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1 Division of Cytokine Signaling, Department of Molecular Microbiology and Immunology, and
3 Department of Investigative Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, 852-8523 Japan
2 First Department of Pathology, Faculty of Medicine, Oita University, Yufu-city, Oita, 879-5593, Japan
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Abstract |
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B plays a critical role by inducing anti-apoptotic molecules. In this study, we firstly carried out an expression-cloning approach to isolate the responsible molecules in the NF-B activation pathway with the defective mutant cell line, COS-A717. This cell line shows reduced constitutive basal activity of NF-B as compared with the parental COS cells. We successfully isolated genes with compensating activity for the pathway, and one gene encoded B-cell activating factor receptor (BAFF-R). However, a Northern blot analysis revealed that the BAFF-R expression is not only limited to cells of B cell origin, but also is found in those with nonhematopoietic origins. In the human fibrosarcoma cell line HT1080, an immunohistochemical analysis revealed that the expression of BAFF-R is not on the cell surface, but in the cytoplasm. The expression of BAFF was also detected, and the reduction of endogenous BAFF or BAFF-R by siRNA decreased the basal NF-B activity. Lastly, from clinicopathological specimens, the associated expression of BAFF-R and BAFF was demonstrated in osteosarcoma. We propose that an aberrant BAFF/BAFF-R-dependent autocrine mechanism may play a role in the development of certain types of cancer cells. |
Introduction |
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B plays important roles in cell survival, proliferation, apoptosis, immune response, and inflammation by regulating the expression of numerous genes. Because of various stimuli, I-B proteins become phosphorylated at serine residues 32 (S32) and 36 (S36) (Brown et al. 1995; Regnier et al. 1997) by the I-B kinase (IKK) complex (Zandi et al. 1997), and are degraded by the ubiquitin-proteasome pathway (Chen et al. 1995). The IKK complex consists of two catalytic units, IKK1 and IKK2 (also referred to IKK
and IKKβ), and a regulatory subunit, NEMO (Yamaoka et al. 1998). Furthermore, some members of the mitogen-activated protein kinases, including NF-B-inducing kinase (NIK) and MEKK1-3, were implicated in the activation of the IKK complex in transient over-expression studies (Zhao & Lee 1999; Yang et al. 2001). The released NF-B is then translocated into the nucleus, where it binds to specific B elements and activates many important genes. On the other hand, in certain cancer cells, constitutive activation of NF-B is involved in development and progression, but this constitutive activation mechanism has not been fully clarified.
To understand the constitutive mechanism, we tried to isolate the molecule(s) involved in the NF-B signaling pathway in cancer cells by an expression cloning method. An attractive approach for expression cloning is to use defective mutant cell lines. Velazquez et al. (1992) and Darnell et al. (1994) identified the Janus kinase family by genetic compensation, using a defective mutant cell line derived from HT1080. Although this approach is labor-intensive, only clones with compensating activity in vivo should be isolated, in contrast to the yeast two-hybrid screening approach, which is usually accompanied by a high background.
