Survival functions of HTLV-1 infected T-cells during the leukemogenic process [Elektronische Ressource] / vorgelegt von Klemens Pichler

De
klemens pichlerS U R V I V A L F U N C T I O N S O F H T L V -1 - I N F E C T E D T C E L L S D U R I N GT H E L E U K E M O G E N I C P R O C E S SS U R V I V A L F U N C T I O N S O F H T L V -1 - I N F E C T E D T C E L L SD U R I N G T H E L E U K E M O G E N I C P R O C E S SDer Naturwissenschaftlichen Fakultätder Friedrich-Alexander-Universität Erlangen-NürnbergzurErlangung des Doktorgradesvorgelegt vonklemens pichleraus RotthalmünsterAls Dissertation genehmigtvon der Naturwissenschaftlichen Fakultätder Universität Erlangen-NürnbergTag der mündlichen Prüfung: 17. Februar 2009Vorsitzender derPromotionskommission: Prof. Dr. Eberhard BänschErstberichterstatter: Prof. Dr. Bernhard FleckensteinZweitberichterstatter: Prof. Dr. Lars NitschkeC O N T E N T Si summary - zusammenfassung . . . . . . . . . . . . . . . . . . . . . . . . . . . 1ii introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Human T-lymphotropic virus type 1 . . . . . . . . . . . . . . . . . . . . . . . . 32 Costimulatory TNF receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 MicroRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Project rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7iii materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Publié le : jeudi 1 janvier 2009
Lecture(s) : 20
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Source : WWW.OPUS.UB.UNI-ERLANGEN.DE/OPUS/VOLLTEXTE/2009/1259/PDF/KLEMENSPICHLERDISSERTATION.PDF
Nombre de pages : 85
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k l emen s p i chler S U R V I V A L F U N C T I O N S O F H T L V -1 E L L S DI N F E C T E D T C-  U R I N G T H E L E U K E M O G E N I C P R O C E S S
S U R V I V A L F U N C T I O N S O F H T L V -1- I N F E C T E D E L L S T C D U R I N G T H E L E U K E M O G E N I C P R O C E S S
Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades
vorgelegt von k l emens p ichler
aus Rotthalmünster
Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung:
Vorsitzender der Promotionskommission: Erstberichterstatter: Zweitberichterstatter:
17. Februar2009
Prof. Dr. Eberhard Bänsch Prof. Dr. Bernhard Fleckenstein Prof. Dr. Lars Nitschke
res u l t s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 21 1exhibit a defined expression pattern in HTLV-Costimulatory TNF receptors transformed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21 2TNFRSF7(CD27) and ligand are not affected by HTLV-1Tax . . . . . . . . . .23 3TNFRSF9(4-1BB) is consistently expressed in HTLV-transformed cell lines .24 4The ligand of4-1BB is present on HTLV-transformed cells . . . . . . . . . . .27 5Tax stimulates gene expression of4-1 . . . . . . . . . . .BB . . .28 . . . . . . . . 6The4-1BB promoter is transactivated by Tax via NF-κB . . . . . . . . . . . . .30 7High4-1 . . . .  .BB expression coincides with a premitotic state. . . . . . .32 8Seven miRNAs are candidates for a role in HTLV pathogenesis . . . . . . . .34 9transcript of the BIC oncogene is up-regulated in HTLV-The primary transformed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35 10A set of regulatory T cell-specific microRNAs is dysregulated in an HTLV context . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .36 11Endogenous miR-146. . . . . . . . . . . . . . . . . . . . a is stimulated by Tax38 12The MIRN146A promoter is transactivated by Tax via NF-κ . . . . . . . .B .38 13 .Target genes display collaborative binding sites for up-regulated miRNAs .39
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summ a r y-z u samm e n fassung
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ma t e r i als and met h o d s. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 8 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell culture .8 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Nucleic acid detection .14 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Nucleic acid manipulation17 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  .Protein detection17 5Promoter analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18 6. . . . . . . . . . . . . . . . . . . . . .Bioinformatic analysis . . . . . . . . . . 19 7Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
