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Gene Therapy with Dominant-negative Stat3 Suppresses Growth of the Murine Melanoma B16 Tumor in Vivo¹

Immunology Program [G. N., H. Y.], Clinical Investigations Program [R. H., D. C., W. D.], and Molecular Oncology Program [R. C-F., R. J.], H. Lee Moffitt Cancer Center and Research Institute, and Departments of Microbiology and Immunology [G. N., R. H., H. Y.], Surgery [R. H., M. J.], Pathology [R. C-F., D. C.], Medicine [W. D.], and Biochemistry and Molecular Biology [R. J.], University of South Florida College of Medicine, Tampa, Florida 33612

	ABSTRACT

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Whereas signal transducers and activators of transcription were originally discovered as mediators of normal cytokine signaling, constitutive activation of certain signal transducer and activator of transcription proteins, including Stat3, has been found in increasing numbers of human cancers. Recently, a causal role for Stat3 activation in oncogenesis has been demonstrated, suggesting that Stat3 represents a novel target for cancer therapy. We report here that in vitro expression of a Stat3 variant with dominant-negative properties, Stat3, induced cell death in murine B16 melanoma cells that harbored activated Stat3. By contrast, expression of Stat3 had no effect on normal fibroblasts or the Stat3-negative murine tumor MethA, suggesting that only tumor cells with activated Stat3 have become dependent on this pathway for survival. Significantly, gene therapy by electroinjection of the Stat3 expression vector into preexisting B16 tumors caused inhibition of tumor growth as well as tumor regression. This Stat3-induced antitumor effect is associated with apoptosis of the B16 tumor cells in vivo. These findings demonstrate for the first time that interfering with Stat3 signaling induces potent antitumor activity in vivo and thus identify Stat3 as a potential molecular target for therapy of human cancers harboring activated Stat3.

	Introduction

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STATs⁴ are latent cytoplasmic transcription factors that function as intracellular effectors of cytokine and growth factor signaling pathways (1) . STAT proteins were originally defined in the context of normal cell signaling, where STATs have been implicated in the control of cell proliferation, differentiation, and apoptosis (1 , 2) . Whereas much progress has been made in understanding the normal roles of STATs in cytokine and growth factor signaling, aberrations in these signaling pathways associated with oncogenesis raise the possibility that unregulated activation of STAT signaling contributes to human cancers. Numerous studies have demonstrated that transformation of cells by oncoproteins, including the Src tyrosine kinase, results in constitutive activation of STATs (3 , 4) . In cells transformed by diverse oncoproteins or tumor viruses, the most frequently activated STAT family members are Stat3, Stat5, and, to a lesser extent, Stat1 (5, 6, 7, 8, 9, 10) . Importantly, Stat3 signaling has been shown to be required for oncogenic transformation by Src, because the expression of dominant-negative forms of Stat3 interferes with Stat3-mediated gene regulation and blocks cell transformation (11 , 12) .

Mounting evidence directly implicates aberrant activation of Stat3 signaling in malignant progression of human cancers. Constitutive activation of Stat3 has been demonstrated in human breast carcinoma, multiple myeloma, lymphomas, leukemias, and head and neck carcinoma (8 , 13, 14, 15, 16, 17) . In the human myeloma cell line U266, interleukin-6-mediated constitutive activation of Stat3 signaling induces elevated expression of the antiapoptotic regulator Bcl-x_L (17) . Blocking Stat3 signaling in these myeloma cells down-regulates Bcl-X_L expression and results in a dramatic induction of programmed cell death in vitro (17) . These findings provide evidence that aberrant Stat3 signaling contributes to malignant progression of multiple myeloma by preventing apoptosis and suggest that Stat3 is a potential target for cancer therapy. However, it has not been determined whether blocking Stat3 signaling is sufficient to inhibit tumor growth in vivo. In this study, we used a gene therapy approach to inhibit activated Stat3 in vivo and assessed its effect on murine melanoma B16 tumor growth. Our results demonstrate that inhibition of activated Stat3 signaling by gene therapy with a dominant-negative Stat3 variant suppresses B16 tumor growth and induces apoptosis of B16 tumor cells in vivo.

