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. 2009 Nov;128(3):360–368. doi: 10.1111/j.1365-2567.2009.03111.x

Cell cycle arrest induced by engagement of B7-H4 on Epstein–Barr virus-positive B-cell lymphoma cell lines

Ga Bin Park 1, Hyunkeun Song 1, Yeong-Seok Kim 1, Minjung Sung 1, Jeoung W Ryu 1, Hyun-Kyung Lee 2, Dae-Ho Cho 3, Daejin Kim 4, Wang J Lee 5, Dae Y Hur 1
PMCID: PMC2770684  PMID: 20067536

Abstract

B7-H4 is a recently discovered B7 family member that has inhibitory effects on T-cell immunity. However, the reverse signalling mechanism of the B7-H4-expressing cells remains unclear. Previous work has shown that B7-H4 expression was enhanced on B cells following Epstein–Barr virus (EBV) infection, and engagement of cell-surface-expressed B7-H4 induces cell death of EBV-transformed B cells. Here we found that B7-H4 was constitutively expressed on EBV-positive lymphoma cells, Raji and IM-9 cells, but was not expressed on EBV-negative lymphoma cells (Ramos). Engagement of B7-H4 significantly reduced cell growth of Raji and IM-9 cells and resulted in cell cycle arrest at G0–G1 phase in a dose- and time-dependent manner. To clarify the mechanism of cell cycle arrest via activation of B7-H4, cell cycle regulatory factors were examined by reverse transcription–polymerase chain reaction and immunoblotting. We found that B7-H4 triggered down-regulation of CDK4/6 and up-regulation of p21 expression at both protein and RNA levels. Furthermore, CDK2 and cyclin E/D expression was down-regulated by B7-H4 triggering. Additionally, the down-regulation of phospho-AKT and phospho-cyclin E were clearly detected in B7-H4-activated Raji cells, but the phosphorylation of p53 was constitutively maintained. These results indicate that B7-H4-mediated signalling on EBV-positive B-cell lymphoma cells modulates the cell cycle through down-regulation of the AKT pathway. Consequently, B7-H4 may be a new potential target for use in EBV-positive lymphoma therapy.

Keywords: apoptosis, B cells, B7-H4, cancer, cell cycle, costimulation, Epstein–Barr virus

Introduction

B7-H4 is a newly identified member of the B7 family and is involved in the negative regulation of T-cell immunity through the inhibition of T-cell function, such as activation, proliferation, cytokine production and cytotoxic activity.1–3 B7-H4 is expressed in human immune cells, such as T cells, B cells, monocytes and dendritic cells. Recently, many studies have reported that B7-H4 is observed in various cancers, such as lung and ovarian cancer.4–7 The level of B7-H4 messenger RNA (mRNA) is high in malignant tumour tissue, but it is not detected in normal tissue. Based on this evidence, some studies have proposed that B7-H4 may have a role in the evasion mechanism of cancer cells through the down-regulation of T-cell immunity. However, whether the engagement of B7-H4 by counter molecules affects the fate of B7-H4-expressing cells is poorly understood. Evidence for a relationship between immune escape of viruses, such as Epstein–Barr virus (EBV) and B7 family members has been proposed.8,9 The activation of T cells in the absence of costimulation with a B7 family member leads to T-cell death. Moreover, B7-1 (CD86) and B7-2 (CD80) deliver negative signals to T cells by binding to cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) on activated T cells.10,11 Epstein–Barr virus is a member of the human gamma herpes virus family and is widespread in approximately 90% of the world’s adult population.12 It preferentially infects B lymphocytes and is associated with malignant human diseases, such as Burkitt’s lymphoma, Hodgkin’s disease and undifferentiated nasopharyngeal carcinoma, and in lymphoproliferative disorders in immunodeficient patients.13 Many studies have reported that CD8+ T cells play a pivotal role in the survival and malignancy of EBV-transformed cells, which restrict the overgrowth of EBV-transformed B cells.14,15 In contrast, some studies revealed that EBV-transformed cells have the ability of immune escape through the modulation of human leucocyte antigen molecules and costimulatory molecules.16,17 However, the detailed mechanisms for the immune evasion of EBV are not fully understood.

