|ognizant Communication Corporation|
VOLUME 13, NUMBERS 4/5
Gene Expression, Vol. 13, pp. 217-226
1052-2166/06 $90.00 + .00
Copyright © 2007 Cognizant Comm. Corp.
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Identification of IL-10 and TGF-β Transcripts Involved in the Inhibition of T-Lymphocyte Proliferation During Cell Contact With Human Mesenchymal Stem Cells
Aisha Nasef,1 Alain Chapel,1 Christelle Mazurier,1 Sandrine Bouchet,1 Manuel Lopez,1 Noelle Mathieu,1 Luc Sensebé,2 Yizhuo Zhang,1 Norbert-Claude Gorin,3 Dominique Thierry,1 and Loïc Fouillard1,3
1EA 1638 Laboratoire de Thérapie Cellulaire et Radioprotection
Accidentelle (LTCRA), Faculté de médecine Saint Antoine,
Université Paris VI, Paris, France
2Etablissement Français du Sang Centre-Atlantique and Inserm ESPRI-EA 3855, Université François Rabelais, Tours, France
3Service d'hématologie, hôpital Saint Antoine, 75012 Paris, France
Mesenchymal stem cells (MSC) inhibit the response of allogeneic T lymphocytes in culture. Because the mechanisms of this effect may differ according to the existence of cell contact, we investigated the differences in gene expression of inhibitory molecules during MSC-T lymphocyte coculture when cell contact does and does not occur. Human MSC and T lymphocytes were cultured together in standard and transwell cultures. MSC gene expression was analyzed by semiquantitative real-time RT-PCR. MSC elicited a high dose-dependent inhibition of T lymphocytes in cultures with cell contact, but inhibition occurred even without cell contact. In both cases, we observed significant upregulation of IDO, LIF, and HLA-G, along with downregulation of HGF and SDF1. In cultures with cell contact, IL-10 and TGF-b transcripts were expressed in a significantly higher level than in cultures without this contact. Furthermore, in the latter, the increased inhibition of T-cell proliferation was positively correlated with IDO gene expression and negatively correlated with SDF1 gene expression. MSC appear to induce T-cell tolerance by two distinct mechanisms. The first of these, which does not require cell contact, induces expression of the tolerogenic genes IDO, LIF, and HLA-G. The second mechanism, which is contact dependent, modulates IL-10 and TGF-b gene expression. These two mechanisms probably play separate roles in MSC-induced tolerance in allogeneic hematopoietic stem cell transplantation.
Key words: Mesenchymal stem cells; T cells; Cell contact; Inhibitory molecules
Address correspondence to Loic Fouillard, Service d'hématologie, hôpital Saint Antoine, 184, rue de faubourg Saint-Antoine, 75012 Paris, France. Tel: 33 1 49 28 32 60; Fax: 33 1 49 28 32 00; E-mail: email@example.com
Antisense Targeting of Thymidylate Synthase (TS) mRNA Increases TS Gene Transcription and TS Protein: Effects on Human Tumor Cell Sensitivity to TS Enzyme-Inhibiting Drugs
Tracey L. H. Jason,1,6 Randal W. Berg,1,2,3 Mark D. Vincent,1,3 and James Koropatnick1,2,3,4,5,6
1The London Regional Cancer Program, London Health Sciences
Centre, London, Ontario, Canada
2Lawson Health Research Institute, London Health Sciences Centre, London, Ontario, Canada
3Department of Oncology, University of Western Ontario, London, Ontario, Canada
4Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada
5Department of Pathology, University of Western Ontario, London, Ontario, Canada
6Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada
Thymidylate synthase (TS) catalyses the only de novo pathway to produce thymidylate for DNA replication and repair and is an important target for cancer chemotherapy. Preexisting or acquired drug resistance in tumor cells limits clinical efficacy of TS-targeting drugs. Cells selected for higher TS protein activity have decreased sensitivity to TS-targeting chemotherapeutic agents (5-FUdR and raltitrexed). New therapeutic strategies are required to overcome treatment resistance. Among these, upregulation of drug resistance mediators in normal, nontarget cells and/or antisense downregulation of those mediators (alone or in combination with protein-targeting drugs) are candidate strategies. We have targeted human TS mRNA with antisense oligodeoxynucleotides (AS ODNs), complementary to the translation start site (TSS), the coding region, and the 3´ untranslated region. We report here that, in response to treatment with a novel TSS-targeting AS ODN 791, TS gene transcription in a human cervical carcinoma cell line (HeLa) was unexpectedly increased by 70%. Interestingly, the increased TS gene transcription and nuclear TS RNA did not elevate levels of total cellular TS mRNA, but did increase TS protein activity by 35% and TS protein level by 150%. Increased TS protein activity and level did not alter proliferation rate or sensitivity to TS-targeting drugs (5-FUdR or raltitrexed). To assess concentration-dependent effects of TS on sensitivity to TS-targeting drugs, incremental increases of TS protein levels were generated by transfection of a mammalian TS expression vector. Increases in TS protein of less than approximately 400% did not significantly affect sensitivity to TS-targeting drugs, while greater TS protein levels did. These data indicate that AS ODNs targeting TS mRNA can upregulate TS expression and activity in a manner dependent on the sequence being targeted, and that there exists a threshold increase (greater than approximately 400-700% in HeLa cells), required to initiate resistance to TS-targeting drugs.
Key words: Antisense oligodeoxynucleotides; Thymidylate synthase; Chemotherapy; Drug resistance; Run-on transcription; Threshold
Address correspondence to James Koropatnick, Victoria Research Laboratories, The London Regional Cancer Program, London Health Sciences Centre, London, Ontario, Canada, N6A 4L6. Tel: (519) 685-8654; Fax: (519) 685-8616; E-mail: firstname.lastname@example.org
The bHLH Domain of Mist1 Is Sufficient to Activate Gene Transcription
Thai Tran, Di Jia, Yan Sun, and Stephen F. Konieczny
Department of Biological Sciences and the Purdue Cancer Center, Purdue University, West Lafayette, IN, USA
Mist1 is a tissue-specific basic helix-loop-helix (bHLH) transcription factor that plays an essential role in maintaining and organizing the exocrine pancreas. Consequently, mice lacking Mist1 exhibit disrupted acinar cellular polarity and defective zymogen granule trafficking. Despite extensive studies demonstrating a requirement for Mist1 in exocrine pancreas development and function, little is known about the molecular targets for Mist1 interaction and the mechanism(s) of how Mist1 regulates gene transcription. To address these deficiencies, a series of molecular studies was performed to identify the preferred Mist1 dimer complex and to establish the preferred DNA binding site for this bHLH factor. In vivo coimmunoprecipitation assays confirmed that the functional Mist1 complex in pancreatic acinar cells was a Mist1 homodimer that bound to a unique DNA target site known as the TA-E-box. Binding of Mist1 to a TA-E-box-regulated promoter led to transcriptional activation of the target gene. Surprisingly, Mist1 truncations containing only the central bHLH domain retained approximately 80% of transcriptional activity. Coimmunoprecipitation studies demonstrated that the bHLH domain interacted with coactivators belonging to the p300/CBP family, suggesting that Mist1 activates exocrine-specific gene transcription through an acetylation mechanism.