	Materials and Methods

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Cell Lines and Culture Medium.
Mouse B16 melanoma cell line, MethA sarcoma cell line, and mammary carcinoma cell lines 4T1 and TSA (kindly provided by Dr. Ning-Sun Yang, University of Wisconsin Medical Center, Madison, WI) were grown in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% MEM nonessential amino acids, and 100 IU/ml penicillin/streptomycin. NIH 3T3 (American Type Culture Collection) cells were grown in DMEM (Life Technologies, Inc.) supplemented with 5% calf serum.

Nuclear Extracts and EMSA.
Nuclear extract preparation and EMSAs were performed essentially as described previously (3 , 8 , 17) .

Plasmids.
Stat3 cDNA was kindly provided by E. Caldenhoven and R. de Groot (University Hospital, Utrecht, the Netherlands; Ref. 18 ). The backbone plasmid, pIRES-EGFP, was obtained from Clontech (Palo Alto, CA). The construction and characterization of pIRES-Stat3 has been described previously (17) . The pcDNA3 plasmid was obtained from Stratagene (La Jolla, CA). Stat3 cDNA was also inserted into the pAdCMV vector (Quantum Biotechnologies, Montreal, Quebec, Canada). The ability of pAdCMV-Stat3 to express Stat3 protein was verified by Western blot analysis after transfection into NIH 3T3 cells.

Transfections and Flow Cytometric Analysis.
Transfections in vitro were performed by the LipofectAMINE-mediated method (Life Technologies, Inc.). To determine transfection efficiency, relative fluorescence intensity was measured by fluorescence-activated cell sorting of both pIRES-EGFP/pSV₂neo- and pIRES-Stat3/pSV₂neo-transfected cells. For stable transfectants, one plate of transfected cells from each group was used to determine the transfection efficiency, and the remaining plates were allowed to grow in medium supplemented with 500 µg/ml G418. Two weeks later, the G418-resistant colonies were fixed in 4% paraformaldehyde, and the number of colonies was counted. GFP-positive colonies were counted (for B16 cells) or estimated (for NIH 3T3 cells) under fluorescence microscopy.

Mice and Tumors.
Six-week-old female C57BL mice were purchased from the National Cancer Institute (Frederick, MD) and maintained in the institutional animal facilities approved by the American Association for Accreditation of Laboratory Animal Care. Mice were shaved in the left flank area and injected s.c. with 2 x 10⁵ B16 cells in 100 µl of PBS. After 7–10 days, B16 tumors with a diameter of 3–6 mm were established. Animals were stratified so that the mean tumor sizes in all treatment groups were nearly identical. Tumor volume was calculated according to the formula V = 0.52 x a² x b (a, smallest superficial diameter; b, largest superficial diameter).

DNA Electroinjection in Vivo.
The gene delivery procedure was performed after the mice were anesthetized in an induction chamber infused with a mixture of 3% isoflurane and 97% oxygen. Procedures were then carried out using a supply of 2% isoflurane in oxygen to a standard rodent mask. One hundred µg of plasmid DNA in 100 µl saline were injected directly into the tumor using a 25-gauge, -inch-long needle. Electric pulses were delivered through custom-designed electrodes that were placed around the tumor using a PA 4000 DC generator (Cyto Pulse Sciences, Inc., Columbia, MD). Electroinjection of the tumor cells was accomplished by applying a total of fourteen 100-µs electric pulses at a nominal field strength of 1500 V/cm at 1-s intervals.

