1: Cancer Res. 2005 Oct 1;65(19):8927-35. Src inhibits adriamycin-induced senescence and G2 checkpoint arrest by blocking the induction of p21waf1. Vigneron A, Roninson IB, Gamelin E, Coqueret O. Institut National de la Sante et de la Recherche Medicale U564, Cancer Center Paul Papin, Angers, France. DNA-damaging drugs stop tumor cell proliferation by inducing apoptosis, necrosis, or senescence. Cyclin-dependent kinase inhibitor p21waf1 is an important regulator of these responses, promoting senescence and preventing aberrant mitosis that leads to cell death. Because tumors expressing oncogenic tyrosine kinases are relatively resistant to DNA-damaging agents, the effects of Src on cellular responses to anticancer drug Adriamycin were investigated. Src expression increased drug survival in HT1080 fibrosarcoma cells, as measured by the colony formation assay, and strongly inhibited Adriamycin-induced senescence. Src also decreased the number of apoptotic cells while increasing the fraction of cells dying through necrosis. In addition, Src inhibited the G2 and G1 tetraploidy checkpoints of Adriamycin-treated cells, permitting these cells to proceed into mitosis and subsequently double their DNA content. Inhibition of senescence and G2-G1 checkpoints in Src-expressing cells was associated with the failure of these cells to up-regulate p21waf1 in response to Adriamycin. The failure of p21waf1 induction, despite increased expression of p53 and its binding to p21waf1 promoter, was mediated by the up-regulation of c-Myc, a negative regulator of p21waf1 transcription. Conversely, ectopic expression of p21waf1 inhibited Myc transcription in Src-expressing cells, an effect that was associated with the interaction of p21waf1 with the STAT3 transcription factor at the Myc promoter. These results reveal a complex effect of Src on cellular drug responses and provide an explanation for the effect of this oncogene on cellular drug resistance. PMID: 16204065 [PubMed - in process] --------------------------------------------------------------- 2: Clin Cancer Res. 2005 Sep 1;11(17):6333-41. Down-regulation of signal transducer and activator of transcription 3 expression using vector-based small interfering RNAs suppresses growth of human prostate tumor in vivo. Gao L, Zhang L, Hu J, Li F, Shao Y, Zhao D, Kalvakolanu DV, Kopecko DJ, Zhao X, Xu DQ. Department of Pathophysiology, School of Basic Medicine, Jilin University, Changchun, PR China. PURPOSE: Signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a variety of cancers and it is a common feature of prostate cancer. Thus, Stat3 represents a promising molecular target for tumor therapy. We applied a DNA vector-based Stat3-specific RNA interference approach to block Stat3 signaling and to evaluate the biological consequences of Stat3 down-modulation on tumor growth using a mouse model. EXPERIMENTAL DESIGN: To investigate the therapeutic potential of blocking Stat3 in cancer cells, three small interfering RNAs (siRNA; Stat3-1, Stat3-2, and Stat3-3) specific for different target sites on Stat3 mRNA were designed and used with a DNA vector-based RNA interference approach expressing short hairpin RNAs to knockdown Stat3 expression in human prostate cancer cells in vitro as well as in vivo. RESULTS: Of the three equivalently expressed siRNAs, only Stat3-3 and Stat3-2, which target the region coding for the SH2 domain and the coiled-coil domain, respectively, strongly suppressed the expression of Stat3 in PC3 and LNCaP cells. The Stat3-1 siRNA, which targeted the DNA-binding domain, exerted no effect on Stat3 expression, indicating that the gene silencing efficiency of siRNA may be dependent on the local structure of Stat3 mRNA. The Stat3 siRNAs down-regulated the expression of Bcl-2 (an anti-apoptotic protein), and cyclin D1 and c-Myc (cell growth activators) in prostate cancer cells. Inhibition of Stat3 and its related genes was accompanied by growth suppression and induction of apoptosis in cancer cells in vitro and in tumors implanted in nude mice. CONCLUSIONS: These data indicate that Stat3 signaling is a promising molecular target for prostate cancer therapy and that vector-based Stat3 siRNA may be useful as a therapeutic agent for treatment of prostate cancer. PMID: 16144938 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Respir Physiol Neurobiol. 2005 Aug 23; [Epub ahead of print] SOCS3 was induced by hypoxia and suppressed STAT3 phosphorylation in pulmonary arterial smooth muscle cells. Bai L, Yu Z, Qian G, Qian P, Jiang J, Wang G, Bai C. Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, PR China. Recently identified suppressors of cytokine signaling (SOCS) have been proposed as negative regulators of cytokine signaling, which have distinct mechanisms of inhibiting JAK-STAT pathway. In this study, using cultures of rat primary pulmonary vascular smooth muscle cells (PASMC), we found that hypoxia induced strongly STAT3 phosphorylation by up to four-fold. At the same time, mRNA for the endogenous cytokine signaling repressor SOCS3, but not SOCS1, was markedly induced in PASMC as early as 2h following hypoxic stimulation. Furthermore, forced expression of SOCS3 gene suppressed tyrosine phosphorylation of STAT3 and transcription of c-myc gene by more than 70% and 60% in PASMC under hypoxic conditions, respectively. Additionally, we showed here that hypoxia enhanced nearly two-fold increase of PASMC proliferation and overexpression of SOCS3 gene downregulated hypoxia-induced PASMC proliferation by about 50%. The finding suggest that STAT3-dependent pathway is involved in the activation and proliferation of PASMC stimulated by hypoxia, and SOCS3 is a rapidly hypoxia-inducible gene and acts to inhibit activation of cellular signaling pathway in a classical negative feedback loop. PMID: 16125475 [PubMed - as supplied by publisher] --------------------------------------------------------------- 4: Curr Pharm Des. 2005;11(22):2841-54. Rational drug design of G-quartet DNA as anti-cancer agents. Jing N, Sha W, Li Y, Xiong W, Tweardy DJ. Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA. njing@bcm.tmc.edu The ability of certain DNA sequences to form G-quartet structures has been exploited recently to develop novel anti-cancer agents including small molecules that promote G-quartet formation within the c-MYC promoter thereby repressing c-MYC transcription and introducing G-quartet-forming oligodeoxynucleotides (GQ-ODN) into cancer cells resulting in p53-dependent cell cycle arrest and inhibition of DNA replication. GQ-ODNs also have been developed as potent inhibitors of signal transducer and activator of transcription (STAT) 3, a critical mediator of oncogenic signaling in many cancers. This review summarizes the rational design of G-quartet forming DNA drugs as Stat3 inhibitors. Topics that are reviewed include the strategy of structure-based drug design, establishment of a structure-activity relationship, development of a novel intracellular delivery system for G-quartet-forming DNA agents and in vivo drug testing to assess the anti-cancer effects of DNA drugs in tumor xenografts. Results to date with GQ-ODN targeting Stat3 are encouraging, and it is hoped that continued pursuit of the methodology outlined here may lead to development of an effective agent for treatment of metastatic cancers, such as prostate and breast, in which Stat3 is constitutively activated. Publication Types: Review PMID: 16101441 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Biochem Biophys Res Commun. 2005 Sep 23;335(2):292-9. Evaluation of potential Stat3-regulated genes in human breast cancer. Hsieh FC, Cheng G, Lin J. Center for Childhood Cancer, Columbus Children's Research Institute, The Ohio State University, Columbus, OH 43205, USA. The constitutive activation of signal transducer and activator of transcription 3 (Stat3) is frequently detected in breast cancer tissues and cell lines. Stat3 has been classified as a proto-oncogene, because an activated form of Stat3 can mediate oncogenic transformation in cultured cells and tumor formation in nude mice. Since Stat3 may play an important role in breast cancer, it is of interest to investigate the expression of phosphorylated Stat3, an activated form of Stat3, and its downstream mediators specifically in breast cancer, and to explore the possible mechanisms of Stat3 signaling pathway in oncogenesis of breast cancer. We analyzed Stat3 phosphorylation and expression of Stat3-regulated genes in breast cancer cell lines as well as invasive breast cancer tissues using tissue microarray slides. Our results showed that elevated levels of phosphorylation of Stat3 protein (Tyr705) were detected in 48 out of total 136 invasive breast tumors (35%) whereas normal breast tissues express much lower levels of Stat3 phosphorylation. The increased levels of Stat3 phosphorylation were associated with the metastasis in regional lymph nodes (P=0.042) and the expression of progesterone receptor (P=0.028) but not with distant metastasis, nor the expression of estrogen receptor. Our results also indicate that elevated levels of Stat3 phosphorylation were significantly associated with increased expression of potential downstream targets of Stat3 which include apoptosis inhibitors (Survivin, Mcl-1, HSP27, Adrenomedullin, and Bcl-xL), cell-cycle regulators (c-Fos, MEK5, and c-Myc), and inducer of tumor angiogenesis (VEGF, COX-2, MMP-2, MMP-10, and MMP-1) in invasive breast cancer tissues. Therefore, our findings suggest that constitutive Stat3 signaling may be one of the key upstream regulators to induce these downstream proteins, which may play important roles in Stat3-mediated oncogenesis in breast cancer. PMID: 16081048 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: J Immunol. 2005 Aug 1;175(3):1450-5. Immunological trigger of mast cells by monomeric IgE: effect on microphthalmia transcription factor, STAT3 network of interactions. Sonnenblick A, Levy C, Razin E. Department of Biochemistry, Hebrew University Hadassah Medical School, Jerusalem, Israel. Microphthalmia transcription factor (MITF) and STAT3 are two transcription factors that play a major role in the regulation of growth and function of mast cells and melanocytes. We have previously provided experimental evidence regarding the functional cross-talk between MITF, protein inhibitor of activated STAT3, and STAT3 in response to cytokine activation of mast cells. Recent studies have demonstrated that binding of different IgE molecules to their FcepsilonRI induces a spectrum of intracellular events in the absence of specific Ag. In this work, we show for the first time that, in mouse bone marrow-derived mast cells and in rat basophilic leukemia cells, monomeric IgE alone can induce the MITF-protein inhibitor of activated STAT3-STAT3 network of interactions and leads to phosphorylation of MITF at S73 and of STAT3 at both tyrosine 705 and S727. This phosphorylation increases the transcriptional activity of MITF and STAT3 as indicated by mRNA accumulation of their target genes such as Bcl-2, granzyme B, and c-Myc. Interestingly, MITF and STAT3 were not found to be obligatory factors in the anti-apoptotic response induced by IgE. Thus, the phenomenon that IgE alone was able to induce transcription factors that are essential for mast cell function could contribute to our understanding of the pathogenesis of allergy and its associated diseases. PMID: 16034081 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Cancer Res. 2005 Jul 1;65(13):5828-34. Malignant transformation but not normal cell growth depends on signal transducer and activator of transcription 3. Schlessinger K, Levy DE. Department of Pathology and New York University Cancer Institute, New York University School of Medicine, New York, New York 10016, USA. Signal transducer and activator of transcription 3 (STAT3) has been indirectly implicated in numerous fundamental cellular processes, including proliferation, survival, and differentiation. We provide genetic evidence from studies of STAT3-null cells that STAT3 is dispensable for normal growth of mouse fibroblasts in culture. STAT3 contributed to the full induction of some (typified by c-fos) but not all (typified by c-myc) immediate early gene expression, but STAT3-independent processes were sufficient to support full cell growth and survival. However, STAT3 was required to manifest a transformed state following expression of v-src, and STAT3-null cells were impaired for anchorage-independent growth as colonies in soft agar and as tumors in mice. The data suggest that STAT3 mediates the maintenance of focal adhesion kinase activity in the absence of cell adhesion by suppressing the action of an inhibitory phosphatase. PMID: 15994959 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Cancer Res. 2005 Jul 1;65(13):5571-7. Proline-, glutamic acid-, and leucine-rich protein-1 is essential in growth factor regulation of signal transducers and activators of transcription 3 activation. Manavathi B, Nair SS, Wang RA, Kumar R, Vadlamudi RK. Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) is a novel estrogen receptor coactivator that plays an important role in the genomic and nongenomic actions of estrogen receptor by interacting with histones and src-mitogen-activated protein kinase pathway, respectively. A great deal of information has emerged in recent years about the possible role of PELP1 in estrogen receptor signaling. However, the participation and significance of PELP1 in other cellular signaling pathways remains unknown. Using a yeast two-hybrid screen, we identified PELP1 as a novel interacting protein of signal transducers and activators of transcription 3 (STAT3) and found evidence of physiologic interaction between PELP1 and STAT3. We also found that these interactions played a mechanistic role in the positive regulation of STAT3 transcription from synthetic promoters and endogenous target genes such as cyclin D1, c-myc, and c-fos. Overexpression of PELP1 enhanced phosphorylation of STAT3 at Ser727 in a src-mitogen-activated protein kinase-sensitive manner and, conversely, down-regulation of PELP1 compromised growth factor-mediated induction of STAT3 target genes. We also discovered that PELP1 interacts with STAT3 in the nuclear compartment and down-regulation of PELP1 interfered with the recruitment of STAT3 to its target gene promoters. In summary, our results highlight a novel role for PELP1 in growth factor signaling and indicate that PELP1-mediated genomic and nongenomic functions play a role in the growth factor-mediated STAT3 transactivation functions. Such regulatory interactions of PELP1 may have important functional implications in the cross-talk of estrogen receptor and growth factor signaling. PMID: 15994929 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Anticancer Drugs. 2005 Jul;16(6):601-7. Targeting Stat3 in cancer therapy. Jing N, Tweardy DJ. aDepartment of Medicine and Cancer Center, Baylor College of Medicine, Houston, TX, USA. Stat3 is constitutively activated in many human cancers where it functions as a critical mediator of oncogenic signaling through transcriptional activation of genes encoding apoptosis inhibitors (e.g. Bcl-xL, Mcl-1 and survivin), cell-cycle regulators (e.g. cyclin D1 and c-Myc) and inducers of angiogenesis (e.g. vascular endothelial growth factor). This article reviews several approaches that have been pursued for targeting Stat3 in cancer therapy including antisense strategies, tyrosine kinase inhibition, decoy phosphopeptides, decoy duplex oligonucleotides and G-quartet oligodeoxynucleotides (GQ-ODN). The GQ-ODN strategy is reviewed in somewhat greater detail than the others because it includes a novel system that effectively delivers drug into cells and tissues, addresses successfully the issue of specificity of targeting Stat3 versus Stat1, and has demonstrated efficacy in vivo. PMID: 15930886 [PubMed - in process] --------------------------------------------------------------- 10: J Child Neurol. 2005 Apr;20(4):330-6. Molecular pathogenesis of focal cortical dysplasia and hemimegalencephaly. Crino PB. PENN Epilepsy Center, Department of Neurology, University of Pennsylvania Medical Center, Philadelphia, PA 19104, USA. crinop@mail.med.upenn.edu My laboratory recently demonstrated that there is selective expression of phosphoribosomal S6 protein in balloon cells in focal cortical dysplasia and hemimegalencephaly but no expression of the upstream kinase, phospho-p70S6 kinase. Two proteins activated by phospho-p70S6 kinase, phospho-STAT3 and phospho-4EBP1, were not detected in balloon cells. Using complementary DNA arrays in hemimegalencephaly specimens, we found increased expression of cyclin D1 and c-myc messenger ribonucleic acids (RNAs). Expression of cyclin D1 and c-myc genes is transcriptionally activated by beta-catenin. Western analysis demonstrated increased levels of nonphosphorylated beta-catenin in hemimegalencephalic cortex. Reduced levels of Ser33, Ser37, and Thr41 phospho-beta-catenin, sites known to be phosphorylated by glycogen synthase kinase 3 and to be essential for beta-catenin inactivation, were detected in hemimegalencephaly. Enhanced transcription of cyclin D1 and c-myc messenger RNAs, increased transcriptionally active beta-catenin, and decreased Ser33/Ser37/Thr41 phospho-beta-catenin suggest activation of the Wnt-1/beta-catenin cascade in hemimegalencephaly, which can lead to aberrant cell proliferation and hemispheric enlargement during brain development. Enhanced activation of phospho-S6 and beta-catenin suggests two converging cell pathways that can be pivotal in the pathogenesis of focal cortical dysplasia and hemimegalencephaly. PMID: 15921235 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Virus Res. 2005 Oct;113(1):26-35. HIV-1 infection initiates changes in the expression of a wide array of genes in U937 promonocytes and HUT78 T cells. Wen W, Chen S, Cao Y, Zhu Y, Yamamoto Y. Center of Clinical Laboratory Medicine, The First Affiliated Hospital, Jinan University, Guangzhou, China. wenwangrong@tom.com Human monocytes/macrophages (M/M) are the major targets for human immunodeficiency virus type 1 (HIV-1) infection. To characterize the global effects of acute HIV-1 infection on gene expression in M/M, the expression levels of 550 host cell RNA transcripts in U937 human promonocytes at 2-3 days after HIV-1 infection were assessed using cDNA microarray analysis and were compared to those in the infected HUT78, a CD4+ T cell line. Confirmed by semiquantitative RT-PCR, our results showed that 12 genes were up-regulated and 26 genes were down-regulated in the infected U937 cells at 2-3 days post-infection, whereas 8 genes were up-regulated and 20 genes were down-regulated in the infected HUT78 cells at 2-3 days post-infection. These genes encode a host of proteins with divergent functions in a variety of cellular processes including apoptosis (FAS, Fas ligand, PIN, HSP90beta, bcl-2, bcl-x), cell signal transduction (Ras, RGS1, IRF-1, STAT3), receptor-mediated signaling transduction (CD71, CD69, CD3delta), cell cycle and growth (c-myc, cytokines, kinase), transcriptional regulation (EWS, CREB-2), and chemotaxis (beta-chemokines, RANTES), supporting the general effects of HIV-1 infection on cells of different origin. Although most identified genes were regulated similarly in both infected cell lines, differences in gene regulation, such as c-myc, CD71, CD69, and beta-chemokines, between the two infected cell lines were also identified in this study. These differences may further our understanding of the pathogenicity of HIV and enable the discovery of novel therapeutic approach for AIDS. PMID: 15885842 [PubMed - in process] --------------------------------------------------------------- 12: Cancer Res. 2005 Apr 1;65(7):2861-71. Farnesyl transferase inhibitor (R115777)-induced inhibition of STAT3(Tyr705) phosphorylation in human pancreatic cancer cell lines require extracellular signal-regulated kinases. Venkatasubbarao K, Choudary A, Freeman JW. Department of Medicine, Division of Medical Oncology, University of Texas Health Center, San Antonio, Texas 78229-3900, USA. In this study, we report that R115777, a nonpeptidomimetic farnesyl transferase inhibitor, suppresses the growth of human pancreatic adenocarcinoma cell lines and that this growth inhibition is associated with modulation in the phosphorylation levels of signal transducers and activators of transcription 3 (STAT3) and extracellular signal-regulated kinases (ERK). Treatment of cells with R115777 inhibited the tyrosine phosphorylation of STAT3((Tyr705)), while increasing the serine phosphorylation of STAT3((Ser727)). We found the differential phosphorylation of STAT3 was due to an increased and prolonged activation of ERKs. The biological significance of ERK-mediated inhibition of STAT3((Tyr705)) phosphorylation was further assessed by treating the cells with an inhibitor (PD98059) of mitogen-activated protein kinase kinase (MEK) or by transfecting the cells with a vector that expresses constitutively active MEK-1. Expression of constitutively active MEK-1 caused an increase of ERK activity and inhibited STAT3((Tyr705)) phosphorylation. Conversely, inhibition of ERK activity by PD98059 reversed the R115777-induced inhibition of STAT3((Tyr705)) phosphorylation. R115777 also caused the inhibition of the binding of STAT3 to its consensus binding element. An increase in the activation of ERKs either by overexpressing MEK-1 or treatment of cells with R115777 caused an up-regulation in the levels of a cyclin-dependent kinase (cdk) inhibitor, p21(cip1/waf1). These observations suggest that R115777-induced growth inhibition is partly due to the prolonged activation of ERKs that mediates an inhibition of STAT3((Tyr705)) phosphorylation and an increase in the levels of p21(cip1/waf1) in human pancreatic adenocarcinoma cell lines. PMID: 15805288 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Cancer Res. 2005 Apr 1;65(7):2532-6. Erratum in: Cancer Res. 2005 Jun 1;65(11):4969. Knockdown of STAT3 expression by RNA interference inhibits the induction of breast tumors in immunocompetent mice. Ling X, Arlinghaus RB. Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA. Constitutively activated STAT3 is involved in the formation of multiple types of tumors including breast cancer. We examined the effects of Stat3 protein knockdown by RNA interference using a dicistronic lentivirus small hairpin (shRNA) delivery system on the growth of mammary tumors in BALB/c mice induced by the 4T1 cell line. A single exposure of 4T1 cells to shRNA/STAT3 lentivirus transduced 75% of the cells with green fluorescent protein (GFP) within 96 hours. In cells selected for GFP expression, neither Stat3 protein nor phosphotyrosine Stat3 was detected. Tumor formation induced by injecting 4T1 cells into the mammary fat pad was blocked by expression of the shRNA for STAT3 whereas all mice injected with 4T1 cells expressing only GFP efficiently formed tumors. c-Myc expression was reduced 75% in cells expressing greatly reduced levels of Stat3 compared with the GFP control. Of interest, the level of activated Src, which is known to activate Stat3, was virtually eliminated but the level of the Src protein itself remained the same. Importantly, expression of Twist protein, a metastatic regulator, was eliminated in STAT3 knockdown cells. Invasion activity of STAT3 knockdown cells was strongly inhibited. However, the proliferation rate of cells in Stat3 knockdown cells was similar to that of the GFP control; the cell cycle was also not affected. We conclude from these studies that activated Stat3 protein plays a critical role in the induction of breast tumors induced by 4T1 cells by enhancing the expression of several important genes including c-Myc and the metastatic regulator Twist. These studies suggest that stable expression of small interfering RNA for STAT3 has potential as a therapeutic strategy for breast cancer. PMID: 15805244 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Development. 2005 Mar;132(5):1147-60. Epub 2005 Feb 2. Erratum in: Development. 2005 Mar;132(6):1475. Developmental stage determines the effects of MYC in the mammary epithelium. Blakely CM, Sintasath L, D'Cruz CM, Hahn KT, Dugan KD, Belka GK, Chodosh LA. Departments of Cancer Biology, Cell and Developmental Biology, Medicine, and The Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6160, USA. Epidemiological findings suggest that the consequences of a given oncogenic stimulus vary depending upon the developmental state of the target tissue at the time of exposure. This is particularly evident in the mammary gland, where both age at exposure to a carcinogenic stimulus and the timing of a first full-term pregnancy can markedly alter the risk of developing breast cancer. Analogous to this, the biological consequences of activating oncogenes, such as MYC, can be influenced by cellular context both in terms of cell lineage and cellular environment. In light of this, we hypothesized that the consequences of aberrant MYC activation in the mammary gland might be determined by the developmental state of the gland at the time of MYC exposure. To test this hypothesis directly, we have used a doxycycline-inducible transgenic mouse model to overexpress MYC during different stages of mammary gland development. Using this model, we find that the ability of MYC to inhibit postpartum lactation is due entirely to its activation within a specific 72-hour window during mid-pregnancy; by contrast, MYC activation either prior to or following this 72-hour window has little or no effect on postpartum lactation. Surprisingly, we find that MYC does not block postpartum lactation by inhibiting mammary epithelial differentiation, but rather by promoting differentiation and precocious lactation during pregnancy, which in turn leads to premature involution of the gland. We further show that this developmental stage-specific ability of MYC to promote mammary epithelial differentiation is tightly linked to its ability to downregulate caveolin 1 and activate Stat5 in a developmental stage-specific manner. Our findings provide unique in vivo molecular evidence for developmental stage-specific effects of oncogene activation, as well as the first evidence linking MYC with activation of the Jak2-Stat5 signaling pathway. PMID: 15689376 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: J Biol Chem. 2005 Apr 22;280(16):15673-81. Epub 2005 Jan 26. The STAT3 transcription factor is a target for the Myc and riboblastoma proteins on the Cdc25A promoter. Barre B, Vigneron A, Coqueret O. INSERM U564, Cancer Center Paul Papin, 49033 Angers, France. The STAT3 (signal transducer and activator of transcription) transcription factor functions as down-stream effector of growth factor signaling. Whereas STAT3 activation is transient in normal cells, constitutively activated forms of the transcription factor have been detected in several cancer cell lines and primary tumors. Through the up-regulation of cell cycle and survival genes, STAT3 plays important roles in cell growth, anti-apoptosis, and cell transformation yet the molecular basis for this behavior is poorly understood. In this study, we show that STAT3 and its transcriptional cofactors are recruited to the promoter of the Cdc25A gene to activate its expression. Using chromatin immunoprecipitations, we observed that Myc is recruited to this promoter following STAT3 DNA binding. Moreover, small interfering RNA-mediated knockdown of Myc specifically inhibits the STAT3-mediated activation of Cdc25A. Reduction in Myc protein level results in defective recruitment of the CREB-binding protein, Cdk9, and RNA polymerase complexes, indicating that Myc is necessary for STAT3 transcription. Surprisingly, the association of STAT3 with the Cdc25A promoter does not necessarily lead to transcriptional induction because this protein also functions as a transcriptional repressor of the Cdc25A gene. Following hydrogen peroxide stimulation, STAT3 forms a repressor complex with the retinoblastoma (Rb) tumor suppressor to occupy the Cdc25A promoter and block its induction. In coimmunoprecipitations and ChIP experiments, Rb was found to associate with STAT3 on DNA and we provide evidence that Rb binds directly to the transcription factor. Thus, we propose that Myc and STAT3 cooperate to induce the expression of Cdc25A and that their transcriptional activity is normally regulated by the Rb tumor suppressor gene. PMID: 15677471 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Development. 2005 Mar;132(5):885-96. Epub 2005 Jan 26. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Cartwright P, McLean C, Sheppard A, Rivett D, Jones K, Dalton S. University of Georgia, Rhodes Center, 425 River Road, Athens, GA 30602-2771, USA. Murine ES cells can be maintained as a pluripotent, self-renewing population by LIF/STAT3-dependent signaling. The downstream effectors of this pathway have not been previously defined. In this report, we identify a key target of the LIF self-renewal pathway by showing that STAT3 directly regulates the expression of the Myc transcription factor. Murine ES cells express elevated levels of Myc and following LIF withdrawal, Myc mRNA levels collapse and Myc protein becomes phosphorylated on threonine 58 (T58), triggering its GSK3beta dependent degradation. Maintained expression of stable Myc (T58A) renders self-renewal and maintenance of pluripotency independent of LIF. By contrast, expression of a dominant negative form of Myc antagonizes self-renewal and promotes differentiation. Transcriptional control by STAT3 and suppression of T58 phosphorylation are crucial for regulation of Myc activity in ES cells and therefore in promoting self-renewal. Together, our results establish a mechanism for how LIF and STAT3 regulate ES cell self-renewal and pluripotency. PMID: 15673569 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Eur J Clin Invest. 2005 Feb;35(2):140-7. Molecular events associated with accelerated proliferative response in rat livers when partial hepatectomy is preceded by a sham operation. Laurent S, Starkel P, Leclercq IA, Lambotte L, Maiter D, Horsmans Y. Department of Gastroenterology, Universite Catholique de Louvain, 1200 Brussels, Belgium. BACKGROUND: When a sham operation is performed 6 h before partial hepatectomy (PH), the regenerative response is accelerated suggesting that sham operation itself contributes to cellular events leading to proliferation. MATERIALS AND METHODS: In order to examine the mechanisms implicated in this acceleration, we compared the activation of several factors associated with the progression through the cell cycle at various times after PH and after PH preceded by sham operation (S6 h + PH). The effect of a single sham (S) and two combined sham operations (S6 h + S) was also examined. Nonoperated rats were used as controls (C). RESULTS: The early factors NF-kappaB and Stat3 were activated after S6 h + PH and S6 h + S. C-jun expression was increased 0.5 h and 2 h after PH and 6 h after sham. There was no further increase in S6 h + PH and S6 h + S. In contrast, c-myc expression returned to baseline levels after S6 h and a new increase was observed 2 h after S6 h + PH but not after S6 h + S. P53 mRNA was significantly expressed 6 h after S6 h + PH, but at a level similar than that observed 6 and 12 h after PH alone. An earlier increase in c-Ha-ras mRNA and cyclin E protein was found in S6 h + PH, in comparison with PH alone. CONCLUSIONS: The first divergent response between the two combined models involved c-myc expression. However, major differences related to the accelerated liver regenerative response observed after S6 h + PH were found at late time points associating an earlier expression of c-Ha-ras and nuclear cyclin E. PMID: 15667586 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Crit Rev Eukaryot Gene Expr. 2004;14(4):287-300. Neuroendocrine cells in prostate cancer. Amorino GP, Parsons SJ. Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA. Neuroendocrine (NE) cells are found in prostate tumors, and their incidence is considered a promising prognostic indicator for the development of androgen-independent disease. NE cells are derived from non-NE prostate cancer cells and secrete factors that can act in a paracrine manner to stimulate the survival, growth, motility, and metastatic potential of prostatic carcinoma cells. Factors such as IL-6, epinephrine, and forskolin induce NE differentiation in prostate cancer cells; the mechanisms involve increases in intracellular cAMP, protein kinase A (PKA) activation and reduced intracellular calcium levels. Transcription factors implicated in the acquisition of NE characteristics by prostate cancer cells include STAT3, CREB, EGR1, c-fos, and NF-kappaB. Expression of Chromogranin A, neuron-specific enolase, bcl-2, and the androgen receptor are modulated during NE differentiation and serve as molecular markers for NE cells. Most importantly, NE cells secrete neuropeptides, such as bombesin, neurotensin, PTHrP, serotonin, and calcitonin, which trigger growth and survival responses in androgen-independent prostate cancer cells. Prostate cancer cell receptors that play a role in these processes include the gastrin-releasing peptide (GRP) receptor, neurotensin receptors, and the epidermal growth-factor receptor (EGFR). Signal-transduction molecules activated by these neuropeptides include Src, focal adhesion kinase (FAK), ERK, and PI3K/Akt, with subsequent activation of Elk-1, NF-kappaB, and c-myc transcription factors. A multitude of genes are then expressed by prostate cancer cells, which are involved in proliferation, anti-apoptosis, migration, metastasis, and angiogenesis. Targeting of these pathways at multiple levels can be exploited to inhibit the process by which NE cells contribute to the progression of androgen-independent, treatment-refractory prostate cancer. Publication Types: Review PMID: 15663358 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Science. 2005 Jan 14;307(5707):269-73. Comment in: Science. 2005 Jan 14;307(5707):217-8. Stat3 dimerization regulated by reversible acetylation of a single lysine residue. Yuan ZL, Guan YJ, Chatterjee D, Chin YE. Department of Surgery, Brown University Medical School-Rhode Island Hospital, Providence, RI 02903, USA. Upon cytokine treatment, members of the signal transducers and activators of transcription (STAT) family of proteins are phosphorylated on tyrosine and serine sites within the carboxyl-terminal region in cells. We show that in response to cytokine treatment, Stat3 is also acetylated on a single lysine residue, Lys685. Histone acetyltransferase p300-mediated Stat3 acetylation on Lys685 was reversible by type I histone deacetylase (HDAC). Use of a prostate cancer cell line (PC3) that lacks Stat3 and PC3 cells expressing wild-type Stat3 or a Stat3 mutant containing a Lys685-to-Arg substitution revealed that Lys685 acetylation was critical for Stat3 to form stable dimers required for cytokine-stimulated DNA binding and transcriptional regulation, to enhance transcription of cell growth-related genes, and to promote cell cycle progression in response to treatment with oncostatin M. PMID: 15653507 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Int J Mol Med. 2005 Feb;15(2):269-75. Functional analysis of the effect of forced activation of STAT3 on M1 mouse leukemia cells. Yoshida T, Iwamoto T, Adachi K, Yokota T, Miyake Y, Hamaguchi M. Department of Ophthalmology, Nagoya University School of Medicine, Showa-ku, Nagoya 466-8550, Japan. M1 mouse myeloid leukemia cells exhibit growth arrest and differentiation to monocytes/macrophages in response to leukemia inhibitory factor (LIF) stimulation. Although recent studies have demonstrated that STAT3 plays a central role in this process, it is unknown whether STAT3 activation alone is sufficient. To address this issue, we have established M1/STAT3ER cells, where STAT3 is selectively activated by 4-hydroxytamoxifen (4HT). 4HT stimulation did not have any effect on growth and morphology of M1/ STAT3ER cells, and did not induce the down-regulation of mRNA of c-myc and c-myb, which is necessary for M1 cell differentiation. On the other hand, mRNA of jun-B, IRF1 and p19 was increased by 4HT. DNA precipitation assay indicated that both stimulation of LIF and 4HT similarly activated STAT3ER. Introduction of a constitutive active MAP kinase kinase (MEK1) into M1/STAT3ER cells did not induce differentiation either. Together, our present data suggest that signaling other than the activation of STAT3 and MEK1 may be necessary for M1 cell-growth arrest and differentiation, while a set of early genes of LIF are induced by only STAT3 activation. PMID: 15647843 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: J Biol Chem. 2005 Feb 25;280(8):6409-15. Epub 2004 Dec 6. Des-gamma-carboxy prothrombin is a potential autologous growth factor for hepatocellular carcinoma. Suzuki M, Shiraha H, Fujikawa T, Takaoka N, Ueda N, Nakanishi Y, Koike K, Takaki A, Shiratori Y. Department of Medicine and Medical Science, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Des-gamma-carboxyl prothrombin (DCP) is a well recognized tumor marker for hepatocellular carcinoma (HCC). In the present study, we demonstrate that DCP has a mitogenic effect on HCC cell lines. Purified DCP stimulated DNA synthesis of Hep3B and SK-Hep-1 cells in a dose-dependent manner. DCP was found to bind with cell surface receptor Met causing Met autophosphorylation and also to activate STAT3 signaling pathway through Janus kinase 1. Luciferase gene reporter analysis showed that DCP induced STAT3-related transcription. Small interfering RNAs against both STAT3 and Met abrogated DCP-induced cell proliferation. DCP did not affect the mitogen-activated protein kinase pathway, Myc signaling pathway, or phosphoinositide 3-kinase/Akt pathway. Based on these results, we believe that DCP acts as an autologous mitogen for HCC cell lines. The Met-Janus kinase 1-STAT3 signaling pathway may be a major signaling pathway for DCP-induced cell proliferation. PMID: 15582995 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Oncogene. 2005 Feb 3;24(6):1075-83. c-Myc activation by Theileria parasites promotes survival of infected B-lymphocytes. Dessauge F, Hilaly S, Baumgartner M, Blumen B, Werling D, Langsley G. Laboratoire de Signalisation Immunoparasitaire, CNRS URA CNRS 2581, Departement de Parasitologie, Institut Pasteur, Batiment Elie Metchnikoff, 25-28 rue du Dr Roux, 75724 Paris Cedex 15, France. Theileria parasites infect and transform bovine lymphocytes, but host cell immortalization is reversible, as upon parasite death the lymphocytes rapidly die of apoptosis. Infection leads to a marked augmentation in the levels of lymphocyte c-Myc, and the parasite achieves this by inducing increased c-myc transcription and by prolonging the half-life of the transcription factor. Reduction in c-Myc turnover can be ascribed to CK2-mediated phosphorylation of the transcription factor. A parasite-dependent GM-CSF autocrine loop activates a JAK2/STAT3 signalling pathway that contributes to heightened c-myc transcription, and inhibition of the pathway leads to caspase 9 activation and apoptosis that can be directly ascribed to a reduction in c-Myc. An antiapoptotic role for c-Myc was clearly demonstrated by specific inhibition of c-myc expression with antisense oligonucleotides, and this correlates with loss of the antiapoptotic protein Mcl-1, and, consistently, ectopic expression of c-Myc abrogates B-cell death induced upon JAK2 inhibition. Thus, Theileria parasites ensure the survival of their host lymphocytes via specific activation of c-Myc. PMID: 15580287 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Blood. 2005 Feb 15;105(4):1777-84. Epub 2004 Oct 26. BCL-6 negatively regulates macrophage proliferation by suppressing autocrine IL-6 production. Yu RY, Wang X, Pixley FJ, Yu JJ, Dent AL, Broxmeyer HE, Stanley ER, Ye BH. Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461, USA. The transcription repressor BCL-6 is known to play critical roles in B-cell lymphomagenesis, germinal center formation, and balanced Th1/Th2 differentiation. In macrophages, although BCL-6 has also been shown to regulate the expression of several chemokine genes, its function in other aspects of macrophage biology has not been studied. In addition, the precise role of BCL-6 in cell proliferation is poorly understood in general. Here we report that BCL-6(-/-) macrophages hyperproliferate due to an accelerated G(1)/S transition accompanied by increased cyclin D2 and c-myc and decreased expression of p27. Crucial to this enhanced proliferation is spontaneous interleukin 6 (IL-6) production and signal transducer and activator of transcription 3 (STAT3) activation in BCL-6(-/-) macrophages. In colony-forming assays, BCL- 6(-/-) bone marrow progenitor cells form spontaneous macrophage colonies that can be inhibited by anti-IL-6 antibodies. Gene expression studies demonstrate that BCL-6 binds to several sequence motifs scattered in the IL-6 locus and can repress IL-6 transcription both in 293T cells and in macrophages. In conclusion, our results indicate that BCL-6 negatively regulates proliferation of the monocytic/macrophage lineage by suppressing an autocrine IL-6/STAT3-mediated gene expression program. Our work also suggests that BCL-6 prevents abnormal Th2 differentiation by suppressing basal level IL-6 production in antigen-presenting cells (APCs). PMID: 15507530 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Expert Opin Ther Targets. 2004 Oct;8(5):409-22. STAT proteins as novel targets for cancer drug discovery. Turkson J. Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, SRB 22214, Tampa, FL 33612, USA. turksonj@moffitt.usf.edu Signal transducer and activator of transcription (STAT) proteins are latent cytoplasmic transcription factors that were discovered in the context of cytokine and growth factor signalling. Normal STAT signalling is tightly controlled with finite kinetics, which is in keeping with standard cellular responses. However, persistent STAT activation has also been observed and is frequently associated with malignant transformation. Constitutive activation of STAT proteins, notably of Stat3 and Stat5, is detected in many human tumour cells and cells transformed by oncoproteins that activate tyrosine kinase signalling pathways. It is well-established that constitutively active Stat3 is one of the molecular abnormalities that has a causal role in oncogenesis. Aberrant Stat3 promotes uncontrolled growth and survival through dysregulation of gene expression, including cyclin D1, c-Myc, Bcl-xL, Mcl-1 and survivin genes, and thereby contributes to oncogenesis. Moreover, recent studies reveal that persistently active Stat3 induces tumour angiogenesis by upregulation of vascular endothelial growth factor induction, and modulates immune functions in favour of tumour immune evasion. Overall, studies have validated Stat3 as a novel target for cancer therapy, and hence provided the rationale for developing small-molecule Stat3 inhibitors. This review will discuss current evidence for the critical role of aberrant STAT signalling in malignant transformation, and examine the validity as well as the therapeutic potential of Stat3 as a cancer target. An update on the efforts to develop novel Stat3 inhibitors for therapeutic application will also be provided. Publication Types: Review PMID: 15469392 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Cancer Res. 2004 Aug 15;64(16):5870-5. Molecular mechanisms involved in the growth stimulation of breast cancer cells by leptin. Yin N, Wang D, Zhang H, Yi X, Sun X, Shi B, Wu H, Wu G, Wang X, Shang Y. Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing, China. Obesity is a risk factor for breast cancer in postmenopausal women. Leptin, an adipocyte-derived cytokine, elicits proliferative effects in some cell types and potentially stimulates the growth of mammary epithelium. Here we show that leptin induced time- and dose-dependent signal transducer and activator of transcription 3 (STAT3) phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 kinase activation in breast carcinoma cells. Blocking STAT3 phosphorylation with a specific inhibitor, AG490, abolished leptin-induced proliferation of MCF-7 cells, whereas blocking ERK1/2 activation by a specific ERK1/2 kinase inhibitor, U0126, did not result in any significant changes in leptin-induced cell proliferation. Our experiments also showed that one member of the p160 family of steroid receptor coactivators, steroid receptor coactivator (SRC)-1, but not glucocorticoid receptor interacting protein 1 (GRIP1) or amplified in breast cancer 1 (AIB1), also functioned in gene transactivation in response to leptin treatment. Glutathione S-transferase pull-down experiments showed that SRC-1 physically interacted with the activation domain of STAT3 and that chromatin immunoprecipitation experiments detected the occupancy of SRC-1, but not GRIP1 or AIB1, on the promoter of STAT3 target genes. Our experiments collectively showed that SRC-1 is involved in STAT3 signaling pathway that is implicated in leptin-stimulated cell growth. PMID: 15313931 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: Mol Cell Biol. 2004 Aug;24(15):6788-98. RACK1 regulates G1/S progression by suppressing Src kinase activity. Mamidipudi V, Zhang J, Lee KC, Cartwright CA. Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5187, USA. Cancer genes exert their greatest influence on the cell cycle by targeting regulators of a critical checkpoint in late G(1). Once cells pass this checkpoint, they are fated to replicate DNA and divide. Cancer cells subvert controls at work at this restriction point and remain in cycle. Previously, we showed that RACK1 inhibits the oncogenic Src tyrosine kinase and NIH 3T3 cell growth. RACK1 inhibits cell growth, in part, by prolonging G(0)/G(1). Here we show that RACK1 overexpression induces a partial G(1) arrest by suppressing Src activity at the G(1) checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and cyclin-dependent kinases 4 and 2 (CDK4 and CDK2, respectively) are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G(1)/S progression is delayed. Conversely, downregulation of RACK1 by short interference RNA activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G(1)/S progression. RACK1 suppresses Src- but not mitogen-activated protein kinase-dependent platelet-derived growth factor signaling. We also show that Stat3 is required for Rac1 induction of Myc. Our results reveal a novel mechanism of cell cycle control in late G(1) that works via an endogenous inhibitor of the Src kinase. PMID: 15254245 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Oncogene. 2004 Jul 15;23(32):5426-34. Selective inhibition of STAT3 induces apoptosis and G(1) cell cycle arrest in ALK-positive anaplastic large cell lymphoma. Amin HM, McDonnell TJ, Ma Y, Lin Q, Fujio Y, Kunisada K, Leventaki V, Das P, Rassidakis GZ, Cutler C, Medeiros LJ, Lai R. Department of Hematopathology, Box 72, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. hamin@mdanderson.org Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is an aberrant fusion gene product expressed in a subset of cases of anaplastic large cell lymphoma (ALCL). It has been shown that NPM-ALK binds to and activates signal transducer and activator of transcription 3 (STAT3) in vitro, and that STAT3 is constitutively active in ALK(+) ALCL cell lines and tumors. In view of the oncogenic potential of STAT3, we further examined its biological significance in ALCL using two ALK(+) ALCL cell lines (Karpas 299 and SU-DHL-1) and an adenoviral vector that carries dominant-negative STAT3 (AdSTAT3DN). Infection by AdSTAT3DN led to the expression of STAT3DN in both ALK(+) ALCL cell lines at a similar efficiency. Subcellular fractionation studies showed that a significant proportion of the expressed STAT3DN protein translocated to the nucleus, despite the fact that STAT3DN has a mutation at residue 705(tyrosine --> phenylalanine), a site that is believed to be crucial for STAT3 activation and nuclear translocation. Introduction of STAT3DN induced apoptosis and G(1) cell cycle arrest. Western blot studies showed that expression of STAT3DN resulted in caspase-3 cleavage, downregulation of Bcl-2, Bcl-xL, cyclin D3, survivin, Mcl-1, c-Myc and suppressor of cytokine signaling 3. These results support the concept that STAT3 activation is pathogenetically important in ALCL cells by deregulating the expression of multiple target proteins that are involved in the control of apoptosis and cell cycle progression. PMID: 15184887 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Hepatology. 2004 May;39(5):1332-42. Interleukin 22 (IL-22) plays a protective role in T cell-mediated murine hepatitis: IL-22 is a survival factor for hepatocytes via STAT3 activation. Radaeva S, Sun R, Pan HN, Hong F, Gao B. Section on Liver Biology, Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892, USA. The central role of T cell activation in hepatocellular injury has been well documented. In this article, we provide evidence suggesting that T cells may also play a protective role in liver disease by releasing interleukin-22 (IL-22), a recently identified T cell-derived cytokine whose biological significance is unclear. IL-22 messenger RNA and protein expression are significantly elevated in T cell-mediated hepatitis induced by concanavalin A (ConA) but are less extensively elevated in the carbon tetrachloride-induced liver injury model. Activated CD3(+) T cells are likely responsible for the production of IL-22 in the liver after injection of ConA. The IL-22 receptor is normally expressed at high levels by hepatocytes and further induced after ConA injection. IL-22 blockade with a neutralizing antibody reduces signal transducer and activator of transcription factor 3 (STAT3) activation and worsens liver injury in T cell-mediated hepatitis, whereas injection of recombinant IL-22 attenuates such injury. In vitro treatment with recombinant IL-22 or overexpression of IL-22 promotes cell growth and survival in human hepatocellular carcinoma HepG2 cells. Stable overexpression of IL-22 in HepG2 cells constitutively activates STAT3 and induces expression of a variety of antiapoptotic (e.g., Bcl-2, Bcl-xL, Mcl-1) and mitogenic (e.g., c-myc, cyclin D1, Rb2, CDK4) proteins. Blocking STAT3 activation abolishes the antiapoptotic and mitogenic actions of IL-22 in hepatic cells. In conclusion, the T cell-derived cytokine IL-22 is a survival factor for hepatocytes; this suggests that T cell activation may also prevent and repair liver injury by releasing hepatoprotective cytokine IL-22 in addition to its previously documented central role in hepatocellular injury. PMID: 15122762 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Mol Biol Rep. 2004 Mar;31(1):13-21. Generation and characterization of a constitutively active Stat3 protein. Barboza JA, Wang S, Schaefer TS. Department of Neurosurgery, UT M.D. Anderson Cancer Center Box 11, 1515 Holcombe Blvd., Houston, TX 77030, USA. jabarboza@mdanderson.org Stats are latent transcription factors involved in normal cellular signaling in response to cytokine or growth factor stimulation. Constitutive activation of Stats (primarily Stat3 and Stat5) has been implicated in growth dysregulation and oncogenesis. Furthermore, increased activation of Stats has been observed in several human tumors and tumor-derived cell lines. To assess the contribution of aberrant Stat activation in oncogenesis, we have created a chimeric molecule between Stat3beta and a portion of the Herpes simplex virus VP16 activation domain. The resulting protein, Stat3beta-VAD (VP16 activation domain), is tyrosine phosphorylated on Y705 and can bind DNA in the absence of upstream activation by c-Src or epidermal growth factor (EGF). Unlike Stat3alpha and Stat3beta, Stat3beta-VAD robustly activates transcription of several reporter genes without cytokine or growth factor stimulation. In addition, we show marked upregulation of the endogenous c-myc and c-fos genes upon inducible expression of Stat3beta-VAD in COS-7 cells. Our protein displays the constitutive transcriptional activation of Stat3alpha seen in human tumors and will be a valuable tool in screens for Stat3-regulated genes. In response to the established Stat3 involvement in human cancers, Stat3beta-VAD will also facilitate assessing the contribution of other cancer signaling cascades in the context of aberrant Stat3alpha activity in cancer development and progression. PMID: 15040450 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: Gene Expr. 2003;11(3-4):149-62. Rapid hepatocyte nuclear translocation of the Forkhead Box M1B (FoxM1B) transcription factor caused a transient increase in size of regenerating transgenic hepatocytes. Wang X, Bhattacharyya D, Dennewitz MB, Kalinichenko VV, Zhou Y, Lepe R, Costa RH. Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, College of Medicine, 900 South Ashland Ave, Chicago, IL 60607-7170, USA. The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA binding domain. Liver regeneration studies with the -3 kb transthyretin (TTR) promoter-driven FoxM1B transgenic (TG) mice demonstrated that premature hepatocyte nuclear localization of the FoxM1B transgene protein at 16 h following partial hepatectomy (PHx) caused an 8-h acceleration in the onset of hepatocyte DNA replication (S-phase) and mitosis by stimulating earlier expression of cell cycle genes. Whether the FoxM1B transgene protein participates in immediate early events during liver regeneration remains to be determined. Here, we found that the FoxM1B transgene protein translocated to hepatocyte nuclei immediately following PHx, that its nuclear staining persisted for the first 6 h after surgery, and that this translocation was associated with an increase in size of regenerating TG hepatocytes. However, regenerating TTR-FoxM1B liver did not exhibit altered expression of proteins that have been implicated in mediating increased cell size, including serum-and-gucocorticoid-inducible protein kinase (SGK), protein kinase-B/Akt, the tumor suppresser gene PTEN (negative regulator of the PI3K/Akt pathway), c-Myc, or peroxisome proliferation. Moreover, we demonstrated that hepatocyte nuclear translocation of the FoxM1B transgene protein was rapidly induced during the hepatic acute phase response, which occurs during the immediate early stages of liver regeneration. Analysis of cDNA expression arrays identified a number of genes such as immediate early transcription factors (ID-3, Stat3, Nur77), matrix metalloproteinase-9 (MMP-9), and several glutathione S-transferase (GST) isoforms and stress response genes, whose expression is elevated in regenerating TTR-FoxM1B TG livers compared with regenerating wild-type (WT) liver. These liver regeneration studies demonstrate that hepatocyte nuclear translocation of the FoxM1B transgene protein was sustained for the first 6 h after PHx, and was associated with transient hypertrophy of regenerating TG hepatocytes and increased expression of genes that may enhance hepatocyte proliferation. PMID: 14686788 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: J Biol Chem. 2003 Dec 19;278(51):50915-22. Epub 2003 Oct 13. Increased expression of Bcl-xL and c-Myc is associated with transformation by Abelson murine leukemia virus. Noronha EJ, Sterling KH, Calame KL. Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA. Transformation mediated by the v-Abl oncoprotein, a tyrosine kinase encoded by the Abelson murine leukemia virus, is a multi-step process requiring genetic alterations in addition to expression of v-Abl. Loss of p53 or p19ARF was previously shown to be required for Abelson murine leukemia virus transformation of primary mouse embryonic fibroblasts (MEFs). By comparing gene expression patterns in primary p53-/- MEFs acutely infected with the v-Abl retrovirus, v-Abl-transformed MEF clones, and v-Abl-transformed MEF clones treated with Abl kinase inhibitor STI 571, we have identified additional genetic alterations associated with v-Abl transformation. Bcl-xL mRNA was elevated in three of five v-Abl-transformed MEF clones. In addition, elevated expression of c-Myc mRNA, caused either by c-myc gene amplification or by enhanced signaling via STAT3, was observed in five v-Abl-transformed MEF clones. The data suggest that increases in cell survival associated with Bcl-xL and increases in cell growth associated with c-Myc facilitate the transformation process dependent on constitutive mitogenic signaling by v-Abl. PMID: 14559912 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: J Cell Physiol. 2003 Nov;197(2):157-68. STAT proteins: from normal control of cellular events to tumorigenesis. Calo V, Migliavacca M, Bazan V, Macaluso M, Buscemi M, Gebbia N, Russo A. Section of Molecular Oncology, Department of Oncology, Regional Reference Center for the Biomolecular Characterization of Neoplasms and Genetic Screening of Hereditary Tumors, University of Palermo, Palermo, Italy. Signal transducers and activators of transcription (STAT) proteins comprise a family of transcription factors latent in the cytoplasm that participate in normal cellular events, such as differentiation, proliferation, cell survival, apoptosis, and angiogenesis following cytokine, growth factor, and hormone signaling. STATs are activated by tyrosine phosphorylation, which is normally a transient and tightly regulates process. Nevertheless, several constitutively activated STATs have been observed in a wide number of human cancer cell lines and primary tumors, including blood malignancies and solid neoplasias. STATs can be divided into two groups according to their specific functions. One is made up of STAT2, STAT4, and STAT6, which are activated by a small number of cytokines and play a distinct role in the development of T-cells and in IFNgamma signaling. The other group includes STAT1, STAT3, and STAT5, activated in different tissues by means of a series of ligands and involved in IFN signaling, development of the mammary gland, response to GH, and embriogenesis. This latter group of STATS plays an important role in controlling cell-cycle progression and apoptosis and thus contributes to oncogenesis. Although an increased expression of STAT1 has been observed in many human neoplasias, this molecule can be considered a potential tumor suppressor, since it plays an important role in growth arrest and in promoting apoptosis. On the other hand, STAT3 and 5 are considered as oncogenes, since they bring about the activation of cyclin D1, c-Myc, and bcl-xl expression, and are involved in promoting cell-cycle progression, cellular transformation, and in preventing apoptosis. Publication Types: Review Review, Tutorial PMID: 14502555 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: J Biol Chem. 2003 Nov 7;278(45):44178-87. Epub 2003 Aug 28. Reciprocal inhibition between MyoD and STAT3 in the regulation of growth and differentiation of myoblasts. Kataoka Y, Matsumura I, Ezoe S, Nakata S, Takigawa E, Sato Y, Kawasaki A, Yokota T, Nakajima K, Felsani A, Kanakura Y. Department of Hematology/Oncology, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan. The development of myoblasts is regulated by various growth factors as well as by intrinsic muscle-specific transcriptional factors. In this study, we analyzed the roles for STAT3 in the growth and differentiation of myoblasts in terms of cell cycle regulation and interaction with MyoD using C2C12 cells. Here we found that STAT3 inhibited myogenic differentiation induced by low serum or MyoD as efficiently as the Ras/mitogen-activated protein kinase cascade. As for this mechanism, we found that STAT3 not only promoted cell cycle progression through the induction of c-myc but also inhibited MyoD activities through direct interaction. STAT3 inhibited not only DNA binding activities of MyoD but also its transcriptional activities. However, the inhibited transcriptional activities were restored by the supplement of p300/CBP and PCAF, suggesting that STAT3 might deprive MyoD of these transcriptional cofactors. In addition, we found that MyoD inhibited DNA binding activities of STAT3, thereby inhibiting STAT3-dependent cell growth and survival of Ba/F3 cells. These results suggest that the development of muscle cells is regulated by the coordination of cytokine signals and intrinsic transcription factors. PMID: 12947115 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: J Biol Chem. 2003 Oct 17;278(42):41270-81. Epub 2003 Aug 4. Functional analysis of interleukin 6 response elements (IL-6REs) on the human gamma-fibrinogen promoter: binding of hepatic Stat3 correlates negatively with transactivation potential of type II IL-6REs. Duan HO, Simpson-Haidaris PJ. Department of Medicine, University of Rochester School of Medicine and Dentistry Rochester, New York 14642, USA. Several families of transcription factors play important roles in modulating liver-specific gene expression during an acute phase response (APR). Stat3/APR factor is the main transactivator of gene expression by the interleukin (IL)-6 family of cytokines signaling through gp130. During an APR, fibrinogen (FBG) genes are coordinately up-regulated by IL-6 and glucocorticoids. Except for rat gammaFBG, attempts to demonstrate direct binding of IL-6-activated Stat3 to FBG CTGGGAA promoter elements have not been successful. Herein we show the presence of three functional type II IL-6 response elements (IL-6REs) on the human gammaFBG promoter and that the magnitude of Stat3 binding to these elements correlates negatively with their functional activity in reporter gene assays. Stat3-specific binding to gammaFBG IL-6REs was confirmed by cross-competition with alpha2-macroglobulin IL-6RE and specific interactions with anti-Stat3 in electrophoretic mobility shift assays. All type II IL-6REs contributed to full promoter activity; however, transactivation from Site II at -306 to -301 was strongest. In contrast to a previous report, IL-6 failed to induce activation of serum amyloid A-activating factor-1/c-Myc-associated zinc finger protein (SAF-1/MAZ), and mutation of the SAF-1RE had little effect on IL-6 induction of gammaFBG promoter activity. In the absence of a functional glucocorticoid receptor response element, dexamethasone potentiated IL-6-induced gammaFBG promoter activity 2-fold, requiring promoter-proximal Site I and Site II; the promoter-distal Site III had no effect on dexamethasone potentiation of IL-6-induced promoter activity. Notably the propensity for Stat3 binding to human gammaFBG IL-6REs was low compared with Stat3 binding to the alpha2-macroglobulin IL-6RE. Together these data suggest that Stat3 transactivation via IL-6REs on FBG promoters likely involves participation of additional transcription factors and/or coactivators to achieve optimal coordinated up-regulation during an APR. PMID: 12900415 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: J Virol. 2003 Apr;77(7):4139-48. A positive autoregulatory loop of LMP1 expression and STAT activation in epithelial cells latently infected with Epstein-Barr virus. Chen H, Hutt-Fletcher L, Cao L, Hayward SD. Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, Maryland 21231, USA. STAT3 and STAT5 are constitutively activated and nuclear in nasopharyngeal carcinoma (NPC) cells. In normal signaling, STATs are only transiently activated. To investigate whether Epstein-Barr virus (EBV), and in particular the protein LMP1, contributes to sustained STAT phosphorylation and activation in epithelial cells, we examined STAT activity in two sets of paired cell lines, HeLa, an EBV-converted HeLa cell line, HeLa-Bx1, the NPC-derived cell line CNE2-LNSX, and an LMP1-expressing derivative, CNE2-LMP1. EBV infection was associated with a significant increase in the tyrosine-phosphorylated forms of STAT3 and STAT5 in HeLa-Bx1 cells. This effect correlated with LMP1 expression, since phosphorylated STAT3 and STAT5 levels were also increased in CNE2-LMP1 cells relative to the control CNE2-LNSX cells. No change was observed in STAT1 or STAT6 phosphorylation in these cell lines, nor was there a significant change in the levels of total STAT3, STAT5, STAT1, or STAT6 protein. Tyrosine phosphorylation allows the normally cytoplasmic STAT proteins to enter the nucleus and bind to their recognition sequences in responsive promoters. The ability of LMP1 to activate STAT3 was further established by immunofluorescence assays in which coexpression of LMP1 in transfected cells was sufficient to mediate nuclear relocalization of Flag-STAT3 and by an electrophoretic mobility shift assay which showed that LMP1 expression in CNE2-LNSX cells was associated with increased endogenous STAT3 DNA binding activity. In addition, the activity of a downstream target of STAT3, c-Myc, was upregulated in HeLa-Bx1 and CNE2-LMP1 cells. A linkage was established between interleukin-6 (IL-6)- and LMP1-mediated STAT3 activation. Treatment with IL-6 increased phosphorylated STAT3 levels in CNE2-LNSX cells, and conversely, treatment of CNE2-LMP1 cells with IL-6 neutralizing antibody ablated STAT3 activation and c-Myc upregulation. The previous observation that STAT3 activated the LMP1 terminal repeat promoter in reporter assays was extended to show upregulated expression of endogenous LMP1 mRNA and protein in HeLa-Bx1 cells transfected with a constitutively activated STAT3. A model is proposed in which EBV infection of an epithelial cell containing activated STATs would permit LMP1 expression. This in turn would establish a positive feedback loop of IL-6-induced STAT activation, LMP1 and Qp-EBNA1 expression, and viral genome persistence. PMID: 12634372 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Eur J Immunol. 2003 Jan;33(1):243-51. Differential requirements for JAK2 and TYK2 in T cell proliferation and IFN-gamma production induced by IL-12 alone or together with IL-18. Sugimoto N, Nakahira M, Ahn HJ, Micallef M, Hamaoka T, Kurimoto M, Fujiwara H. Department of Oncology, Osaka University Graduate School of Medicine, Osaka, Japan. IL-12 activates TYK2 and Janus kinase (JAK)-2 to induce the phosphorylation of various signal transducers and activators of transcription (STAT) proteins. However, little is known regarding how these JAK exhibit the distinct biological effects of IL-12. Using two JAK inhibitors, tyrphostin A1 (A1) for TYK2 and tyrphostin B42 (B42) for JAK2, we investigated the involvement of JAK2 and TYK2 in IL-12-induced T cell proliferation and IFN-gamma production. B42, but not A1, inhibited T cell proliferation along with down-regulation of IL-12-induced c-Myc expression and STAT5 phosphorylation. In contrast, A1 but not B42 inhibited STAT4/STAT3 phosphorylation and IFN-gamma production. IL-18, but not IL-12, induced activator protein-1 (AP-1) responsible for high levels of IFN-gamma promoter activation. However, this IL-18 effect depended on the interaction of AP-1 with STAT4. A1 prevented AP-1 binding by inhibiting STAT4 involvement and down-regulated synergistic IFN-gamma promoter activation. These results indicate that JAK2 activation is required for IL-12-mediated T cell growth, whereas the TYK2-STAT4 signaling pathway is critical for IFN-gamma expression that is mediated by IL-12 alone and enhanced synergistically by combination with IL-18. PMID: 12594853 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: Oncogene. 2003 Feb 13;22(6):894-905. Delineating an oncostatin M-activated STAT3 signaling pathway that coordinates the expression of genes involved in cell cycle regulation and extracellular matrix deposition of MCF-7 cells. Zhang F, Li C, Halfter H, Liu J. Department of Veterans Affairs Palo Alto Health Care System, CA 94304, USA. A number of studies have demonstrated that the STAT pathway is an important signaling cascade utilized by the IL-6 cytokine family to regulate a variety of cell functions. However, the downstream target genes of STAT activation that mediate the cytokine-induced cellular responses are largely uncharacterized. The aims of the current study are to determine whether the STAT signaling pathway is critically involved in the oncostatin M (OM)-induced growth inhibition and morphological changes of MCF-7 cells and to identify STAT3-target genes that are utilized by OM to regulate cell growth and morphology. We show that expression of a dominant negative (DN) mutant of STAT3 in MCF-7 cells completely eliminated the antiproliferative activity of OM, whereas expression of DN STAT1 had no effect. The growth inhibition of breast cancer cells was achieved through a concerted action of OM on cell cycle components. We have identified four cell cycle regulators including c-myc, cyclin D1, c/EBPdelta, and p53 as downstream effectors of the OM-activated STAT3 signaling cascade. The expression of these genes is differentially regulated by OM in MCF-7 cells, but is unaffected by OM in MCF-7-dnStat3 stable clones. We also demonstrate that the OM-induced morphological changes are correlated with increased cell motility in a STAT3-dependent manner. Expression analysis of extracellular matrix (ECM) proteins leads to the identification of fibronectin as a novel OM-regulated ECM component. Our studies further reveal that STAT3 plays a key role in the robust induction of fibronectin expression by OM in MCF-7 and T47D cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of OM on cell growth and migration. PMID: 12584569 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: J Biol Chem. 2003 Mar 28;278(13):11197-204. Epub 2003 Jan 27. Tip60 is a co-repressor for STAT3. Xiao H, Chung J, Kao HY, Yang YC. Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA. Tip60 (Tat-interactive protein, 60 kDa), a cellular protein with intrinsic histone acetyltransferase activity, is involved in DNA damage repair and apoptosis. Recent studies have suggested that Tip60 acts either as a co-activator or a co-repressor to modulate transcription. In this study, we demonstrate that Tip60 represses reporter gene expression when it is fused to the Gal4 DNA binding domain. We also show that Tip60 associates with histone deacetylase 7 (HDAC7) through its N-terminal zinc finger-containing region and that HDAC7 activity is required for the repressive effect of Tip60. Because endogenous Tip60 interacts with STAT3, we hypothesized that Tip60 might complex with STAT3 and HDAC7 and modulate STAT3-mediated trans-activation. Consistent with this hypothesis, the overexpression of Tip60 represses STAT3-driven reporter gene expression, which can be further potentiated by the co-transfection of HDAC7. Furthermore, interleukin-9-induced c-myc expression, which depends on STAT3 activity, is abrogated by exogenous expression of Tip60. This is the first demonstration of which Tip60 represses STAT3 activity in part through the recruitment of HDAC7. PMID: 12551922 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: J Biol Chem. 2003 Mar 28;278(13):11281-8. Epub 2002 Dec 30. Interleukin-6/glycoprotein 130-dependent pathways are protective during liver regeneration. Wuestefeld T, Klein C, Streetz KL, Betz U, Lauber J, Buer J, Manns MP, Muller W, Trautwein C. Department of Gastroenterology, Hepatology, and Endocrinology, Medizinische Hochschule Hannover, 30625 Hannover, Germany. After tissue loss the liver has the unique capacity to restore its mass by hepatocyte proliferation. Interleukin-6 (IL6)-deficient mice show a lack in DNA synthesis after partial hepatectomy (PH). To define better the role of IL6 and its family members for liver regeneration after PH, we used conditional knockout mice for glycoprotein 130 (gp130), the common signal transducer of all IL6 family members. We show that gp130-dependent pathways control Stat3 activation after PH. By using gene array analysis, we demonstrate that c-jun, NF-kappa B, c-myc, and tumor necrosis factor receptor expression is gp130-dependent. However, in gp130-deleted mice only minor effects on cell cycle and on the maximum of DNA synthesis after PH were found compared with controls. As in conditional gp130 animals, the acute phase response was completely abolished, we considered that other means are essential to define the role of gp130-dependent pathways for liver regeneration. LPS stimulation in gp130-deleted and also IL6 -/- animals after PH leads to a significant reduction in survival and DNA synthesis, which was associated with decreased Bcl-xL expression and higher apoptosis in the liver. These results indicate that the phenotype concerning the reduction in DNA synthesis might be linked to the degree of infection after PH. Thus our results suggest that the role of gp130-dependent signaling is not a direct influence on cell cycle progression after partial hepatectomy but is to activate protective pathways important to enable hepatocyte proliferation. PMID: 12509437 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: J Immunol. 2002 Dec 1;169(11):6610-6. Rheumatoid arthritis synoviocyte survival is dependent on Stat3. Krause A, Scaletta N, Ji JD, Ivashkiv LB. Department of Medicine, Hospital for Special Surgery, Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10021, USA. Rheumatoid arthritis (RA) synovial fibroblasts (SFs) are relatively resistant to apoptosis and exhibit dysregulated growth secondary to production of autocrine-acting growth factors and the accumulation of cell-autonomous defects. Many of the cytokines and growth factors expressed during RA synovitis, including IL-6, epidermal growth factor (EGF), and platelet-derived growth factor, activate the transcription factor Stat3 that has been implicated in promoting cell growth and survival. We analyzed the role of Stat3 in mediating the abnormal growth and survival properties of RA synoviocytes using retroviral-mediated gene transfer of a dominant negative mutant of Stat3, termed Stat3-YF. Approximately 3- to 5-fold overexpression of Stat3-YF effectively blocked endogenous Stat3 activation and Stat3-dependent gene expression, including expression of the socs3 and myc genes. Stat3-YF-transduced RA synoviocytes failed to grow in culture, exhibited markedly diminished [(3)H]thymidine incorporation (>90% decreased), and died spontaneously. Cell death occurred by apoptosis, as confirmed by annexin V staining, propidium iodide exclusion, and identification of cells with subdiploid levels of DNA. In marked contrast to control cells, EGF accelerated death of Stat3-YF-transduced SFs, such that >90% of cells were dead within 24-48 h of transduction. These results indicate that ablation of Stat3 function converts EGF from a growth/survival factor for RA synoviocytes to a death factor. Stat3-YF also induced apoptosis in osteoarthritis synoviocytes, and levels of apoptosis were increased by exogenous EGF. Apoptosis in Stat3-YF-transduced osteoarthritis synoviocytes was suppressed when Stat1 activity was blocked using a dominant negative Stat1 mutant. Our results identify Stat3 as an important molecule for RA SF survival, and suggest that Stat3 may represent a good target for gene therapy. PMID: 12444174 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: J Biol Chem. 2003 Jan 31;278(5):2990-6. Epub 2002 Nov 15. Opposite regulation of myc and p21waf1 transcription by STAT3 proteins. Barre B, Avril S, Coqueret O. INSERM U564, 4 rue Larrey, Centre Hospitalier Universitaire Angers, 49033 Angers Cedex, France. Activated forms of STAT3 transcription factors are often found in various cancers and tumor cell lines, indicating that this signaling pathway is involved in tumorogenesis. At the molecular level, STAT3 proteins function as transcriptional activators and up-regulate several growth-promoting genes such as myc, pim-1, or cyclin D1. However, these transcription factors have also proapoptotic functions and can activate the expression of the cell-cycle inhibitor p21(waf1), suggesting that STAT3 can also block cell-cycle progression and prevent abnormal cell proliferation. To reconcile these observations, one would predict that the STAT3-mediated activation of p21(waf1) is lost during cell transformation. In this study, we show that upon IL-6 stimulation of glioblastoma cells, STAT3 does not activate the expression of the p21(waf1) gene, whereas the expression of the myc gene remains unaltered. Chromatin immunoprecipitation experiments show that STAT3 and its cofactor NcoA/SRC1a are effectively recruited to the p21(waf1) promoter but that this is not followed by the association of the CREB-binding protein (CBP) histone acetylase and the type II RNA polymerase as normally seen on the myc promoter. Whereas the PI-3K/Akt pathway is constitutively activated in these cells, inactivation of this pathway restores the loading of CBP and the RNA polymerase and the expression of the p21(waf1) gene without having any effect on myc regulation. Moreover, this effect was recapitulated in HepG2 cells expressing an activated form of the Akt kinase. In these cells, the kinase blocked the STAT3-mediated expression of the p21(waf1) gene by inhibiting the recruitment of CREB-binding protein and the type II RNA polymerase, without having any effects on the loading of STAT3 and its cofactor NcoA/SRC1a. Together, these findings suggest that the phosphatidylinositol 3-kinase/Akt pathway inhibits the transcriptional activation of the p21(waf1) gene by STAT3 proteins without altering the regulation of the myc promoter. PMID: 12438313 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: FASEB J. 2002 Oct;16(12):1642-4. Epub 2002 Aug 7. Codon 12 and codon 13 mutations at the K-ras gene induce different soft tissue sarcoma types in nude mice. Guerrero S, Figueras A, Casanova I, Farre L, Lloveras B, Capella G, Trias M, Mangues R. Laboratori d'Investigacio Gastrointestinal, Institut de Recerca, Hospital de Sant Pau, Barcelona, Spain. K-ras codon 12 mutation is more oncogenic in in vitro and in vivo experimental systems than K-ras codon 13 mutation. Moreover, human colorectal tumors bearing a codon 12 mutation are more aggressive, invasive, and metastatic than the same tumor types carrying a codon 13 mutation. However, despite the association between specific sarcoma types and codon 12 or codon 13 mutations, the relationship between the position of the mutated codon at ras genes and tumor aggressiveness has not been studied in this tumor type. Here, we used a nude mice model to evaluate the tumorogenic capacity of stable transfectants of NIH3T3 fibroblasts, expressing K-ras mutated at codon 12 (K12) or 13 (K13), and morphologically, functionally, and molecularly compared these tumors. We found histopathological differences between them, K12-derived tumors showing fibrosarcoma-like features, whereas K13-derived tumors resembled malignant fibrous histiocytomas. Moreover, K12 tumors showed shorter latency of appearance, lower apoptotic and mitotic rates, and higher expression of markers for sarcoma aggressiveness (Ki67, p53 and c-myc) than K13 tumors. They also showed differences in the expression or activation of Ras, Ras downstream pathways [c-Jun N-terminal kinase (JNK), MAPK and AKT], and apoptotic [AKT, Bcl-2, Focal adhesion kinase (FAK)] and mitotic (cyclin B1) regulators, which could explain their functional differences. Most remarkably, the significantly diminished apoptotic rate observed in K12-derived tumors was associated with enhanced antiapoptotic signaling through the AKT pathway. These morphological, functional, and molecular differences demonstrate that codon 12 and codon 13 mutations in the K-ras oncogene can induce two different soft tissue sarcoma types in our in vivo model. PMID: 12207005 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: Eur J Biochem. 2002 Aug;269(15):3732-41. N-myc oncogene overexpression down-regulates leukemia inhibitory factor in neuroblastoma. Hatzi E, Murphy C, Zoephel A, Ahorn H, Tontsch U, Bamberger AM, Yamauchi-Takihara K, Schweigerer L, Fotsis T. Laboratory of Biological Chemistry, Medical School, University of Ioannina, Greece. Amplification of N-myc oncogene is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis and enhanced neovascularization. Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies, which is modulated by tumor suppressors and oncogenes. We have addressed the possibility that N-myc oncogene might regulate angiogenesis in neuroblastoma. Here, we report that experimental N-Myc overexpression results in down-regulation of leukemia inhibitory factor (LIF), a modulator of endothelial cell proliferation. Reporter assays using the LIF promoter and a series of N-Myc mutants clearly demonstrated that down-regulation of the LIF promoter was independent of Myc/Max interaction and required a contiguous N-terminal N-Myc domain. STAT3, a downstream signal transducer, was essential for LIF activity as infection with adenoviruses expressing a phosphorylation-deficient STAT3 mutant rendered endothelial cells insensitive to the antiproliferative action of LIF. LIF did not influence neuroblastoma cell proliferation suggesting that, at least in the context of neuroblastoma, LIF is involved in paracrine rather than autocrine interactions. Our data shed light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastoma. PMID: 12153570 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: Mol Biol Cell. 2002 Jul;13(7):2233-44. Epidermal growth factor receptor dependence of radiation-induced transcription factor activation in human breast carcinoma cells. Amorino GP, Hamilton VM, Valerie K, Dent P, Lammering G, Schmidt-Ullrich RK. Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, USA. Ionizing radiation (1-5 Gy) activates the epidermal growth factor receptor (EGFR), a major effector of the p42/44 mitogen-activated protein kinase (MAPK) pathway. MAPK and its downstream effector, p90 ribosomal S6 kinase (p90RSK), phosphorylate transcription factors involved in cell proliferation. To establish the role of the EGFR/MAPK pathway in radiation-induced transcription factor activation, MDA-MB-231 human breast carcinoma cells were examined using specific inhibitors of signaling pathways. Gel-shift analysis revealed three different profile groups: 1) transcription factors that responded to both radiation (2 Gy) and epidermal growth factor (EGF) (CREB, Egr, Ets, and Stat3); 2) factors that responded to radiation, but not EGF (C/EBP and Stat1); and 3) those that did not respond significantly to either radiation or EGF (AP-1 and Myc). Within groups 1 and 2, a two- to fivefold maximum stimulation of binding activity was observed at 30-60 min after irradiation. Interestingly, only transcription factors that responded to EGF had radiation responses significantly inhibited by the EGFR tyrosine kinase inhibitor, AG1478; these responses were also abrogated by farnesyltransferase inhibitor (FTI) or PD98059, inhibitors of Ras and MEK1/2, respectively. Moreover, radiation-induced increases in CREB and p90RSK phosphorylation and activation of Stat3 and Egr-1 reporter constructs by radiation were all abolished by AG1478. These data demonstrate a distinct radiation response profile at the transcriptional level that is dependent on enhanced EGFR/Ras/MAPK signaling. PMID: 12134064 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: Leukemia. 2002 Jun;16(6):1182-8. Distinct IL-6 signal transduction leads to growth arrest and death in B cells or growth promotion and cell survival in myeloma cells. Cheung WC, Van Ness B. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. In B cell development, interleukin-6 (IL-6) induces terminal maturation of B lymphocytes into antibody producing plasma cells. Terminal differentiated B cells cell cycle arrest and death follows. In contrast, IL-6 acts as a growth factor for malignant myeloma plasma cells and in some cases protects them from therapeutic treatment. In this study, we examined two cell lines that show different responses to IL-6. Lymphoblastoid CESS cells respond to IL-6 by terminally differentiating into antibody producing plasma cells, cell cycle arrest, and undergo cell death. Continuous addition of IL-6 to these cells induces transient activation of STAT3, SHP-2 phosphorylation, and does not alter bcl-X(L) and c-myc expression. In contrast, the myeloma line ANBL6 proliferates when stimulated with IL-6 and this correlates with prolonged STAT3 activation and up-regulation of bcl-X(L) and c-myc. Interestingly, gp130-associated SHP-2 phosphorylation was detected in the IL-6-induced CESS cells but not myeloma cell lines. The data show a very distinct IL-6 signal transduction and kinetics in these cell lines and the distinct molecular events correlate closely to the cell fate of the lymphoblast and myeloma cell lines. PMID: 12040451 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: Oncogene. 2002 May 16;21(22):3552-61. N-myc oncogene overexpression down-regulates IL-6; evidence that IL-6 inhibits angiogenesis and suppresses neuroblastoma tumor growth. Hatzi E, Murphy C, Zoephel A, Rasmussen H, Morbidelli L, Ahorn H, Kunisada K, Tontsch U, Klenk M, Yamauchi-Takihara K, Ziche M, Rofstad EK, Schweigerer L, Fotsis T. Laboratory of Biological Chemistry, Medical School, University of Ioannina, Ioannina, Greece. Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies. Tumor angiogenesis appears to be governed by alterations of tumor suppressor or oncogenes operant in a broad range of tumors. We have addressed this issue in neuroblastoma, a malignancy characterized by the near-exclusive amplification and overexpression of the N-Myc oncogene. Here, we report that N-Myc overexpression results in down-regulation of interleukin-6 (IL-6) and that IL-6 is an inhibitor of endothelial cell proliferation and VEGF-induced rabbit corneal angiogenesis. STAT3 is instrumental for IL-6 activity as infection with adenoviruses expressing a phosphorylation deficient STAT3 mutant renders endothelial cells insensitive to the antiproliferative action of IL-6. Finally, though IL-6 does not influence neuroblastoma cell growth, IL-6-expressing xenograft tumors in mice exhibit reduced neovascularization and suppressed growth. Our data shed new light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastomas. PMID: 12032857 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: Am J Physiol Lung Cell Mol Physiol. 