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Cancer Gene Therapy (2006) 13, 194–202 All rights reserved 0929-1903/06 $30.00

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ORIGINAL ARTICLE

Suppressor of cytokine signaling-1 expression by infectivity-enhanced adenoviral vector inhibits IL-6-dependent proliferation of multiple myeloma cells M Yamamoto1, N Nishimoto2, J Davydova1, T Kishimoto2 and DT Curiel1 1

Division of Human Gene Therapy, Department of Medicine, Pathology and Surgery, and the Gene Therapy Center at UAB, University of Alabama at Birmingham, Birmingham, AL, USA and 2Laboratory of Immune Regulation, Graduate School of Frontier Bioscience, Osaka University, Suita, Osaka, Japan Multiple myeloma (MM) accounts for 10% of hematological malignant disorders. Its refractory nature indicates the necessity of developing novel therapeutic modalities. Since interleukin 6 (IL-6) is one of the major growth factors for MM cells, we expressed suppressor of cytokine signaling-1 (SOCS-1), one of the blockades of IL-6 receptor downstream signaling, to suppress the proliferation of MM cells. Because MM cells are resistant to conventional adenoviral vector infection, we utilized infectivityenhanced adenoviral vectors with an RGD4C motif in the adenoviral fiber-knob region (RGD-modified vector). In infectivity analysis, RGD-modified vectors were superior to unmodified controls in the majority of the MM cell lines tested. The overexpression of SOCS-1 using infectivity-enhanced adenoviral vectors achieved growth suppression in IL-6-dependent MM cells, but not in the IL-6-independent cells. IL-6-induced STAT3 phosphorylation was suppressed in IL-6-dependent cells, indicating that the signal transduction cascade of the IL-6 receptor signaling was blocked. In aggregate, SOCS-1 overexpression with RGDmodified adenoviral vectors achieved the antiproliferative effect in IL-6-dependent MM cells. These results provide an initial proofof-principle of the anticancer effect of SOCS-1 expression vector as well as a promise for the future development of therapeutic modality for MM based on this vector. Cancer Gene Therapy (2006) 13, 194–202. doi:10.1038/sj.cgt.7700873; published online 5 August 2005 Keywords: SOCS; myeloma; infectivity enhancement; adenoviral vector; IL-6; proliferation

Introduction

Multiple myeloma (MM) accounts for 1–2% of all malignancies and 10% of hematological malignant disorders.1,2 The median survival with standard therapy is approximately 3 years.3 Because this disease is refractory to conventional therapies, novel therapeutic strategies have been sought. While advanced chemotherapies combined with auto- or allo- hematopoietic stem cell transplantation show promise, there remain issues with respect to relapse or treatment-related deaths.4,5 These considerations suggest the need for a novel approach for MM therapy. In this regard, gene therapy represents one novel therapeutic modality that has been proposed in the context of MM.6,7 Characterization of key facts of MM pathobiology may allow selective molecular interventions via gene therapy approach. Interleukin 6 (IL-6) is one of the major growth factors for MM cells, and in vitro studies indicate that Correspondence: Professor N Nishimoto, Laboratory of Immune Regulation, Graduate School of Frontier Bioscience, Osaka University, 1-3 Yamada-Oka, Suita City, Osaka 565-0871, Japan. E-mail: [email protected] Received 14 July 2004; revised 17 April 2005; accepted 27 April 2005; published online 5 August 2005

IL-6 is a growth and antiapoptotic factor for freshly isolated MM plasma cells and for MM-derived cell lines.8,9 We have reported that humanized anti-IL-6 receptor (IL-6R) antibody (Ab) possesses in vivo therapeutic potency for the treatment of MM.10–12 In MM cells, the signal from IL-6R is transduced through the JAK–STAT3 (JAK: Janus kinase; STAT3: signal transducer and activator of transcription 3) cascade13,14 and Ras/Raf/MEK/MAPK pathway.15,16 Interestingly, some cell signaling cascades have intrinsic negative feedback mechanisms, and the JAK–STAT system is known to have such a group of proteins called SOCS.17 Among them, we have identified STAT-induced STAT inhibitor-1 (SSI-1), also called suppressor of cytokine signaling-1 (SOCS-1)/JAK binding protein (JAB).18–20 Overexpression of SOCS may inhibit the proliferation of MM cells by blocking the IL-6 signal transduction cascade, which is critical for the viability of these cells.21–23 While the negative feedback of IL-6-induced JAK-STAT signaling is mediated through SOCS-3,24–26 the inhibitory effect of SOCS-1 onto JAK-STAT signaling is reported to be stronger than that of SOCS-3.17,27 Thus, as a tool for developing a therapeutic modality, SOCS-1 expression vectors may provide a promise in this tumor context. These considerations thus suggest the concept of gene