Histochemistry and Immunohistochemistry.
Electroinjection with pIRES-EGFP or pIRES-Stat3 was carried out in 4–5-mm B16 tumors. Three days after in vivo transfection, mice were euthanized, and the tumors were excised and frozen immediately in liquid N₂. Serial sections of tumors were also fixed in formalin, stained with H&E, and processed for routine histological examination. The anti-GFP monoclonal antibody (Clontech) was applied to 3-µm sections from frozen sections of tumors using the avidin-biotin-peroxidase complex method (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). All slides were lightly counterstained with Mayer’s hematoxylin for 30 s before dehydration and mounting. Nonimmune protein (mouse IgG) negative controls were used for each section. For -gal staining, tumor tissues were excised and fixed in 0.5% gluteraldehyde 3 days after electroinjection of either the -gal or pcDNA3 plasmids. Cryostat sections were mounted on poly-L-lysine-coated slides and fixed briefly in 0.5% glutaraldehyde. The X-gal reaction was carried out according to the supplier’s instructions (Boehringer Mannheim, Indianapolis, IN).

TUNEL Assay.
B16 tumors that received either pIRES-EGFP or pIRES-Stat3 electroinjections were used for this assay. Three-µm sections from paraffin-embedded tissues were dewaxed and rehydrated according to standard protocols. After incubation with proteinase K (30 min at 21°C), the TUNEL reaction mixture (Boehringer Mannheim) was added to rinsed slides and incubated in a humidified chamber for 60 s at 37°C. This was followed by an incubation with Converter-AP (50 µl) and substrate solution (50 µl). The reaction was visualized by light microscopy.

	Results

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Stat3 Is Constitutively Activated in Murine Tumor Cells.
Four murine tumor cell lines, including melanoma B16, mammary carcinomas TSA and 4T1, and sarcoma MethA, were evaluated for STAT DNA-binding activity by EMSA. An oligonucleotide probe corresponding to a high-affinity mutant of the sis-inducible element (hSIE), which binds activated Stat1 and Stat3 (3 , 8 , 17) , was used to determine whether the nuclear extracts from these tumor cells contain constitutively activated Stat3 protein. With the exception of MethA, all of the other murine tumor cells contained elevated hSIE binding activity corresponding to Stat3 homodimers (Fig. 1, a and b) . Because B16 is a poorly immunogenic s.c. tumor, it provides an excellent model for testing local gene therapy. We therefore focused our studies on this tumor model to evaluate whether Stat3 could be used as a molecular target for the therapy of cancers with constitutively activated Stat3.

View larger version (104K): [in this window] [in a new window]   Fig. 1. Stat3 is activated in murine tumor cell lines. a, nuclear extracts prepared from the indicated mouse tumor cells and human U266 myeloma cells were incubated with the 32P-labeled hSIE oligonucleotide probe and analyzed by EMSA. b, nuclear extracts from B16 cells were preincubated with excess unlabeled hSIE (Lane 2) or FIRE (irrelevant oligonucleotide; Lane 3) probes and anti-Stat1 (Lane 5) or anti-Stat3 (Lane 6) antibodies to confirm specific Stat3 activation. Human myeloma U266 cells are used here as a positive control for Stat3 activation (17) . ST 3/3, Stat3 homodimers.

Overexpression of a Stat3 Dominant-negative Protein, Stat3, Induces Cell Death in B16 Tumor Cells in Vitro.

In all of the experiments shown in Table 1 , transfection efficiencies with pIRES-EGFP or pIRES-Stat3 vectors were very similar, as determined by the percentage of cells that exhibit green fluorescence at 36 h after transfection (fluorescence-activated cell-sorting analysis). The remaining transfected plates were selected in medium supplemented with G418. Because the transfection efficiencies of the two constructs in each experiment were nearly the same, >95% of the B16 cells that received the Stat3 construct did not survive (only 6 colonies survived, as compared with 138 colonies in B16 cells transfected with the empty vector). In the six surviving colonies, the intensity of green fluorescence was also much dimmer than that seen in those transfected with the empty vector (data not shown). To determine whether expression of Stat3 could mediate the cell death of other murine tumor cells with activated Stat3, transfection was carried out in the TSA murine breast carcinoma cell line. Consistent with the B16 cells, a marked reduction in the number of viable cells was observed in Stat3-transfected TSA tumor cells when compared with empty vector-transfected control cells (data not shown).