Previously, our study reported that B7-H4 expression was enhanced by EBV infection. Moreover, activation of B7-H4 molecules on EBV-transformed B cells significantly induced Fas-mediated apoptosis.18 These findings elucidate a possible relationship between B7-H4 and the immune evasion of EBV-transformed B cells in EBV infection, and suggest that the engagement of EBV-infected B cells by anti-B7-H4 antibody can be applied as a potential therapeutic tool for malignant diseases with EBV involvement.

To confirm the possible role of B7-H4 on EBV-infected B cells, we investigated the expression and function of B7-H4 on EBV-positive lymphoma cell lines Raji and IM-9. Interestingly, we found that B7-H4 was constitutively expressed in both of these EBV-positive lymphoma cell lines but was not expressed on the EBV-negative lymphoma cell line, Ramos. Moreover, we observed that engagement of B7-H4 molecules on Raji and IM-9 cells significantly induced cell cycle arrest of G0/G1 through up-regulation of p21waf1 and p27kip1 and down-regulation of AKT activation.

Materials and methods

Cell line and culture

Raji cells, an EBV-positive human Burkitt’s lymphoma cell line, IM-9, an EBV-positive human B lymphoblastoid cell line, and Ramos, an EBV-negative human Burkitt’s lymphoma cell line, were obtained from the American Type Culture Collection (Rockville, MD) and were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and antibiotics in a 5% CO2 atmosphere at 37°.

Antibodies and reagents

Anti-B7-H4 [B7H4, immunoglobulin G1κ (IgG1κ)] was purchased from eBioscience (San Diego, CA). Phospho-p53 (Ser15), p-53, p2lWaf1, CDK4, CDK6, phospho-cyclin E (Thr62), cyclin E, phospho-Akt (Ser473), Akt and β-actin were purchased from Cell Signaling (Beverly, MA). MOPC21 (IgG1k, isotype control antibody) was purchased from Sigma-Aldrich (St Louis, MO) as the isotype-control antibody for anti-B7H4 antibody. DiOC6 (3,3′-dihexyloxacarbocyanineiodide) and DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) were purchased from Molecular Probes (Eugene, OR). Alamar Blue reagent was purchased from Serotec (Raleigh, NC). The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was purchased from Cell Signaling.

Alamar Blue assay

The inhibition of cell growth was determined using an Alamar Blue assay. Raji Burkitt’s lymphoma cells were plated on 96-well plates (105 and 5 × 104 cells/well) and treated with 0, 1, 2, 5, 10 and 20 μg/ml anti-B7-H4 antibody for 72 hr before adding Alamar Blue solution. Alamar Blue was added to each well in an amount equal to 1/10 volume of the total culture medium and fluorescence was measured at 570 nm (excitation) and 600 nm (emission) using a Fluorometer (Synergy HT; Bio-Tek instruments Inc., Winnoski, VT) 4 hr after addition of the dye. Each experiment was performed in triplicate.

EBV-positive Burkitt’s lymphoma cell line, Raji, death following B7-H4 activation and measurement of mitochondrial membrane potential (△ψ)

For the activation of B7-H4, Raji cells (5 × 105 cells/well, 200 μl) were harvested in tubes and antibody was added (2 μg/ml). The cells were collected, washed twice with phosphate-buffered saline (PBS), and then resuspended in 100 μl of annexin V-binding buffer (10 mm HEPES/NaOH pH 7·4, 140 mm NaCl, 2·5 mm CaCl2). After the addition of 2 μl fluorescein isothiocyanate-conjugated annexin V (BD Pharmingen, San Diego, CA) and 2 μl propidium iodide (BD Pharmingen), the cells were incubated in a dark room at room temperature for 15 min. Finally, 400 μl annexin V binding buffer was added to each tube and the cells were analysed using a FACSCalibur (BD Pharmingen). To measure mitochondrial potential, cells were collected and further incubated in 100 μl PBS containing 20 nm DiOC6 at 37° for 15 min. Cells were then harvested and washed with cold PBS, and reactive oxygen species levels and △ψ were determined by FACSCalibur (BD Pharmingen).