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Table 1 Infectivity enhancement with RGD modification Infectivitya

Cell type

S6B45 KPMM2 IM-9 ARH77 MH60 B9 KT3 CESS HS-Saltan RPMI8226 U266 KMS5 a b

MM (IL-6 dependent) MM MM MM Mouse hybridoma Mouse hybridoma Lennert’s T-cell lymphoma B-cell lymphoma Burkitt’s lymphoma MM MM MM

Enhancementb

Ad

RGD

7 7  + + +   + ++ ++ ++

++ + + ++ +++ ++ + + + ++ ++ ++

(++) (+) (++) (++) (++) (+) (++) (++) () () () ()

Infectivity (at 10 000 vp/cell): : 0–5%; 7: 5–15%; +: 15–80%; ++: 80–100%. Enhancement (dose reduction): (): 0–2.5 times; (+): 2.5–10 times; (++): 410 times.

therapy for MM by employing a SOCS-1 expression vector. The feasibility of this concept however has been limited by the absence of a vector system that confers efficient gene expression in MM cells. While adenoviral (Ad) vector has been used for this purpose in various tumor contexts, and many established cell lines show relatively good infectivity, the infectivity of MM is frequently poor.28–31 This Ad resistance is due to the absence of the coxsackie adenovirus receptor (CAR), which mediates the initial viral binding to the cell surface.28–30 We have endeavored to develop a modality to circumvent this issue by achieving CAR-independent infection28,32 through infectivity-enhanced Ad vectors, which incorporate an RGD4C motif in the HI-loop of Ad fiber-knob region (RGD modification).33,34 These modified vectors show highly efficient gene delivery in a variety of cancer cells that are poorly infectable with unmodified Ad vectors. Notably, most MM cells are known to bind to fibronectin-coated surfaces,35 mediated by the common RGD motif of fibronectin.36 Thus, it is rational to hypothesize that the RGD-modified Ad would be of utility in enhancing target cell infectivity to realize Ad gene therapy of MM (Table 1). In this study, we initially evaluated the infectivity enhancement of Ad vectors with an RGD4C motif in the fiber-knob region, followed by construction of infectivityenhanced SOCS-1-expressing Ad vectors. The vectors were evaluated in the context of growth suppression and the inhibition of IL-6 receptor downstream cascade in MM cells. These results provide an initial proof-ofprinciple for the future development of therapeutic modality for MM based on the SOCS-1 expression vector.

Methods

Cell lines RPMI8226 (myeloma), U266 (myeloma), HS-Saltan (Burkitt’s lymphoma), IM-9 (Epstein–Barr virus-trans-

formed B lymphocyte), CESS (IgG-bearing human Blymphoblastoid cell line), and ARH-77 (plasma cell leukemia) cells were obtained from American Type Culture Collection (Manassas, VA). S6B45 (myeloma),37 MH60 (hybridoma),38 B9 (hybridoma, Dr Lucien Aarden, The Netherlands Red Cross Blood Transfusion Service, Amsterdam, the Netherlands),39 KPMM2 (myeloma),40 KMS-5 (myeloma),41 and KT-3 (Lennert’s lymphoma)42 were provided by the establisher. All cell lines were cultivated with RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, antibiotics (100 IU/ml penicillin, 100 U/ml streptomycin), and 50 mM 2-mercaptoethanol. IL-6-dependent myeloma cells were also supplemented with a proper concentration of IL-6 (S6B45: 10 ng/ml; KT-3 and KPMM2: 4 ng/ml; B9 and KMS-5: 1 ng/ml; and MH60: 0.1 ng/ml). A549 and 293 cells (ATCC) were maintained in RPMI 1640 and DMEM supplemented with 5% FBS, L-glutamine, and antibiotics, respectively. For infection, Dulbecco’s modified Eagle’s medium supplemented with 5% FBS and antibiotics was used. Cells were incubated in a 371C and 5% CO2 environment under humidified conditions.