To establish a mutant cell line defective in the NF-B signaling pathway, we exposed several transformed cell lines to the frame-shift mutagen ICR191, using a similar procedure to that reported by Darnell et al. (1994). We then established a mutant cell line, designated COS-A717, which showed reduced constitutive basal activity of NF-B as compared with the parental COS cells. The COS cell line was derived from an African monkey kidney fibroblast-like cell line, CV-1, by transformation with an origin-defective mutant of SV40 encoding wild-type T antigen. Using the COS-A717 clone, we isolated genes that allowed the compensating NF-B activity, and one of them encoded BAFF receptor (BAFF-R), which is a member of the tumor necrosis factor receptor family (Thompson et al. 2001), like BCMA (Laabi et al. 1994) and TACI (von Bülow & Bram 1997). BAFF-R expression was detected in various B cell lines, B cells and T cells (Yan et al. 2001; Mackay et al. 2003). BCMA and TACI signaling reportedly activate NF-B through the canonical pathway (von Bülow & Bram 1997; Hatzoglou et al. 2000; Xia et al. 2000). In B cells, BAFF-R activation promotes the processing of NF-B2/p100 to p52, which is called the alternative pathway (Claudio et al. 2002; Kayagaki et al. 2002). BAFF-R signaling plays crucial roles in B cell survival, germinal center maintenance, and class switch recombination (Mackay et al. 2003; Castigli et al. 2005; Kalled 2006). The specific ligand for BAFF-R is BAFF, which is produced by macrophages, dendritic cells, T cells, and epithelial cells (Schneider et al. 1999; Craxton et al. 2003; Huard et al. 2004; Daridon et al. 2007). After cloning BAFF-R, we investigated its expression as well as that of BAFF in several cell lines, and found that some cells possibly constitute an autocrine or paracrine pathway between BAFF and BAFF-R. This possibility was assessed by the effect of BAFF siRNA on the basal NF-B activity. Finally, our examination of clinicopathological specimens revealed that osteosarcoma expressed both BAFF/BAFF-R.
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B signaling
The overall protocol for establishing the mutant cell line, COS-A717, is shown in Fig. 1A. The HIV-LTR-
Sp1-Hook construct was transfected into COS cells, and a stable expression cell line, COS-Hook, was established. The mutagenesis was carried out according to the method of Pellegrini et al. (Pellegrini et al. 1989). Briefly, the cells were plated in a 10-cm dish, and ICR 191 (2 µg per mL) was added for 2 h. The cells were rinsed twice with serum-free medium, and then were incubated in medium with 10% FBS. This treatment killed 70% to 90% of the cells. After a recovery for 7–9 days, the treatment was repeated twice more. After the treatment, the cells were allowed to recover, and those with reduced NF-B activity were selected by using the Capture-Tec system. The HIV-LTR-Sp1pHook, which is driven by HIV-LTR-Sp1, is designed to express a unique single chain antibody (sFv) on the cell surface. As sFv only recognizes specific haptens coated on magnetic beads, the cells selected with the beads are regarded as NF-B active. After the mutagen treatment, the cells that did not bind the magnetic beads were collected and plated at a relatively low density for cloning. The mutant clone A7 was isolated and treated with ICR 191 again, for the further reduction of NF-B activity. After the recovery of the cells, clones with further decreased NF-B activity were isolated, based on the luciferase activity of the 5x
B reporter plasmid described above, and one of these clones was COS-A717.
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B in COS-A717 cells was reduced as much as 7-fold, as compared with that in COS cells (Fig. 1B). Accordingly, the DNA binding activity of NF-B analyzed by EMSA was reduced approximately 3-fold (Fig. 1C). The NF-B activation level of each cell was analyzed by flow cytometry 2 days after the HIV-LTR-Sp-1GFP transfection, and revealed that most of the COS-A717 cells exhibited reduced fluorescence intensity levels as compared with the parental COS cells (Fig. 1D).
Expression cloning of an NF-B activation gene
The HIV-LTR-Sp1-GFP construct was transfected into COS-A717 cells to produce the stable expression cell line, COS-A717-GS. The cloning of genes with compensating activity for the NF-B activation pathway was carried out according to the method of Barton et al. (Barton et al. 2001). Briefly, COS-A717-GS cells were transfected with a human spleen cDNA library (Life Technologies, Gaithersburg, MD) using the FuGene 6 reagent. After 48 h of incubation, the top 0.5% of fluorescent cells were collected using a FACStar Plus (Becton, Dickinson and Co., Franklin Lakes, NJ). The plasmids were extracted from the sorted cells (Hirt 1967), amplified in bacteria, and used in three subsequent rounds of flow cytometry-based enrichment (Fig. 2A). Individual bacterial colonies obtained from the third sorting were grouped into pools of 50 colonies. The plasmids recovered from each pool were used to transfect COS-A717-GS cells. Positive pools were defined as those conferring a greater than 1.5% increase in the number of maximally-fluorescent cells, as compared with cells transfected with the control plasmid. Each positive pool was subdivided into 10 subpools of 20 colonies, and the plasmids from the subpools were tested for the ability to confer enhanced GFP in transfected COS-A717-GS cells. Positive subpools were further subdivided into 10 subpools with half the number of colonies, and were subjected to repeated screening. This process finally yielded independent clones that conferred compensation for the NF-B activation pathway in COS-A717. A sequencing analysis of the cloned cDNA revealed that it perfectly matched the BAFF-receptor (BAFF-R).