2002 Jun;282(6):L1296-304. Role of the JAK-STAT pathway in PDGF-stimulated proliferation of human airway smooth muscle cells. Simon AR, Takahashi S, Severgnini M, Fanburg BL, Cochran BH. Pulmonary and Critical Care Division, Tupper Research Institute, New England Medical Center, Boston, Massachusetts 02111, USA. Airway remodeling, as manifested by an increase in airway smooth muscle mass, mucous gland hyperplasia, and subepithelial fibrosis, contributes to the airway hyperresponsiveness and fixed obstruction seen in some asthmatic patients. Here we investigated whether the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway contributes to platelet-derived growth factor (PDGF)-stimulated mitogenesis of human airway smooth muscle cells (HASMC). PDGF treatment of quiescent HASMC resulted in the rapid tyrosine phosphorylation and DNA binding of STAT1 and STAT3. This phosphorylation was blocked by inhibition of Src and JAK2 kinases. In addition, STAT activation by PDGF was found to be redox dependent. Moreover, PDGF-induced thymidine uptake was completely blocked by pretreatment of HASMC with the STAT kinase inhibitors AG-490, SU-6656, and PP2. Interestingly, the JAK pathway was required for HASMC mitogenesis independently of mitogen-activated protein kinase activation. Inhibition of the Src and JAK kinases blocked PDGF-stimulated gene expression of the STAT target genes cyclin D1 and c-myc. These results indicate that the JAK-STAT pathway contributes to PDGF-induced mitogenesis, and thus this pathway may be important in the airway remodeling seen in some asthmatic patients. PMID: 12003786 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: Clin Cancer Res. 2002 Apr;8(4):945-54. Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention. Buettner R, Mora LB, Jove R. Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, College of Medicine, Tampa, Florida 33612, USA. The signal transducers and activators of transcription (STAT)factors function as downstream effectors of cytokine and growth factor receptor signaling. Compared with normal cells and tissues, constitutively activated STATs have been detected in a wide variety of human cancer cell lines and primary tumors. STATs are activated by tyrosine phosphorylation, which is normally a transient and tightly regulated process. In tumor cells, constitutive activation of STATs is linked to persistent activity of tyrosine kinases, including Src, epidermal growth factor receptor, Janus kinases, Bcr-Abl, and many others. Such oncogenic tyrosine kinases are often activated as a consequence of permanent ligand/receptor engagement in autocrine or paracrine cytokine and growth factor signaling or represent autonomous constitutively active enzymes as a result of genetic alterations found in tumor but not normal cells. Persistent signaling of specific STATs, in particular Stat3 and Stat5, has been demonstrated to directly contribute to oncogenesis by stimulating cell proliferation and preventing apoptosis. STATs participate in oncogenesis through up-regulation of genes encoding apoptosis inhibitors and cell cycle regulators such as Bcl-x(L), Mcl-1, cyclins D1/D2, and c-Myc. Inhibition of constitutively active STAT signaling pathways has been shown repeatedly to inhibit tumor cell growth in vitro and in vivo and provides a novel means for therapeutic intervention in human cancer. In this review, we will: (a) explain the mechanisms of STAT activation in normal and malignant signaling; (b) summarize recent evidence for the critical role of constitutively activated Stat3 and Stat5 in oncogenesis; (c) identify candidate STAT target genes implicated in tumor progression; and (d) discuss molecular and pharmacological strategies to interfere with STAT signaling for potential therapeutic intervention in human cancer. Publication Types: Review Review, Tutorial PMID: 11948098 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: Gastroenterology. 2002 Apr;122(4):1020-34. Interferon-alpha activates multiple STAT signals and down-regulates c-Met in primary human hepatocytes. Radaeva S, Jaruga B, Hong F, Kim WH, Fan S, Cai H, Strom S, Liu Y, El-Assal O, Gao B. Section on Liver Biology, Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892, USA. BACKGROUND & AIMS: Interferon (IFN)-alpha therapy is currently the primary choice for viral hepatitis and a promising treatment for hepatocellular carcinoma (HCC). Primary mouse and rat hepatocytes respond poorly to IFN-alpha stimulation. Thus, it is very important to examine the IFN-alpha signal pathway in primary human hepatocytes. METHODS: The IFN-alpha-activated signals and genes in primary human hepatocytes and hepatoma cells were examined by Western blotting and microarray analyses. RESULTS: Primary human hepatocytes respond very well to IFN-alpha stimulation as shown by activation of multiple signal transducer and activator of transcription factor (STAT) 1, 2, 3, 5, and multiple genes. The differential response to IFN-alpha stimulation in primary human and mouse hepatocytes may be caused by expression of predominant functional IFN-alpha receptor 2c (IFNAR2c) in primary human hepatocytes vs. expression of predominant inhibitory IFNAR2a in mouse hepatocytes. Microarray analyses of primary human hepatocytes show that IFN-alpha up-regulates about 44 genes by over 2-fold and down-regulates about 9 genes by 50%. The up-regulated genes include a variety of antiviral and tumor suppressors/proapoptotic genes. The down-regulated genes include c-myc and c-Met, the hepatocyte growth factor (HGF) receptor. Down-regulation of c-Met is caused by IFN-alpha suppression of the c-Met promoter through down-regulation of Sp1 binding and results in attenuation of HGF-induced signals and cell proliferation. CONCLUSIONS: IFN-alpha directly targets human hepatocytes, followed by activation of multiple STATs and regulation of a wide variety of genes, which may contribute to the antiviral and antitumor activities of IFN-alpha in human liver. PMID: 11910354 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 50: Oncogene. 2002 Mar 7;21(11):1668-78. Activation of STAT5 triggers proliferation and contributes to anti-apoptotic signalling mediated by the oncogenic Xmrk kinase. Morcinek JC, Weisser C, Geissinger E, Schartl M, Wellbrock C. Department of Physiological Chemistry I, Biocenter (Theodor-Boveri Institut), University of Wurzburg, Am Hubland, 97074 Wurzburg, Germany. Extensive studies of primary tumors and tumor derived cell lines revealed that inappropriate activation of specific STATs (particularly of STAT3 and STAT5) occurs with high frequency in a wide variety of human cancers. We reported recently that the melanoma inducing EGFR-related receptor Xmrk specifically induces constitutive activation of STAT5 in fish melanoma cells. However, little is known about the role of STAT5 in solid tumours in general and its function in melanoma in particular. Recent examinations suggest that activated STAT signalling participates in oncogenesis by stimulating cell proliferation and preventing apoptosis. As an initial approach to understanding the consequences of Xmrk induced STAT5 signalling we used the well characterized pro B-cell line Ba/F3 as a sensitive system to analyse mitogenic as well as anti-apoptotic signalling. We identified STAT5 activation as being involved in both growth and survival signalling triggered by the Xmrk kinase possibly due to STAT5 induced expression of pim-1 and bcl-x. We also found a new mechanism of activation of STAT5 by receptor tyrosine kinases, whereby direct interaction of the receptor kinase domain with the STAT protein in a phosphotyrosine independent way led to activation of STAT5 in terms of DNA binding and target gene expression. PMID: 11896598 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 51: J Biol Chem. 2002 May 17;277(20):17638-48. Epub 2002 Mar 7. Distinct role of phosphatidylinositol 3-kinase and Rho family GTPases in Vav3-induced cell transformation, cell motility, and morphological changes. Sachdev P, Zeng L, Wang LH. Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029, USA. Vav3 is a member of the Vav family of guanine nucleotide exchange factors (GEFs) for the Rho family GTPases. Deleting the N-terminal calponin homology (CH) domain to generate Vav3-(5-10) or deleting both the CH and the acidic domain to generate Vav3-(6-10) results in activating the transforming potential of Vav3. Expression of either the full-length Vav3 or its truncation mutants led to activation of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), and Stat3. We investigated the requirement of these signaling molecules for Vav3-induced focus formation and found that PI3K and its downstream signaling molecules, Akt and p70 S6 kinase, are required, albeit to varying degrees. Inhibition of PI3K had a more dramatic effect than inhibition of MAPK on Vav3-(6-10)-induced focus formation. Activated PI3K enhanced the focus-forming activity of Vav3-(6-10). Wild type FAK but not Y397F mutant FAK enhanced Vav3-(6-10)-induced focus formation. Dominant negative (dn) mutant of Stat3 resulted in a 60% inhibition of the focus-forming activity of Vav3-(6-10). Moreover, Rac1, RhoA, and to a lesser extent, Cdc42, are important for Vav3-(6-10)-induced focus formation. Constitutively activated (ca) Rac synergizes with Vav3-(6-10) in focus formation. This synergy requires signaling via Rho-associated kinase (ROK) and p21-activated kinase (PAK), downstream effectors of Rac. Consistently, a ca PAK mutant enhanced, whereas a dn PAK mutant inhibited the focus-forming ability of Vav3-(6-10). Despite having potent focus-forming ability, Vav3-(6-10) has very weak colony-forming ability. This colony-forming ability of Vav3-(6-10) can be enhanced dramatically by co-expressing an activated PI3K and to some extent by co-expressing an activated PAK mutant or c-Myc. Interestingly, inhibition of PI3K and MAPK had no effect on the ability of either wild type or Vav3-(6-10) to induce cytoskeletal changes including formation of lamellipodia and filopodia in NIH 3T3 cells. Over expression of Vav3 or Vav3-(6-10) resulted in an enhancement of cell motility. This enhancement was dependent on PI3K, Rac1, and Cdc42 but not on Rho. Overall, our results show that signaling pathways of PI3K, MAPK, and Rho family GTPases are differentially required for Vav3-induced focus formation, colony formation, morphological changes, and cell motility. PMID: 11884391 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 52: Int J Hematol. 2002 Jan;75(1):51-4. Constitutive activation of Stat1 and Stat3 in primary erythroleukemia cells. Kirito K, Nagashima T, Ozawa K, Komatsu N. Department of Medicine, Jichi Medical School, Tochigi, Japan. Signal transducers and activators of transcription (Stat) proteins play important roles in the regulation of hematopoiesis as downstream molecules of cytokine signal transduction. Previously, we demonstrated that Stat1 and Stat3 are activated by erythropoietin (EPO) in a human EPO-dependent erythroleukemia cell line UT-7/EPO. We report here that Stat1 and Stat3 are constitutively activated in freshly isolated erythroleukemia cells. In addition, EPO promoted cell growth of these cells, accompanied by enhanced activities of Stat1 and Stat3. Furthermore, mutation in the Statl/Stat3-binding sites of the c-myc gene promoter clearly blocked its promoter activity in EPO-stimulated primary erythroleukemia cells. Thus, Stat1 and Stat3 may support cell growth in part via c-myc gene activation in primary erythroleukemia cells. Publication Types: Case Reports PMID: 11843291 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 53: Blood. 2002 Jan 1;99(1):102-10. Identification of the human erythropoietin receptor region required for Stat1 and Stat3 activation. Kirito K, Nakajima K, Watanabe T, Uchida M, Tanaka M, Ozawa K, Komatsu N. Division of Hematology, Department of Medicine, Jichi Medical School, Tochigi, Japan. Signal transducers and activators of transcription (Stat) proteins play important roles in the regulation of hematopoiesis as downstream molecules of cytokine signal transduction. It was previously demonstrated that erythropoietin (EPO), a major regulator of erythropoiesis, activates 3 different Stat members, Stat1, Stat3, and Stat5, in a human EPO-dependent cell line, UT-7/EPO. To clarify the mechanism by which EPO activates Stat1 and Stat3 via the EPO receptor (EPOR), a series of chimeric receptors was constructed bearing the extracellular domain of the granulocyte colony-stimulating factor receptor linked to the transmembrane domain of EPOR and the full length or several mutants of the cytoplasmic domain of EPOR, and these chimeric receptor complementary DNAs were introduced into UT-7/EPO cells. Tyr432 on human EPOR was important for activation of Stat1 and Stat3 and c-myc gene induction. In addition, Jak2 and Fes tyrosine kinases were involved in EPO-induced activation of Stat1 and Stat3. These results indicate that Stat1 and Stat3 are activated by EPO via distinct mechanisms from Stat5. PMID: 11756159 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 54: Exp Hematol. 2001 Oct;29(10):1177-84. Promegapoietin, a family of chimeric growth factors, supports megakaryocyte development through activation of IL-3 and c-Mpl ligand signaling pathways. Doshi PD, Giri JG, Abegg AL, Favara JP, Huynh MS, Kahn LE, Minnerly JC, Pegg LE, Villani-Price D, Siegel NR, Staten NR, Thomas JW, McKearn JP, Smith WG. Pharmacia Discovery Research, Chesterfield, Mo. 63198, USA. parul.doshi@pharmacia.com OBJECTIVE: The signaling pathways induced by promegapoietin (PMP), a family of chimeric growth factors that activate the human IL-3 and c-Mpl receptors, were investigated. METHODS: The biological activity of PMP was examined by receptor binding, cell proliferation, ex vivo expansion of hematopoietic progenitor cells, and in vivo production of platelets. The activation of signaling pathways was examined by Western blot and Northern blot analyses. RESULTS: Two PMP molecules, PMP-1 and PMP-1a, induced proliferation of cells expressing the IL-3 receptor, c-Mpl, or both receptors and bound to the IL-3 receptor and c-Mpl with high affinity. Ex vivo expansion assays using human bone marrow CD34(+) cells suggested that PMP-1 induced greater total cellular expansion as well as expansion of CD41(+) megakaryocytic precursor cells than IL-3 or c-Mpl ligand alone. Subcutaneous administration of 50 microg/kg of PMP-1 for 10 days to rhesus monkeys resulted in increased platelet production in vivo from a baseline of 357 +/- 45 x 10(3) cells/mL to 1376 +/- 151 x 10(3) cell/mL. PMP-1 induced phosphorylation of the beta(c) subunit of IL-3 receptor and c-Mpl, JAK2, and STAT5b, but not STAT3. PMP-1 induced greater expression of Pim-1, c-Myc, and cyclin D2 than did either an IL-3 receptor agonist or c-Mpl receptor agonist alone. The magnitude of induction of early response genes was similar for PMP and the coaddition of IL-3 receptor agonist and c-Mpl receptor agonist. CONCLUSION: PMP combines the biological activities of IL-3 and c-Mpl ligand in a single molecule that can simultaneously activate signaling pathways induced by both these ligands. PMID: 11602319 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 55: Lab Invest. 2001 Sep;81(9):1299-307. Expression of presumed specific early and late factors associated with liver regeneration in different rat surgical models. Laurent S, Otsuka M, De Saeger C, Maiter D, Lambotte L, Horsmans Y. Gastroenterology Laboratories, Universite Catholique de Louvain, Brussels, Belgium. Experiments performed on the portal branch ligation (PBL) model indicate that early changes observed after surgery are not related to the regenerative process because they also occur in atrophying lobes. To further confirm the lack of specificity of the early events and to exclude the influence of circulatory factors released by proliferating lobes on their occurrence, we investigated this response after sham operation (SO) and portacaval shunt (PCS), a model characterized by liver atrophy. We also attempted to determine expression of later events associated specifically with regeneration, ie, expression of p53 or c-Ha-ras, or inhibition of proliferation, ie, interleukin-1beta (IL-1beta) and transforming growth factor-beta1 (TGF-beta1) after partial (PH) and temporary partial (TPH) hepatectomy, SO and PCS. Nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription 3 (STAT3) DNA binding were assessed by electrophoretic mobility shift assay (EMSA), interleukin-6 (IL-6) mRNA by reverse transcription-polymerase chain reaction (RT-PCR), c-myc and c-jun mRNAs by Northern blot analysis at 0.5 and 2 hours, p53 and c-Ha-ras mRNAs by Northern blot analysis at 8 and 24 hours, and IL-1beta and TGF-beta1 by RT-PCR at 24 hours. The early response including an increase of NF-kappaB, STAT3, IL-6, and immediate-early genes expression was present after PH, PCS, and SO. In SO, slight differences were observed in comparison with PH: no NF-kappaB p65/p50 DNA binding was observed, only three of six SO rats were positive for IL-6, and immediate-early genes induction showed differences in the intensity of the response. At later times, p53 mRNA increased at 8 hours after PH and TPH, c-Ha-ras mRNA at 24 hours after PH, and IL-1beta mRNA at 24 hours after PCS. Early events are not specifically associated with the reduction of liver mass or with the regenerative process, are not predictive of future cell fate, and are most likely related to surgical stress. p53 and c-Ha-ras induction is closely associated with cell cycle progression whereas IL-1beta, but not TGF-beta1, appears to be one of the negative growth regulators that might play an important role in atrophy. PMID: 11555677 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 56: Blood. 2001 Aug 15;98(4):897-905. Granulocyte colony-stimulating factor regulates myeloid differentiation through CCAAT/enhancer-binding protein epsilon. Nakajima H, Ihle JN. Howard Hughes Medical Institute, Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA. hnakajim@med.keio.ac.jp Granulocyte colony-stimulating factor (G-CSF) is a major cytokine that regulates proliferation and differentiation of myeloid cells, although the underlying mechanisms by which G-CSF controls myeloid differentiation are largely unknown. Differentiation of hematopoietic cells is regulated by lineage-specific transcription factors, and gene-targeting studies previously revealed the critical roles of CCAAT/enhancer-binding protein (C/EBP) alpha and C/EBP epsilon, respectively, in the early and mid-late stages of granulocyte differentiation. The expression of C/EBP epsilon in 32Dcl3 cells and FDCP1 cells expressing mutant G-CSF receptors was examined and it was found that G-CSF up-regulates C/EBP epsilon. The signal for this expression required the region containing the first tyrosine residue of G-CSF receptor. Dominant-negative signal transducers and activators of transcription 3 blocked G-CSF--induced granulocytic differentiation in 32D cells but did not block induction of C/EBP epsilon, indicating that these proteins work in different pathways. It was also found that overexpression of C/EBP epsilon greatly facilitated granulocytic differentiation by G-CSF and, surprisingly, that expression of C/EBP epsilon alone was sufficient to make cells differentiate into morphologically and functionally mature granulocytes. Overexpression of c-myc inhibits differentiation of hematopoietic cells, but the molecular mechanisms of this inhibition are not fully understood. In 32Dcl3 cells overexpressing c-myc that do not differentiate by means of G-CSF, induction of C/EBP epsilon is completely abrogated. Ectopic expression of C/EBP epsilon in these cells induced features of differentiation, including changes in nuclear morphologic characteristics and the appearance of granules. These data show that C/EBP epsilon constitutes a rate-limiting step in G-CSF-regulated granulocyte differentiation and that c-myc antagonizes G-CSF-induced myeloid differentiation, at least partly by suppressing induction of C/EBP epsilon. (Blood. 2001;98:897-905) PMID: 11493431 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 57: Rinsho Ketsueki. 2001 May;42(5):393-6. [Erythropoiesis and signal transduction: basic and clinical aspects] [Article in Japanese] Komatsu N. PMID: 11452458 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 58: Oncogene. 