Cell cycle analysis

The cells were harvested after treatment, washed twice in PBS (2% FBS), fixed with 70% cold aqueous ethanol and stored at 4° for at least 24 hr. Cells were then washed in PBS, and the cell pellets were stained with a propidium iodide (PI) stain (10 mg/ml RNAse A and 2 mg/ml PI in PBS). The cell suspension was then incubated in the dark at room temperature for 30 min, and DNA content was determined using a FACSCalibur (BD Pharmingen).

Reverse-transcription polymerase chain reaction (RT-PCR)

The cells were harvested after treatment and washed twice in cold PBS. RNA was extracted using an RNeasy mini kit (Qiagen, Valencia, CA), and complementary DNA was produced using RT premix (Bioneer, Daejeon, South Korea). The PCR amplification was performed using specific primer sets (Bioneer) for p27kip1 [upstream primer; 5′-TGG AGA AGC ACT GCA GAG AC, downstream primer; 5′-GCG TGT CCT CAG AGT TAG CC, 252-base-pair (bp) product], p53 (upstream primer; 5′-CAT GAG CGC TGC TCA GAT AG, downstream primer; 5′-CTG AGT CAG GCC CTT CTG TC, 543-bp product), p21waf1 (upstream primer; 5′-AGC TCA ATG GAC TGG AAG GG, downstream primer; 5′-GAG CTG GAA GGT GTT TGG GG, 457-bp product), CDK2 (upstream primer; 5′-CAT GGA GAA CTT CCA AAA G, downstream primer; 5′-CTA TCA GAG TCG AAG ATG GG, 799-bp product), CDK4 (upstream primer; 5′-CAG GAC CTA AGG ACA TAT CTG GA, downstream primer; 5′-CTC GGT ACC AGA GTG TAA CAA CC, 253-bp product), CDK6 (upstream primer; 5′-CCG AGT AGT GCATCG CGA TCT AA, downstream primer; 5′-CTT TGC CTA GTT CAT CGA TAT C, 407-bp product), cyclin E1 (upstream primer; 5′-GGA AGG CAA ACG TGA CCG TT, downstream primer; 5′-GGG ACT TAA ACG CCA CTT AA, 638-bp product), cyclin D1 (upstream primer; 5′-CTG GAG CCC GTG AAA AAG AGC, downstream primer; 5′-CTG GAG AGG AAG CGT GTG AGG, 434-bp product) and cyclin D2 (upstream primer; 5′-TAC TTC AAGTGC GTG CAG AAG GAC, downstream primer; 5′-TCC CAC ACT TCC AGT TGC GATCAT, 498-bp product). For control, a specific primer set for β-actin (upstream primer; 5′-ATC CAC GAA ACT ACC TTC AA, downstream primer; 5′-ATC CAC ACG GAG TAC TTG C) was used, which yielded a 200-bp product. The PCR (25 cycles; 20 seconds at 94°, 10 seconds at 60°, 30 seconds at 72°) was performed using AccuPower PCR premix (Bioneer) and PCR products were analysed by agarose gel electrophoresis and visualized with ethidium bromide under ultraviolet light using a Multiple Gel-DOC system (Fujifilm, Tokyo, Japan). Densitometry was performed using multi-gauge version 2·3 (Fujifilm).