Key words: Coactivator; Pancreas; Gene regulation; Transcription factor; CBP
Address correspondence to Stephen F. Konieczny, Department of Biological Sciences and the Purdue Cancer Center, Purdue University, Hansen Life Sciences Research Building, 201 South University Street, West Lafayette, IN 47907-2064, USA. Tel: 765-494-7976; Fax: 765-496-2536; E-mail: email@example.com
Transcription Coactivator PRIP, the Peroxisome Proliferator-Activated Receptor (PPAR)-Interacting Protein, Is Redundant for the Function of Nuclear Receptors PPARa and CAR, the Constitutive Androstane Receptor, in Mouse Liver
Joy Sarkar,1 Chao Qi,1 Dongsheng Guo,1 Mohamed R. Ahmed,1 Yuzhi Jia,1 Nobuteru Usuda,2 Navin Viswakarma,1 M. Sambasiva Rao,1 and Janardan K. Reddy1
1Department of Pathology, Northwestern University, Feinberg
School of Medicine, Chicago, IL 60611, USA
2Department of Anatomy, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
Disruption of the genes encoding for the transcription coactivators, peroxisome proliferator-activated receptor (PPAR)-interacting protein (PRIP/ASC-2/RAP250/ TRBP/NRC) and PPAR-binding protein (PBP/TRAP220/DRIP205/MED1), results in embryonic lethality by affecting placental and multiorgan development. Targeted deletion of coactivator PBP gene in liver parenchymal cells (PBPLiv-/-) results in the near abrogation of the induction of PPARa and CAR (constitutive androstane receptor)-regulated genes in liver. Here, we show that targeted deletion of coactivator PRIP gene in liver (PRIPLiv-/-) does not affect the induction of PPARa-regulated pleiotropic responses, including hepatomegaly, hepatic peroxisome proliferation, and induction of mRNAs of genes involved in fatty acid oxidation system, indicating that PRIP is not essential for PPARa-mediated transcriptional activity. We also provide additional data to show that liver-specific deletion of PRIP gene does not interfere with the induction of genes regulated by nuclear receptor CAR. Furthermore, disruption of PRIP gene in liver did not alter zoxazolamine-induced paralysis, and acetaminophen-induced hepatotoxicity. Studies with adenovirally driven EGFP-CAR expression in liver demonstrated that, unlike PBP, the absence of PRIP does not prevent phenobarbital-mediated nuclear translocation/retention of the receptor CAR in liver in vivo and cultured hepatocytes in vitro. These results show that PRIP deficiency in liver does not interfere with the function of nuclear receptors PPARa and CAR. The dependence of PPARa- and CAR-regulated gene transcription on coactivator PBP but not on PRIP attests to the existence of coactivator selectivity in nuclear receptor function.
Key words: Nuclear receptor coactivators; PRIP; PBP/TRAP220/MED1; CAR; PPARa
Address correspondence to Janardan Reddy, Department of Pathology, Northwestern University, Feinberg School of Medicine, 303 East Chicago Avenue, Chicago, IL 60611, USA. Tel: 312-503-7948; Fax: 312-503-8249; E-mail: firstname.lastname@example.org
Widespread, Exceptionally High Levels of Histone H3 Lysine 4 Trimethylation Largely Mediate "Privileged" Gene Expression
Li Chen,1 Pervez Firozi,1 Michelle Barton,3 and Nancy Smyth Templeton1,2
1Center for Cell and Gene Therapy, Baylor College of Medicine,
Houston, TX, 77030, USA
2Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, 77030, USA
3Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
We examined the molecular determinants for sustained high-level expression of "privileged" genes, defined as the 0.03% most highly expressed genes within any specific cell. We identified histone modifications by chromatin immunoprecipitation analyses on Keratin 8, the most highly expressed gene in the human breast cancer cell line, MCF-7, based on serial analysis of gene expression. Quantitative comparisons to the "normal" counterpart cell line, MCF-10A, expressing 350-fold lower levels of Keratin 8 and other breast cancer cell lines expressing higher levels were performed using real-time PCR. Extraordinarily high levels of trimethyl histone H3 lysine 4 (H3K4) were found primarily in the first intron of the Keratin 8 gene stretching from 400 to 2000 bp downstream from the promoter in all breast cancer cells lines but not in MCF-10A cells. The highest levels of histone H3K4 trimethylation in MCF-7 cells ranged from 70% to 80% over input within 1200 bp of this region. Knockdown of mixed-lineage leukemia (MLL), the specific methyltransferase for histone H3K4, with MLL-specific siRNA decreased histone H3K4 trimethylation on the Keratin 8 gene and decreased Keratin 8 mRNA levels. Histone H3K4 trimethylation mediates approximately 86% of the elevated, sustained expression of the Keratin 8 gene in MCF-7 cells.
