1: Endocr Relat Cancer. 2005 Jun;12(2):319-34. RET/PTC-induced gene expression in thyroid PCCL3 cells reveals early activation of genes involved in regulation of the immune response. Puxeddu E, Knauf JA, Sartor MA, Mitsutake N, Smith EP, Medvedovic M, Tomlinson CR, Moretti S, Fagin JA. Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA. RET/PTC rearrangements represent key genetic events involved in papillary thyroid carcinoma (PTC) initiation. The aim of the present study was to identify the early changes in gene expression induced by RET/PTC in thyroid cells. For this purpose, microarray analysis was conducted on PCCL3 cells conditionally expressing the RET/PTC3 oncogene. Gene expression profiling 48 h after activation of RET/PTC3 identified a statistically significant modification of expression of 270 genes. Quantitative PCR confirmation of 20 of these demonstrated 90% accuracy of the microarray. Functional clustering of genes with greater than or less than 1.75-fold expression change (86 genes) revealed RET/PTC3-induced regulation of genes with key functions in apoptosis (Ripk3, Tdga), cell-cell signaling (Cdh6, Fn1), cell cycle (Il24), immune and inflammation response (Cxcl10, Scya2, Il6, Gbp2, Oas1, Tap1, RT1Aw2, C2ta, Irf1, Lmp2, Psme2, Prkr), metabolism (Aldob, Ptges, Nd2, Gss, Gstt1), signal transduction (Socs3, Nf1, Jak2, Cpg21, Dusp6, Socs1, Stat1, Stat3, Cish) and transcription (Nr4a1, Junb, Hfh1, Runx1, Foxe1). Genes coding for proteins involved in the immune response and in intracellular signal transduction pathways activated by cytokines and chemokines were strongly represented, indicating a critical role of RET/PTC3 in the early modulation of the immune response. PMID: 15947106 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: 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] --------------------------------------------------------------- 3: Antimicrob Agents Chemother. 2000 Jul;44(7):1869-73. Correlation between pretreatment levels of interferon response genes and clinical responses to an immune response modifier (Imiquimod) in genital warts. Arany I, Tyring SK, Brysk MM, Stanley MA, Tomai MA, Miller RL, Smith MH, McDermott DJ, Slade HB. Departments of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, USA. iarany@utmb.edu Imiquimod (IQ) has been successfully used in treatment of genital warts. In clinical settings, patients responded well but wart reduction rates varied. Our aim was to find a correlation between clinical responses and pretreatment (constitutive) levels of genes that might be involved in the molecular action of IQ. Since IQ is a cytokine inducer, we analyzed levels of expression of genes of the JAK/STAT signaling pathway and their inhibitors as well as interferon response factors (IRFs) in pretreatment biopsy specimens from complete responders (99 to 100% wart reduction rate) versus incomplete responders (75 to 92% wart reduction rate) by reverse transcription-PCR. We found that mRNA levels of signal transducer and activator of transcription 1 (STAT1) and IRF1 were higher in complete responders than in incomplete responders. Incomplete responders expressed larger amounts of STAT3, IRF2, and protein inhibitor of activated STAT1 (PIAS1) mRNAs compared to complete responders before IQ treatment. We hypothesize that high-level expression of STAT1 and IRF1 is advantageous for a better IQ response. The observed differences in constitutive mRNA levels of these genes may be the consequence of alterations in cellular differentiation and/or variable expression of endogenous interferons. Previous in vitro studies showed that keratinocyte differentiation coordinates the balance between positive and negative signals along the JAK/STAT pathway by regulating the IRF1:IRF2 and STAT1:PIAS1 ratios and thus affecting induction of IQ-inducible genes. Specifically, differentiation supports constitutive expression of STAT1 and IRF1 mRNAs but not expression of IRF2 and PIAS1. Our data are in good agreement with studies that showed the importance of STAT1 in cytokine induction and activation of interferon-responsive genes by IQ. Publication Types: Clinical Trial Randomized Controlled Trial PMID: 10858346 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: 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] --------------------------------------------------------------- 5: 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] --------------------------------------------------------------- 6: Nucleic Acids Res. 1995 Sep 11;23(17):3539-46. 5' upstream sequences of MyD88, an IL-6 primary response gene in M1 cells: detection of functional IRF-1 and Stat factors binding sites. Harroch S, Gothelf Y, Revel M, Chebath J. Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, Israel. Transcription regulatory elements have been analyzed in upstream sequences of an Interleukin-6 (Il-6) primary response gene, MyD88. MyD88 2.3 kb mRNA is strongly and persistently induced in the course of myeloleukemic M1 cells differentiation with Il-6. MyD88 cDNA sequences were found in a region of 12 kb of mouse genomic DNA. Using Il-6 treated M1 cell RNAs, two transcription start sites have been localized, approximately 100 bp upstream from the 5' end of the cloned cDNA. We sequenced 1.4 kb of 5' genomic DNA including the first exon. In 5' of mRNA transcription start site, MyD88 nucleotidic sequence is 85% identical to 5' complementary sequences of the rat 3'-ketoacetyl CoA thiolase gene, over 1.2 kb. A DNA element conferring Il-6-inducible transcription to reporter genes, and localized 30 bp upstream of MyD88 first RNA start site, contains overlapping binding sites for cytokine activated transcription factors Stat and for the Interferon Regulatory Factor-1 and -2 (IRF-1 and IRF-2). In vitro binding assays showed that attachment of Stat factors to this element early in Il-6 treatment requires tyrosine kinase activation. IRF1, an activator of transcription, is also induced to bind to this sequence at later times. A model of persistent activation of MyD88 gene through these two types of factors is proposed. PMID: 7567467 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: EMBO J. 1995 Apr 3;14(7):1421-9. A major role for the protein tyrosine kinase JAK1 in the JAK/STAT signal transduction pathway in response to interleukin-6. Guschin D, Rogers N, Briscoe J, Witthuhn B, Watling D, Horn F, Pellegrini S, Yasukawa K, Heinrich P, Stark GR, et al. Imperial Cancer Research Fund, London, UK. The protein tyrosine kinases JAK1, JAK2 and Tyk2 and STATs (signal transducers and activators of transcription) 1 and 3 are activated in response to interleukin-6 (IL-6) in human fibrosarcoma cells. In mutant cells lacking JAK1, JAK2 or Tyk2, the absence of one kinase does not prevent activation of the others; activation does not, therefore, involve a sequential three-kinase cascade. In the absence of JAK1, the phosphorylation of the gp130 subunit of the IL-6 receptor and the activation of STATs 1 and 3 are greatly reduced. JAK1 is also necessary for the induction of IRF1 mRNA, thus establishing a requirement for the JAK/STAT pathway in the IL-6 response. JAK2 and Tyk2 although activated cannot, in the absence of JAK1, efficiently mediate activation of STATs 1 and 3. A kinase-negative mutant of JAK2 can, however, inhibit such activation, and ancillary roles for JAK2 and Tyk2 are not excluded. A major role for JAK1 and the nonequivalence of JAK1 and JAK2 in the IL-6 response pathway are, nevertheless, clearly established for these cells. PMID: 7537214 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------