Cancer Research The Future of Cancer Research: Science and Patient Impact AACR Conference on Molecular Diagnostics - 2008
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Cancer Research 66, 9903-9912, October 15, 2006. doi: 10.1158/0008-5472.CAN-06-0002
© 2006 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Cleavage of Misfolded Nuclear Receptor Corepressor Confers Resistance to Unfolded Protein Response–Induced Apoptosis

Angela Ping Ping Ng1,4, Jek Howe Fong1, Dawn Sijin Nin1, Jayshree L. Hirpara2, Norio Asou5, Chien-Shing Chen1,3, Shazib Pervaiz1,2,4 and Matiullah Khan1,3

1 Oncology Research Institute, Departments of 2 Physiology and 3 Medicine, Yong Loo Lin School of Medicine and 4 NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore; and 5 Department of Hematology, Kumamoto University School of Medicine, Kumamoto, Japan

Requests for reprints: Matiullah Khan, Oncology Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Clinical Research Center, Block MD11, 10 Medical Drive, Singapore 117597. Phone: 65-6874-8055; Fax: 65-6873-9664; E-mail: nmimmk{at}nus.edu.sg or Shazib Pervaiz. Phone: 65-6874-6602; Fax: 65-6778-8161; E-mail: phssp{at}nus.edu.sg.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have recently reported that accumulation of misfolded nuclear hormone receptor corepressor (N-CoR) as insoluble protein aggregates in acute promyelocytic leukemia (APL) cells induces endoplasmic reticulum (ER) stress and activates unfolded protein response (UPR). Although accumulation of misfolded proteins is known to trigger UPR-induced cytotoxic cell death in several neurodegenerative disorders, APL cells are notably resistant to UPR-induced apoptosis. The molecular basis for the paradoxical response of APL cells to UPR is not known. Here, we report that a glycoprotease, selectively expressed in APL cells, regulates the response of APL cells to UPR-induced apoptosis through processing of misfolded N-CoR protein. Results show that misfolded N-CoR is cleaved selectively in APL cells, and cellular extracts of APL cells and human primary APL cells contain activity that cleaves N-CoR protein. Purification and spectrometric analysis of N-CoR cleaving activity from an APL cell line reveals that it is a glycoprotein endopeptidase known as OSGEP. Furthermore, the cleavage of N-CoR in APL cells could be blocked by the broad-spectrum protease inhibitor AEBSF and by RNA interference–mediated down-regulation of OSGEP expression. AEBSF selectively inhibits growth and promotes apoptosis of APL cells possibly through a mechanism involving AEBSF-induced accumulation of insoluble N-CoR protein and by triggering ER stress. Taken together, these findings suggest that selective induction of protease activity in APL cells may represent a novel cytoprotective component of UPR, which could be exploited by tumor cells to survive the toxic insult of misfolded protein(s). (Cancer Res 2006; 66(20): 9903-12)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acute promyelocytic leukemia (APL) resulting from the fusion of the promyelocytic leukemia (PML) protein and the retinoic acid receptor (RAR)-{alpha} is a subtype of acute myelogenous leukemia (AML; refs. 1–3). In APL cells, the nuclear PML oncogenic domains (POD) are dispersed across the nucleus and cytoplasm as microspeckles. These microspeckles rearrange back into normal dot-like pattern in APL patients treated with the differentiating agent all-trans-retinoic acid (4–6). We have previously reported that the nuclear hormone receptor corepressor (N-CoR) is a component of PODs and that PML-RAR-mediated misfolding and cytosolic localization of N-CoR contributes to the disintegration of PODs in APL (7, 8). First identified as a corepressor of RAR and thyroid hormone receptor (9, 10), N-CoR was later shown to be essential for transcriptional repression mediated by the tumor suppressor Mad and by other sequence-specific transcription factors (11, 12). Reports also suggest an active role for N-CoR in preventing tamoxifen-induced proliferation of breast cancer cells by repressing a subset of target genes involved in cell proliferation (13). Interestingly, recent findings provide evidence that N-CoR recruitment by PML-RAR contributes to abnormal growth and transformation of APL cells by repressing retinoic acid–responsive genes essential for the maturation of promyelocytic cells (14, 15). Retinoic acid relieves this repression by inducing dissociation of N-CoR from PML-RAR and in turn promotes differentiation of APL cells (16, 17). We have previously reported an unexpected role for N-CoR in the pathogenesis of APL; PML-RAR-mediated accumulation of insoluble N-CoR protein in the endoplasmic reticulum (ER) induced ER stress and activated unfolded protein response (UPR), which contributed to differentiation arrest of APL cells (8).

UPR, triggered by misfolded proteins in the ER and initiated by proteolytic processing of activating transcription factor 6 (ATF6), involves the coordinated transcriptional activation of a set of genes encoding ER chaperones and cell death pathways, such as GRP78 and protein disulfide isomerase (PDI; refs. 18, 19). The accumulation of misfolded protein(s) initially triggers the chaperone-mediated or ER-associated degradation (ERAD)–mediated cytoprotective branch of UPR, but if the accumulation of misfolded protein(s) and the ensuing stress persists, the proapoptotic limb of UPR takes over (18). Thus, UPR plays a critical role in protecting cells from the harmful effect(s) of misfolded proteins by promoting chaperone-mediated refolding or death of terminally damaged cells. To that end, tumor cells somehow acquire escape mechanisms that prevent ER stress from reaching the threshold for triggering apoptotic death. For instance, in vitro studies have shown that treatment of ER-stressed myeloma cells with protease inhibitors greatly promotes UPR and ultimately leads to the activation of apoptosis (20). This finding clearly implicates proteases in stress tolerance as well as in resistance to apoptosis in myeloma cells.