Western blots

The cells (1 × 107 cells/sample) were washed in cold PBS and lysed in RIPA buffer (EIPiS, Daejeon, South Korea) on ice for 10 min. The lysates were centrifuged at 15 000 g for 15 min at 4°, and the proteins (10 μg/sample) were immediately heated for 1 min at 100° after addition of sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer. Each cell lysate was separated by 15% SDS–PAGE under reducing conditions and transferred to a nitrocellulose membrane using semidry technique at 80 mA for 2 hr. Membranes were blocked by treatment with 5% skim milk in Tris-buffered saline supplemented with 0·1% Tween-20 (TBST) for 1 hr, and subsequently incubated with the primary monoclonal or polyclonal antibodies at a final concentration of 1 μg/ml or at a final dilution of 1 : 1000, respectively, overnight in TBS. After three washes in TBST, membranes were incubated with peroxidase-conjugated secondary antibodies (final dilution, 1 : 3000) in TBS (5% skim milk) for 1 hr and subsequently washed as described above. Detection was performed by chemiluminescence using an ECL kit (Enhanced ChemiLuminescence; Amersham Life Science, Braunschweig, Germany) and the Multiple Gel-DOC system (Fujifilm). The following primary antibodies were used: phospho-p53 (Ser15), p-53, p21Waf1, CDK4, CDK6, phospho-cyclin E (Thr62), cyclin E, phospho-Akt (Ser473), Akt and β-actin from Cell Signaling.

Statistical analysis

Results are representative of at least three separate experiments. Statistical significance was calculated using Student’s t-test. P-values < 0·05 were considered significant.

Results

Effects of B7-H4 activation on the cell viability of EBV-positive lymphoma cells

We have previously shown that EBV-infected primary B cells enhanced the expression of surface B7-H4 and that activation of B7-H4 induced by EBV infection significantly increased the rate of cell death of transformed B cells.18 We next determined whether expression of B7-H4 affects the EBV-positive and -negative pattern of B lymphoma cell lines. Using flow cytometric analysis, we observed that B7-H4 was significantly expressed on EBV-positive lymphoma cells, Raji and IM-9 cells, but not on the EBV-negative lymphoma cell line, Ramos (Fig. 1a). Based on our previous studies, we sought to determine the signalling mechanism of surface-expressed B7-H4, and used Burkitt’s lymphoma cell line (EBV-positive) Raji cells as a model for this purpose. Raji cells were plated in 96-well plates and treated with various concentrations of anti-B7-H4 antibody (0–10 μg/ml), and their cell growth was determined by using the Alamar Blue assay. After 72 hr treatment with anti-B7-H4 antibody, Raji cells showed significantly reduced cell growth compared to the MOPC control antibody treatment group (Fig. 1b). In contrast, B7-H4 activation using anti-B7-H4 antibody did not show a dramatic change in the rate of apoptosis or mitochondrial membrane potential disruption in a dose-dependent manner (Fig. 1c). Overall, these data suggest that activation of B7-H4 on Raji cells has strong effects on cell growth inhibition.

Figure 1.

Figure 1

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B7-H4 expression on Epstein–Barr virus (EBV)-positive and -negative lymphoma cells and the effect of B7-H4 activation on cell viability and mitochondrial potential of EBV-positive Raji cells. (a) EBV-positive lymphoma cell lines, Raji and IM-9, and EBV-negative lymphoma cell line, Ramos, were stained with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human B7-H4 antibody. (b) To examine the effect of B7-H4 activation on cell viability of Raji cells, we treated cells (1 × 105 and 5 × 104 cells/well) with various concentrations of anti-B7-H4 antibody for 72 hr. Cell proliferation was determined by an Alamar Blue assay. (c) The cells were washed with phosphate-buffered saline and treated with the indicated dose of anti-B7-H4 antibody for 24 hr. The cells were stained with a FITC-conjugated anti-annexin V monoclonal antibody and propidium iodide (PI). DiOC6 staining represents the mitochondrial potential in each condition. Results are representative of three independent experiments. *P<0·05 versus control; **P<0·01 versus control.

