ag-490 and Cell-Transformation--Neoplastic

The somatic Jak2 mutation (V617F) was identified in most patients with polycythemia vera (PV). Here, we show that the activating Jak2 V617F mutant completely protected Ba/F3 cells from cytokine withdrawal-induced apoptotic cell death. Interestingly, Ba/F3 cells expressing Jak2 V617F mutant induced rapid tumorigenesis in nude mice, leading to rapid death. Whereas an injection of Ba/F3 cells expressing wild-type Jak2 had no effect, an injection of Ba/F3 cells expressing Jak2 V617F mutant promptly invaded and spread into various distinct organs, such as the liver and spleen. Strikingly, Jak2 inhibitor, AG490 potently inhibited cytokine-independent cell growth induced by the Jak2 V617F mutant. Also, treatment with AG490 effectively delayed Jak2 V617F mutant-induced tumorigenesis in nude mice. Thus, our results both in vitro and in vivo suggest that Jak2 harboring V617F mutation is a potent oncogene able to promote cell transformation and tumorigenesis.

Topics: Animals; Apoptosis; Cell Line; Cell Transformation, Neoplastic; Genetic Predisposition to Disease; Hematopoietic Stem Cells; Janus Kinase 2; Liver; Mice; Mice, Nude; Mutation; Neoplasm Invasiveness; Neoplasm Transplantation; Polycythemia Vera; Polymorphism, Genetic; Spleen; Transfection; Tyrphostins

Signal transducers and activators of transcription (STATs) are involved in growth regulation of cells. They are usually activated by phosphorylation at specific tyrosine residues. In neoplastic cells, constitutive activation of STATs accompanies growth dysregulation and resistance to apoptosis through changes in gene expression, such as enhanced anti-apoptotic gene expression or reduced pro-apoptotic gene expression. Activated STAT3 is thought to play an important role in prostate cancer (PCA) progression. Because we are interested in how persistently-activated STAT3 changes the cellular phenotype to a malignant one in prostate cancer, we used expression vectors containing a gene for constitutively-activated STAT3, called S3c, into NRP-152 rat and BPH-1 human benign prostatic epithelial cells.. We observed that prostatic cell lines stably expressing S3c required STAT3 expression for survival, because they became sensitive to antisense oligonucleotide for STAT3. However, S3c-transfected cells were not sensitive to the effects of JAK inhibitors, meaning that STAT3 was constitutively-activated in these transfected cell lines. NRP-152 prostatic epithelial cells lost the requirement for exogenous growth factors. Furthermore, we observed that NRP-152 expressing S3c had enhanced mRNA levels of retinoic acid receptor (RAR)-alpha, reduced mRNA levels of RAR-beta and -gamma, while BPH-1 cells transfected with S3c became insensitive to the effects of androgen, and also to the effects of a testosterone antagonist. Both S3c-transfected cell lines grew in soft agar after stable transfection with S3c, however neither S3c-transfected cell line was tumorigenic in severe-combined immunodeficient mice.. We conclude, based on our findings, that persistently-activated STAT3 is an important molecular marker of prostate cancer, which develops in formerly benign prostate cells and changes their phenotype to one more closely resembling transformed prostate cells. That the S3c-transfected cell lines require the continued expression of S3c demonstrates that a significant phenotypic change occurred in the cells. These conclusions are based on our data with respect to loss of growth factor requirement, loss of androgen response, gain of growth in soft agar, and changes in RAR subunit expression, all of which are consistent with a malignant phenotype in prostate cancer. However, an additional genetic change may be required for S3c-transfected prostate cells to become tumorigenic.

Topics: Androgens; Animals; Cell Line; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Dihydrotestosterone; DNA-Binding Proteins; Enzyme Inhibitors; Epithelial Cells; Gene Expression; Growth Substances; Humans; Janus Kinase 2; Male; Mutation; Phenotype; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Rats; Receptors, Retinoic Acid; RNA, Messenger; STAT3 Transcription Factor; Trans-Activators; Transfection; Tyrphostins