It was recently reported that several proteases (i.e., neutrophil elastase and cathepsin G) play significant role in the pathogenesis of murine APL via cleavage of the PML-RAR protein (21, 22). Although PML-RAR is a potent oncogene associated with APL, its oncogenic potential is strongly influenced by the cell type and its subcellular localization. For example, in in vitro studies and in transgenic mice models of APL, the expression of PML-RAR in hematopoietic cells (other than promyelocytes) and in nonhematopoietic cells is cytotoxic (23–26). These data suggest that PML-RAR, despite being a bona fide oncogene in promyelocytic cells, paradoxically functions as a "tumor suppressor" on expression in nonpromyelocytic cells. However, the molecular events underlying the cell type–specific effect of PML-RAR are poorly understood. Our recent data showed that expression of PML-RAR in non-APL embryonic kidney cells (293T) promoted accumulation of N-CoR protein as insoluble aggregates in the ER, whereas in an APL cell line, NB4, insoluble N-CoR accumulated mostly in the Golgi (8). We hypothesize that ER-accumulated misfolded N-CoR cooperates with PML-RAR to trigger cell death in non-APL cells, whereas in APL cells the toxic effect is neutralized due to the Golgi-specific structural and functional modification of misfolded N-CoR. To investigate how cell type–specific conformation dynamics of N-CoR protein could possibly influence the resistance of APL cells to UPR, here we set out to study the conformational status of N-CoR in selected APL and non-APL cells and its implication in the response of the cells to UPR-induced apoptosis. Our results link a glycoprotein endopeptidase, OSGEP, selectively expressed in APL cells, to the regulation of the response of APL cells to UPR-induced apoptosis via processing of the misfolded N-CoR protein.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents. The retinoic acid–sensitive APL cell line NB4 and the retinoic acid–resistant APL cell line UF-1 (27) were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) with 10% or 15% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), 100 units/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere of 5% CO2. The non-APL cell lines HL60, K562, and U937 were similarly maintained in RPMI 1640, whereas 293T cells were cultivated in DMEM enriched with 10% FBS. The N-CoR (C-20), PML, GRP78, PDI, and CAAT/enhancer binding protein homologous protein antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used as described before (8). The ER stress-inducing agents thapsigargin, DTT, and tunicamycin were used as described elsewhere (28).

Protease purification and in vitro cleavage assay. NB4 whole-cell extract in cleavage assay buffer was loaded onto a Superose 12 gel filtration column of high-performance liquid chromatography (HPLC), and purified fractions were incubated with recombinant N-CoR protein under optimized assay conditions. To make crude cellular extracts containing active proteases, NB4 cells or cryopreserved human APL primary cells were incubated in RSB buffer [10 mmol/L Tris (pH 8.0), 10 mmol/L NaCl, 3 mmol/L MgCl2, 0.1% NP40] at 4°C for 10 minutes and nuclei were removed by centrifugation. The supernatants were harvested, and protein content was determined. N-CoR substrate was prepared from 293T cells transfected with N-CoR and PML-RAR expression plasmids. Optimized cleavage assays were done in 300 mmol/L NaCl and 50 mmol/L Tris (pH 8.0) at 25°C or 37°C for 60 minutes. The reaction was stopped by heating at 50°C in SDS sample buffer, and proteins were resolved with SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes for Western blotting. Purified human neutrophil elastase, OSGEP, and aminopeptidase N were purchased from Elastin Products Co. (Owensville, MO).

N-CoR deglycosylation assay. NB4 cells were seeded at 15 x 104/mL and treated with 100 µmol/L AEBSF for 48 hours at 37°C with 5% CO2. Cells were harvested with a buffer mixture containing 5x reaction buffer, denaturing solution, and double-distilled water in proportions as described in Sigma enzymatic deglycosylation kit (EDEGLY, Sigma, St. Louis, MO). The sample was heated at 95°C for 5 minutes, and Triton X-100 was added to a final concentration of 0.75%. This was then divided into four portions: one as control and the rest sequentially digested with PNGase F, neuraminidase, and O-glycosidase at 37°C for 6 hours. The reaction was stopped by heating at 50°C for 10 minutes in SDS loading buffer. The proteins were resolved with 6% SDS-PAGE and transferred to PVDF membrane. Western blotting was carried out using anti-N-CoR (C-20).

N-CoR solubility assay and preparation of cytosolic fractions. N-CoR solubility assay was done as described previously (8). Soluble and insoluble fractions of cytosolic extracts of NB4 cells were prepared as described elsewhere (28). Briefly, NB4 cells were homogenized in a hypotonic buffer [10 mmol/L HEPES (pH 8.0), 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT, protease inhibitor cocktail] to prepare cytosolic and nuclear fractions. Aliquots of the cytosolic fraction were treated on ice with 1/10 volume of H2O, 5 mol/L NaCl, or 1 mol/L Na2CO3 for 1 hour and centrifuged 40,000 rpm for 1 hour. The resulting soluble and insoluble fractions were resolved by SDS-PAGE and stained with anti-N-CoR (C-20).

