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TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue

Dean Kujubu et al.Jul 1, 1991

TIS10 is a primary response gene whose cDNA was cloned as a result of its rapid, superinducible expression in Swiss 3T3 cells in response to 12-O-Tetradecanoylphorbol-13-acetate. The 5'-untranslated region of the 3.9-kilobase TIS10 message contains only 124 nucleotides, whereas the 3'-untranslated region is almost 2 kilobases in length. Within this long 3' region, there are multiple repeats of the sequence ATTTA, a sequence often present in rapidly degraded mRNA species. Primer extension revealed that the TIS10 cDNA begins 16 base pairs downstream of the transcription start site for the TIS10 gene. The TIS10 cDNA encodes a predicted protein of 604 amino acids. A computer search identified striking similarities between the predicted TIS10 protein product and the murine, sheep, and human prostaglandin synthase/cyclooxygenase proteins. The TIS10 protein has many of the same conserved amino acids that are thought to be important for cyclooxygenase function. TIS10 mRNA is undetectable by Northern analysis in quiescent 3T3 cells. The TIS10 gene is rapidly and transiently induced by forskolin and serum, as well as by 12-O-tetradecanoylphorbol-13-acetate, in Swiss 3T3 cells. These agents elicit far more dramatic changes in TIS10 mRNA levels than in cyclooxygenase mRNA levels. The expression of the TIS10 gene appears to be highly cell type-restricted in cultured cell lines; of 12 cell lines tested under superinducing conditions, only the rodent embryonic Swiss 3T3 and Rat1 cell lines expressed TIS10 gene.

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Arginine Methylation of STAT1 Modulates IFNα/β-Induced Transcription

Kerri Mowen et al.Mar 1, 2001

Transcriptional induction by interferons requires the tyrosine and serine phosphorylation of STAT transcription factors. The N-terminal region is highly homologous among the STAT proteins and surrounds a completely conserved arginine residue. Here we demonstrate arginine methylation of STAT1 by the protein arginine methyl-transferase PRMT1 as a novel requirement for IFNalpha/beta-induced transcription. Methyl-thioadenosine, a methyl-transferase inhibitor that accumulates in many transformed cells, inhibits STAT1-mediated IFN responses. This inhibition arises from impaired STAT1-DNA binding due to an increased association of the STAT inhibitor PIAS1 with phosphorylated STAT1 dimers in the absence of arginine methylation. Thus, arginine methylation of STAT1 is an additional posttranslational modification regulating transcription factor function, and alteration of arginine methylation might be responsible for the lack of interferon responsiveness observed in many malignancies.

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Imaging adenoviral-directed reporter gene expression in living animals with positron emission tomography

Sanjiv Gambhir et al.Mar 2, 1999

We are developing quantitative assays to repeatedly and noninvasively image expression of reporter genes in living animals, using positron emission tomography (PET). We synthesized positron-emitting 8-[18F]fluoroganciclovir (FGCV) and demonstrated that this compound is a substrate for the herpes simplex virus 1 thymidine kinase enzyme (HSV1-TK). Using positron-emitting FGCV as a PET reporter probe, we imaged adenovirus-directed hepatic expression of the HSV1-tk reporter gene in living mice. There is a significant positive correlation between the percent injected dose of FGCV retained per gram of liver and the levels of hepatic HSV1-tk reporter gene expression ( r 2 > 0.80). Over a similar range of HSV1-tk expression in vivo , the percent injected dose retained per gram of liver was 0–23% for ganciclovir and 0–3% for FGCV. Repeated, noninvasive, and quantitative imaging of PET reporter gene expression should be a valuable tool for studies of human gene therapy, of organ/cell transplantation, and of both environmental and behavioral modulation of gene expression in transgenic mice.