B7-H4 ligation induced cell cycle arrest/block of EBV-positive Raji cells

The cell cycle was analysed by flow cytometry to investigate the effects of B7-H4 triggering on Raji cells. Raji cells were treated with different concentrations of anti-B7-H4 antibody for 24 hr (Fig. 2(a,b)). B7-H4 activation-induced growth inhibition was associated with cell cycle arrest in G0/G1 phase following treatment with 1 and 10 μg/ml anti-B7-H4. Compared with the untreated and MOPC groups (40·02% and 39·68% of cells in G0/G1 phase, respectively), the percentage of Raji cells in the G0/G1 phase of the cell cycle reached a maximum peak at 2 μg/ml. Next, we performed a time kinetic assay. Raji cells were treated with 2 μg/ml anti-B7-H4 for 2, 4, 8, 16 and 24 hr. Raji cells that were treated with anti-B7-H4 antibody for 16 hr demonstrated a significant increase in percentage of cells in the G0/G1 phase (65·66%) compared with untreated, MOPC-treated, 2-hr-treated and 4-hr-treated groups (39·24%, 44·51%, 37·05% and 40·38%, respectively) (Fig. 2c,d). Although the level of G0/G1 phase cells increased in accordance with time treatment of anti-B7-H4 antibody, the level of G0/G1 phase cells reached a maximum at 2 μg/ml anti-B7-H4. These results demonstrate that the cross-linking of B7-H4-induced growth-arrest of EBV-positive Raji cells accompanies the accumulation of cells in the G0/G1 phase.

Figure 2.

Figure 2

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Cell cycle distribution of Epstein–Barr virus (EBV)-positive Raji cells according to dose- and time-dependent activation of B7-H4. (a) Flow cytometric analysis showing dose-dependent B7-H4-induced cell cycle arrest of EBV-positive Raji cells. Raji cells were incubated with the indicated dose of anti-B7-H4 or an isotype-matched control monoclonal antibody for 24 hr, and flow cytometry after propidium iodide (PI) staining of nuclei was performed as described in the Materials and methods. The percentages of cells in the sub-G1, G0/G1, S and G2/M phases were calculated using the modfit computer software program and are summarized in (b). The data are representative examples of duplicate tests. (c) Time-dependent effect of B7-H4 stimulation on the cell cycle distribution of Raji cells. Cells were treated with anti-B7-H4 (2 μg/ml) for the indicated time and then were stained with PI and analysed by flow cytometry. The percentage of cells in the sub-G1, G0/G1, S and G2/M phases were calculated using the modfit computer software program and are summarized in (d).

Expression of cell cycle regulator gene after B7-H4 ligation

It is well known that the relationship of cyclin–cyclin-dependent kinase (CDK) plays a critical role in the progression of the cell cycle. To further understand the effect of B7-H4 activation on cell cycle progression, the expression of cell cycle regulatory protein was investigated by RT-PCR (Fig. 3). As shown in Fig. 3, the mRNA levels of CDK2, CDK4, CDK6, cyclin E1 and cyclin D1, but not of cyclin D2, were significantly decreased following B7-H4 activation in a dose-dependent manner. These results indicate that B7-H4 stimulation targets cyclin D, cyclin E, CDK4/6 and CDK2, which subsequently leads to G0/G1 arrest. Next we investigated the change in p53, p21waf1 and p27kip1 mRNA expression. The expression levels of p21waf1 and p27kip1 were constitutively expressed following B7-H4 activation in a dose-dependent manner. In contrast, we found that the stimulation of B7-H4 significantly decreased the level of p53 mRNA in Raji cells (Fig. 3a) but did not change the level of p53 mRNA in IM-9 cells (Fig. 3b). Taken together, our data show that the cross-linking of these B7-H4 molecules leads to cell cycle arrest at the G0/G1 phase of EBV-positive B cells via down-regulating gene expression of CDK and cyclin.

Figure 3.

Figure 3

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Messenger RNA (mRNA) expression of cell cycle regulatory protein in B7-H4-induced cell cycle arrest at G0/G1 phase of Epstein–Barr virus (EBV)-positive Raji (a) and IM-9 (b) cells. EBV-positive Raji and IM-9 cells (1 × 106 cell/ml) were washed with phosphate-buffered saline and treated with the indicated dose of anti-B7-H4 antibody for 8 hr. Total RNA was isolated using an RNeasy mini kit (Qiagen) and complementary DNA was obtained using RT premix (Bioneer). The polymerase chain reaction product was analysed by electrophoresis on 1% agarose gel. β-Actin was included as an internal control. Results are representative of three independent experiments.