Protease inhibitor/EDTA assay. Preparation of N-CoR and NB4 lysates: 293T cells (1.4 x 106) were plated overnight and then transfected with N-CoR and PML-RAR using the Fugene transfection reagent (Roche, Penzberg, Germany). After 48 hours of incubation, cells were harvested using 2x NT, heated at 50°C for 10 minutes, and stored at –20°C. NB4 cells were harvested with RSB buffer, kept on ice for 10 minutes, mixed, and spun down at 800 x g for 5 minutes. The supernatant was transferred to a new tube for the experiment. N-CoR and NB4 lysates were mixed in equal portions. Various protease inhibitors and EDTA (12.5-100 mmol/L) were added as described elsewhere (21). The reaction mixtures were incubated at 25°C for 1 hour. Reaction was stopped with 4x SDS and heated at 50°C for 10 minutes. The samples were subjected to 6% SDS-PAGE.

O-sialoglycoprotein endopeptidase digestion assay. N-CoR in 2x NT was diluted with equal volume of RSB buffer. O-sialoglycoprotein endopeptidase (Gcp) from Mannheimia haemolytica (Cedarlane, Hornby, Ontario, Canada) was added at 10-fold dilution (0.01-1 µg), and one set of samples was each incubated at 25°C and 37°C for 1 hour concurrently. The reactions were stopped with 4x SDS loading buffer, heated at 50°C for 10 minutes, and subjected to 6% SDS-PAGE.

AEBSF inhibition assay. AEBSF inhibition assays were done in an equal mixture of N-CoR and RSB buffer at 37°C for 90 minutes with 4 µg Gcp in the presence or absence of AEBSF. The reactions were stopped by heating at 50°C for 10 minutes in SDS loading buffer. The proteins were resolved with 6% SDS-PAGE and transferred to PVDF membranes. Western blotting was carried out using anti-Flag (M2, Sigma).

OSGEP short hairpin RNA. The human OSGEP short hairpin RNA (shRNA) sequence cloned in pShag Magic version 2.0 vector (pSM2c) was purchased from Open Biosystems (Huntsville, AL). Approximately 2 x 106 NB4 cells were electroporated with 2 µg OSGEP shRNA vector using Amaxa (Cologne, Germany) electroporation kit following the protocol supplied by the company. Western blotting and apoptosis assays of electroporated cells were done as described elsewhere in this article. The rabbit polyclonal anti-OSGEP was raised against the human OSGEP peptide sequence CHRTPLSDSGVTQ and purified using an affinity column.

Cell proliferation assay. The cell proliferation assay was carried out using the Cell Proliferation kit I [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); Roche] as described by the manufacturer. Briefly, cells were seeded into 96-well plates at 5 x 103 per well in 100 µL culture medium containing different concentrations of AEBSF. Cells were then incubated at 37°C and 5% CO2 for the durations stated. After the incubation period, MTT labeling reagent was added and incubated at 37°C for 5 hours. This was followed by the addition of the solubilization reagent for 18 hours at 37°C. The spectrophotometric absorbance was measured using a microplate reader (Ultramark, Bio-Rad, Hercules, CA) using a wavelength of 595 nm with a reference wavelength of 655 nm.

Immunostaining and confocal microscopy. For immunodetection of endogenous N-CoR protein, various APL and non-APL cells were fixed onto glass slides by centrifugation and addition of 3% paraformaldehyde at 25°C and permeabilized using 0.2% Triton X-100 in PBS at 4°C. After blocking with 2% milk and 2% bovine serum albumin (BSA) in PBS, slides with untreated cells were incubated in goat polyclonal anti-N-CoR (C-20) diluted 1:100 in 2% milk and 2% BSA in PBS. The secondary chicken anti-goat Alexa Fluor 594 (Molecular Probes, Eugene, OR) was used at a dilution of 1:200 in 1% milk and 1% BSA in PBS. The cells were then stained with 4',6-diamidino-2-phenylindole (DAPI) and visualized using confocal microscopy. For the detection of exogenous N-CoR protein, 293T cells transfected with N-CoR and PML-RAR or PML-RAR (ring finger mutant) were fixed with cold methanol at –20°C for 7 minutes and then stained with anti-Flag antibody as described above.

Determination of apoptosis. Detection of phosphatidylserine on the outer leaflet of apoptotic cells was done using Annexin V-FITC (PharMingen, San Diego, CA) and propidium iodide according to the manufacturer's recommendations. Flow cytometry was done with a FACSCalibur. For morphologic analysis of NB4 cells treated with AEBSF, cytospun slides were stained with Wright-Giemsa stain and examined under light microscopy. The morphologic features considered consistent with apoptosis were cell shrinkage, nuclear condensation, nuclear fragmentation, and formation of apoptotic bodies.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Misfolded N-CoR is cleaved in APL cells. To investigate whether conformation of N-CoR protein plays any role in the resistance of APL cells to UPR-induced apoptosis, we first analyzed cell type–dependent conformation of N-CoR protein in APL cell line NB4 (which expresses PML-RAR) and in three non-APL cell lines, HL60 (AML), K562 (chronic myelogenous leukemia), and U937 (AML), as well as in human embryonic kidney cells 293T. A significant amount of N-CoR protein expressed with PML-RAR in 293T cells was found in the insoluble fraction as full-length protein aggregates, although a cleaved fragment of N-CoR protein with apparent molecular mass of 100 kDa was detected in the soluble fraction (Fig. 1A, left ). In contrast, very little full-length N-CoR was found in APL cells. In NB4 cells, virtually all of the N-CoR present in insoluble fraction as well as a proportion of N-CoR in the soluble fraction were found as the cleaved fragment (Fig. 1A, middle). Of note, the soluble fraction of NB4 cells contained a single cleaved N-CoR fragment with apparent molecular mass of ~100 kDa, whereas two N-CoR fragments of ~90 and ~110 kDa were present in the insoluble fraction (Fig. 1A, middle). On the contrary, almost all the N-CoR present in the non-APL cells (HL60 and K562) was present in the soluble fraction and appeared at a molecular mass indicative of intact full-length protein (Fig. 1A, right). When subcellular distribution of cleaved N-CoR fragments in NB4 cells was analyzed, the ~100-kDa fragment was found to be localized to the nucleus, whereas insoluble ~90- and ~110-kDa fragments were found in the cytosolic fraction (Fig. 1B, left). To further examine whether cleavage of N-CoR was linked to its deregulated subcellular distribution, we studied the subcellular localization of endogenous N-CoR protein(s) in APL and selected non-APL cells. As we reported previously, a significant amount of endogenous N-CoR protein was localized to the cytosol of APL cells, whereas in non-APL cells N-CoR was mostly confined to the nucleus (Fig. 1B, right).