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A mutant herpes simplex virus type 1 thymidine kinase reporter gene shows improved sensitivity for imaging reporter gene expression with positron emission tomography

Sanjiv Gambhir et al.Mar 14, 2000

We are developing assays for noninvasive, quantitative imaging of reporter genes with positron emission tomography (PET), for application both in animal models and in human gene therapy. We report here a method to improve the detection of lower levels of PET reporter gene expression by utilizing a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) as a PET reporter gene. The HSV1-sr39tk mutant was identified from a library of site-directed mutants. Accumulation (net uptake) of the radioactively labeled substrates [8- 3 H]penciclovir ([8- 3 H]PCV), and 8-[ 18 F]fluoropenciclovir (FPCV) in C6 rat glioma cells expressing HSV1-sr39tk is increased by a factor of ≈2.0 when compared with C6 cells expressing wild-type HSV1-tk. The increased imaging sensitivity of HSV1-sr39tk when FPCV is used is also demonstrated in vivo both with tumor cells stably transfected with either HSV1-tk or HSV1-sr39tk, and after hepatic delivery of HSV1-tk or HSV1-sr39tk by using adenoviral vectors. The use of HSV1-sr39tk as a PET reporter gene and FPCV as a PET reporter probe results in significantly enhanced sensitivity for imaging reporter gene expression in vivo .

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PRMT1 Is the Predominant Type I Protein Arginine Methyltransferase in Mammalian Cells

Jie Tang et al.Mar 1, 2000

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The Mammalian Immediate-early TIS21 Protein and the Leukemia-associated BTG1 Protein Interact with a Protein-arginine -Methyltransferase