2000 Dec 27;19(56):6613-26. STAT proteins: novel molecular targets for cancer drug discovery. Turkson J, Jove R. H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, USA. Signal Transducers and Activators of Transcription (STATs) are a family of cytoplasmic proteins with roles as signal messengers and transcription factors that participate in normal cellular responses to cytokines and growth factors. Frequently, however, abnormal activity of certain STAT family members, particularly Stat3 and Stat5, is associated with a wide variety of human malignancies, including hematologic, breast, head and neck, and prostate cancers. Application of molecular biology and pharmacology tools in disease-relevant models has confirmed Stat3 as having a causal role in oncogenesis, and provided validation of Stat3 as a target for cancer drug discovery and therapeutic intervention. Furthermore, a constitutively-active mutant form of Stat3 is sufficient to induce oncogenic transformation of cells, which form tumors in vivo. Constitutive activation of Stat3 signaling is accompanied by upregulation of cyclin D1, c-Myc, and Bcl-x, changes consistent with subversion of normal cellular growth and survival control mechanisms. Block of constitutive Stat3 signaling results in growth inhibition and apoptosis of Stat3-positive tumor cells in vitro and in vivo. The observed dependence of certain tumors on constitutive Stat3 signaling for growth and survival has wide implications for cancer therapy, offering the potential for preferential tumor cell killing. This review evaluates constitutive Stat3 activation as a 'cancer-causing' factor, and proposes a number of molecular strategies for targeting Stat3 signaling for therapeutic intervention. Publication Types: Review PMID: 11426647 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 59: Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7319-24. Epub 2001 Jun 12. Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis. Bowman T, Broome MA, Sinibaldi D, Wharton W, Pledger WJ, Sedivy JM, Irby R, Yeatman T, Courtneidge SA, Jove R. Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida College of Medicine, 12902 Magnolia Drive, Tampa, FL 33612, USA. Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3beta protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c-myc gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3beta protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF --> Src --> Stat3 --> Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation. PMID: 11404481 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 60: Blood. 2001 Jun 1;97(11):3559-67. Signal transducer and activator of transcription 3 activation is required for Asp(816) mutant c-Kit-mediated cytokine-independent survival and proliferation in human leukemia cells. Ning ZQ, Li J, Arceci RJ. Division of Hematology/Oncology, Children's Hospital Medical Center, Cincinnati, OH, USA. Activating mutations of c-kit at codon 816 (Asp(816)) have been implicated in a variety of malignancies, including acute myeloid leukemia (AML). The mutant c-Kit receptor confers cytokine-independent survival of leukemia cells and induces tumorigenicity. Changes in the signal transduction pathways responsible for Asp(816) mutant c-Kit-mediated biologic effects are largely undefined. The results of this study show that Asp(816) mutant c-Kit induces constitutive activation of signal transducer and activator of transcription 3 (STAT3) and STAT1, and up-regulates STAT3 downstream targets, Bcl-x(L) and c-myc. The phosphatidylinositol-3-kinase (PI-3K)/Akt pathway, but not the Ras-mediated mitogen-activated protein (MAP) kinase pathway, is also constitutively activated by Asp(816) mutant c-Kit. Suppression of STAT3 activation by a dominant negative molecule in MO7e leukemia cells transduced with mutant c-kit inhibits stem cell factor (SCF)-independent survival and proliferation, accompanied by the down-regulation of Bcl-x(L) and c-myc. However, activated STAT3 does not appear to be the sole mediator that is responsible for the phenotypic changes induced by Asp(816) mutant c-Kit, because expression of constitutively activated STAT3 in MO7e cells does not completely reconstitute cytokine independence. Activation of other signaling components by mutant c-Kit, such as those in the PI-3K/Akt pathway, is demonstrated and may also be needed for the mutant c-Kit-mediated biologic effects. The investigation of altered signal transduction pathways and the resulting functional consequences mediated by Asp(816) mutant c-Kit should provide important information for the characterization of subsets of leukemia and potential molecular pathways for therapeutic targeting. (Blood. 2001;97:3559-3567) PMID: 11369651 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 61: Int J STD AIDS. 2001 Apr;12(4):229-33. The endogenous interferon system in anal squamous epithelial lesions with different grades from HIV-positive individuals. Arany I, Muldrow M, Tyring SK. Department of Microbiology, The University of Texas Medical Branch, Galveston, TX 77555-1070, USA. iarany@utmb.edu Anal intraepithelial lesions (ASILs) are considered as precursors of anal cancer. The incidence of high-grade ASIL (HSIL) and progression of low-grade ASIL (LSIL) to HSIL are high in HIV-positive men. Endogenous cytokines, such as interferons (IFNs) play an important role in the regulation of proliferation and immune responses in epithelial cells, and thus, they might control the above-mentioned progression events. Accordingly, we determined mRNA levels of IFN-gamma and IFN-gamma receptors, levels of IFN-gamma receptor-associated kinases (JAK1 and TYK2) and signalling molecules (signal transducer and activator of transcription-1 [STAT1], STAT3, interferon-responsive-factor-1 [IRF-1] and IRF-2) as well as inhibitors of cytokine signalling (protein inhibitor of activated STAT1 [PIAS1] and suppressor of cytokine signalling 2 [SOCS2]) in biopsies of anal condylomas, LSILs as well as HSILs from HIV-positive individuals by a semi-quantitative reverse transcribed polymerase chain reaction (RT-PCR) method. We found that HSIL significantly differs in expression of these genes from LSIL and condylomas. Expression profile of HSIL samples showed activation of STAT3 signalling, probably accounting for the observed high levels of genes that support cellular proliferation (IRF-2, c-fos and c-myc). Decreases in levels of suppressors (IFN-gamma and IRF-1) and JAK1 kinase, but increases in levels of inhibitors of cytokine signalling (PIAS1 and SOCS2) might also contribute to the altered cytokine signalling in HSIL biopsies. These findings might reveal important molecular events associated with progression of LSIL to HSIL in HIV-infected men. PMID: 11319973 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 62: FASEB J. 2001 Apr;15(6):1006-13. Cyclin D1 is an early target in hepatocyte proliferation induced by thyroid hormone (T3). Pibiri M, Ledda-Columbano GM, Cossu C, Simbula G, Menegazzi M, Shinozuka H, Columbano A. Department of Toxicology, Oncology and Molecular Pathology Unit, University of Cagliari, Italy. The thyroid hormone (T3) affects cell growth, differentiation, and regulates metabolic functions via its interaction with the thyroid hormone nuclear receptors (TRs). The mechanism by which TRs mediate cell growth is unknown. To investigate the mechanisms responsible for the mitogenic effect of T3, we have determined changes in activation of transcription factors, mRNA levels of immediate early genes, and levels of proteins involved in the progression from G1 to S phase of the cell cycle. We show that hepatocyte proliferation induced by a single administration of T3 to Wistar rats occurred in the absence of activation of AP-1, NF-kappa B, and STAT3 or changes in the mRNA levels of the immediate early genes c-fos, c-jun, and c-myc. These genes are considered to be essential for liver regeneration after partial hepatectomy (PH). On the other hand, T3 treatment caused an increase in cyclin D1 mRNA and protein levels that occurred much more rapidly compared to liver regeneration after 2/3 PH. The early increase in cyclin D1 expression was associated with accelerated onset of DNA synthesis, as demonstrated by a 20-fold increase of bromodeoxyuridine-positive hepatocytes at 12 h after T3 treatment and by a 20-fold increase in mitotic activity at 18 h. An early increase of cyclin D1 expression was also observed after treatment with nafenopin, a ligand of a nuclear receptor (peroxisome proliferator-activated receptor alpha) of the same superfamily of steroid/thyroid receptors. T3 treatment also resulted in increased expression of cyclin E, E2F, and p107 and enhanced phosphorylation of pRb, the ultimate substrate in the pathway leading to transition from G1 to S phase. The results demonstrate that cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by T3 and suggest that this cyclin might be a common target responsible for the mitogenic activity of ligands of nuclear receptors. PMID: 11292661 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 63: J Biol Chem. 2001 May 25;276(21):17800-7. Epub 2001 Feb 15. Bcl-XL expression correlates with primary macrophage differentiation, activation of functional competence, and survival and results from synergistic transcriptional activation by Ets2 and PU.1. Sevilla L, Zaldumbide A, Carlotti F, Dayem MA, Pognonec P, Boulukos KE. Institute of Signalisation, Developmental Biology and Cancer, INSERM 470, Centre de Biochimie, Universite de Nice, Faculte des Sciences, 06108 Nice, France. Depriving primary bone marrow-derived macrophages of colony-stimulating factor-1 (CSF-1) induces programmed cell death by apoptosis. We show that cell death is accompanied by decreases in the expression of anti-apoptotic Bcl-x(L) protein and the Ets2 and PU.1 proteins of the Ets transcription factor family. Macrophages require both priming and triggering signals independent of CSF-1 to kill neoplastic cells or microorganisms, and this activation of macrophage competence is accompanied by increased expression of bcl-x(L), ets2, and PU.1. Furthermore, we show that only Ets2 and PU.1, but not Ets1, function in a synergistic manner to transactivate the bcl-x promoter. The synergy observed between PU.1 and Ets2 is dependent on the transactivation domains of both proteins. Although other transcription factors like Fos, c-Jun, Myc, STAT3, and STAT5a are implicated in the activation of macrophage competence or in CSF-1 signaling, no synergy was observed between Ets2 and these transcription factors on the bcl-x promoter. We demonstrate that the exogenous expression of both Ets2 and PU.1 in macrophages increases the number of viable cells upon CSF-1 depletion and that Ets2 and PU.1 can functionally replace Bcl-x(L) in inhibiting Bax-induced apoptosis. Together, these results demonstrate that PU.1 and Ets2 dramatically increase bcl-x activation, which is necessary for the cytocidal function and survival of macrophages. PMID: 11278399 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 64: J Surg Res. 2001 Apr;96(2):289-95. Activation of interleukin-6/STAT3 and liver regeneration following transplantation. Debonera F, Aldeguer X, Shen X, Gelman AE, Gao F, Que X, Greenbaum LE, Furth EE, Taub R, Olthoff KM. Department of Surgery, University of Pennsylvania, Philadelphia 19104, USA. BACKGROUND: Every liver that is procured, stored, and transplanted experiences injury from cold ischemia and reperfusion. Most recover quickly, but some grafts sustain enough injury to result in prolonged organ dysfunction or require retransplantation. The molecular mechanisms involved in early graft function and recovery following cold ischemia and reperfusion (I/R) after liver transplantation have not been well defined. Interleukin (IL)-6 is a critical factor in the mitogenic response within the liver, and is important for cell cycle progression and protection from injury. Activation of the latent transcription factor, STAT3, is dependent on IL-6 release. The role of the IL-6/STAT3 pathway and hepatocellular regeneration in graft recovery and cell cycle progression following cold ischemia and reperfusion was studied in a rat liver transplant orthotopic (OLT) model. Methods. Rat OLT was performed in a syngeneic model. The presence, time course, and magnitude of expression of IL-6, STAT3 activation, and upregulation of target immediate early genes were determined in liver grafts with minimal (<1 h) and prolonged (12 h) cold preservation times followed by transplantation. Progression of the cell cycle and replication was confirmed by BrdU uptake. RESULTS: Prolonged cold ischemia resulted in increased IL-6 expression and STAT3 activation. This correlated with upregulation of junB, c-fos, c-myc, and c-jun, immediate early genes associated with hepatic regeneration. Extensive DNA replication was present in livers with 12-h ischemia, demonstrating successful completion of the cell cycle. CONCLUSIONS: The participation of the IL-6/STAT3 pathway leading to cell cycle progression and regeneration is an important component in the recovery of organs immediately following cold preservation and transplantation. Copyright 2001 Academic Press. PMID: 11266286 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 65: Blood. 2000 Apr 15;95(8):2577-85. Induction of apoptosis by extracellular ubiquitin in human hematopoietic cells: possible involvement of STAT3 degradation by proteasome pathway in interleukin 6-dependent hematopoietic cells. Daino H, Matsumura I, Takada K, Odajima J, Tanaka H, Ueda S, Shibayama H, Ikeda H, Hibi M, Machii T, Hirano T, Kanakura Y. Department of Hematology/Oncology, Biomedical Research Center, Osaka University Medical School, Osaka, Japan. The ubiquitin-proteasome pathway is responsible for selective degradation of short-lived cellular proteins and is critical for the regulation of many cellular processes. We previously showed that ubiquitin (Ub) secreted from hairy cell leukemia cells had inhibitory effects on clonogenic growth of normal hematopoietic progenitor cells. In this study, we examined the effects of exogenous Ub on the growth and survival of a series of human hematopoietic cells, including myeloid cell lines (HL-60 and U937), a B-cell line (Daudi), and T-cell lines (KT-3, MT-4, YTC-3, and MOLT-4). Exogenous Ub inhibited the growth of various hematopoietic cell lines tested, especially of KT-3 and HL-60 cells. The growth-suppressive effects of Ub on KT-3 and HL-60 cells were almost completely abrogated by the proteasome inhibitor PSI or MG132, suggesting the involvement of the proteasome pathway in this process. Furthermore, exogenous Ub evoked severe apoptosis of KT-3 and HL-60 cells through the activation of caspase-3. In interleukin-6 (IL-6)-dependent KT-3 cells, STAT3 was found to be conjugated by exogenous biotinylated Ub and to be degraded in a proteasome-dependent manner, whereas expression levels of STAT1, STAT5, or mitogen-activated protein kinase were not affected. Moreover, IL-6-induced the up-regulation of Bcl-2 and c-myc, and JunB was impaired in Ub-treated KT-3 cells, suggesting that the anti-apoptotic and mitogenic effects of IL-6 were disrupted by Ub. These results suggest that extracellular Ub was incorporated into hematopoietic cells and mediated their growth suppression and apoptosis through proteasome-dependent degradation of selective cellular proteins such as STAT3. (Blood. 2000;95:2577-2585) PMID: 10753837 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 66: Mol Cell Biol. 2000 Dec;20(24):9271-80. v-Src generates a p53-independent apoptotic signal. Webb BL, Jimenez E, Martin GS. Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA. Evasion of apoptosis appears to be a necessary event in tumor progression. Some oncogenes, such as c-myc and E1A, induce apoptosis in the absence of survival factors. However, others, such as bcl-2 and v-src, activate antiapoptotic pathways. For v-Src, these antiapoptotic pathways are dependent on the function of Ras, phosphatidylinositol (PI) 3-kinase, and Stat3. Here we asked whether v-Src can activate a proapoptotic signal when survival signaling is inhibited. We show that when the functions of Ras and PI 3-kinase are inhibited, v-src-transformed Rat-2 fibroblasts undergo apoptosis, evidenced by loss of adherence, nuclear fragmentation, and chromosomal DNA degradation. The apoptotic response is dependent on activation of caspase 3. Under similar conditions nontransformed Rat-2 cells undergo considerably lower levels of apoptosis. Apoptosis induced by v-Src is accompanied by a loss of mitochondrial membrane potential and release of cytochrome c and is blocked by overexpression of bcl-2, indicating that it is mediated by the mitochondrial pathway. However apoptosis induced by v-Src is not accompanied by an increase in the level of p53 and is not dependent on p53 function. Thus v-Src generates a p53-independent proapoptotic signal. PMID: 11094078 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 67: J Biol Chem. 2000 Aug 4;275(31):24096-105. Full oncogenic activities of v-Src are mediated by multiple signaling pathways. Ras as an essential mediator for cell survival. Odajima J, Matsumura I, Sonoyama J, Daino H, Kawasaki A, Tanaka H, Inohara N, Kitamura T, Downward J, Nakajima K, Hirano T, Kanakura Y. Department of Hematology/Oncology and Molecular Oncology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Tyrosine kinase oncoproteins cause simultaneous activation of multiple intracellular signaling pathways. However, the precise mechanisms by which individual pathways induce oncogenesis are not well understood. We have investigated the roles of individual signaling pathways in v-Src-dependent cell growth and survival by inhibiting one particular pathway. v-Src induced constitutive activation of signal transducers and activators of transcription 3 (STAT3), phosphatidylinositol 3-kinase, and Ras in murine Ba/F3 cells and led to factor-independent proliferation. Dominant-negative mutants of STAT3 (STAT3D) and phosphatidylinositol 3-kinase (Deltap85) inhibited v-Src-dependent growth by approximately 60 and approximately 40%, respectively. Moreover, dominant-negative Ras (N17) induced severe apoptosis, which was accompanied by down-regulation of Bcl-2 and activation of caspase-3. Although cells overexpressing Bcl-2 or caspase-3 inhibitors remained viable even when N17 was expressed, the growth was reduced by approximately 85%. During N17- and STAT3D-induced growth suppression, expression of cyclin D2, cyclin D3, c-myc, and c-fos was suppressed by N17, whereas that of cyclin D2, cyclin E, and c-myc was suppressed by STAT3D. Thus, v-Src-activated Ras and STAT3 are involved in distinct but partly overlapping transcriptional regulation of cell cycle regulatory molecules. These results suggest that the full oncogenic activity of v-Src requires simultaneous activation of multiple signalings, in which Ras is particularly required for survival. PMID: 10918073 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 68: Cell Immunol. 2000 Jun 15;202(2):124-35. STAT3 regulates the growth and immunoglobulin production of BCL(1) B cell lymphoma through control of cell cycle progression. Karras JG, McKay RA, Lu T, Pych J, Frank DA, Rothstein TL, Monia BP. Department of Molecular and Cellular Pharmacology, Isis Pharmaceuticals, Inc., 2292 Faraday Avenue, Carlsbad, California 92008, USA. STAT3 is constitutively phosphorylated on tyrosine(705) in self-renewing, CD5(+) murine B-1 lymphocytes. Nuclear extracts from untreated primary B-1 or CD5(+) BCL(1) B lymphoma cells were found to contain immunoreactive STAT3 protein that binds to a sis-inducible element present in the promoter of the p21(waf1/cip1) tumor suppressor gene and is constitutively phosphorylated on serine(727). To determine the functional significance of constitutive STAT3 activation in B lymphoma cells, a specific STAT3 antisense oligonucleotide was developed and used to examine basal BCL(1) cell growth and IgM production. Abrogating STAT3 expression in BCL(1) cells inhibited their proliferative capacity and induced a corresponding decrease in secretion of IgM. Cell cycle analysis showed a block in progression through G1 in BCL(1) cells treated with the STAT3 antisense oligonucleotide. These results indicate that STAT3 controls cell growth and immunoglobulin secretion by enhancing progression through the G1 phase of the cell cycle in BCL(1) B cell lymphoma. Copyright 2000 Academic Press. PMID: 10896772 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 69: Oncogene. 2000 May 15;19(21):2548-56. Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Hirano T, Ishihara K, Hibi M. Division of Molecular Oncology C-7, Biomedical Research Center, Osaka University Graduate School of Medicine, 2-2 Yamada-oko, Suita, Osaka 565-0871, Japan. Members of the IL-6 cytokine family are involved in a variety of biological responses, including the immune response, inflammation, hematopoiesis, and oncogenesis by regulating cell growth, survival, and differentiation. These cytokines use gp130 as a common receptor subunit. The binding of ligand to gp130 activates the JAK/STAT signal transduction pathway, where STAT3 plays a central role in transmitting the signals from the membrane to the nucleus. STAT3 is essential for gp130-mediated cell survival and G1 to S cell-cycle-transition signals. Both c-myc and pim have been identified as target genes of STAT3 and together can compensate for STAT3 in cell survival and cell-cycle transition. STAT3 is also required for gp130-mediated maintenance of the pluripotential state of proliferating embryonic stem cells and for the gp130-induced macrophage differentiation of M1 cells. Furthermore, STAT3 regulates cell movement, such as leukocyte, epidermal cell, and keratinocyte migration. STAT3 also appears to regulate B cell differentiation into antibody-forming plasma cells. Since the IL-6/gp130/STAT3 signaling pathway is involved in both B cell growth and differentiation into plasma cells it is likely to play a central role in the generation of plasma cell neoplasias. Oncogene (2000). Publication Types: Review PMID: 10851053 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 70: Am J Physiol Cell Physiol. 