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B activation in COS-A717 cells
To confirm that BAFF-R induced the NF-B activation observed in COS-A717 cells, we carried out a luciferase reporter assay and an EMSA analysis in COS-A717 cells. The level of constitutive NF-B activation was approximately 7-fold elevated in COS cells, as compared with that in COS-A717 cells. In BAFF-R transfected COS-A717 cells, the NF-B promoter activity was increased to approximately the same constitutive level as in COS cells (Fig. 2B). On the other hand, the level of NF-B activation was approximately 2-fold elevated in BAFF-R transfected COS cells (Fig. 2B). This result suggested that the NF-B activity in COS cells might reach an upper limit.
In BAFF-R transfected COS-A717 cells, the DNA binding activity of NF-B was 6.5-fold elevated. The complex was supershifted in the presence of p65 or p50 Abs (Fig. 2C). The results of the EMSA analysis agreed well with those from the luciferase reporter assay in COS-A717 cells. These results indicated that BAFF-R mediated the NF-B activation in COS-A717 cells. Furthermore, we investigated which molecules were related to the NF-B activating pathway, using an IB super-repressor (IB SR) and dominant-negative mutants of IKK1 (IKK1.DN) and IKK2 (IKK2.DN). IB SR and IKK2.DN reduced the BAFF-R induced NF-B activation in a dose-dependent fashion in COS-A717 cells, while IKK1.DN did not (Fig. 2D).
Function of BAFF receptor for cell survival
The constitutive activity of NF-B is reduced in COS-A717 cells as compared with that in COS cells. Furthermore, TNF- induced NF-B activation in COS cells, but not in COS-A717 cells. The TNF-receptor adaptor molecules, TRADD, RAIDD, and RIP, were expressed in COS-A717 cells (data not shown). Even in BAFF-R transfected COS-A717 cells, TNF- did not induce NF-B activation, as revealed by an EMSA (Fig. 3A). The complex was supershifted in the presence of p65 or p50 Abs (data not shown). In a luciferase assay, the TNF--induced NF-B activity was poor in COS-A717 and BAFF-R transfected COS-A717 cells (Fig. 3A). NF-B activation plays essential roles for preventing TNF- induced cell death (Beg & Baltimore 1996; Wang et al. 1996). These results suggested that COS-A717 cells might be susceptible to TNF-. As expected, COS-A717 cells were susceptible to TNF-, whereas COS cells were resistant to it. In total, 98% of the COS cells survived with 1 ng/mL TNF-, in contrast to the COS-A717 cells, with less than 10% survival. Incubating COS-A717 cells with TNF- for 24 h caused concentration-dependent cell death (Fig. 3B). In B cells, BAFF induced NF-B activation may regulate survival-related genes (Claudio et al. 2002; Hsu et al. 2002). Therefore, we investigated whether over-expressing BAFF-R in COS-A717 cells increased the expression of survival-related genes. In COS-A717 cells, the bcl-2 mRNA expression level was lower than that in COS cells. The over-expression of BAFF-R increased the expression level of bcl-2 mRNA in COS-A717 cells (Fig. 3C). To examine the role of BAFF-R in COS-A717 cells, a Flag-tagged BAFF-R construct was transfected into COS-A717, and the stable expression cell lines COS-A717/B1and COS-A717/B2 were established. The expression of BAFF-R in COS-A717/B1 and COS-A717/B2 was determined by immunoblotting (Fig. 4A). Interestingly, the expression level of bcl-2 mRNA was correlated with the BAFF-R expression levels in the COS-A717/B1 and COS-A717/B2 cell lines (Fig. 3C). The NF-B promoter activity was increased by approximately 3-fold and 4-fold in COS-A717/B1 and COS-A717/B2, respectively, as compared with that in COS-A717 cells (Fig. 4B). The NF-B activation levels were approximately 2.1-fold and 3.5-fold higher in COS-A717/B1 