View this table: [in this window] [in a new window]   Table 1 STAT3 kills B16 tumor cells but not normal NIH 3T3 fibroblasts

Intratumoral Electroinjection of Stat3 Vector Leads to Suppression of Tumor Growth in Vivo.
Electroinjection for gene delivery in vivo has been reported previously (19 , 20) . We first determined the efficacy of gene delivery into 4–5-mm (average) B16 tumors by examining the percentage of tumor cells positive for GFP or -gal after electroinjection with the respective vectors. Approximately 15% of the tumor cells were scored as positive for -gal expression (Fig. 2a) , and similar results were obtained for GFP expression (data not shown).

View larger version (49K): [in this window] [in a new window]   Fig. 2. Expression of Stat3 inhibits B16 tumor growth in vivo. a, -gal staining of tumor cells electroinjected with the -gal plasmid. Overall, 15% of the tumor cells were -gal positive (semiquantitative analysis). Little or no staining was observed in the negative control (pcDNA3-transfected tumor section receiving the same staining; data not shown). b, summary of several independent Stat3 gene therapy experiments in vivo. Data shown represent the average tumor volumes in three experiments on the day when one or more animals in each experiment was sacrificed due to oversized or ulcerated tumors (on days 28, 20, and 22, respectively). A total of 15 control animals were treated with empty vector (), and 20 mice were treated with Stat3 (), either pIRES-Stat3 or pAdCMV-Stat3 (*). For one representative experiment, growth kinetics of B16 tumors treated with either pIRES-EGFP (c) or pIRES-Stat3 (d) are shown. The first gene electroinjection was performed on day 8, when tumors reached an average diameter of 3–6 mm. Tumor measurements and the gene therapies indicated above were performed every 3–4 days. Four of five pIRES-Stat3-treated tumors regressed.

Stat3-mediated Tumor Suppression Involves Apoptosis in Vivo.
To determine the mechanism of tumor cell killing in vivo, B16 tumors (from experiment 3 in Fig. 2b ) treated with either the empty vector or the Stat3 vectors were excised for H&E staining and TUNEL assays. All 5 of the control tumors and all 10 of the Stat3-treated tumors were stained with H&E. Whereas none of the five control tumors showed more than 10% apoptotic cells, many of the Stat3-treated tumors had undergone massive apoptosis (Fig. 3, a and b) . Of the 10 Stat3-treated tumors, 5 regressing tumors had more than 50% apoptotic cells (2 of them had greater than 90%). TUNEL/alkaline phosphatase assays for apoptosis confirmed that Stat3 treatment induced extensive apoptosis in B16 tumors (Fig. 3, c and d) . In addition to apoptosis, infiltrating inflammatory cells in the apoptotic tumors were observed in Stat3-treated tumors (data not shown).

View larger version (124K): [in this window] [in a new window]   Fig. 3. Gene therapy with Stat3 induces apoptosis in B16 tumors in vivo. H&E staining of B16 tumors treated with either the empty vector (a) or the Stat3 vector (b). TUNEL assay of the B16 tumors electroinjected with either the empty vector (c) or the Stat3 vector (d).

	Discussion

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The high incidence of Stat3 activation in human cancers from diverse origins implicates Stat3 signaling in neoplastic transformation (4 , 8 , 13, 14, 15, 16, 17) . Although the mechanisms of Stat3 activation in most cancers are not known, our results indicate that Stat3 is also constitutively activated with high incidence in murine tumors, highlighting the importance of Stat3 signaling in oncogenesis. Compared with the human myeloma cell line U266, the levels of activated Stat3 in the B16 tumor cell line are relatively low (Fig. 1) . Such low levels of Stat3 activation have also been observed frequently in human tumor cell lines and tissues, including myeloma and breast cancer (8 , 17) . The fact that expression of Stat3 killed nearly all of the B16 tumor cells in vitro suggests that low levels of constitutively activated Stat3 are sufficient to maintain tumor cell survival. These results also imply that human tumors with low levels of constitutively activated Stat3 are potential candidates for Stat3-targeted therapy. In contrast to B16 and TSA tumor cells, expression of Stat3 had no detectable effect on the survival of normal NIH 3T3 fibroblasts or the Stat3-negative MethA tumor cells, suggesting that cells lacking constitutively activated Stat3 are resistant to Stat3-targeted therapy. We speculate that in contrast to normal cells or Stat3-negative tumor cells, cells with constitutively activated Stat3 have become dependent on this pathway for survival.