B7-H4 triggering signals through the PI3K/AKT pathway for G0/G1 cell cycle progression

To gain insight into the involvement of the PI3K/Akt pathway in the arrest of G0/G1 cell cycle through the triggering of B7-H4, we analysed the expression pattern of signalling molecule known to be important in the PI3K/Akt pathway by Western blot analysis. As shown in Fig. 4(a,b), the activation of B7-H4 induces Akt activation in a dose-dependent manner in Raji and IM-9 cells. Additionally, CDK4/6 expression and phosphorylation of cyclin E was markedly decreased after B7-H4 activation. By contrast, p53 phosphorylation and p53-dependent p21 expression increased in Raji and IM-9 cells following activation of B7-H4 (Fig. 4a,b). We next investigated the effects of Ly294002, a specific inhibitor of PI3K, on expression patterns of cell cycle regulator proteins in Raji cells. As shown in Fig. 4(c), Ly294002 treatment significantly reduced phosphorylation of Akt and cyclin E. Moreover, treatment with Ly294002 decreased expression of CDK2, -4 and -6, but increased phosphorylation of p53 and p21waf1.

Figure 4.

Figure 4

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Western blot analysis of cell cycle regulatory protein after activation of B7-H4 and treatment with a phosphoinositide 3-kinase (PI3K)-specific inhibitor on Epstein–Barr virus (EBV)-positive lymphoma cells. Raji (a) and IM-9 (b) cells (5 × 106 cells/ml) were washed with phosphate-buffered saline (PBS) and treated with the indicated dose of anti-B7-H4 antibody for 24 hr. Western blot analysis was performed with antibodies recognizing human phosphorylated-Akt, Akt, phosphorylated-p53, p21waf1, CDK4, CDK6, phosphor-cyclin E and cyclin E. β-Actin was included as an internal control. A representative result from three independent experiments is shown. (c) Raji cells (5 × 106 cells/ml) were washed with PBS and treated with 25 μm Ly294002, a PI3K-specific inhibitor. Western blot analysis was performed using antibodies recognizing human phosphor-Akt, Akt, phosphor-p53, p21waf1, CDK4, CDK6, phosphor-cyclin E and cyclin E. β-Actin was included as an internal control. A representative result from three independent experiments is shown.

These results indicate that cell cycle arrest and apoptotic signalling mediated via the activation of B7-H4 occurs through the PI3K/Akt pathway.

Discussion

B7-H4 has been shown to negatively regulate the T-cell immune response via the inhibition of T-cell activation, proliferation and cytokine production.1–3 Previously, we have shown that EBV-infected B cells significantly express B7-H4 and that stimulation of B7-H4 on EBV-infected B cells leads to apoptosis through the Fas-mediated pathway.18 In this report, we investigated the molecular mechanism associated with B7-H4 signalling in an EBV-positive Burkitt’s lymphoma cell line, Raji cells, as a model system and found that B7-H4 activation leads to cell growth arrest and apoptosis in Raji cells (Figs 1 and 2). Additionally, we found that activation of B7-H4 induced cell cycle arrest and apoptosis in another EBV-positive B lymphoblast cell line, IM-9 (Figs 3 and 4). These results contradict our previous finding that the activation of B7-H4 on EBV-transformed B cells enhanced apoptosis induced by Fas through rapid reactive oxygen species-mediated Fas ligand expression. The reasonable explanation for this difference is unclear, but it may be related to the specific cell type of EBV-transformed B cells and EBV-positive cell line (Fig. S1).

It is well known that the PI3K/Akt pathway is important for controlling signals in cell cycle and apoptosis.19–21 To determine the association of the PI3K/Akt pathway in B7-H4 activation-induced cell cycle arrest and apoptosis, we analysed Akt phosphorylation after B7-H4 stimulation. We observed that Akt phosphorylation significantly decreased in a dose-dependent manner (Fig. 4a,b). Furthermore, we found that LY294002, a PI3K-specific inhibitor, inhibited Akt phosphorylation in a dose-dependent manner and showed the same pattern of expression of cell cycle regulatory genes as was seen after B7-H4 activation (Fig. 4c). Additionally, we observed that the mRNA level of p27kip1 was constitutively maintained after B7-H4 treatment. Thus, p27kip1, a cyclin-dependent kinase inhibitor, can block cyclin A and cyclin E, and prevent Akt activation.22,23 These results indicate that stimulation of B7-H4 in Raji cell induces cell cycle arrest at G0/G1 phase through down-regulation of the PI3K/Akt pathway.