Adenoviral vectors Recombinant Ad vectors were constructed through homologous recombination in Escherichia coli using the AdEasy system43 (Figure 1). To generate the SOCS-1expressing nonreplicative Ad vectors, the cytomegalovirus immediate-early promoter, SOCS-1/SSI-1/JAB cDNA (GenBank acc. no. AB005043),18 and simian virus 40 polyadenylation signal were cloned into the shuttle vector in this order. For detection of transgene expression, a version with FLAG epitope on the 30 end was made by inserting the corresponding sequence of pFLAG-CMV-5 (Sigma-Aldrich, St Louis, MO) in-frame. The resultant shuttle vectors linearized with PmeI were recombined with pAdEasy-143,44 and pVK50333 to generate vectors with wild-type and RGD-modified fibers, respectively. The desired recombinant plasmids were cut with PacI and transfected into 293 cells for vector production. The

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Figure 1 Structure of SOCS-1-expressing Ad vectors. The Ad vectors were constructed based on an E1-deleted Ad backbone. The cDNAs of SOCS-1 and SOCS-1-FLAG fusion protein were placed downstream of the cytomegalovirus immediate-early promoter. In the context of the Ad fiber region, versions with wild-type fiber and the fiber with RGD-4C motif insertion in the HI-loop of the fiber-knob region (RGD modification) were constructed.

viruses were amplified with 293 cells and purified by double CsCl density gradient centrifugation, followed by dialysis against phosphate-buffered saline (PBS) with 10% glycerol. The vectors were titrated by a plaque assay, and viral particle (vp) number was determined by optical density.44 The viruses were stored at 801C until usage. The absence of replication-competent Ad was confirmed by polymerase chain reaction of the E1 region. To analyze infectivity, GFP expression vectors with wildtype and RGD-modified fibers (AdGFP and RGDGFP) were also generated in parallel. The vp/plaque forming unit (PFU) ratio was approximately 500.

Analysis of infectivity The percentage of infected cells was determined by microscopic counting of GFP-expressing cells after infection with wild-type and RGD-modified GFP expression vectors (AdGFP and RGDGFP). A total of 50 000 cells of each line were suspended in 100 ml of infection medium and mixed with various amounts (10–10 000 vp/cell) of each vector in 100 ml infection medium. After 2 h of incubation, complete medium with an appropriate concentration of IL-6 was added and the cells were incubated for 48 h. The cells were then observed with an inverted fluorescent microscope (IX70, Olympus America, Melville, NY), and the dark and bright field images of three random fields were captured by MagnaFire imaging system (Optronics, Goleta, CA). The percentage of each field was calculated using the GFP-expressing cell number in the dark field and total cell number in the bright field. An average of three fields was used as a percentage of infected cells. Analysis of SOCS-1 expression To confirm the expression of SOCS-1 from the vectors, infected cells were analyzed for the expression of SOCS-1 and FLAG epitope. The A549 cells, which exhibit IL-6independent growth, were used for this assay. The cells (1.0  106) were inoculated on a 10 cm dish, and 18 h later