Whereas the precise mechanism by which Stat3 mediates apoptosis in B16 cells is still under investigation, our recent studies in human myeloma cells demonstrated that Stat3 inhibits expression of the Bcl-x_L protein (17) . These experiments have provided evidence that Stat3 can be a proapoptosis regulator in cells that require Stat3 function for survival. The tumor suppressor protein p53 is another apoptosis regulator capable of mediating repression of Bcl-2 family genes (21) . Ectopic expression of high levels of wild-type p53 has been shown to induce apoptosis of various tumor cell lines in vitro. Gene therapy (predominantly using adenoviral vectors) with wild-type p53 has been shown to inhibit tumor growth and, in some cases, induce complete tumor regression in vivo (22, 23, 24) . However, most of these experiments were performed with human tumor cells in nude mice. In one syngeneic murine model of breast cancer, p53 gene therapy resulted in tumor growth delay, but not in tumor regression (25) . The antitumor effect mediated by Stat3 that we report here is more pronounced compared with p53 gene therapy in the murine breast cancer model. Nonetheless, initial reports of p53 gene therapy in clinical trials have described antitumor responses in patients with advanced non-small cell lung cancer (26) . Therefore, it is conceivable that Stat3 gene therapy in cancer patients could be successful, as seen for p53 gene therapy.

Although antitumor bystander effects have been observed in tumors treated with p53 gene therapy (24 , 27) , in vivo studies demonstrating the precise mechanism of the p53-mediated bystander effect are lacking. Nevertheless, a recent report demonstrated 29% growth inhibition of nontransduced cells after p53-transduced and nontransduced cells were cocultured in vitro (27) . In the case of B16 tumors treated with the Stat3 gene via electroinjection, the number of apoptotic cells also exceeds the number of cells transfected, consistent with antitumor bystander effects. Experiments are under way to investigate the mechanism by which Stat3 gene therapy may induce antitumor bystander effects. It is also notable that tumor infiltration by acute and chronic inflammatory cells was observed after Stat3 expression.⁵ These inflammatory cells may participate in killing of residual tumor cells, suggesting that one strategy to improve the efficacy of anti-Stat3-based therapy is to combine it with immunotherapy. Whereas p53 treatment alone failed to induce impressive tumor regression in a murine breast cancer model, gene therapy with combined interleukin 2 and p53 treatment has been shown to achieve long-term and significantly greater antitumor effects than either one alone (25) .

Our results provide in vivo proof of principle that Stat3 is a valid molecular target for developing novel therapies against cancers harboring constitutively activated Stat3. In addition to gene therapy, the development of small molecule drugs that specifically inhibit Stat3 signaling would also be highly desirable.

	ACKNOWLEDGMENTS

 
We thank E. Caldenhoven and R. de Groot for the Stat3 cDNA and the members of the Moffitt Cancer Center’s Flow Cytometry Core and Pathology Core for assistance.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by NIH Grants CA75243 (to H. Y.), CA55652 (to R. J.), and CA77859 (to W. D.); American Cancer Society Grant RPG-97-031-01 (to R. H.); and the Dr. Tsai-Fan Yu Cancer Research Endowment.

² G. N. and R. H. contributed equally to this work.

³ To whom requests for reprints should be addressed, at Immunology Program, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612.

⁴ The abbreviations used are: STAT, signal transducer and activator of transcription; EMSA, electrophoretic mobility shift assay; hSIE, high-affinity sis-inducible element; EGFP, enhanced green fluorescence protein; -gal, -galactosidase; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling.

⁵ Unpublished data.