Wey‐Jinq Lin et al.Jun 1, 1996

The TIS21 immediate-early gene and leukemia-associated BTG1 gene encode proteins with similar sequences. Two-hybrid analysis identified a protein that interacts with TIS21 and BTG1. Sequence motifs associated with S-adenosyl-L-methionine binding suggested this protein might have methyltransferase activity. A glutathione S-transferase (GST) fusion of the putative methyltransferase modifies arginine residues, in appropriate protein substrates, to form NG-monomethyl and NG,NG-dimethylarginine (asymmetric). We term the protein- arginine N-methyltransferase (EC) gene “PRMT1,” for rotein-aginine ethylransferase 1. GST-TIS21 and GST-BTG1 fusion proteins qualitatively and quantitatively modulate endogenous PRMT1 activity, using control and hypomethylated RAT1 cell extracts as methyl-accepting substrates. PRMT1 message appears ubiquitous, and is constitutive in mitogen-stimulated cells. Modulation of PRMT1 activity by transiently expressed regulatory subunits may be an additional mode of signal transduction following ligand stimulation. The TIS21 immediate-early gene and leukemia-associated BTG1 gene encode proteins with similar sequences. Two-hybrid analysis identified a protein that interacts with TIS21 and BTG1. Sequence motifs associated with S-adenosyl-L-methionine binding suggested this protein might have methyltransferase activity. A glutathione S-transferase (GST) fusion of the putative methyltransferase modifies arginine residues, in appropriate protein substrates, to form NG-monomethyl and NG,NG-dimethylarginine (asymmetric). We term the protein- arginine N-methyltransferase (EC) gene “PRMT1,” for rotein-aginine ethylransferase 1. GST-TIS21 and GST-BTG1 fusion proteins qualitatively and quantitatively modulate endogenous PRMT1 activity, using control and hypomethylated RAT1 cell extracts as methyl-accepting substrates. PRMT1 message appears ubiquitous, and is constitutive in mitogen-stimulated cells. Modulation of PRMT1 activity by transiently expressed regulatory subunits may be an additional mode of signal transduction following ligand stimulation. INTRODUCTIONThe protein products of the immediate-early/primary response genes are thought to act as “third messengers,” mediating phenotypic alterations in cells in response to ligands such as growth factors, hormones, neurotransmitters, cytokines, and neurotrophins. Many immediate-early genes encode transcription factors (e.g. Fos, Jun, Egr-1) that initiate transcriptional cascades required for proliferation or differentiation (Herschman, 25Herschman H.R. Annu. Rev. Biochem. 1991; 60: 281-319Google Scholar). Other ligand-induced immediate-early genes encode paracrine mediators of cellular communication whose products (e.g. prostaglandin synthase-2, inducible nitric- oxide synthase, and cytokines such as MCP-1) modulate the behavior of neighboring cells (Smith and Herschman, 60Smith J.B. Herschman H.R. J. Biol. Chem. 1995; 270: 16756-16765Google Scholar).Because immediate-early/primary response genes have been cloned on the basis of their induction characteristics, rather than the functions of their protein products, a number of these genes encode proteins whose biological roles have not yet been determined. One such immediate-early gene is TIS21. The TIS21 cDNA was cloned by differential screening, both from a cDNA library prepared from mitogen-treated, quiescent murine Swiss 3T3 cells (Fletcher et al., 18Fletcher B.S. Lim R.W. Varnum B.C. Kujubu D.A. Koski R.K. Herschman H.R. J. Biol. Chem. 1991; 266: 14511-14518Google Scholar) and from a cDNA library prepared from nerve growth factor-treated rat PC12 pheochromocytoma cells (Bradbury et al., 5Bradbury A. Possenti R. Shooter E.M. Tirone F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3353-3357Google Scholar). The predicted rat and mouse TIS21 proteins differ at only four out of 158 amino acid residues. We demonstrated, by metabolic labeling followed by immunoprecipitation, that maximal TIS21 protein synthesis occurs within the first hour after exposure to ligand, both in mitogen-stimulated Swiss 3T3 cells and in nerve growth factor-stimulated PC12 cells (Varnum et al., 65Varnum B.C. Reddy S.T. Koski R.A. Herschman H.R. J. Cell. Physiol. 