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infected with 5000 vp/cell of SOCS-1 expression vectors and a mock vector encoding luciferase in 2 ml of infection medium for 2 h, followed by 48 h of incubation with complete medium. Pelleted cells were dispersed with trypsin and washed with PBS. The pellets were then resuspended in 100 ml of RIPA buffer (1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 10 mM Tris, pH 7.4, 150 mM NaCl) and placed on ice for 30 min, followed by centrifugation at 14 000 r.p.m. for 30 min at 41C and recovery of the supernatant. Protein concentration was determined by a Dc Protein Assay (Bio-Rad, Hercules, CA). For Western blot analysis of SOCS-1, 40 mg of lysates was resolved by SDS-PAGE under reducing conditions, transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad), and immunoblotted with anti-SOCS-1 monoclonal Ab (mAb 1262B45). Blots were visualized with horseradish peroxidase (HRP)-labeled anti-mouse Ig Ab (Dako, Carpinteria, CA) using the ECL detection system (Amersham, Piscataway, NJ). For the analysis of FLAG epitope, 40 mg of lysates was immunoprecipitated with anti-FLAG M2 Ab (Sigma, St Louis, MO) and protein G agarose (Santa Cruz Biotechnology, Santa Cruz, CA). The immunoprecipitate was resolved by SDS-PAGE under reducing conditions, and blotted as described above. The blot was developed with biotinylated anti-SOCS-1 mAb and the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) as described above.

Analysis of cell proliferation To analyze the effect of SOCS-1 expression vectors on the viability of MM cells, MM cells were infected with the vectors and the number of viable cells was analyzed. Following incubation without IL-6 for 24 h, the cells were infected at 10 000 and 1000 vp/cell for 2 h and inoculated onto 96-well plates at a density of 10 000 cells/well (containing 50 ml of infection medium). After 18 h of incubation, complete medium with different concentrations of IL-6 (0, 0.2, and 2.0 ng/ml final) was added and incubation resumed for another 5 days. The cell number was determined using the CellTiter 96 AQueous NonRadioactive Cell Proliferation Assay (Promega, Madison, WI). Absorbance at 490 nm was converted to cell number using constants determined with the absorbance of the wells with a known number of cells for each cell line. As controls, the same assay was performed in an IL-6-dependent mouse hybridoma cell line (MH60) and in IL-6-independent cell lines (U266: myeloma; HS-Saltan: Burkitt’s lymphoma). Analysis of STAT3 and phosphorylated STAT3 STAT3 and phosphorylated STAT3 levels after SOCS-1expressing vector infection were analyzed by Western blot analysis. Two million cells were infected at 5000 vp/cell for 2 h and incubated for an additional 22 h in medium lacking IL-6, followed by cultivation in medium containing IL-6 (0.05 ng/ml for MH60 and 0.2 ng/ml for S6B45) for 24 h. The cells were harvested and lysed on ice for 30 min with RIPA buffer containing 1 mM Na3VO4,

Infectivity-enhanced SOCS-1-expressing adenoviral vector for myeloma M Yamamoto et al

5 mg/ml aprotinin (1 mM), and phenylmethylsulfonyl fluoride. After centrifugation at 14 000 r.p.m. for 30 min at 41C, the supernatant was recovered. SDS-PAGE and Western blotting were performed in the same way as described above, except that a nitrocellulose membrane was used for the detection of phosphorylated STAT3. The detection was performed using the PhosphoPlus Stat3 (Tyr705) Antibody Kit (Cell Signaling Technology, Beverly, MA) and ECL kit (Amersham) as described by the manufacturer.

Results

Incorporation of the RGD4C motif can enhance the infectivity of the adenoviral vectors in many hematopoietic cells including MM Infectivity enhancement via the incorporation of an RGD4C motif was evaluated in 12 hematopoietic cell lines using GFP expression vectors with wild-type and RGD-modified fibers. As indicated in Figure 2, the RGDmodified vector was superior to the unmodified Ad in eight out of 12 cell lines tested. While all 12 cell lines were infected efficiently with the RGD-modified vector, four cell lines did not show a difference in transduction efficiency, reflecting relatively high infectivity with the vectors with the wild-type fiber. These studies indicate that RGD modification is a promising strategy to achieve augmented infectivity for MM cells. Induction of SOCS-1 employing expression vectors To confirm the expression of SOCS-1 and SOCS-1-FLAG fusion protein from the vectors, A549 cells were infected with each vector and analyzed using Western blot. After infection, A549 cells expressed SOCS-1 protein of the expected size, while cells expressing SOCS-1 with the FLAG epitope showed a slightly larger size due to the additional peptides on the carboxyl terminus (Figure 3a). Following immunoprecipitation with an anti-FLAG Ab, signal was detected only from cells infected with vectors expressing the SOCS-1-FLAG fusion protein (Figure 3b). These data confirm that the entire sequence of SOCS-1, with or without the FLAG epitope, was expressed correctly in mammalian cells. Between the vectors with wild-type and RGD-modified fibers, there was no significant difference in SOCS-1 expression in A549 cells. Infectivity-enhanced SOCS-1 expression vectors suppress the proliferation of MM cells The antiproliferative effects of infectivity-enhanced SOCS-1 expression vectors in MM cells were analyzed in two IL-6-dependent MM cells and IL-6-dependent Lennert’s T-leukemia cells. In all three cell lines, infectivity-enhanced SOCS-1-expressing Ad vectors demonstrated an antiproliferative effect under conditions of various concentrations of IL-6. S6B45 cells, which are the most dependent on IL-6, were the most sensitive to SOCS-1 expression vectors (Figure 4a). The same experiment performed in MH60 (IL-6-dependent mouse hybrid