Figure 1
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  		Figure 1. Analysis of N-CoR cleavage in APL and non-APL cells. A, N-CoR protein in soluble (S) and insoluble (I) fractions of whole-cell extracts of APL tumor cells (NB4) or non-APL cells (293T, HL60, and K562) as well as in nuclear or cytosolic fractions of NB4 cells was determined by Western blotting assay using anti-Flag or anti-N-CoR antibodies as described elsewhere (8). 293T cells were cotransfected with PML-RAR and Flag-N-CoR expression plasmids. B, solubility and subcellular location of cleaved N-CoR fragments and subcellular distribution of endogenous N-CoR protein in APL as well as in non-APL cells were determined. C, NB4 and UF-1 cells, two APL-derived cells, contain N-CoR cleaving activity, whereas non-APL cells lack it. Flag-linked N-CoR protein prepared from 293T cells, which also expressed PML-RAR, was used as substrate with extracts of UF-1, NB4, HL60, K562, or U937 cells in optimized cleavage condition. To inactivate the cleaving activity, NB4 and HL60 cell extracts used in lanes 4 and 8 were preheated at 100°C for 10 minutes before incubating with N-CoR substrate. Cleaved N-CoR protein fragment was detected using anti-Flag antibody. D, human primary APL cells contain N-CoR cleaving activity. Flag-linked N-CoR protein substrate was incubated with extracts of human primary APL cells, and cleavage assay was done as described above.

 
Assuming that the processing of N-CoR protein in NB4 cells was most likely a post-translational event, we reconstituted an in vitro N-CoR cleavage assay using the recombinant Flag-tagged N-CoR protein expressed in 293T cells and cellular extracts of APL cells or those of non-APL cells. Indeed, when Flag-tagged N-CoR was incubated with cellular extracts of two established APL cell lines NB4 or UF-1 at 37°C, N-CoR was cleaved to a 100-kDa fragment, whereas N-CoR incubated with buffer or extracts of non-APL cells showed no evidence of cleavage (Fig. 1C, left and middle). These data suggested that an activity present in APL cells promoted cleavage of N-CoR protein. Furthermore, the N-CoR cleavage to 100-kDa fragment was completely blocked when N-CoR was incubated with NB4 cell extracts that had been preheated at 100°C for 10 minutes, thus indicating that the N-CoR cleaving activity in NB4 cells was a function of a heat labile protease (Fig. 1C, left, lane 4). The 100-kDa cleaved fragment of N-CoR was also generated when the recombinant N-CoR was incubated with NB4 cells extract at 25°C; however, under these conditions, only a small fraction of full-length N-CoR was cleaved (Fig. 1C, right). To confirm that N-CoR cleaving activity was not an artifact restricted to NB4 cells but an inherent property of human promyelocytic cells, we did N-CoR cleavage assay using cellular extracts from three primary human APL samples. Cellular extracts of primary human APL cells were prepared in RSB buffer, and cleavage assay was done under the same optimized condition using the Flag-tagged recombinant N-CoR protein. Indeed, similar to the results obtained with extracts of APL cell lines, the human APL cell extracts contained activity that cleaved N-CoR protein and produced COOH-terminal fragments with an apparent molecular mass of 100 kDa (Fig. 1D).

N-CoR cleavage is inhibited by protease inhibitors or EDTA. To characterize the nature of N-CoR cleaving activity of NB4 cells, we first determined the optimum amount of NB4 cell extracts required for the complete cleavage of N-CoR protein. Equal amounts of recombinant N-CoR protein were incubated at 25°C with increasing amounts of NB4 cell extracts under optimized condition. The cleavage of N-CoR protein was enhanced in a dose-dependent manner as reflected by the gradual decrease in full-length N-CoR protein and the reciprocal increase in the amount of the cleaved 100-kDa N-CoR fragment (Fig. 2A ); cell extract (200 µg) was required to completely cleave full-length N-CoR (Fig. 2A). To identify the biochemical properties of the N-CoR cleaving activity, we repeated N-CoR cleavage assay with 100 µg NB4 cell extracts (50% cleaving activity) and various protease inhibitors or EDTA at the optimum concentration. From the various protease inhibitors tested, only AEBSF and phenylmethylsulfonyl fluoride (PMSF) blocked in vitro cleavage of N-CoR protein in a dose-dependent manner (Fig. 2B and C). More importantly, AEBSF also blocked the processing of endogenous N-CoR protein in NB4 cells in a dose-dependent manner, which led to the stabilization of full-length N-CoR protein along with several higher molecular weight (HMW) N-CoR protein bands (Fig. 2D). However, the inhibitory effect of EDTA on N-CoR cleaving activity was observed at relatively higher (50-100 mmol/L) concentrations (Fig. 2C).