1994; 158: 205-213Google Scholar). Moreover, the half-life of both mitogen- and nerve growth factor-induced TIS21 protein is less than 15 min (Varnum et al., 65Varnum B.C. Reddy S.T. Koski R.A. Herschman H.R. J. Cell. Physiol. 1994; 158: 205-213Google Scholar). Despite substantial investigation into both the structure of the TIS21 gene and the induced expression of the TIS21 message and protein, no function has been identified for this protein.The human BTG1 gene was cloned and characterized (Rimokh et al., 49Rimokh R. Rouault J.P. Wahbi K. Gadoux M. Lafage M. Archimbaud E. Charrin C. Gentilhomme O. Germain D. Genes Chromosomes Cancer. 1991; 3: 24-36Google Scholar) because of its proximity to a chromosome translocation breakpoint (8;12) associated with a chronic lymphocytic leukemia. BTG1 overexpression in murine 3T3 cells decreases growth rate and reduces colony formation, leading to the suggestion that BTG1 is an “antiproliferative gene” whose loss of function contributes to uncontrolled growth (Rouault et al., 51Rouault J.P. Rimokh R. Tessa C. Paranhos G. Ffrench M. Duret L. Garoccio M. Germain D. Samarut J. Magaud J.P. EMBO J. 1992; 11: 1663-1670Google Scholar). The human BTG1 and rodent TIS21 open reading frames share 59% identity and 75% conserved sequence similarity at the amino acid level. Moreover, the exon/intron structures of the TIS21 and BTG1 genes are remarkably similar (Varnum et al., 1991; Rimokh et al., 49Rimokh R. Rouault J.P. Wahbi K. Gadoux M. Lafage M. Archimbaud E. Charrin C. Gentilhomme O. Germain D. Genes Chromosomes Cancer. 1991; 3: 24-36Google Scholar). However, BTG1 is not the human TIS21 orthologue; the murine BTG1 cDNA has been cloned and identified as a distinct entity with an amino acid sequence identical to human BTG1 (Rouault et al., 52Rouault J.P. Samarut C. Duret L. Tessa C. Samarut J. Magaud J.P. Gene (Amst.). 1993; 129: 303-306Google Scholar). TIS21 and BTG1 are, therefore, members of a gene family.The stringent regulation of TIS21 gene expression, the short time window for the presence of TIS21 protein following ligand stimulation, and the similarities between the TIS21 and BTG1 proteins suggest that these proteins play important roles in mediating ligand-induced biological responses. We previously speculated that, during its brief availability in mitogen-stimulated cells, TIS21 might reverse a BTG1 antiproliferative barrier by formation of a TIS21-BTG1 heterodimer (Varnum et al., 65Varnum B.C. Reddy S.T. Koski R.A. Herschman H.R. J. Cell. Physiol. 1994; 158: 205-213Google Scholar). Alternatively, TIS21 and BTG1 might serve as regulatory partners that modulate the function of a common catalytic subunit. To test these hypotheses, we used the yeast two-hybrid interaction system (Fields and Song, 16Fields S. Song O. Nature. 1989; 240: 245-246Google Scholar; Fields and Sternglanz, 17Fields S. Sternglanz R. Trends Genet. 1994; 10: 286-292Google Scholar) to determine (i) whether TIS21 and BTG1 proteins can form heterodimers and/or homodimers, and (ii) whether there exist proteins that interact with both TIS21 and BTG1. INTRODUCTIONThe protein products of the immediate-early/primary response genes are thought to act as “third messengers,” mediating phenotypic alterations in cells in response to ligands such as growth factors, hormones, neurotransmitters, cytokines, and neurotrophins. Many immediate-early genes encode transcription factors (e.g. Fos, Jun, Egr-1) that initiate transcriptional cascades required for proliferation or differentiation (Herschman, 25Herschman H.R. Annu. Rev. Biochem. 