plasmacytoma cells), which has been used for the bioassay of IL-6 concentration, indicated a similar effect, except that the effect at 10 000 vp/cell infection without IL-6 could not be evaluated due to poor viability of these cells without IL-6 (Figure 4b). Vectors expressing SOCS-1FLAG fusion protein demonstrated antiproliferative effects comparable to vectors expressing SOCS-1 without FLAG. In IL-6-independent cells U226 and HS-Sultan, overexpression of SOCS-1 did not induce an antiproliferative effect (Figure 5).

SOCS-1 overexpression suppresses STAT3 cascade activation To analyze the STAT3 activation status after SOCS-1 overexpression, the STAT3 expression level and its phosphorylation at Tyr705 were analyzed by Western blotting with anti-STAT3 Ab and anti-phospho STAT3 Ab. The S6B45 MM cell line and MH60 IL-6-dependent hybridoma were analyzed after infection with RGDmodified SOCS-1 expression vectors and an RGDmodified mock vector expressing luciferase (Figure 6). STAT3 was detected in all samples, and the level was slightly lower in cells infected with SOCS-1 expression vectors. On the other hand, while phosphorylated STAT3 was detected in uninfected and mock-infected cells, it was completely suppressed in cells infected with SOCS-1expressing vectors regardless of the existence of the FLAG epitope. This result indicates that STAT3 phosphorylation (Tyr705) was suppressed by overexpression of SOCS-1 in these cells.

Discussion

IL-646 is one of the predominant factors controlling the proliferation of MM cells through autocrine and paracrine mechanisms.47–50 Since inhibition of IL-6 receptor suppress MM cell proliferation in vitro and in vivo,13,22,51 a gene therapy employing overexpression of SOCS-1, which blocks the IL-6 receptor downstream signaling from gp130,15,16,52,53 represents a rational approach. Fundamentally, all gene therapy approaches must embody a requisite level of transduction to achieve an effective expression of the gene of interest. For MM, various vectors have been tested with various levels of efficacy reported (vesicular stomatitis virus glycoprotein pseudo-typed lentiviral vector,7 herpes simplex virus,54 fiber-modified Ad,55,56 adeno-associated virus,57 and measles virus57). With respect to Ad vectors, DF3/ MUC-1 promoter-driven herpes simplex virus thymidine kinase expression vector has been applied to MM for ex vivo purging of MM cells.6 However, primary MM cells were marginally transducible with vectors containing unmodified fibers.31 These studies suggest barriers for achievement of the needed level of transduction to implement the therapeutic utility of SOCS-1 in MM. In this study, we used vectors with an RGD4C motif in the HI-loop of the fiber-knob region as a means to circumvent this obstacle.33 These infectivity-enhanced

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Figure 2 Infectivity of unmodified and RGD-modified Ad vectors in MM and other hematopoietic malignant cells. To assess infectivity enhancement of Ad vector system by RGD modification, MM and other hematopoietic malignant cells were infected with GFP expression vectors containing wild-type and RGD-modified fibers at different viral doses. The origin of each cell line is shown in Methods. The percentage of GFPexpressing cells was analyzed with an inverted fluorescent microscope (& GFP expression vector with wild-type fiber, ’ GFP expression vector with RGD-modified fiber).