Key words: Histone modifications; H3K4 trimethylation; Gene expression; SAGE; Mixed-lineage leukemia (MLL)
Address correspondence to Nancy Smyth Templeton, Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Alkek Bldg., Rm. N1010, Houston, TX 77030, USA. Tel: (713) 798-1231; Fax: (713) 798-1230; E-mail: NANCYT@bcm.edu
Expression of the Prion Protein Gene (PRNP) and Cellular Prion Protein (PrPc) in Cattle and Sheep Fetuses and Maternal Tissues During Pregnancy
Patama Thumdee,1 Siriluck Ponsuksili,2 Eduard Murani,2 Korakot Nganvongpanit,1 Bernhard Gehrig,3 Dawit Tesfaye,1 Markus Gilles,1 Michael Hoelker,1 Danyel Jennen,1 Josef Griese,1 Karl Schellander,1 and Klaus Wimmers2
1Institute of Animal Science, Animal Breeding and Husbandry
Group, University of Bonn, Bonn, Germany
2Research Institute for the Biology of Farm Animals, Dummerstorf, Germany
3Institute of Animal Physiology, Biochemistry and Hygiene, University of Bonn, Bonn, Germany
We investigated the expression of prion protein gene both on mRNA and protein levels in bovine and ovine female reproductive organs during gestation and various tissues of their fetuses. The fetal tissues of both species included brain, cotyledon, heart, intestine, kidney, liver, lung, and muscle. In cattle, prion protein gene (PRNP) transcripts were detected by semiquantitative RT-PCR in reproductive tissues such as ovary, oviduct, endometrium, myometrium, follicles, and granulosa cells. In various tissues of 2-month-old fetuses, higher expression levels were found in brain and cotyledon compared to the other tissues. To detect the expression of the gene transcript in in vivo preimplantation embryos and 1-month-old fetuses, real-time PCR was performed showing that the level of gene expression in zygote stage was significantly higher (p < 0.05) than that of the other stages. Sheep were categorized as resistant (R1) or high susceptible (R5) to scrapie according to their PRNP genotype. In both genotype groups, the PRNP mRNA was detectable in all tissues studied including ovary, oviduct, endometrium, myometrium, and caruncle of ewes and all tissues of 2-month-old fetuses of both groups. Comparison between reproductive organs demonstrates the highest expression level in caruncle tissue of R1 ewes, whereas the level was high in brain and low in liver of both R1 and R5 fetuses. In addition, real-time RT-PCR was performed in immature oocytes, mature oocytes, in vivo embryos at morula stage, and 1-month-old fetuses. The results showed that the relative expression levels of the ovine PRNP mRNA in mature oocytes and morula stage embryos were significantly lower than those in immature oocytes and 1-month-old fetuses (p < 0.05). Western blot analyses revealed the immunoreactive bands corresponding to the cellular prion protein (PrPc) in all maternal and fetal tissues examined of both cattle and sheep. Moreover, immunohistochemical staining implicated localization of the PrPc in ovarian cortex and ovarian medulla of both species. However, PrPc was not detected in oocyte, granulosa cells, theca cells, and corpus luteum in this study.
Key words: Prion protein gene (PRNP); Cellular prion protein (PrPc); Prion; Preimplantation embryo; Reproductive organs; Pregnancy; Fetus
Address correspondence to Klaus Wimmers, Ph.D., Research Institute for the Biology of Farm Animals, Molecular Biology Research Division, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany. Tel: (+49) 038208 68700; Fax: (+49) 038208 68702; E-mail: email@example.com