Figure 2
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  		Figure 2. N-CoR protein cleavage in APL cells. A, dose-dependent effect of N-CoR cleaving activity. Fixed amount of N-CoR substrate was incubated with increasing amount of NB4 cell extracts in optimum cleavage condition. B and C, effect of various protease inhibitors on in vitro cleavage of N-CoR protein. Flag-linked N-CoR protein substrate prepared from 293T cells was incubated with 100 µg of NB4 cell extract and the following protease inhibitors and EDTA: 5 µg/mL aprotinin, 5 µg/mL chymostatin, 5 µg/mL leupeptin, 5 µg/mL pepstatin, 1 to 5 mmol/L PMSF, 200 µmol/L AEBSF, 20 µmol/L MG132, or 12.5 to 100 mmol/L EDTA. After 1 hour of incubation in 25°C, the reaction was stopped by adding SDS sample buffer, and inhibition of N-CoR cleavage was analyzed by Western blotting using anti-Flag antibody that recognizes both the full-length and the cleaved 100-kDa fragment. D, AEBSF inhibited N-CoR cleavage and stabilized the full-length HMW N-CoR protein in vivo. NB4 cells were cultured with increasing dose of AEBSF for 72 hours, and extracts were prepared in SDS sample buffer. N-CoR cleavage was analyzed by Western blotting using anti-N-CoR antibody that recognizes the full-length protein and the cleaved fragments.

 
N-CoR cleaving activity is a glycoprotein endopeptidase. Having confirmed that N-CoR cleaving activity present in NB4 cells was a function of protease activation, we next set out to identify and characterize the protease involved. To do so, we adopted the approach described by Lane and Ley (21). Briefly, whole-cell extracts from NB4 cells prepared in RSB buffer were loaded onto HPLC Superose 12 column. The column-purified fractions from exclusion chromatography were incubated with recombinant Flag-linked N-CoR protein at the optimal cleavage conditions. Of the 50 fractions collected, fractions ranging from numbers 14 to 24 contained varying degrees of N-CoR cleaving activity, whereas fractions 19 to 23 exhibited peak activity as evidenced by the amount of cleaved 100-kDa N-CoR fragment (Fig. 3A ). Next, the protein content from fractions 19 to 23 was separated by SDS-PAGE, and the bands corresponding to the peak protease activity were excised and subjected to mass spectrometry (MS). Interestingly, two peptide sequences determined by MS showed homology to known proteases. One of the peptides exhibited partial homology to the sequence of Gcp, the Pasteurella haemolytica homologue of human Gcp, OSGEP, and the other matched to peptide sequence of human aminopeptidase N. To investigate their N-CoR-specific protease activity, we obtained highly purified Gcp and aminopeptidase N from a commercial vendor and did dose-dependent in vitro cleavage assay using full-length recombinant N-CoR protein as substrate. Purified Gcp cleaved recombinant N-CoR in a dose-dependent manner and generated a cleaved COOH-terminal fragment with an apparent molecular mass of ~100 kDa, which was almost identical to that generated by the extracts of NB4 cells or human APL primary cells (Fig. 3B). Moreover, AEBSF (100-400 µmol/L) inhibited Gcp-mediated site-specific cleavage of N-CoR, which resulted in stabilization of full-length N-CoR protein (Fig. 3C). In contrast, purified aminopeptidase N cleaved N-CoR in a nonspecific manner and did not produce the expected 100-kDa fragment (data not shown).


Figure 3
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  		Figure 3. Purification of N-CoR cleaving activity from NB4 cell extracts. A, Western blotting of Flag-linked N-CoR protein substrate treated with fractions of NB4 cell extracts eluted from gel filtration chromatography column. B, the highly purified Gcp did dose-dependent cleavage of N-CoR protein and generated the 100-kDa N-CoR fragment. C, AEBSF-mediated dose-dependent inhibition of Gcp activity leads to stabilization of full-length N-CoR protein. D, N-CoR is modified by O-linked sialic acid side chain in NB4 cells. Extracts of AEBSF-treated NB4 cells were incubated with buffer, PNGase F (removes N-glycan), neuraminidase (removes sialic acid side chain), or O-glycosidase (removes O-glycan) at 37°C for 6 hours. The reaction was stopped by heating at 50°C for 10 minutes in SDS loading buffer. The proteins were resolved in 6% SDS-PAGE and transferred to PVDF membrane. Western blotting was carried out with anti-N-CoR antibody (C-20). Asterisk, position of lighter deglycosylated N-CoR protein band. E, shRNA-mediated abrogation of OSGEP activity inhibited N-CoR cleavage. NB4 cells were electroporated with or without shRNA vector designed against human OSGEP mRNA, and N-CoR protein level in electroporated cells was analyzed by Western blotting assay using anti-N-CoR antibody.

 
Cleavage of N-CoR in NB4 cells by a glycoprotease suggested that misfolded N-CoR was most likely modified by glycan side chains and that selective inhibition of glycoprotease activity by AEBSF could stabilize glycan-modified N-CoR protein bands. Indeed, when NB4 cells were treated with increasing concentrations of AEBSF, cleavage of N-CoR was inhibited at concentrations ranging from 50 to 200 µmol/L (Fig. 2D). Interestingly, at higher concentration (100 µmol/L) of AEBSF, several HMW bands of N-CoR protein were revealed on Western blotting (Fig. 2D). To confirm that HMW N-CoR bands observed after AEBSF treatment were indeed modified by glycan side chains, we did deglycosylation assay using full-length N-CoR from AEBSF-treated NB4 cells. When extracts of AEBSF-treated NB4 cells were incubated with neuraminidase or O-glycosidase enzymes that remove sialic acid side chain and O-linked glycan, respectively, N-CoR was deglycosylated and a faster-migrating N-CoR band was obtained (Fig. 3D). Under similar conditions, incubation of N-CoR with buffer or PNGase F, an enzyme that removes N-linked glycan, did not produce faster-migrating bands (Fig. 3D). These findings confirmed that N-CoR was indeed modified by O-linked sialic acid side chains in NB4 cells.