2000 Feb;278(2):C331-5. Inhibition of ornithine decarboxylase induces STAT3 tyrosine phosphorylation and DNA binding in IEC-6 cells. Pfeffer LM, Yang CH, Pfeffer SR, Murti A, McCormack SA, Johnson LR. Department of Pathology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA. lpfeffer@utmem.edu Polyamines are required for the proliferation of the rat intestinal mucosal IEC-6 cell line. Ornithine decarboxylase (ODC) is the enzyme that catalyzes the first step in polyamine synthesis. ODC inhibition not only leads to polyamine depletion but also leads to inhibition of cell proliferation and regulates the expression of the immediate-early genes c-fos, c-myc, and c-jun. Members of the signal transducers and activators of transcription (STAT) transcription factor family bind to the sis-inducible element (SIE) present in the promoters to regulate the expression of a variety of important genes. In the present study, we tested the hypothesis that the STAT3 transcription factor, which is responsible for activation of the acute phase response genes, is activated after inhibition of ODC. We found that inhibition of ODC rapidly induces STAT3 activation as determined by STAT3 tyrosine phosphorylation, translocation of STAT3 from the cytoplasm into the nucleus, and the presence of STAT3 in SIE-dependent DNA-protein complexes. STAT3 activation upon inhibition of ODC was accompanied by the activation of a STAT3-dependent reporter construct. Moreover, prolonged polyamine depletion resulted in downregulation of cellular STAT3 levels. PMID: 10666028 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 71: Immunity. 1999 Dec;11(6):709-19. Synergistic roles for Pim-1 and c-Myc in STAT3-mediated cell cycle progression and antiapoptosis. Shirogane T, Fukada T, Muller JM, Shima DT, Hibi M, Hirano T. Division of Molecular Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Japan. The activation of STAT3 by the cytokine receptor gp130 is required for both the G1 to S cell cycle transition and antiapoptosis. We found that Pim-1 and Pim-2 are targets for the gp130-mediated STAT3 signal. Expression of a kinase-defective Pim-1 mutant attenuated gp130-mediated cell proliferation. Constitutive expression of Pim-1 together with c-myc, another STAT3 target, fully compensated for loss of the STAT3-mediated cell cycle progression, antiapoptosis, and bcl-2 expression. We also identified valosine-containing protein (VCP) as a target gene for the Pim-1-mediated signal. Expression of a mutant VCP led cells to undergo apoptosis. These results indicate that Pim-family proteins play crucial roles in gp130-mediated cell proliferation and explain the synergy between Pim and c-Myc proteins in cell proliferation and lymphomagenesis. PMID: 10626893 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 72: Cell Growth Differ. 1999 Dec;10(12):819-28. Tumor necrosis factor induces DNA replication in hepatic cells through nuclear factor kappaB activation. Kirillova I, Chaisson M, Fausto N. Department of Pathology, University of Washington School of Medicine, Seattle 98195-7470, USA. Tumor necrosis factor (TNF) signaling through TNF receptor 1 (TNFR1) with downstream participation of nuclear factor kappaB (NFkappaB), interleukin 6 (IL-6), and signal transducers and activators of transcription 3 (STAT3) is required for initiation of liver regeneration. It is not known whether the proliferative effect of TNF on hepatocytes is direct or requires the participation of Kupffer cells, the liver resident macrophages. Moreover, it has not been determined whether NFkappaB activation is an essential step in TNF-induced proliferation. To answer these questions, we conducted studies in LE6 cells, a rat liver epithelial cell line with hepatocyte progenitor capacity. We report that TNF induces DNA replication in growth-arrested LE6 cells and that its effect involves the activation of NFkappaB and STAT3 and an increase in c-myc and IL-6 mRNAs. All of these effects, which mimic the events that initiate liver regeneration in vivo, are blocked if NFKB activation is inhibited by expression of a dominant-inhibitor IkappaBalpha mutant (deltaN-IkappaBalpha). Although NFkappaB blockage by deltaN-IkappaBalpha causes caspase activation and massive death of cells stimulated by TNF, inhibition of NFkappaB and STAT3 binding by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone results in G0-G1 cell cycle arrest without death. We conclude that NFkappaB is an essential component of the TNF proliferative pathway and that TNF-induced changes in IL-6 mRNA, STAT3, and c-myc mRNA are dependent on NFkappaB activation. Blockage of NFkappaB inhibits TNF-induced proliferation but does not necessarily cause cell death. PMID: 10616907 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 73: Prostate. 2000 Feb 1;42(2):88-98. STAT3 mediates IL-6-induced growth inhibition in the human prostate cancer cell line LNCaP. Spiotto MT, Chung TD. Department of Radiation and Cellular Oncology, University of Chicago, and the Pritzker School of Medicine, Chicago, Illinois 60637, USA. BACKGROUND: In prostate cancer, we and others have observed distinct phenotypic responses to interleukin-6 (IL-6), which acts either as a paracrine growth inhibitor in the LNCaP cell line or as an autocrine growth stimulator in PC-3, DU145, and TSU cell lines. To understand the underlying mechanism responsible for this phenotypic difference, we investigated differences in the IL-6-induced Janus kinase-signal transducers and activators of transcription (JAK-STAT) signal transduction pathway between these two phenotypes. METHODS: Prostate cancer cell lines were assayed for STAT3 activity by immunoblotting, electrophoretic gel shift assays (EMSA), and a luciferase reporter assay to test for STAT3 protein expression, phosphorylation, DNA binding, and transcriptional activity. To address the physiological role of STAT3, we introduced a dominant-negative mutant of STAT3 into LNCaP cells and assayed the effects of IL-6 on cell growth of this stable transfectant by cell counting, clonogenic assays, and c-myc expression. RESULTS: IL-6 induced transcriptional activity of STAT3 only in LNCaP. STAT3 was transcriptionally inactive in PC-3, TSU, and DU145 at the level of protein expression, tyrosine phosphorylation, and DNA binding/transcriptional activity, respectively. An isolated LNCaP subclone containing a dominant-negative mutant of STAT3, LNCaP-SF, did not show STAT3-DNA binding or transcriptional activity. LNCaP-SF exhibited a proliferative response to IL-6 as compared to the control LNCaP-neo clone, which underwent growth arrest. Unlike LNCaP-neo, LNCaP-SF was able form colonies and to maintain c-myc expression in the presence of IL-6. CONCLUSIONS: STAT3 transcriptional activation correlates with the growth-inhibitory signal of IL-6 in LNCaP, suggesting that STAT3 transcriptional activity is an important determinant in the different phenotypic responses to IL-6 in prostate cancer. Copyright 2000 Wiley-Liss, Inc. PMID: 10617865 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 74: Oncogene. 1999 Jun 17;18(24):3583-92. STAT activation by the PDGF receptor requires juxtamembrane phosphorylation sites but not Src tyrosine kinase activation. Sachsenmaier C, Sadowski HB, Cooper JA. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA. Activation of the platelet-derived growth factor (PDGF) receptor tyrosine kinase induces tyrosine phosphorylation of Signal Transducer and Activator of Transcription (STAT) proteins. Since the PDGF receptor also activates the Src tyrosine kinase, it is possible that Src mediates tyrosine phosphorylation of STATs in PDGF-treated cells. Consistent with a role for Src in STAT activation, we found that a PDGF receptor juxtamembrane tyrosine residue required for Src activation is necessary and sufficient for activation of STATs 1 and 3. To test the Src requirement further, we made other mutations in the PDGF receptor juxtamembrane region that increased or decreased Src binding. In epithelial and fibroblast cells, PDGF activated STAT1, 3 and 6 in the absence of detectable binding and activation of Src. In addition, PDGF induced c-myc RNA expression and DNA synthesis even though Src was not detectably activated. The activation of MAP kinase and the induction of c-fos gene expression both correlated with STAT but not Src activation by the receptor. We conclude that juxtamembrane tyrosine phosphorylation is necessary for both Src tyrosine kinase and STAT activation by the betaPDGF receptor, but that both processes are regulated independently by this region. PMID: 10380880 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 75: Hepatology. 1999 May;29(5):1463-70. After portal branch ligation in rat, nuclear factor kappaB, interleukin-6, signal transducers and activators of transcription 3, c-fos, c-myc, and c-jun are similarly induced in the ligated and nonligated lobes. Starkel P, Horsmans Y, Sempoux C, De Saeger C, Wary J, Lause P, Maiter D, Lambotte L. Laboratories of Gastroenterology, Catholic University of Louvain Medical School, Brussels, Belgium. Several studies have emphasized the involvement of transcription factors, cytokines, and proto-oncogenes in initiating the regenerative process after partial hepatectomy. To assess whether these events do specifically occur in a cellular system undergoing regeneration, we studied the induction of nuclear factor kappaB (NFkappaB), interleukin-6 (IL-6), signal transducers and activators of transcription 3 (Stat3), c-fos, c-myc, c-jun, after portal branch ligation (PBL), which produces atrophy of the deprived lobes (70% of the liver parenchyma), whereas the perfused lobes undergo compensatory regeneration. Nuclear extracts and total RNA were prepared from control livers as well as from atrophying and regenerating lobes at 0.5, 1, 2, 5, and 8 after PBL. NFkappaB and Stat3 induction were studied by electrophoretic mobility shift assays and Western blotting. IL-6 and proto-oncogenes expressions were assessed by reverse transcription polymerase chain reaction and Northern blotting, respectively. Assays were also performed after a sham operation. NFkappaB and Stat3 protein expression and DNA binding were rapidly and similarly induced in nuclear extracts from the atrophying and regenerating lobes. IL-6 was elevated in both lobes from 1 to 8 hours after PBL as well as c-fos, c-myc, and c-jun during the first 2 hours. IL-6 and Stat3 but not NFkappaB were also elevated after a sham operation. These findings suggest that the cellular and molecular changes occurring early in a regenerating liver are nonspecific, possibly stress-induced, cellular responses. They do not indicate the future evolution towards atrophy or regeneration. PMID: 10216130 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 76: Blood. 1999 Mar 15;93(6):1980-91. Interferon-alpha activates multiple STAT proteins and upregulates proliferation-associated IL-2Ralpha, c-myc, and pim-1 genes in human T cells. Matikainen S, Sareneva T, Ronni T, Lehtonen A, Koskinen PJ, Julkunen I. Department of Virology, National Public Health Institute, Helsinki, Finland. sampsa.matikainen@ktl.fi Interferon-alpha (IFN-alpha) is a pleiotropic cytokine that has antiviral, antiproliferative, and immunoregulatory functions. There is increasing evidence that IFN-alpha has an important role in T-cell biology. We have analyzed the expression of IL-2Ralpha, c-myc, and pim-1 genes in anti-CD3-activated human T lymphocytes. The induction of these genes is associated with interleukin-2 (IL-2)-induced T-cell proliferation. Treatment of T lymphocytes with IFN-alpha, IL-2, IL-12, and IL-15 upregulated IL-2Ralpha, c-myc, and pim-1 gene expression. IFN-alpha also sensitized T cells to IL-2-induced proliferation, further suggesting that IFN-alpha may be involved in the regulation of T-cell mitogenesis. When we analyzed the nature of STAT proteins capable of binding to IL-2Ralpha, pim-1, and IRF-1 GAS elements after cytokine stimulation, we observed IFN-alpha-induced binding of STAT1, STAT3, and STAT4, but not STAT5 to all of these elements. Yet, IFN-alpha was able to activate binding of STAT5 to the high-affinity IFP53 GAS site. IFN-alpha enhanced tyrosine phosphorylation of STAT1, STAT3, STAT4, STAT5a, and STAT5b. IL-12 induced STAT4 and IL-2 and IL-15 induced STAT5 binding to the GAS elements. Taken together, our results suggest that IFN-alpha, IL-2, IL-12, and IL-15 have overlapping activities on human T cells. These findings thus emphasize the importance of IFN-alpha as a T-cell regulatory cytokine. PMID: 10068671 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 77: Exp Hematol. 1999 Jan;27(1):139-46. Distinct actions of interleukin-9 and interleukin-4 on a hematopoietic stem cell line, EMLC1. Wang XY, Gelfanov V, Sun HB, Tsai S, Yang YC. Department of Medicine (Hematology/Oncology), Walther Oncology Center, Indiana University Medical Center, Indianapolis, USA. EMLC1 is a hematopoietic stem cell line that depends on stem cell factor (SCF) for growth and generates lymphoid, erythroid and myeloid progenitors in the presence of different cytokines. We have studied signaling events leading to cell proliferation and differentiation of EMLC1 mediated by interleukin (IL)-4 and IL-9. It was found that IL-9 enhances SCF-induced cell proliferation and promotes erythropoietin (EPO)-dependent erythroid differentiation of EMLC1 cells. However, IL-9 alone cannot support the growth of this cell line. In contrast, IL-4 by itself is sufficient to promote the growth of EMLC1 cells, even in the absence of SCF. Antiphosphotyrosine immunoblots of total cell lysates demonstrated that IL-4 and IL-9 induce tyrosine phosphorylation of different cellular substrates. Both IL-4 and IL-9 stimulated tyrosine phosphorylation of SHP-2, whereas the 90-kD tyrosine phosphorylated protein induced by IL-9 stimulation is Stat3. We have also shown that IL-4 is much more potent than IL-9 in inducing the expression of primary response gene c-myc. It was further determined that c-myc antisense oligodeoxynucleotide blocked IL-4 supported cell growth. Taken together, these results indicate that IL-4 may serve as a growth-promoting factor for hematopoietic stem cells, and IL-9 enhances both growth and erythroid differentiation of primitive hematopoietic progenitors. The results also suggest that differences in tyrosine phosphorylation induced by IL-4 and IL-9 may in part determine their distinct biological functions. PMID: 9923452 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 78: J Exp Med. 1999 Jan 4;189(1):63-73. STAT3 is required for the gp130-mediated full activation of the c-myc gene. Kiuchi N, Nakajima K, Ichiba M, Fukada T, Narimatsu M, Mizuno K, Hibi M, Hirano T. Division of Molecular Oncology, Biomedical Research Center, Osaka University Medical School, Suita, Osaka 565-0871, Japan. The signal transducers and activators of transcription (STAT) family members have been implicated in regulating the growth, differentiation, and death of normal and transformed cells in response to either extracellular stimuli, including cytokines and growth factors, or intracellular tyrosine kinases. c-myc expression is coordinately regulated by multiple signals in these diverse cellular responses. We show that STAT3 mostly mediates the rapid activation of the c-myc gene upon stimulation of the interleukin (IL)-6 receptor or gp130, a signal transducing subunit of the receptor complexes for the IL-6 cytokine family. STAT3 does so most likely by binding to cis-regulatory region(s) of the c-myc gene. We show that STAT3 binds to a region overlapping with the E2F site in the c-myc promoter and this site is critical for the c-myc gene promoter- driven transcriptional activation by IL-6 or gp130 signals. This is the first identification of the linkage between a member of the STAT family and the c-myc gene activation, and also explains how the IL-6 family of cytokines is capable of inducing the expression of the c-myc gene. PMID: 9874564 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 79: Am J Physiol. 1998 Dec;275(6 Pt 1):C1640-52. Activation of the JAK-STAT pathway by reactive oxygen species. Simon AR, Rai U, Fanburg BL, Cochran BH. Pulmonary and Critical Care Division, Tupper Research Institute, New England Medical Center, Boston 02111, Massachusetts, USA. Reactive oxygen species (ROS) play an important role in the pathogenesis of many human diseases, including the acute respiratory distress syndrome, Parkinson's disease, pulmonary fibrosis, and Alzheimer's disease. In mammalian cells, several genes known to be induced during the immediate early response to growth factors, including the protooncogenes c-fos and c-myc, have also been shown to be induced by ROS. We show that members of the STAT family of transcription factors, including STAT1 and STAT3, are activated in fibroblasts and A-431 carcinoma cells in response to H2O2. This activation occurs within 5 min, can be inhibited by antioxidants, and does not require protein synthesis. STAT activation in these cell lines is oxidant specific and does not occur in response to superoxide- or nitric oxide-generating stimuli. Buthionine sulfoximine, which depletes intracellular glutathione, also activates the STAT pathway. Moreover, H2O2 stimulates the activity of the known STAT kinases JAK2 and TYK2. Activation of STATs by platelet-derived growth factor (PDGF) is significantly inhibited by N-acetyl-L-cysteine and diphenylene iodonium, indicating that ROS production contributes to STAT activation in response to PDGF. These findings indicate that the JAK-STAT pathway responds to intracellular ROS and that PDGF uses ROS as a second messenger to regulate STAT activation. PMID: 9843726 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 80: Hepatology. 1998 Oct;28(4):959-70. Comment on: Hepatology. 1998 Oct;28(4):906-13. Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 receptor. Yamada Y, Webber EM, Kirillova I, Peschon JJ, Fausto N. Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195-7705, USA. We used KO mice lacking either TNF receptor 1 (TNFR-1) or receptor 2 (TNFR-2) to determine whether signaling at the start of liver regeneration after partial hepatectomy (PH) involves only one or both TNF receptors and to analyze in more detail the abnormalities caused by lack of TNFR-1 receptor, which is required for the initiation of liver regeneration. Lack of TNFR-2 had little effect on NF-kappaB and STAT3 binding, and no effect in interleukin-6 production after PH, but caused a delay in AP-1 and C/EBP binding and in the expression of c-jun and c-myc messenger RNA (mRNA). In contrast to mice lacking TNFR-1, which had deficient hepatocyte DNA synthesis and massive lipid accumulation in hepatocytes, TNFR-2 KO mice had normal liver structure and similar levels of hepatocyte DNA replication as those of wild type mice. We conclude that TNFR-1, but not TNFR-2, is necessary for liver regeneration, and that NF-kappaB and STAT3 binding are activated by signals transduced by TNFR-1. Inhibition of AP-1 and C/EBP binding and in the expression of c-jun and c-myc mRNA in the first 4 hours after PH, as well as the apparent lack of Fos in AP-1 complexes, had no effect on the timing or extent of DNA replication. Publication Types: Comment PMID: 9755232 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 81: EMBO J. 1998 Oct 1;17(19):5588-97. Coexpression of IL-6 and soluble IL-6R causes nodular regenerative hyperplasia and adenomas of the liver. Maione D, Di Carlo E, Li W, Musiani P, Modesti A, Peters M, Rose-John S, Della Rocca C, Tripodi M, Lazzaro D, Taub R, Savino R, Ciliberto G. Istituto Ricerche di Biologia Molecolare (IRBM) P.Angeletti, Via Pontina Km 30.600, 00400 Pomezia, Rome. Studies with tumor necrosis factor p55 receptor- and interleukin-6 (IL-6)-deficient mice have shown that IL-6 is required for hepatocyte proliferation and reconstitution of the liver mass after partial hepatectomy. The biological activities of IL-6 are potentiated when this cytokine binds soluble forms of its specific receptor subunit (sIL-6R) and the resulting complex interacts with the transmembrane signaling chain gp130. We show here that double transgenic mice expressing high levels of both human IL-6 and sIL-6R under the control of liver-specific promoters spontaneously develop nodules of hepatocellular hyperplasia around periportal spaces and present signs of sustained hepatocyte proliferation. The resulting picture is identical to that of human nodular regenerative hyperplasia, a condition frequently associated with immunological and myeloproliferative disorders. In high expressors, hyperplastic lesions progress with time into discrete liver adenomas. These data strongly suggest that the IL-6/sIL-6R complex is both a primary stimulus to hepatocyte proliferation and a pathogenic factor of hepatocellular transformation. PMID: 9755159 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 82: J Biol Chem. 1998 Mar 20;273(12):6776-85. The Src and signal transducers and activators of transcription pathways as specific targets for low molecular weight phosphotyrosine-protein phosphatase in platelet-derived growth factor signaling. Chiarugi P, Cirri P, Marra F, Raugei G, Fiaschi T, Camici G, Manao G, Romanelli RG, Ramponi G. Dipartimento di Scienze Biochimiche, viale Morgagni 50, 50134 Firenze, Italy. The low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) is a cytosolic phosphotyrosine-protein phosphatase specifically interacting with the activated platelet-derived growth factor (PDGF) receptor through its active site. Overexpression of the LMW-PTP results in modulation of PDGF-dependent mitogenesis. In this study we investigated the effects of this tyrosine phosphatase on the signaling pathways relevant for PDGF-dependent DNA synthesis. NIH 3T3 cells were stably transfected with active or dominant negative LMW-PTP. The effects of LMW-PTP were essentially restricted to the G1 phase of the cell cycle. Upon stimulation with PDGF, cells transfected with the dominant negative LMW-PTP showed an increased activation of Src, whereas the active LMW-PTP induced a reduced activation of this proto-oncogene. We observe that c-Src binding to PDGF receptor upon stimulation is prevented by overexpression of LMW-PTP. These effects were associated with parallel changes in myc expression. Moreover, wild-type and dominant negative LMW-PTP differentially regulated STAT1 and STAT3 activation and tyrosine phosphorylation, whereas they did not modify extracellular signal-regulated kinase activity. However, these modifications were associated with changes in fos expression despite the lack of any effect on extracellular signal-regulated kinase activation. Other independent pathways involved in PDGF-induced mitogenesis, such as phosphatidylinositol 3-kinase and phospholipase C-gamma1, were not affected by LMW-PTP. These data indicate that this phosphatase selectively interferes with the Src and the STATs pathways in PDGF downstream signaling. The resulting changes in myc and fos proto-oncogene expression are likely to mediate the modifications observed in the G1 phase of the cell cycle. PMID: 9506979 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 83: Int J Cancer. 