and COS-A717/B2 cells, respectively, relative to the control clone, as determined by EMSAs (Fig. 4C). However, the NF-B activation level of COS-A717/B1 and COS-A717/B2 was lower than that of COS cells. Because NF-B activation plays crucial roles in TNF- resistance, we investigated whether the increased NF-B activity in COS-A717/B1 and COS-A717/B2 influenced the resistance to TNF-. The percents of cell viability of COS-A717/B1 and COS-A717/B2 cells were higher than that of the COS-A717 control cells, and the criterion for statistical significance was the 0.01 level (Fig. 4D). Thus, COS-A717/B1 and COS-A717/B2 cells were more resistant to TNF- than the COS-A717 control cells. Because neither the COS-A717 nor parental COS cells originally express the BAFF-R mRNA (Fig. 5A, lanes 1 and 2), these results suggested that the ectopic expression of BAFF-R activates NF-B and endows the cells with resistance to the apoptotic stimuli.
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Because it seemed possible that the ectopic expression of BAFF-R could be a mechanism in the constitutive activation of NF-B in some cancer cells, we next examined the BAFF-R expression in various cell lines. Consistent with the previous reports, BAFF-R is expressed in B cells (Ramos) and T cells (HUT102, MT4, MT2, MOLT4, Jurkat). However, BAFF-R is also expressed in myeloid cells, such as K562 and HL60. Furthermore, BAFF-R is even expressed in nonhematopoietic cells, HeLa and HT1080. This indicates that the expression of BAFF-R mRNA is broader than previously reported. To confirm that HT1080 cells express the authentic BAFF-R, we sequenced the entire coding region of the gene by RT-PCR, and it perfectly matched the published sequence of BAFF-R (data not shown). We next detected the BAFF-R protein in HT1080 cells by an immunohistochemical analysis. In contrast to the expression in B cells, BAFF-R was not expressed on the cell surface, but in the cytoplasm (Fig. 5B). A flow cytometric analysis produced a similar result (Fig. 5C).
Taken together, these results suggested that the ectopically expressed BAFF-R protein is confined to the cytoplasm, and is not present on the cell surface. Recently, several reports indicated that intracytoplasmic receptors recognize their cognate cytokines, such as vascular endothelial growth factor (VEGF) (Gerber et al. 2002), angiopoietin-2 (Scharpfenecker et al. 2005), and CCR6 (Inoue et al. 2006), and constitute an internal autocrine loop. Thus, we next examined the expression of BAFF. Indeed, HT1080 cells express BAFF mRNA (Fig. 6A). In contrast, COS and COS-A717 cells do not express BAFF-mRNA (data not shown). Because BAFF also exerts its effects through binding to other receptors, such as BCMA and TACI, we examined the expression of these genes by RT-PCR. The expression of BCMA mRNA, but not TACI mRNA, was detected in HT1080 cells (Fig. 6A). Because HT1080 cells have significant endogenous NF-B activity (Park et al. 2005), we used this line to evaluate the existence of an internal autocrine loop by inhibiting it with BAFF-specific small interference RNAs (siRNAs). After transfection with the BAFF, BCMA or BAFF-R siRNA, the expression level of each gene was reduced (Fig. 6A). Under these conditions, the 5x B reporter activity was reduced to approximately half of the control (Fig. 6A). In agreement with this, an EMSA revealed the reduced DNA binding activity of NF-B in BAFF siRNA-transfected cells (Fig. 6B). The complex was supershifted in the presence of p65 or p50 Abs (data not shown). Taken together, these results suggested that an internal autocrine loop of BAFF and its receptors contributes, at least in part, to the endogenous NF-B activity seen in HT1080 cells.