The t(2;5) translocation results in a 80-kDa oncogenic fusion protein consisting of NPM and the kinase domain of the tyrosine kinase ALK and is present in over half the cases of anaplastic large cell lymphoma (ALCL). NPM/ALK exerts its transforming potential via activation of multiple signaling pathways promoting growth factor independence and protection from apoptosis. Jak/Stat signaling is aberrantly activated in several human hematopoietic malignancies. We investigated the role of Jak2 in the context of NPM/ALK-mediated oncogenesis.. Constitutive tyrosine phosphorylation of Jak2 was analyzed by Jak2 immunoprecipitation and subsequent anti-phosphotyrosine Western blotting. NPM/ALK-transformed cells were treated with the Jak2 inhibitor AG490 or transfected with wild-type or dominant-negative Jak2 expression constructs to measure 3[H]-thymidine incorporation. Apoptosis was assessed by flow cytometric analysis of annexin V-stained cells. The effect of Jak2 on Stat5-dependent transcriptional activity was measured by beta-casein promoter-dependent luciferase expression.. Jak2 was found to be constitutively tyrosine phosphorylated in ALCL cells and in NPM/ALK-transformed hematopoietic cells. Also, NPM/ALK was present in immunoprecipitates of Jak2. Inhibition of Jak2 led to a reduction of NPM/ALK-mediated proliferation and induced apoptosis. Stat5-dependent transcriptional activity was inhibited by transfection of NPM/ALK-transformed cells with a dominant-negative Jak2 expression construct or treatment with AG490.. Constitutive activation of Jak2 constitutes a pro-proliferative, anti-apoptotic signaling pathway in NPM/ALK-transformed hematopoietic cells.

Topics: Animals; Apoptosis; B-Lymphocytes; Caseins; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Gene Expression; Hematopoietic Stem Cells; Immunosorbent Techniques; Janus Kinase 2; Luciferases; Mice; Milk Proteins; Phosphorylation; Phosphotyrosine; Promoter Regions, Genetic; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; STAT5 Transcription Factor; Trans-Activators; Transcription, Genetic; Transfection; Tyrphostins

Human T cell leukemia virus type 1 (HTLV-1) transforms cytokine-dependent T lymphocytes and causes adult T cell leukemia. Janus tyrosine kinase (Jak)3 and transcription factors Stat5a and Stat5b are essential for the proliferation of normal T cells and are constitutively hyperactivated in both HTLV-1-transformed human T cell lines and lymphocytes isolated from HTLV-1-infected patients; therefore, a critical role for the Jak3-Stat5 pathway in the progression of this disease has been postulated. We recently reported that tyrphostin AG-490 selectively blocked IL-2 activation of Jak3/Stat5 and growth of murine T cell lines. Here we demonstrate that disruption of Jak3/Stat5a/b signaling with AG-490 (50 microM) blocked the proliferation of primary human T lymphocytes, but paradoxically failed to inhibit the proliferation of HTLV-1-transformed human T cell lines, HuT-102 and MT-2. Structural homologues of AG-490 also inhibited the proliferation of primary human T cells, but not HTLV-1-infected cells. Disruption of constitutive Jak3/Stat5 activation by AG-490 was demonstrated by inhibition of 1) tyrosine phosphorylation of Jak3, Stat5a (Tyr(694)), and Stat5b (Tyr(699)); 2) serine phosphorylation of Stat5a (Ser(726)) as determined by a novel phosphospecific Ab; and 3) Stat5a/b DNA binding to the Stat5-responsive beta-casein promoter. In contrast, AG-490 had no effect on DNA binding by p50/p65 components of NF-kappaB, a transcription factor activated by the HTLV-1-encoded phosphoprotein, Tax. Collectively, these data suggest that the Jak3-Stat5 pathway in HTLV-1-transformed T cells has become functionally redundant for proliferation. Reversal of this functional uncoupling may be required before Jak3/Stat5 inhibitors will be useful in the treatment of this malignancy.

Topics: Cell Line, Transformed; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA-Binding Proteins; Enzyme Inhibitors; Growth Inhibitors; Human T-lymphotropic virus 1; Humans; Immunosuppressive Agents; Interleukin-15; Interleukin-2; Janus Kinase 3; Lymphocyte Activation; Milk Proteins; NF-kappa B; Nuclear Proteins; Phosphorylation; Phytohemagglutinins; Protein-Tyrosine Kinases; Serine; Signal Transduction; STAT5 Transcription Factor; T-Lymphocytes; Trans-Activators; Tumor Cells, Cultured; Tumor Suppressor Proteins; Tyrosine; Tyrphostins