vectors outperformed unmodified Ad vectors in the majority of MM cell lines (Figure 2). While most MM cells lack expression of avb3 integrin, which is capable of binding to the Ad RGD motif in the penton base region,35,58–60 a4b1 (VLA-4) and a5b1 (VLA-5) integrins are significantly positive in MM cells.35,58,59,61 These two

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types of integrins have the ability to bind to the RGD motif.62 Notably, a4b1 mediates the adhesion of MM cells onto fibronectin-coated plates.35 These observations suggest that a4b1 and a5b1 integrins have the features to mediate binding of the RGD-modified vectors to the cell surface.

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Tropism-modified SOCS-1 expression vectors, which incorporate the RGD4C motif, showed an antiproliferative effect in three IL-6-dependent MM and lymphoma cells (S6B45, KPMM2, and KT-3) and IL-6-dependent mouse hybridoma (MH60) (Figure 4). The growth suppression was not detected in IL-6-independent cells (U266 and HS-Saltan; Figure 5). This strongly indicates

Figure 3 SOCS-1 expression in A549. (a) Cells were infected with 5000 vp/cell of each vector and mock luciferase expression vector. The expression of SOCS-1 was analyzed with Western blotting. Each vector expressed the SOCS-1 protein with the expected sizes. Those with FLAG epitope showed a larger molecular weight corresponding to the attached peptide. (b) In immunoprecipitation with anti-FLAG M2 Ab, only vectors expressing the FLAG fusion protein exhibited a signal after the development with biotinylated anti-SOCS-1 Ab.

that the antiproliferative effect of SOCS-1 overexpression is related to the activated IL-6 signal transduction cascade in target cells. Interestingly, in most cell lines, the effect of SOCS-1 expression vectors was relatively independent of IL-6 concentration. This could be explained by the fact that the mechanism of SOCS-1 function is via noncompetitive inhibition, since SOCS-1 inhibits the IL-6 signal transduction by binding and blocking JAK activity via the kinase inhibitory region.16,63,64 The IL-6 signal transduction cascade includes the Ras/Raf/MEK/MAPK pathway as well as the JAK/ STAT pathway,15,65,66 and each is mainly responsible for cell proliferation and survival, respectively.67–69 Activation of both pathways is mediated by the phosphorylation of Shc and gp130 by JAKs.15,16 Thus, SOCS-1, which binds to JAKs and blocks their kinase activity, should have the ability to block both Ras/Raf/MEK/MAPK and JAK/STAT pathways, preventing Shc and gp130 phosphorylation. Therefore, it is rational that SOCS-1 overexpression can inhibit the growth of MM cells. Interestingly, the growth suppression (Figure 4) was stronger than expected from transduction efficiency (Figure 2). This suggests the existence of a mechanism that causes growth suppression or death of bystander cells. While the mechanism of the bystander effect has not been elucidated yet, CD8 þ CD44high memory T cells from SOCS-1/INF-g/ mice show elevated bystander growth compared to cells from SOCS-1 þ / þ INF-g/ mice.70 We might have observed the reverse of the similar phenomenon in MM cells by overexpressing SOCS-1. In the context of SOCS-1 effect on the IL-6 receptor signaling, we analyzed the activation of STAT3, which plays a key role in the IL-6-induced JAK/STAT cascade

Figure 4 Antiproliferative effect of SOCS-1 expression vectors in IL-6-dependent target cells. (a) MM cells were infected with infectivityenhanced Ad vectors expressing SOCS-1 and SOCS-1-FLAG fusion protein at 10 000 and 1000 vp/cell under different concentrations of IL-6 (0, 0.05, and 0.5 ng/ml). The number of viable cells was determined by an MTS assay. The results are indicated as percentages of the cell number obtained with a mock vector (GFP-expressing vector). (b) A typical IL-6-dependent cell MH60 (mouse hybrid plasmacytoma cells) was also analyzed in the same way. The growth of these cell lines was suppressed by overexpression of SOCS-1 with Ad vector (& RGDCMVLuc, RGDCMVSOCS-1, ’ RGDCMVSOCS-1 FLAG).