To further confirm the involvement of OSGEP in the cleavage of endogenous N-CoR protein in NB4 cells, we knocked down OSGEP expression in NB4 cells using shRNA and asked whether this could block the processing of endogenous N-CoR protein. Indeed, shRNA-mediated down-regulation of OSGEP in NB4 cells resulted in stabilization of N-CoR, thus strongly implicating OSGEP in the in vivo cleavage of endogenous N-CoR protein (Fig. 3E).

AEBSF promotes growth arrest of APL cells. To investigate the link, if any, between N-CoR cleavage and the cell growth, we tested the effect of AEBSF on the proliferative ability of NB4 cells. Results showed that AEBSF inhibited the proliferation of NB4 cells in a dose-dependent manner, with the effective dose being ≥100 µmol/L (Fig. 4A ). The growth-inhibitory effect of AEBSF was selective for APL cells, as several non-APL cell lines were resistant to this effect of AEBSF (Fig. 4B). Stimulated by these data, we investigated the effect of AEBSF on the processes of cellular differentiation, apoptosis, and cell cycle progression in NB4 cells. Data show that AEBSF promoted apoptosis of NB4 cells in a dose-dependent manner as evidenced by an increase in the percentage of Annexin V–positive cells (indicative of phosphatidylserine exposure; Fig. 4C and D). In contrast, AEBSF had no effect on differentiation or cell cycle progression in the same cell line (data not shown). Similar to data obtained with AEBSF, shRNA-mediated knockdown of OSGEP in NB4 cells also resulted in a significant increase in Annexin V–positive cells (Fig. 4E), thus indicating that down-regulation of OSGEP favored apoptosis in APL cells.


Figure 4
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  		Figure 4. Selective inhibition of growth of APL cells by AEBSF. A and B, APL (NB4) cells and various non-APL cells were treated with various concentration of AEBSF for the duration indicated, and the growth of treated cells was measured by the MTT assay. C and D, morphologic and molecular features suggestive of apoptosis in AEBSF-treated (50-200 µmol/L) NB4 cells were monitored by Giemsa staining (C) and Annexin V assay (D). E, OSGEP down-regulation in NB4 cells by shRNA leads to induction of apoptosis. NB4 cells were electroporated with or without shRNA vector designed against human OSGEP mRNA, and apoptotic change in electroporated cells was monitored by Annexin V assay.

 
AEBSF-induced apoptosis is a function of ER stress. To investigate the effect of insoluble N-CoR cleavage in the resistance of APL cells to ER stress-induced apoptosis, we first tested the effect of insoluble N-CoR protein on the fate of 293T cells. We previously reported that, unlike wild-type PML-RAR, coexpression of N-CoR with a ring finger mutant of PML-RAR (PML-RAR-RFM) in 293T cells generated misfolded N-CoR protein, which was detergent soluble (8). Using a similar system, we compared the effect of insoluble and soluble variants of misfolded N-CoR protein on the fate of 293T cells. Data show that 293T cells expressing the insoluble N-CoR protein exhibited morphologic features of apoptosis, such as condensation of chromatin and DNA fragmentation (Fig. 5A, left ). Moreover, cells expressing the insoluble N-CoR protein exhibited significantly higher level of GRP78 expression, a marker of ER stress in mammalian cells (Fig. 5A, right). In comparison, 293T cells expressing the soluble variant of misfolded N-CoR, localized to the Golgi, failed to undergo apoptosis (Fig. 5B). It should be pointed out that the apoptotic effect of insoluble N-CoR was not due to its higher level of accumulation because both variants of N-CoR were expressed at equal levels on transfection (Fig. 5C). These findings suggest that accumulation of insoluble N-CoR protein may promote apoptosis of 293T cells via mechanism(s) involving amplification of ER stress. To test whether AEBSF-induced apoptosis of NB4 cells also involved accumulation of full-length insoluble N-CoR protein and ER stress, we analyzed the solubility of the full-length N-CoR protein and expression of ER stress markers GRP78 and PDI. We showed that AEBSF treatment led to the accumulation of detergent-insoluble full-length N-CoR protein in the cytosolic fraction of NB4 cells (Fig. 5D). Moreover, AEBSF promoted the formation of HMW detergent-resistant GRP78 and PDI bands (Fig. 5E), which were also induced by thapsigargin and, to a lesser extent, by tunicamycin (Fig. 5F), two known inducers of ER stress. These data agree with recent reports suggesting that GRP78 accumulates as HMW detergent-resistant bands in mammalian cells treated with tunicamycin (29, 30).