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B (Munk Pedersen & Reed 2004). In the present study, we demonstrated that BAFF-R and its ligand BAFF are expressed aberrantly in cancer cells and confer the activation of NF-B.
It is noteworthy that the expression of BAFF-R mRNA is not limited to lymphocytes, but is broader than previously thought. However, among the cell lines studied, the BAFF-R protein was only detected in HT1080, indicating the strict regulation of the posttranscriptional steps for BAFF-R mRNA. There are several mechanisms proposed for the control of translation in cancer, and one of the candidates is a subunit of the E3 ubiquitin ligase complex, VHL. However, this type of control is not likely in HT1080, because this cell line has wild type VHL (Ogura et al. 2005). We also tried to identify the BAFF-R expressing tumors in clinicopathological specimens; however, none of the tumors except osteosarcoma produced the protein. We also detected BAFF in osteosarcoma specimens as well as in HT1080 cells, which suggested the existence of an autocrine loop or a paracrine loop that dysregulates the BAFF signaling pathway. Consistent with this speculation, we found that siRNA-targeted to BAFF, BAFF-R, or BCMA expression decreased the NF-B basal activity.
The aberrant expression of BAFF-R endowed the cells with activated NF-B; however, it might use a different pathway from that of B cells. In the present study, we used the mutant cell line COS-A717 to isolate BAFF-R. Theoretically, the defect of COS-A717 in the NF-B signaling pathway would be rescued by exogenously introduced cDNAs that encode molecules not only compensating for the primary defect but also bypassing the activation pathway. In B cells, BAFF-R activation promotes the processing of NF-B2/p100 to p52, which is referred to as the alternative pathway (Claudio et al. 2002; Kayagaki et al. 2002). However, the aberrantly expressed BAFF-R seems to activate NF-B through the canonical pathway, for the following reasons. First, when we examined the involvement of IB by co-transfection of the IB SR cDNA, IB SR abolished the BAFF-R-induced NF-B activation in a dose-dependent fashion. IB SR is resistant to phosphorylation-induced degradation, and thereby prevents NF-B activation through the canonical pathway. Second, NF-B2/p100 processing is mediated by NIK and IKK1 (Claudio et al. 2002; Kayagaki et al. 2002). However, we could not detect p52 in parental COS and COS-A717 cells (data not shown). We instead detected the DNA binding complex composed of p65/p50, which are components of the classical pathway. Third, it has been reported that in the alternative pathway, the processing of the NF-B2 protein p100 to p52 is mediated by NIK and IKK1 (Claudio et al. 2002; Kayagaki et al. 2002). However, when we examined whether IKK1 and IKK2 were involved by using dominant negative forms of IKK1 and IKK2, IKK2 DN, but not IKK1 DN, inhibited the exogenously expressed BAFF-R-mediated NF-B activation. Because we could not detect the involvement of an alternative NF-B activating pathway in COS-A717 and HT1080 cells, the aberrantly expressed BAFF-R signaling in tumor cells may use a different pathway from that of primary B cells. It remains to be solved how the constitutive NF-B activity is defective in COS-A717. In parallel experiment, we have identified another gene that seems to be involved in this pathway, because this gene is lost in COS-A717 and exogenously induction of this gene into COS-A717 compensates the constitutive NF-B activity. It also seems the induction of NF-B activity by TNF- is defective in COS-A717. However, in the reporter assay COS-A717 responds to TNF- to some extent even though the constitutive activity is limited (Fig. 3A, right panel). The further analysis of COS-A717 is in progress in our laboratory.