1991; 60: 281-319Google Scholar). Other ligand-induced immediate-early genes encode paracrine mediators of cellular communication whose products (e.g. prostaglandin synthase-2, inducible nitric- oxide synthase, and cytokines such as MCP-1) modulate the behavior of neighboring cells (Smith and Herschman, 60Smith J.B. Herschman H.R. J. Biol. Chem. 1995; 270: 16756-16765Google Scholar).Because immediate-early/primary response genes have been cloned on the basis of their induction characteristics, rather than the functions of their protein products, a number of these genes encode proteins whose biological roles have not yet been determined. One such immediate-early gene is TIS21. The TIS21 cDNA was cloned by differential screening, both from a cDNA library prepared from mitogen-treated, quiescent murine Swiss 3T3 cells (Fletcher et al., 18Fletcher B.S. Lim R.W. Varnum B.C. Kujubu D.A. Koski R.K. Herschman H.R. J. Biol. Chem. 1991; 266: 14511-14518Google Scholar) and from a cDNA library prepared from nerve growth factor-treated rat PC12 pheochromocytoma cells (Bradbury et al., 5Bradbury A. Possenti R. Shooter E.M. Tirone F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3353-3357Google Scholar). The predicted rat and mouse TIS21 proteins differ at only four out of 158 amino acid residues. We demonstrated, by metabolic labeling followed by immunoprecipitation, that maximal TIS21 protein synthesis occurs within the first hour after exposure to ligand, both in mitogen-stimulated Swiss 3T3 cells and in nerve growth factor-stimulated PC12 cells (Varnum et al., 65Varnum B.C. Reddy S.T. Koski R.A. Herschman H.R. J. Cell. Physiol. 1994; 158: 205-213Google Scholar). Moreover, the half-life of both mitogen- and nerve growth factor-induced TIS21 protein is less than 15 min (Varnum et al., 65Varnum B.C. Reddy S.T. Koski R.A. Herschman H.R. J. Cell. Physiol. 1994; 158: 205-213Google Scholar). Despite substantial investigation into both the structure of the TIS21 gene and the induced expression of the TIS21 message and protein, no function has been identified for this protein.The human BTG1 gene was cloned and characterized (Rimokh et al., 49Rimokh R. Rouault J.P. Wahbi K. Gadoux M. Lafage M. Archimbaud E. Charrin C. Gentilhomme O. Germain D. Genes Chromosomes Cancer. 1991; 3: 24-36Google Scholar) because of its proximity to a chromosome translocation breakpoint (8;12) associated with a chronic lymphocytic leukemia. BTG1 overexpression in murine 3T3 cells decreases growth rate and reduces colony formation, leading to the suggestion that BTG1 is an “antiproliferative gene” whose loss of function contributes to uncontrolled growth (Rouault et al., 51Rouault J.P. Rimokh R. Tessa C. Paranhos G. Ffrench M. Duret L. Garoccio M. Germain D. Samarut J. Magaud J.P. EMBO J. 1992; 11: 1663-1670Google Scholar). The human BTG1 and rodent TIS21 open reading frames share 59% identity and 75% conserved sequence similarity at the amino acid level. Moreover, the exon/intron structures of the TIS21 and BTG1 genes are remarkably similar (Varnum et al., 1991; Rimokh et al., 49Rimokh R. Rouault J.P. Wahbi K. Gadoux M. Lafage M. Archimbaud E. Charrin C. Gentilhomme O. Germain D. Genes Chromosomes Cancer. 1991; 3: 24-36Google Scholar). However, BTG1 is not the human TIS21 orthologue; the murine BTG1 cDNA has been cloned and identified as a distinct entity with an amino acid sequence identical to human BTG1 (Rouault et al., 52Rouault J.P. Samarut C. Duret L. Tessa C. Samarut J. Magaud J.P. Gene (Amst.). 1993; 129: 303-306Google Scholar). TIS21 and BTG1 are, therefore, members of a gene family.The stringent regulation of TIS21 gene expression, the short time window for the presence of TIS21 protein following ligand stimulation, and the similarities between the TIS21 and BTG1 proteins suggest that these proteins play important roles in mediating ligand-induced biological responses. We previously speculated that, during its brief availability in mitogen-stimulated cells, TIS21 might reverse a BTG1 antiproliferative barrier by formation of a TIS21-BTG1 heterodimer (Varnum et al., 65Varnum B.C. Reddy S.T. Koski R.A. Herschman H.R. J. Cell. Physiol. 1994; 158: 205-213Google Scholar). Alternatively, TIS21 and BTG1 might serve as regulatory partners that modulate the function of a common catalytic subunit. To test these hypotheses, we used the yeast two-hybrid interaction system (Fields and Song, 16Fields S. Song O. Nature. 1989; 240: 245-246Google Scholar; Fields and Sternglanz, 17Fields S. Sternglanz R. Trends Genet. 1994; 10: 286-292Google Scholar) to determine (i) whether TIS21 and BTG1 proteins can form heterodimers and/or homodimers, and (ii) whether there exist proteins that interact with both TIS21 and BTG1.