Received 7/26/99. Accepted 8/30/99.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 

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				K. Selvendiran, H. Koga, T. Ueno, T. Yoshida, M. Maeyama, T. Torimura, H. Yano, M. Kojiro, and M. Sata Luteolin Promotes Degradation in Signal Transducer and Activator of Transcription 3 in Human Hepatoma Cells: An Implication for the Antitumor Potential of Flavonoids. Cancer Res., May 1, 2006; 66(9): 4826 - 4834. [Abstract] [Full Text] [PDF]

				T.-x. Xie, F.-J. Huang, K. D. Aldape, S.-H. Kang, M. Liu, J. E. Gershenwald, K. Xie, R. Sawaya, and S. Huang Activation of Stat3 in Human Melanoma Promotes Brain Metastasis. Cancer Res., March 15, 2006; 66(6): 3188 - 3196. [Abstract] [Full Text] [PDF]

				S. Nabarro, N. Himoudi, A. Papanastasiou, K. Gilmour, S. Gibson, N. Sebire, A. Thrasher, M. P. Blundell, M. Hubank, G. Canderan, et al. Coordinated oncogenic transformation and inhibition of host immune responses by the PAX3-FKHR fusion oncoprotein J. Exp. Med., November 21, 2005; 202(10): 1399 - 1410. [Abstract] [Full Text] [PDF]

				D. YU, D. COZMA, A. PARK, and A. THOMAS-TIKHONENKO Functional Validation of Genes Implicated in Lymphomagenesis: An in Vivo Selection Assay Using a Myc-Induced B-Cell Tumor Ann. N.Y. Acad. Sci., November 1, 2005; 1059(1): 145 - 159. [Abstract] [Full Text] [PDF]

				Y. Nefedova, S. Nagaraj, A. Rosenbauer, C. Muro-Cacho, S. M. Sebti, and D. I. Gabrilovich Regulation of Dendritic Cell Differentiation and Antitumor Immune Response in Cancer by Pharmacologic-Selective Inhibition of the Janus-Activated Kinase 2/Signal Transducers and Activators of Transcription 3 Pathway Cancer Res., October 15, 2005; 65(20): 9525 - 9535. [Abstract] [Full Text] [PDF]

				J. Turkson, S. Zhang, L. B. Mora, A. Burns, S. Sebti, and R. Jove A Novel Platinum Compound Inhibits Constitutive Stat3 Signaling and Induces Cell Cycle Arrest and Apoptosis of Malignant Cells J. Biol. Chem., September 23, 2005; 280(38): 32979 - 32988. [Abstract] [Full Text] [PDF]

				C. Proietti, M. Salatino, C. Rosemblit, R. Carnevale, A. Pecci, A. R. Kornblihtt, A. A. Molinolo, I. Frahm, E. H. Charreau, R. Schillaci, et al. Progestins Induce Transcriptional Activation of Signal Transducer and Activator of Transcription 3 (Stat3) via a Jak- and Src-Dependent Mechanism in Breast Cancer Cells Mol. Cell. Biol., June 15, 2005; 25(12): 4826 - 4840. [Abstract] [Full Text] [PDF]

				D. Yu, M. Dews, A. Park, J. W. Tobias, and A. Thomas-Tikhonenko Inactivation of Myc in Murine Two-Hit B lymphomas Causes Dormancy with Elevated Levels of Interleukin 10 Receptor and CD20: Implications for Adjuvant Therapies Cancer Res., June 15, 2005; 65(12): 5454 - 5461. [Abstract] [Full Text] [PDF]

				K. Venkatasubbarao, A. Choudary, and J. W. Freeman Farnesyl Transferase Inhibitor (R115777)-Induced Inhibition of STAT3(Tyr705) Phosphorylation in Human Pancreatic Cancer Cell Lines Require Extracellular Signal-Regulated Kinases Cancer Res., April 1, 2005; 65(7): 2861 - 2871. [Abstract] [Full Text] [PDF]

