated after loss of E-protein activity due to growth arrest caused by loss of multiple proteins required for cell proliferation.

Our previous data combined with data presented in this study demonstrate that the threshold of E-protein activity in BLPs critically determines the fate of these cells. At high levels of activity cells are able to activate genes involved in differentiation and proliferation. A decrease in E-protein activity, for example by loss of E2A but not HEB and E2-2, results in a decline in N-myc expression and failure to proliferate optimally in response to IL-7, whereas a further decline in activity results in catastrophic growth arrest, activation of caspase-2, and consequent apoptosis. Therefore, the level of E-protein activity functions as a major integrator of differentiation, proliferation, and survival of B lineage cells.


   Acknowledgments
 
I am grateful to Kees Murre, in whose lab many of the initial observations for these studies were made; Rachel Brumbaugh, Laura Glasgow, Christopher Seet, Latishya Steele, and Christina Spaulding for technical assistance; Xinmin Li in the Functional Genomic Facility for screening the gene arrays; James Marvin and Ryan Duggan for cell sorting; and Markus Boos for comments on the manuscript. I also thank Marcus Peter and Bryan Barnhart for assistance setting up the caspase assays, use of their fluorometer, and helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported in part by an award to the Division of Biological Sciences at University of Chicago under the Research Resources Program for Medical Schools of the Howard Hughes Medical Institute, an Institutional Research Award from the American Cancer Society, the Edward Mallinckrodt, Jr. Foundation, and the National Cancer Institute (R01 CA99978). Back

2 Address correspondence and reprint requests to Dr. Barbara L. Kee, Department of Pathology, University of Chicago, 5841 South Maryland Avenue, MC1089, Chicago, IL 60637. E-mail address: bkee{at}bsd.uchicago.edu Back

3 Abbreviations used in this paper: EBF, early B cell factor; BM, bone marrow; KL, c-kit ligand; WT, wild type; BLP, B lymphocyte progenitor; HLH, helix-loop-helix; DHE, dihydroethidium; PS, phosphadidylserine; L, long; S, short. Back

Received for publication January 6, 2005. Accepted for publication July 22, 2005.


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 Materials and Methods
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