In conclusion, we found the aberrant expression of BAFF-R and its ligand in cells of nonhematopoietic origins, and the internal autocrine loop contribute the constitutive NF-B activity in malignant cells. The aberrant expression of BAFF-R may use a canonical NF-B activating pathway and play a role in the development of certain types of cancer cells. These results were mainly obtained by the analysis of the NF-B mutant cell line, COS-A717. We believe that this cell line will provide a powerful tool for scientists working on the NF-B signaling pathway.
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Experimental procedures |
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The human spleen cDNA library was purchased from Life Technologies (Rockville, MD). The Capture-Tec kit was purchased from Invitrogen (Carlsbad, CA). The plasmid, HIV-LTR-Sp1-pHook, was constructed by replacing the cytomegalovirus (CMV) promoter of the pHook-1 vector with the HIV-LTR lacking the Sp1 site. The green fluorescent protein (GFP) expression vector was constructed by ligating HIV-LTR-Sp1 upstream from GFP. The five-tandem B luciferase reporter vector (5x
B) was purchased from Stratagene (La Jolla, CA). The I-B superrepressor (I-BSR) expression plasmid was described previously (Sugita et al. 2002). The expression vectors for the dominant negative mutants of IKK1 (IKK1.DN) and IKK2 (IKK2.DN) were a kind gift from Dr Yamaoka (Tokyo Medical and Dental University, Tokyo, Japan) (Hironaka et al. 2004). ICR191 was purchased from Sigma (St. Louis, MO). TNF- was purchased from Pepro Tech EC (London, UK).
Cell culture
COS, COS-A717, HeLa, and HT1080 cultures were maintained in Dulbecco's modified Eagle's medium, containing 10% heat-inactivated fetal bovine serum (FBS), at 37 °C in a humidified 5% CO2 atmosphere. HUT102, MT4, Molt4, Jurkat, and Ramos cultures were maintained in RPMI1640 containing 10% FBS, and K562 and HL60 cells were cultured in Iscove's medium with 10% FBS, at 37 °C in a humidified 5% CO2 atmosphere.
Transfection and luciferase assay
Cells were transfected with the 5x B-luciferase reporter and the BAFF-R expression vectors, as indicated in the text and figure legends. Transient transfections were carried out using the FuGene 6 reagent (Roche, Indianapolis, IN). When necessary, additional DNA (pcDNA3) was added to equalize the amount of transfected DNA in each sample. At 48 h post-transfection, the B-directed expression of firefly luciferase was determined, using luciferase assay reagents from Promega (Madison, WI), and the luciferase activities were measured with a BioOrbit 1254 luminometer (Turku, Finland). The relative transfection efficiency in each sample was determined by measuring the Renilla luciferase activity. The data were normalized per transfection efficiency.
Northern blot analysis
Total RNA was extracted with the ISOGEN (NipponGene, Tokyo, Japan) reagent, according to the manufacturer's protocol. Total RNA (10 µg) was fractioned by electrophoresis through 1% agarose, transferred to a Gene Screen Plus hybridization transfer membrane (NEN Life Science Products, Boston, MA), and hybridized with 32P-labeled cDNA probes for BAFF-R and GAPDH. The membrane was autoradiographed, and the image was quantified using an image analyzer, BAS-5000 (Fuji Photo Film, Tokyo, Japan).