1998 Jan 5;75(1):64-73. Oncostatin M induces the differentiation of breast cancer cells. Douglas AM, Grant SL, Goss GA, Clouston DR, Sutherland RL, Begley CG. Rotary Bone Marrow Research Laboratory, Royal Melbourne Hospital, Parkville, Australia. douglas@wehi.edu.au We have recently described the action of Oncostatin M (OSM) to inhibit the proliferation of breast cancer cells. In this study we examined the action of OSM on 2 breast cancer cell lines to further characterize the nature of OSM inhibition of cellular proliferation. Treatment with OSM for 6 days resulted in an approximately 2- to 5-fold decrease in cell number, which was independent of estrogen receptor status. Consistent with this, colony formation was reduced to approximately 50% when cells were exposed to OSM in primary agar cultures. Clonogenicity was further inhibited following 7 days treatment with OSM in monolayer cultures: the total number of clonogenic cells was suppressed approximately 10-fold. Analysis of cell cycle status in OSM-treated cells demonstrated a 40% reduction in the proportion of cells in S phase within 12 hr, with an increase in cells in G0/G1. After 6 days, there was a 10-fold reduction in the absolute number of cells in S phase in OSM-treated cultures. These changes were associated with striking changes in cellular morphology, including disruption of intercellular junctions and the production of lipid droplets. There was a 5-fold increase of c-fos and c-myc mRNA within 30 min of commencing treatment with OSM. In addition, in the ER positive cells there was a decrease in ER mRNA (evident within approximately 2 hr) and ER protein expression following treatment with OSM. Conversely, there was a 5-fold increase in epidermal growth factor receptor (EGFR) mRNA within 4 hr, and a 2.5-fold rise in mRNA for transforming growth factor alpha (TGF alpha). Thus, the inhibition of breast cancer cells by OSM was associated with decreased clonogenicity, a decrease in S phase cells and a variety of phenotypic changes, all consistent with the induction of differentiation. PMID: 9426692 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 84: Biochem Biophys Res Commun. 1997 Sep 29;238(3):764-8. Association of Stat3-dependent transcriptional activation of p19INK4D with IL-6-induced growth arrest. Narimatsu M, Nakajima K, Ichiba M, Hirano T. Department of Oncology, Biomedical Research Center, Osaka University Medical School, Suita, Japan. Signal transducer and activator of transcription 3 (Stat3) is the major mediator of the IL-6-induced signals regulating growth and differentiation. In the M1 myeloleukemic cell line, Stat3 is a critical transcription factor causing repression of c-myc and c-myb genes, expression of junB and IRF1, growth arrest at G1, and subsequent macrophage differentiation. To understand the mechanisms by which Stat3 causes such effects, we searched for other Stat3-regulated genes possibly involved in growth arrest. We identified this inducible molecule as p19INK4D using a specific antibody. Both p19INK4D mRNA and protein were rapidly induced by IL-6 treatment without requiring de novo protein synthesis and the induction was fully suppressed by dominant-negative forms of Stat3. Thus both Stat3-regulated events, repressions of c-myc and c-myb and induction of p19INK4D, are likely to be involved in IL-6-induced growth arrest in M1 cells. PMID: 9325164 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 85: J Biol Chem. 1997 Oct 3;272(40):25184-9. Involvement of STAT3 in the granulocyte colony-stimulating factor-induced differentiation of myeloid cells. Shimozaki K, Nakajima K, Hirano T, Nagata S. Department of Genetics, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan. Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation and differentiation of the progenitor cells of neutrophilic granulocytes. The binding of G-CSF to its receptor specifically activates JAK1 and JAK2 kinases, as well as STAT3, a signal transducer and activator of transcription (STAT). To examine the role of STAT3 in G-CSF receptor-mediated signal transduction, two different forms of the dominant negative STAT3 were introduced into a mouse myeloid cell line that exogenously expresses the mouse G-CSF receptor. In response to G-CSF, the parental myeloid cells grew for about 4 days, and then they stopped dividing and differentiated into cells with lobulated nuclei. During this period, the expression of the myeloperoxidase (MPO) gene was induced, while c-myc gene expression was down-regulated. In contrast, in the cells expressing the dominant negative STAT3, G-CSF could induce neither growth arrest nor morphological change. However, the induction of the MPO gene by G-CSF was not affected by the dominant negative STAT3. These results indicate that STAT3 activation is responsible for part of the G-CSF-induced differentiation of neutrophils but that another pathway, involving the expression of the MPO gene, that does not utilize the activated STAT3, is also required to fully differentiate the cells. PMID: 9312131 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 86: J Biol Chem. 1997 Aug 22;272(34):21334-40. Critical cytoplasmic domains of human interleukin-9 receptor alpha chain in interleukin-9-mediated cell proliferation and signal transduction. Zhu YX, Sun HB, Tsang ML, McMahel J, Grigsby S, Yin T, Yang YC. Department of Medicine (Hematology/Oncology), Indianapolis, Indiana 46202, USA. Interleukin-9 receptor (IL-9R) complex consists of a ligand-specific alpha chain and IL-2R gamma chain. In this study, two regions in the cytoplasmic domain of human IL-9Ralpha were found to be important for IL-9-mediated cell growth. A membrane-proximal region that contains the BOX1 consensus sequence is required for IL-9-induced cell proliferation and tyrosine phosphorylation of Janus kinases (JAKs). Deletion of this region or internal deletion of the BOX1 motif abrogated IL-9-induced cell proliferation and signal transduction. However, substitution of the Pro-X-Pro in the BOX1 motif with Ala-X-Ala failed to abolish IL-9-induced cell proliferation but decreased IL-9-mediated tyrosine phosphorylation of JAK kinases, insulin receptor substrate-2, and signal transducer and activator of transcription 3 (STAT3) and expression of c-myc and junB. Another important region is downstream of the BOX1 motif and contains a STAT3 binding motif YLPQ. Deletion of this region significantly impaired IL-9-induced cell growth, activation of JAK kinases, insulin receptor substrate-2, and STAT3 and expression of early response genes. A point mutation changing YLPQ into YLPA greatly reduced IL-9-induced activation of STAT3 and expression of c-myc but did not affect cell proliferation. These results suggest that cooperation or cross-talk of signaling molecules associated with different domains of IL-9Ralpha other than STAT3 is essential for IL-9-mediated cell growth. PMID: 9261146 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 87: Endocrinology. 1997 Jul;138(7):2740-6. Transcriptional regulation of Sertoli cell immediate early genes by interleukin-6 and interferon-gamma is mediated through phosphorylation of STAT-3 and STAT-1 proteins. Jenab S, Morris PL. The Population Council, New York, New York 10021, USA. The immediate early genes are regulated by a variety of extracellular signals, including pleiotropic cytokines. The effects of the testicular cytokines, interleukin-6 (IL-6) and interferon-gamma (IFN-gamma), on signal transducers and activators of transcription 3 and 1 (STAT-3 and STAT-1) and on c-fos gene expression in primary Sertoli cells are suggestive of their roles in differential function. Using the tyrosine phosphorylation inhibitor, genistein, and electrophoretic mobility shift assay, we show that IL-6 and IFN-gamma induce nuclear factor STAT-3 and STAT-1 DNA-binding activity to the sis-inducible element of c-fos in a genistein-dependent pathway. Quantitative solution hybridization, Northern blot, and nuclear run-on analysis show that differential induction of c-fos, junB, and c-myc messenger RNA (mRNA) by these cytokines occur at transcriptional levels. IL-6 stimulates c-fos mRNA levels by 6-fold while increasing junB levels by 2-fold. IFN-gamma increases c-fos message 2-fold, but has no effect on junB mRNA levels. Furthermore, genistein treatment blocks the induction of c-fos and junB gene expression, demonstrating that tyrosine phosphorylation of STAT proteins is involved in the cytokine regulation of the Sertoli immediate early genes. H7, a serine/threonine phosphorylation inhibitor, also blocks c-fos gene induction by IL-6 and IFN-gamma, but does not affect the DNA-binding activities of STAT-3 and STAT-1. Finally, IL-6 treatment of Sertoli cells (3-6 h) increases the amounts of activating protein-1 binding to activating protein-1 element and c-myc transcription. PMID: 9202212 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 88: Science. 1996 Nov 22;274(5291):1379-83. Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Cressman DE, Greenbaum LE, DeAngelis RA, Ciliberto G, Furth EE, Poli V, Taub R. Department of Genetics and Medicine, University of Pennsylvania School of Medicine, 705a Stellar-Chance, 422 Curie Boulevard, Philadelphia, PA 19104-6145, USA. Liver regeneration stimulated by a loss of liver mass leads to hepatocyte and nonparenchymal cell proliferation and rapid restoration of liver parenchyma. Mice with targeted disruption of the interleukin-6 (IL-6) gene had impaired liver regeneration characterized by liver necrosis and failure. There was a blunted DNA synthetic response in hepatocytes of these mice but not in nonparenchymal liver cells. Furthermore, there were discrete G1 phase (prereplicative stage in the cell cycle) abnormalities including absence of STAT3 (signal transducer and activator of transcription protein 3) activation and depressed AP-1, Myc, and cyclin D1 expression. Treatment of IL-6-deficient mice with a single preoperative dose of IL-6 returned STAT3 binding, gene expression, and hepatocyte proliferation to near normal and prevented liver damage, establishing that IL-6 is a critical component of the regenerative response. PMID: 8910279 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 89: Immunity. 1996 Nov;5(5):449-60. Two signals are necessary for cell proliferation induced by a cytokine receptor gp130: involvement of STAT3 in anti-apoptosis. Fukada T, Hibi M, Yamanaka Y, Takahashi-Tezuka M, Fujitani Y, Yamaguchi T, Nakajima K, Hirano T. Department of Molecular Oncology, Biomedical Research Center, Osaka University Medical School, Japan. gp130 is a common signal transducer for the interleukin-6-related cytokines. To delineate the gp130-mediated growth signal, we established a series of pro-B cell lines expressing chimeric receptors composed of the extracellular domain of the granulocyte colony-stimulating factor receptor and the transmembrane and cytoplasmic domains of gp130. The second tyrosine (from the membrane) of gp130, which was required for the tyrosine phosphorylation of SHP-2, its association with GRB2, and activation of a MAP kinase, was essential for mitogenesis, but not for anti-apoptosis. On the other hand, the tyrosine in the YXXQ motifs essential for STAT3 activation was required for bcl-2 induction and anti-apoptosis. Furthermore, dominant-negative STAT3 inhibited anti-apoptosis. These data demonstrate that two distinct signals, mitogenesis and anti-apoptosis, are required for gp130-induced cell growth and that STAT3 is involved in anti-apoptosis. PMID: 8934572 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 90: EMBO J. 1996 Jul 15;15(14):3651-8. A central role for Stat3 in IL-6-induced regulation of growth and differentiation in M1 leukemia cells. Nakajima K, Yamanaka Y, Nakae K, Kojima H, Ichiba M, Kiuchi N, Kitaoka T, Fukada T, Hibi M, Hirano T. Department of Molecular Oncology, Osaka University Medical School, Japan. Interleukin-6 (IL-6) induces either differentiation or growth of a variety of cells. Little is known about the molecular basis of this cellular decision. The family of signal transducer and activator of transcription (Stat) proteins are involved in signaling through a variety of cytokine and growth factor receptors, although their biological roles have not been established. To address whether Stat proteins play roles in IL-6-induced growth or differentiation, we introduced two types of mutant Stat3 acting in a dominant-negative manner into M1 leukemic cells which respond to IL-6 with growth arrest and terminal differentiation. We show that dominant-negative forms of Stat3 inhibited both IL-6-induced growth arrest at G(0)/G1 and macrophage differentiation in the M1 transformants. Blocking of Stat activation resulted in inhibition of IL-6-induced repression of c-myb and c-myc. Furthermore, IL-6 enhanced the growth of M1 cells primarily through shortening the length of the G1 period when Stat3 was suppressed. Thus IL-6 generates both growth-enhancing signals and growth arrest- and differentiation-inducing signals at the same time. Stat3 may be a key molecule which determines the cellular decision from cell growth to differentiation in M1 cells. PMID: 8670868 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 91: Immunity. 1996 Jul;5(1):81-9. Distinct tumorigenic potential of abl and raf in B cell neoplasia: abl activates the IL-6 signaling pathway. Hilbert DM, Migone TS, Kopf M, Leonard WJ, Rudikoff S. Laboratory of Genetics, National Cancer Institute, Bethesda, Maryland 20892, USA. The development of murine plasma cell tumors induced by raf/myc containing retroviruses is facilitated by T cells and completely dependent on IL-6. To determine whether kinases with differing specificities reflect alternative biochemical pathways in B cell tumorigenesis, we have employed an abl/myc containing retrovirus to assess neoplastic development. In contrast with raf/myc, abl/myc disease is T cell and IL-6 independent. An examination of the IL-6 signal transduction pathway reveals that this pathway, as defined by activation of Stat3, is inducible by IL-6 in raf/myc tumors but constitutively activated in abl/myc tumors. These findings provide a mechanism for the derivation of cytokine-independent plasma cell tumors and suggest that both IL-6-dependent and independent tumors may arise in vivo depending on the particular mutational events incurred during tumorigenesis. PMID: 8758897 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 92: Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):3963-6. STAT3 activation is a critical step in gp130-mediated terminal differentiation and growth arrest of a myeloid cell line. Minami M, Inoue M, Wei S, Takeda K, Matsumoto M, Kishimoto T, Akira S. Institute for Molecular and Cellular Biology, Osaka University, Japan. Myeloid leukemia M1 cells can be induced for growth arrest and terminal differentiation into macrophages in response to interleukin 6 (IL-6) or leukemia inhibitory factor (LIF). Recently, a large number of cytokines and growth factors have been shown to activate the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. In the case of IL-6 and LIF, which share a signal transducing receptor gp130, STAT3 is specifically tyrosine-phosphorylated and activated by stimulation with each cytokine in various cell types. To know the role of JAK-STAT pathway in M1 differentiation, we have constructed dominant negative forms of STAT3 and established M1 cell lines that constitutively express them. These M1 cells that overexpressed dominant negative forms showed no induction of differentiation-associated markers including Fc gamma receptors, ferritin light chain, and lysozyme after treatment with IL-6. Expression of either c-myb or c-myc was not downregulated. Furthermore, IL-6- and LIF-mediated growth arrest and apoptosis were completely blocked. Thus these findings demonstrate that STAT3 activation is the critical step in a cascade of events that leads to terminal differentiation of M1 cells. PMID: 8632998 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 93: EMBO J. 1996 Apr 1;15(7):1557-65. Differentiation and growth arrest signals are generated through the cytoplasmic region of gp130 that is essential for Stat3 activation. Yamanaka Y, Nakajima K, Fukada T, Hibi M, Hirano T. Department of Molecular Oncology, Osaka University Medical School, Japan. Interleukin-6 (IL-6) induces growth arrest and macrophage differentiation through its receptor in a murine myeloid leukaemic cell line, M1, although it is largely unknown how the IL-6 receptor generates these signals. By using chimeric receptors consisting of the extracellular domain of growth hormone receptor and the transmembrane and cytoplasmic domain of gp130 with progressive C-terminal truncations, we showed that the membrane-proximal 133, but not 108, amino acids of gp130 could generate the signals for growth arrest, macrophage differentiation, down-regulation of c-myc and c-myb, induction of junB and IRF1 and Stat3 activation. Mutational analysis of this region showed that the tyrosine residue with the YXXQ motif was critical not only for Stat3 activation but also for growth arrest and differentiation, accompanied by down-regulation of c-myc and c-myb and immediate early induction of junB and IRF1. The tight correlation between Stat3 activation and other IL-6 functions was further observed in the context of the full-length cytoplasmic region of gp130. The result suggest that Stat3 plays an essential role in the signals for growth arrest and differentiation. PMID: 8612579 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 94: FASEB J. 1996 Mar;10(4):413-27. Liver regeneration 4: transcriptional control of liver regeneration. Taub R. Department of Genetics and Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6145, USA. Determining what factors are responsible for initiating regeneration following partial hepatectomy or toxic damage, and how the liver maintains differentiated functions while the hepatocytes are undergoing cellular proliferation are central issues in understanding the molecular bases of liver regeneration. Examination of the transcriptional milieu in the regenerating liver provides clues to the answers to these questions. Growth factor-generated intracellular signals that trigger liver regeneration result in activation via posttranslational modifications of latent, normally inactive transcription factors that preexist in the liver. Two transcription factors that are activated by this mechanism include posthepatectomy factor/nuclear factor-kappa B) and Stat3. Because cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-l (IL-1), and IL-6 can induce these factors in the liver, the finding of activated Stat3 and PHF/NF-kappa B suggests that these cytokines may play a role in some aspects of growth regulation during liver regeneration. Rapidly induced transcription factors, Stat3, PHF/NF-kappa B, and others are responsible for activation of the primary growth response or immediate-early genes, which play a role in regulating later phases of cell growth in regenerating liver and other mitogen-activated cells. Immediate-early genes encode many members of diverse transcription factor families including the Jun-Fos-LRF-1, nuclear receptor, and myc families to name a few. In this way a transcriptional cascade is established during the G1 phase of liver regeneration. Coexisting with these induced factors are liver-specific transcription factors such as the CAAT enhancer binding proteins and hepatocyte nuclear factors, which may interact with growth-induced factors to help the liver maintain metabolic homeostasis during regeneration. As a result the liver is able to accomplish the goals of reestablishing its mass while it maintains its functional capacity during regeneration. Publication Types: Review Review, Tutorial PMID: 8647340 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------