Figure 5
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  		Figure 5. Enhancement of ER stress in APL cells by AEBSF. A and B, morphology of 293T cells expressing the insoluble or the soluble version of misfolded N-CoR proteins. 293T cells were transfected with N-CoR and PML-RAR (A) or N-CoR and a ring finger mutant of PML-RAR (B). A, transfected cells were stained with anti-Flag antibody to visualize N-CoR (red; left), with anti-GRP78 antibody (red; right), or with anti-Flag antibody against N-CoR (green; right). Blue, DNA was stained with DAPI. Arrows, cells expressing misfolded N-CoR and exhibiting evidence of apoptosis (e.g., condensed nuclei). B, green, transfected cells were stained with anti-Flag antibody against N-CoR; red, Golgi was visualized by coexpressing a DsRed-Golgi plasmid containing Golgi localization signals; blue, nucleus was counterstained with DAPI. C, whole-cell extracts were prepared from 293T cells transfected with N-CoR and PML-RAR or PML-RAR (RFM), and level of N-CoR protein was determined by Western blotting. D, AEBSF treatment leads to accumulation of full-length insoluble N-CoR protein in the cytosol of NB4 cells. E, AEBSF promotes formation of HMW detergent-resistant GRP78 and PDI protein bands. NB4 cells were treated with AEBSF, and whole-cell extracts prepared in SDS sample buffers and heated at 100°C were stained with GRP78 and PDI antibodies. F, thapsigargin and tunicamycin (to some extent) promote formation of detergent-resistant GRP78 and PDI protein bands. NB4 cells were treated with ER stress-inducing agents DTT, thapsigargin (Tg), or tunicamycin (Tm), and whole-cell extracts prepared in SDS sample buffer and heated at 100°C were stained with GRP78 and PDI antibodies.

 
Schematic model for the role of proteases in the resistance of APL cells to ER-induced apoptosis. In light of the data presented in this report, a schematic model for the role of protease-mediated cleavage of misfolded proteins in tumor cell resistance to UPR-induced apoptosis is presented in Fig. 6 . Immediately after translation, N-CoR may associate with PML-RAR and become misfolded. The misfolded PML-RAR/N-CoR complex would accumulate as toxic insoluble protein aggregates in the ER and stimulate the UPR-induced apoptosis pathway in non-APL cells. In APL cells, however, toxicity associated with misfolded PML-RAR/N-CoR would be neutralized through Golgi-specific post-translational modification and selective induction of aberrant protease activity. Aberrant proteases, such as neutrophil elastase or OSGEP, induced or activated as cellular defense mechanism against PML-RAR/N-CoR protein aggregates, may selectively cleave the misfolded PML-RAR or N-CoR proteins resulting in reduction of ER stress and stress tolerance and thus protect tumor cells from the toxic insults of misfolded PML-RAR/N-CoR protein aggregates. Treatment of APL cells with protease inhibitors would inhibit the activity of neutrophil elastase and OSGEP, resulting in further accumulation of misfolded PML-RAR/N-CoR protein aggregates and activation of ER stress, which will ultimately trigger the UPR-induced apoptosis signaling. The red lines and arrows represent the cytotoxic branch of UPR, which we consider as antioncogenic because it would eliminate the transformed cells through apoptosis. The cytoprotective branch of UPR, represented by green arrows, would allow the transformed cell to remain viable and, with the help of additional growth-promoting stimuli, to proliferate.


Figure 6
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  		Figure 6. Schematic representation of the regulation of ER stress and UPR in APL cells. Red letters and arrows, cytotoxic branch of the UPR. Green, cytoprotective branch of UPR. NE, neutrophil elastase.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cytotoxicity of PML-RAR in non-APL cells is mediated by misfolded N-CoR. A cell type–specific effect of PML-RAR on cellular growth and survival has been reported in multiple in vivo and in vitro studies (23–26). PML-RAR was found to induce apoptosis in nonhematopoietic as well as in most hematopoietic cell lines. In contrast, the growth-promoting effect of PML-RAR protein was observed only in a limited set of hematopoietic cell lines. The molecular basis for this cell type–specific effect of PML-RAR remains to be elucidated. Our findings strongly suggest that PML-RAR-induced cell death in many non-APL cells could be mediated, at least in part, by misfolded N-CoR protein. We have previously shown that, in its native conformation, N-CoR is a soluble nuclear protein that resides in small dot-like nuclear structures surrounding the PODs. Although PML-RAR could promote misfolding or insolubility of N-CoR protein in hematopoietic as well as nonhematopoietic cells, a cell type–specific difference in PML-RAR-induced subcellular distribution of N-CoR protein was observed; misfolded N-CoR accumulated preferentially in the ER in non-APL cells, whereas in APL cells it was mostly targeted to the Golgi apparatus (8). Moreover, the toxicity associated with ER-localized insoluble N-CoR protein, which promotes apoptosis in non-APL cells, is neutralized in APL cells via Golgi-specific O-linked glycosylation and subsequent cleavage of insoluble N-CoR protein aggregates. However, the mechanism by which specific subcellular localization of N-CoR protein is differentially regulated in APL and non-APL cells is far from clear.

A careful examination of N-CoR peptide sequence reveals two putative ER targeting sequences in the NH2-terminal domain, which could be masked when the protein is in the native conformation. ER targeting of misfolded N-CoR in the presence of PML-RAR could occur as a result of unmasking of ER retention signals in the misfolded conformation. In APL cells, selective glycosylation of misfolded N-CoR may facilitate its transport across the ER-Golgi network, and the glycan-modified N-CoR could be preferentially cleaved by OSGEP in the Golgi or lysosomal vesicles.