Reverse transcription/polymerase chain reaction (RT/PCR)
Reverse transcription/polymerase chain reaction was carried out using a commercial kit (RT-PCR kit, Stratagene, La Jolla, CA) according to the instructions provided by the manufacturer. PCR was accomplished using a DNA Thermal Cycler (TaKaRa, Kyoto, Japan). The parameters for 30-cycles of PCR were as follows: denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min for BAFF-R, denaturation at 94 °C for 1 min, annealing at 62 °C for 1 min, and extension at 72 °C for 1 min for BAFF, and denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 1 min for BCMA. The parameters for 22-cycles of PCR were as follows: denaturation at 94 °C for 1 min, annealing at 62 °C for 1 min, and extension at 72 °C for 1 min for β-actin. The primers for BAFF-R were 5'-AATTCATATGAGGCGAGGGCC-3' (forward primer), 5'-CGGAGACAGAATGATGACCT-3' (reverse primer), the primers for BAFF were 5'-CACAGAAAGGGAGCAGTCAC-3' (forward primer), 5'-CTGAACGGCACGCTTATTTC-3' (reverse primer), the primers for BCMA were 5'-CTGGAAAAGAG C AGGACTGG-3' (forward primer), 5'-ACTGCTCGAGTCG AAATGGT-3' (reverse primer), and the primers for β-actin were 5'-AAGAGAGGCATC CTCACCCT-3' (forward primer), 5'-TACATCGCT GGGGTGTTGAA-3' (reverse primer). The PCR products were resolved in a 1.5% agarose gel and were visualized by ethidium bromide staining.
Real-time polymerase chain reaction for bcl-2
The first strand of the cDNA was described above. PCR primer pairs were as follows: bcl-2, 5'-GAGGATTGTGGCCTTC TTTG-3', and 5'-ACAGTTCCACAAAGGCATCC-3'; β-actin, 5'-AAGAGAGGCATCCTCACCCT-3', and 5'-TACATCGCTGGGGTGTTGAA-3'. PCR was carried out using QuantiTect SYBR Green (Qiagen, Hilden, Germany) with a LightCycler QuickSystem 330 system (Roche Molecular Biochemicals, Mannheim, Germany). All data were normalized to β-actin measured in the same samples.
Western blot analysis
Cell extracts were prepared from the cells transfected for the luciferase assay. Cell lysates were resolved by 12.5% SDS PAGE, transferred onto an Immobilon-P membrane (Millipore, Bedford, MA), and blocked with 5% non-fat dry milk in TBS with 0.5% Tween 20. The blot was incubated with anti-Flag-antibody (M2) (Sigma) and anti-β-actin antibody (Chemicon, Temecula, CA), followed by an incubation with horseradish peroxidase-conjugated goat anti-mouse Ig (Amersham Pharmacia Biotech, Uppsala, Sweden). The blot was visualized with the ECL detection system (Amersham Pharmacia Biotech).
Electrophoretic mobility shift assays (EMSAs)
The COS-A717 and parent cells were cultured in 100-mm cell culture dishes. Preparation of nuclear extracts for electrophoretic mobility shift assays (EMSAs) was carried out as described previously (Sugita et al. 2002). Nuclear extracts were prepared 24 h post-transfection. Consensus NF-B site 5'-AGTTGAGGGGACT TTCCCAGGC-3' and mutant 5'-AGTTGAGGCGACTTTCC CAGGC-3' oligonucleotides were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The double stranded oligonucleotides were end labeled with [
-32P] ATP, using T4 polynucleotide kinase (TaKaRa, Kyoto, Japan). The reaction was conducted in a total volume of 10 µL, using 10 µg of nuclear extract, 1 µg of poly(dI-dC), 20 mM HEPES-NaOH (pH 7.6), 100 mM NaCl, 1 mM DTT, 1 mM PMSF, and 2% glycerol. The binding reaction mixture was incubated with 10 000 cpm of radiolabeled probe for 15 min. For the competition and supershift assays, a 20-fold excess of unlabeled or mutant oligonucleotide and the antibodies to p65, p50, and cRel (Santa Cruz Biotechnology) were added to the reactions, respectively. The samples were loaded onto a 5% nondenaturing polyacrylamide gel and run in 0.5x TBE buffer. After electrophoresis, the gel was dried and processed for autoradiography.