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Dexamethasone inhibits mitogen induction of the TIS10 prostaglandin synthase/cyclooxygenase gene.

Dean Kujubu et al.Apr 1, 1992

Glucocorticoids block the induced secretion of prostaglandins in a variety of biological contexts. We have identified a primary response gene, TIS10, which encodes a mitogen-inducible prostaglandin synthase/cyclooxygenase in Swiss 3T3 cells. TIS10 is distinct from prostaglandin synthase/cyclooxygenase. (EC 1.14.99.1), previously cloned from mouse, man, and sheep. Dexamethasone blocks prostaglandin E2 synthesis by 3T3 cells in response to tetradecanoylphorbol acetate. Dexamethasone also blocks both phorbol ester- and forskolin-induced TIS10 mRNA accumulation. In contrast, phorbol esters, forskolin, and dexamethasone have little or no effect on the levels of prostaglandin synthase/cyclooxygenase mRNA in 3T3 cells. Moreover, dexamethasone does not inhibit induction of TIS8/egr-1, another primary response gene. Inhibition of the synthesis of TIS10 prostaglandin synthase/cyclooxygenase may be a principal mechanism by which glucocorticoids block prostaglandin synthesis and secretion.

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Epidermal growth factor. Relationship between receptor regulation and mitogenesis in 3T3 cells.

Aharon Aharonov et al.Jun 1, 1978

Exposure of confluent nondividing 3T3 cells to 10 nm mouse epidermal growth factor (EGF) at 37/sup 0/, followed by incubation for 4.5 h at 37/sup 0/, leads to a 70 to 85% reduction in the binding capacity for /sup 125/I-EGF. Scatchard analysis of the binding data indicate that the reduction of /sup 125/I-EGF binding is due to a decrease in the number of available EGF-receptors per cell, without any change in the affinity of the receptors for EGF. This modulation of the EGF-receptor by the growth factor, termed ''down regulation,'' is dependent on temperature, EGF concentration, time, and the physiological state of the cell. Receptor loss occurs at physiological EGF concentrations (0.1 to 10 nm) which span the concentration range which is mitogenic for 3T3 cells. Maximal stimulation of either (/sup 3/H)thymidine uptake or cell division occurs at 1 nm EGF, a concentration at which only 20% of the EGF-receptor sites are occupied and down regulation is only 55% complete. Low EGF concentrations (< or = to 1 nm) result in down regulation of unoccupied EGF-receptors. Down regulation of the EGF-receptor also occurs in SV 40-transformed 3T3 cells. Growing 3T3 cells exposed to EGF also loose available EGF-receptors. In contrastmore » to confluent cells, dividing 3T3 cells rapidly replace EGF-receptors on the surface of the cell, in the presence of EGF.« less

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Cancer-Stimulated Mesenchymal Stem Cells Create a Carcinoma Stem Cell Niche via Prostaglandin E2 Signaling

Hua‐Jung Li et al.Jul 4, 2012

Abstract Mesenchymal cells of the tumor-associated stroma are critical determinants of carcinoma cell behavior. We focus here on interactions of carcinoma cells with mesenchymal stem cells (MSC), which are recruited to the tumor stroma and, once present, are able to influence the phenotype of the carcinoma cells. We find that carcinoma cell–derived interleukin-1 (IL-1) induces prostaglandin E2 (PGE2) secretion by MSCs. The resulting PGE2 operates in an autocrine manner, cooperating with ongoing paracrine IL-1 signaling, to induce expression of a group of cytokines by the MSCs. The PGE2 and cytokines then proceed to act in a paracrine fashion on the carcinoma cells to induce activation of β-catenin signaling and formation of cancer stem cells. These observations indicate that MSCs and derived cell types create a cancer stem cell niche to enable tumor progression via release of PGE2 and cytokines. Significance: Although PGE2 has been implicated time and again in fostering tumorigenesis, its effects on carcinoma cells that contribute specifically to tumor formation are poorly understood. Here we show that tumor cells are able to elicit a strong induction of the COX-2/microsomal prostaglandin-E synthase-1 (mPGES-1)/PGE2 axis in MSCs recruited to the tumor-associated stroma by releasing IL-1, which in turn elicits a mesenchymal/stem cell–like phenotype in the carcinoma cells. Cancer Discov; 2(9); 840–55. ©2012 AACR. Read the Commentary on this article by Räsänen and Herlyn, p. 775. This article is highlighted in the In This Issue feature, p. 753.

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Variants of 3T3 cells lacking mitogenic response to epidermal growth factor.

Rebecca Pruss et al.Sep 1, 1977

We have previously demonstrated that epidermal growth factor (EGF) can serve as a potent mitogen for 3T3 cells. We have now selected variant 3T3 cell lines unable to respond to EGF, in order to define cellular events unique to the EGF response and to distinguish which of these events are necessary and which are merely correlative to mitogenesis. By simultaneously treating cells with EGF and colchicine, we eliminated those cells stimulated by EGF to enter mitosis. Of the eight clonal EGF nonresponder variants selected by this procedure, none retains a functional EGF receptor. The EGF nonresponsive va-riant lines still retain the ability to respond to other mitogens.

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