Tumour suppressor p53 regulates the G0/G1 and G2/M phases of the cell cycle and causes growth arrest.24,25 Akt down-regulation leads to stabilization of p53 protein, which leads to increased p21waf1 expression.26 Our results show that mRNA levels of p21waf1 are constitutively expressed, but p53 decreased in a dose-dependent manner after B7-H4 activation (Fig. 3). However, Western blot analysis showed that activation of B7-H4 induced a detectable increase in phosphorylated p53 protein and increased p21waf1 protein in Raji and IM-9 cells (Fig. 4). These findings suggest that p53 stabilization, not expression, plays a critical role in cell cycle arrest at the G0/G1 phase following B7-H4 stimulation. Recently, several studies reported that deletion of phosphatase and tensin (PTEN) homologue, a tumour suppressor gene, is involved in enhancing p21waf1 expression and stabilization of p53 through the prevention of the downstream cascade mediated by the PI3K/Akt pathway.26–28 Further studies are necessary to understanding the function of PTEN following B7-H4 activation in Raji and IM-9 cells.

Based on this evidence, we propose that two branched signalling pathways are involved in B7-H4-induced G0/G1 cell cycle arrest; one pathway is a p53-independent p27kip1-mediated pathway and the other is a p53-dependent p21waf1-mediated pathway.

Generally, mammalian cell growth is regulated by the association of cyclin-dependent kinase (CDK) and cyclin protein.29,30 The D-type cyclins (cyclin D1, D2, D3) and CDK 4/6 association is important for triggering the DNA synthesis of S phase.31 Moreover, CDK2 binds cyclin E, while CDK4/6 interacts with D-type cyclin for initiation of S phase.32,33 Here, we have shown that ligation of B7-H4 leads to decreased expression levels of CDKs/cyclins protein and mRNA. These data suggest that expression and activation of CDK2, -4, -6 and D/E-type cyclin proteins might be tightly regulated by the activity of B7-H4. Many studies have reported that CDK protein interacts with retinoblastoma (Rb) protein, a tumour suppressor protein, and that the expression of the CDK gene is positively regulated by Rb.29,34,35 However, we did not test Rb and CDK interaction following stimulation of B7-H4. It would be interesting to characterize Rb signalling pathways leading to CDK expression in EBV-positive lymphoma cells following activation of B7-H4 in future studies. Taken together, we conclude that B7-H4 engagement induces cell cycle arrest at the G0/G1 phase by down-regulating the PI3K/Akt pathway and modulating the protein of cell cycle regulators. These results indicate that B7-H4 would be useful for therapeutic approaches in treating B7-H4-expressing cancers.

Acknowledgments

This work was supported by the SRC/ERC programme of MOST/KOSEF (grant #R11-2005-017-02002-0) and the 2008 Inje University Research Grant.

Glossary

Abbreviations:

CDK

cyclin-dependent kinase

EBV

Epstein–Barr virus

FBS

fetal bovine serum

LCL

lymphoblastoid cell line

mRNA

messenger RNA

PBS

phosphate-buffered saline

PI

propidium iodide

PI3K

phosphoinositide-3 kinase

RT–PCR

reverse transcription–polymerase chain reaction

SDS–PAGE

sodium dodecyl sulphate–polyacrylamide gel electrophoresis

Disclosures

The authors have no financial conflicts of interest..

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1. B7-H4 expression on Epstein-Barr virus (EBV)-transformed B cells. The cells were stained with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human B7-H4 and phycoerythrin (PE)-conjugated mouse anti-human CD19. Numbers in all quadrants epresent the percentages. This figure was published in reference 18.

imm0128-0360-SD1.tif (4.3MB, tif)

Please note: Wiley-Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than about missing material) should be directed to the corresponding author for the article.

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