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Figure 5 Effect of SOCS-1 expression vectors in IL-6-independent cells. IL-6-independent MM cell line (U266) and Burkitt’s lymphoma cell line (HS-Saltan) were infected with infectivity-enhanced SOCS-1 expression vectors and analyzed in the same way as in Figure 4. The growth of these IL-6-independent cell lines was not affected by overexpression of SOCS-1 with Ad vector (& RGDCMVLuc, RGDCMVSOCS-1, ’ RGDCMVSOCS-1 FLAG).

Figure 6 Effect of SOCS-1 overexpression on STAT3 signaling. IL-6-dependent MM (S6B45) and mouse hybridoma cells (MH60) were infected with SOCS-1-expressing infectivity-enhanced Ad vectors, and the lysates were analyzed by Western blot and immunochemistry using anti-STAT3 and anti-phospho-STAT3 Abs. STAT3 (phosphorylated and nonphosphorylated) was detected with antiSTAT3 Ab (both bands represent STAT3), showing some decrement for cells infected with SOCS-1 expression vectors (upper panels). In contrast, phosphorylated STAT3 (Tyr705) was significantly suppressed by SOCS-1 overexpression (lower panels).

activation.15,16 SOCS-1 expression vectors dramatically inhibited the phosphorylation of STAT3 (Tyr705) in S6B45 MM cells and MH60 IL-6-dependent mouse hybridoma. This observation indicates that STAT3 activation was blocked since STAT3 (Tyr705) phosphorylation represents activation of the JAK/STAT pathway in IL-6 receptor signaling.15,16 Although the JAK/STAT

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cascade is not necessarily the dominant pathway controlling IL-6-dependent MM cell growth, these data clearly indicate that SOCS-1 overexpression can modulate IL-6 receptor signaling. While the amount of STAT3 slightly declined after the infection with SOCS-1 expression vectors, this decrement is in the range explainable by the fact that SOCS-1 overexpression suppresses viability of IL-6-dependent cells, as shown in Figure 4. In aggregate, these data indicate that overexpression of SOCS-1 using RGD-modified Ad vector system has a potential to suppress the cell proliferation in IL-6dependent cell lines by reducing the level of STAT-3 activation. While SOCS-3 is mainly induced with IL-6 receptor signaling and knockout of this gene affects IL-6-induced STAT3 activation in various ways,24–26 these data do not conflict with our observation of STAT-3 inhibition by SOCS-1 overexpression. The SOCS-1 and SOCS-3 show closest similarity in the structure, including interchangeability of their N-terminal sequence.27,71 Moreover, SOCS-1 blocks JAK activity more efficiently than SOCS-3 does.17,27 The effect in normal hematopoietic progenitor cells (HPC) remains a potential issue for clinical implication, although this study established an antiproliferative effect of SOCS-1 overexpression in MM cells. If there is an undesired effect on SOCS-1 overexpression in this context, utilizing a selective promoter that exhibits a differential between MM cells and HPC (e.g. DF3/MUC-1 promoter6) may be required to reduce toxicity via minimizing the transgene expression in HPC. In this study, we have established that the incorporation of an RGD4C motif in the HI-loop of the Ad fiberknob region confers effective transgene expression in MM cells. Using this strategy, we overexpressed an intrinsic negative feedback factor of the IL-6 signal transduction pathway, SOCS-1, in MM cells, and achieved an antiproliferative effect. This study establishing a proof-ofprinciple of the anticancer effect of SOCS-1 expression vector provides a promise in the future development of gene therapy modality for MM with this vector. Acknowledgements

This work was supported in part by the National Organization for Rare Disorders, Research Grant Program for Multiple Myeloma to Masato Yamamoto, and by a Grant-in-Aid of the Ministry of Health, Labor and Welfare of Japan to Norihiro Nihsimoto. We thank Dr Victor Krasnykh for providing the Ad backbone plasmid for infectivity-enhanced vectors, and Dr Kazuyuki Yoshizaki and Long Le for excellent advice.

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