Misfolded N-CoR in APL cells is processed by a glycoprotein endopeptidase. The involvement of a specific protease in the cleavage of PML-RAR and its association with APL was first reported in U937 myelomonocytic cells (21). The protease was described as neutrophil elastase, and neutrophil elastase–mediated cleavage of PML-RAR was shown to be important for APL pathogenesis in mice. However, our initial data suggested that the protease that cleaved N-CoR in APL cells was distinct from neutrophil elastase, as N-CoR protein incubated with neutrophil elastase under optimum cleavage conditions failed to produce site-specific cleavage of N-CoR; rather, it cleaved N-CoR protein in a nonspecific manner (data not shown). We have purified the N-CoR cleaving activity from NB4 cells by size exclusion chromatography using HPLC and present evidence to support that the protease involved in N-CoR cleavage is a Gcp. As the activity of this protease is highly sensitive to pH and salt content, further purification of the protease from the eluates of size exclusion chromatography was unsuccessful. To that end, earlier reports have shown a 70-fold increase in specific Gcp activity on purification by gel exclusion chromatography; however, the activity was significantly diminished by anion exchange chromatography (31). Gcp was originally identified in culture supernatant of the bacterium P. haemolytica and belongs to a family of metallopeptidase (32). Gcp has been linked to the cleavage of cell surface antigens and transmembrane glycoproteins, which bear sialic acid side chains attached to serine/threonine residues.

Role of N-CoR cleavage in malignant growth and transformation of APL cells. How does processing of misfolded N-CoR, a protein already incapacitated by conformational change, possibly contribute to the pathogenesis of APL? Misfolded N-CoR may cooperate with PML-RAR, fusion protein that also accumulates as insoluble aggregates in the ER, to create cytotoxic insults subversive to the survival of APL cells. The cleavage of N-CoR by OSGEP, together with the cleavage of PML-RAR by neutrophil elastase, would keep the stress generated by these proteins within the tolerable limits and help APL cells survive the cytotoxic effects of UPR. In addition, proteolytic processing of N-CoR could contribute further to the leukemogenic potential of misfolded protein via a dominant-negative gain-of-function mechanism as observed in amyloidosis, a classic disease caused by accumulation of misfolded protein. In amyloidosis, limited proteolysis of serum amyloid A protein creates a 76-residue fragment that is more prone to self-association than the parent molecule (33). N-CoR fragments generated by cleavage may have increased propensity for self-aggregation due to the exposure of hydrophobic domains. Although neutralization of toxicity associated with N-CoR protein aggregates may confer resistance to UPR-induced apoptosis and favor survival of promyelocytic cells, additional growth-promoting stimuli must work in concert to promote uncontrolled proliferation or differentiation arrest in these cells. The cleaved N-CoR fragments that accumulate in the nucleus and the cytosol may provide these additional growth-promoting stimuli by dint of their unique gain-of-function properties or dominant-negative effect.

Therapeutic potential of AEBSF in APL. A possible link between N-CoR cleavage and survival of APL cells is also supported by the selective proapoptotic effect of AEBSF in APL cells. Interestingly, in an earlier report, AEBSF was found to protect HL60 cells from DNA damage-induced apoptosis (34). Therefore, the selective proapoptotic effect of AEBSF observed in APL cells is probably mediated by amplification of ER stress resulting from accumulation of insoluble N-CoR protein aggregates. It is likely that only those cells at the threshold of ER stress are sensitized to UPR-induced apoptosis by AEBSF. Aside from its inhibitory effect on N-CoR cleavage, AEBSF may also contribute to apoptosis of APL cells by inducing ER stress through its ability to block proteases involved in ATF6 activation. AEBSF was found to inhibit ER stress-induced cleavage of ATF6 in HeLa cells, resulting in inhibition of transcriptional induction of ATF6 target gene GRP78 (35). By inhibiting the proteases that cleave ATF6, AEBSF may indirectly dismantle the stress-sensing mechanism located within the ER. Thus, accumulation of misfolded proteins in the ER of AEBSF-treated cells would fail to activate ATF6, resulting in further accumulation of misfolded protein and enhancement of ER stress and ultimately leading to apoptosis.

Dynamic regulation of UPR in tumor cells. Cellular responses to ER stress and UPR induce expression of molecular chaperones that refold the misfolded proteins or activate the ERAD pathway to promote degradation of misfolded proteins. However, in certain conformational diseases, toxic buildup of misfolded proteins inhibits the ERAD pathway, leading to further accumulation of misfolded proteins in the ER (36, 37). This may create a potentially challenging situation for a cell already under stress. Under this stressful state, what could be the possible way out for a cell to survive the toxic insult of misfolded proteins? Our findings suggest that a tumor cell may induce proteases as a final thrust to offset the toxic insult of misfolded proteins and gain a survival advantage. Thus, activation of proteases may represent a novel cytoprotective component of UPR in tumor cells subjected to sustained ER stress. It is likely that different branches of the UPR might have distinct roles at different stages of carcinogenesis, and induction of specific downstream targets through the dynamic regulation of UPR may determine the fate of tumor cells by tipping the balance between survival and apoptosis.


   Acknowledgments
 
Grant support: National Medical Research Council of Singapore grant NMRC/0790/2003 (M. Khan and S. Pervaiz) and Academic Research Fund of National University of Singapore (M. Khan). A.P.P. Ng is a recipient of Agency of Science, Technology, and Research graduate scholarship.

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.

We thank Dr. Ryosuke Takahashi (Laboratory for Motor System Neurodegeneration, RIKEN Brain Science Institute, Saitama, Japan) for the gift of DsRed-Golgi plasmids, Dr. Masahiro Kizaki (Keo University School of Medicine, Tokyo, Japan) for retinoic acid–resistant APL cell UF-1, and Dr. Shunsuke Ishii (Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Japan) for his continued support and encouragements.


   Footnotes
 
Note: J.H. Fong and D.S. Nin contributed equally to this work.

Received 1/ 3/06. Revised 6/23/06. Accepted 8/ 3/06.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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