				L. Burdelya, M. Kujawski, G. Niu, B. Zhong, T. Wang, S. Zhang, M. Kortylewski, K. Shain, H. Kay, J. Djeu, et al. Stat3 Activity in Melanoma Cells Affects Migration of Immune Effector Cells and Nitric Oxide-Mediated Antitumor Effects J. Immunol., April 1, 2005; 174(7): 3925 - 3931. [Abstract] [Full Text] [PDF]

				G. Waris, J. Turkson, T. Hassanein, and A. Siddiqui Hepatitis C Virus (HCV) Constitutively Activates STAT-3 via Oxidative Stress: Role of STAT-3 in HCV Replication J. Virol., February 1, 2005; 79(3): 1569 - 1580. [Abstract] [Full Text] [PDF]

				R. Christine, R. Sylvie, B. Erik, P. Genevieve, R. Amelie, R. Gerard, B. Marc, G. Christian, and A. Samir Implication of STAT3 Signaling in Human Colonic Cancer Cells during Intestinal Trefoil Factor 3 (TFF3) - and Vascular Endothelial Growth Factor-Mediated Cellular Invasion and Tumor Growth Cancer Res., January 1, 2005; 65(1): 195 - 202. [Abstract] [Full Text] [PDF]

				J. Turkson, S. Zhang, J. Palmer, H. Kay, J. Stanko, L. B. Mora, S. Sebti, H. Yu, and R. Jove Inhibition of constitutive signal transducer and activator of transcription 3 activation by novel platinum complexes with potent antitumor activity Mol. Cancer Ther., December 1, 2004; 3(12): 1533 - 1542. [Abstract] [Full Text] [PDF]

				B. E. Barton, T. F. Murphy, P. Shu, H. F. Huang, M. Meyenhofen, and A. Barton Novel single-stranded oligonucleotides that inhibit signal transducer and activator of transcription 3 induce apoptosis in vitro and in vivo in prostate cancer cell lines Mol. Cancer Ther., October 1, 2004; 3(10): 1183 - 1191. [Abstract] [Full Text] [PDF]

				K. Nagel-Wolfrum, C. Buerger, I. Wittig, K. Butz, F. Hoppe-Seyler, and B. Groner The Interaction of Specific Peptide Aptamers With the DNA Binding Domain and the Dimerization Domain of the Transcription Factor Stat3 Inhibits Transactivation and Induces Apoptosis in Tumor Cells Mol. Cancer Res., March 1, 2004; 2(3): 170 - 182. [Abstract] [Full Text] [PDF]

				B. E. Barton, J. G. Karras, T. F. Murphy, A. Barton, and H. F-S. Huang Signal transducer and activator of transcription 3 (STAT3) activation in prostate cancer: Direct STAT3 inhibition induces apoptosis in prostate cancer lines Mol. Cancer Ther., January 1, 2004; 3(1): 11 - 20. [Abstract] [Full Text]

				C. M. Ulane, J. J. Rodriguez, J.-P. Parisien, and C. M. Horvath STAT3 Ubiquitylation and Degradation by Mumps Virus Suppress Cytokine and Oncogene Signaling J. Virol., June 1, 2003; 77(11): 6385 - 6393. [Abstract] [Full Text] [PDF]

				M. Benekli, M. R. Baer, H. Baumann, and M. Wetzler Signal transducer and activator of transcription proteins in leukemias Blood, April 15, 2003; 101(8): 2940 - 2954. [Abstract] [Full Text] [PDF]

				C. Richardson, C. Fielding, M. Rowe, and P. Brennan Epstein-Barr Virus Regulates STAT1 through Latent Membrane Protein 1 J. Virol., April 1, 2003; 77(7): 4439 - 4443. [Abstract] [Full Text] [PDF]

P. L. Leong, G. A. Andrews, D. E. Johnson, K. F. Dyer, S. Xi, J. C. Mai, P. D. Robbins, S. Gadiparthi, N. A. Burke, S. F. Watkins, et al. Targeted inhibition of Stat3 with a decoy oligonucleotide abrogates head and neck cancer cell growth PNAS, April 1,