Immunohistochemistry
Using formalin-fixed, paraffin-embedded tissue sections and cell lines, we carried out an immunohistochemical analysis. Surgical specimens prepared for immunohistochemistry were as follows: three cases of angiosarcoma, 7 cases of chondrosarcoma, 3 cases of Ewing's sarcoma, 6 cases of leiomyosarcoma, 4 cases of liposarcoma, 8 cases of osteosarcoma (seven conventional osteosarcomas and one periosteal osteosarcoma), 3 cases of rhabdomyosarcoma, 6 cases of synovial sarcoma, 7 cases of malignant fibrous histiocytoma, and 3 cases of malignant peripheral nerve sheath tumor. The cell lines were fixed in cold acetone for 10 min. The tissue sections and cell lines were incubated with a rabbit anti-human BAFF-R antibody and an anti-BAFF antibody (Abcam, Cambridge, UK) at dilutions of 1 : 500 and 1 : 200, respectively. After washing the unbound antibody, the specimens were treated with a biotinylated secondary antibody followed by streptavidin conjugated with horseradish peroxidase, according to the instructions included with the DAKO LSAB kit (DAKO A/S Glostrup, Denmark). Immunohistochemical reactions were developed with diaminobenzidine as the chromogen.
Flow cytometric analysis
The expression of BAFF-R on the cell surface was analyzed by flow cytometry. The cells (5 x 105) were washed twice with PBS containing 2% FBS (PBS/FBS), and then were incubated for 30 min at 4 °C with a mouse monoclonal anti-human BAFF-R (Abcam) or control antibody. After washing twice with PBS, the cells were suspended in PBS/FBS and were incubated with a fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody (PharMingen, San Diego, CA) at 4 °C. To analyze the intracellular expression of BAFF-R, stainings of the intracellular antigen were carried out using the fixation and permeabilization reagents in a Cytofix/Cytoperm Kit (Becton-Dickinson, San Jose, CA). The washed cells were suspended in the fixation/permeabilization solution for 20 min at 4 °C. After washing twice with 1 x BD Perm/wash buffer, the cells were incubated at 4 °C with a mouse monoclonal anti-human BAFF-R (Abcam) or control antibody. After two washes with the 1 x BD Perm/wash buffer, the cells were incubated with an FITC-conjugated anti-rabbit antibody at 4 °C. The cells were then washed again, resuspended in PBS/FBS and analyzed by FACScan using the CELLQuest software (Becton-Dickinson, San Jose, CA).
Cytotoxicity assay
The sensitivity of these cells to TNF- was determined using the tetrazolium salt assay. The cells were placed in 100 µL of medium/well in 96-well plates. Twenty-four hours after the addition of various concentrations of TNF-, the cells were incubated for 4 h at 37 °C with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (5 mg/mL). The cells were lysed with 100 µL of isopropanol containing HCl per well. Wells without cells served as blanks.
siRNA
The sequences of the targeted genes are as follows: sense 5'-CAUAUCGUGAUCAAGUCUUUG-3', anti-sense 5'-AAGACUU GAUCACGAUAUGGG-3' for BAFF, and sense 5'-GGCUACGUC CAGGAGCGCATT-3', anti-sense 5'-UGCGCUCCUGGACGU AGCCTT-3' for GFP. The BAFF-R siRNAs were purchased from Santa Cruz Biotechnology. The siTRIO BCMA siRNAs were obtained from B-Bridge International (Sunnyvale, CA). The annealed oligonucleotides were transfected using Lipofectamine 2000 (Invitrogen, San Diego, CA). The cells were harvested for further analyses after 24 h (HT1080 cells). For the luciferase assay, the cells were transfected with the 5x B-luciferase reporter, using the FuGene 6 reagent, at 24 h after the siRNA transfection.
Statistical analysis
Statistical comparisons were made using the Student's t-test. Differences were considered to be statistically significant at P < 0.05.
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Acknowledgements |
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Footnotes |
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* Correspondence: tosim{at}net.nagasaki-u.ac.jp
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References |
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Received: 27 February 2008
Accepted: 23 July 2008
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