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int r o d uction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 1Human T-lymphotropic virus type1. . . . . . . . . . . . . . . . .. . . . . . . 3 2. . . . . . . . . . . . . . . . . . . . . . . . . . .  .Costimulatory TNF receptors4 3MicroRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 4Project rationale. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
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L I S T
O F
Figure1
Figure2 Figure3
Figure4 Figure5
Figure6 Figure7 Figure8 Figure9 Figure10
Figure11 Figure12
Figure13
Figure14 Figure15 Figure16
L I S T O F
Table1 Table2 Table3 Table4
Table5 Table6
F I G U R E S
The TNF receptor superfamily . . . . . . . . . . . . . . . . . . . . . . .5 Biogenesis and functions of microRNAs . . . . . . . . . . . . . . . . . .6 Lack of CD27expression and repressive Tax effect in HTLV-transformed cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23 Tax-independent presence of CD70on HTLV-transformed cells . . . .24 Up-regulation of4-1BB mRNA in HTLV-1 . . . ./Tax-positive cells .25 Surface expression of4-1 .BB on transformed T cells . . . . . . . . . .26 Presence of4-1BBligand (4-1BBL . . . .) mRNA in transformed cells27 Surface expression of4-1BB ligand on transformed cells . . . . . . . .28 Kinetics of4-1BB. . . . . . . . . . . . . . . . . . . . . induction by Tax28 Induction of4-1BB .in T cells by Tax. . . . . . . . . . . . . . . . . . . 29 Transactivation of the4-1BBpromoter by Tax . . . . . . . . . . . . . .31 Increased frequency of a premitotic state in transformed,4-1BBhigh cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33 Expression of pri-miR-155in transformed cells . . . . . . . . . . . . . .35 Dysregulation of oncogenic microRNAs . . . . . . . . . . . . . . . . . .37 Stimulation of endogenous miR-146 . . . . .  .a by Tax. . . . . . . . .39 Transactivation of MIRN146 . . . . . . . . . . . .A promoter by Tax .40
T A B L E S
Cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 Sequences of oligonucleotides . . . . . . . . . . . . . . . . . . . . . . .15 Expression of TNF(R) superfamily members . . . . . . . . . . . . . . .22 MicroRNAs selected for potential involvement in HTLV pathogenesis34 Correlation of provirus copy number and microRNA expression . . .38 MicroRNA target predictions. . . . . . . . . . . . . . . . . . . . . . . . .41
v
S U M M A R Y
s u mmar y
-
Z U S A M M E N F A S S U N G
I
Human T-lymphotropic virus type1(HTLV-1), the cause of adult T cell leukemia, stim-ulates the growth of infected T cells in cultures and in non-leukemic patients. In the latter, HTLV-1is found in long-term persisting T cell clones. The persistence of normal T cells is controlled by the growth-stimulating and antiapoptotic functions of costimula-tory receptors, while the growth-stimulating HTLV-1functions are mediated by the viral oncoprotein Tax. Among costimulatory receptors of the tumor necrosis factor receptor su-perfamily,4-1BB(TNFRSF9/CD137/ILA) was strongly expressed in HTLV-transformed cells. Upregulated4-1BBexpression was a consistent feature of all HTLV-1-infected cell lines, whether patient-derived orin-vitro-transformed. Tax was sufficient to induce the expression of the endogenous4-1BBgene in uninfected T cells and it strongly activated (45-fold) the4-1BBpromoter via a single NF-κB site. The ligand of4-1BB was also found on transformed T cell lines opening the way for autostimulatory events. Moreover,4-1BB expression in patients’ lymphocytesex vivocorrelated with Tax expression, strongly sug-gesting Tax-mediated4-1BBactivationin vivo. Simultaneous determination of4-1BB ex-pression and cell cycle phase revealed that HTLV-transformed cells exhibiting high4-1BB expression appeared more often in a premitotic state, i.e., in G2and M phase. Thus, 4-1BB upregulation by Tax could contribute to growth, survival and clonal expansion of the infected cells during persistence and disease. MicroRNAs are regulators of gene expression and, as such, also impinge upon growth and apoptosis of cells. Since HTLV-1does not encode any microRNAs of its own, it has to reuse cellular microRNAs to access their regulatory potential. Based on pheno-typic parallels to regulatory T cells and links to oncogenicity, seven cellular microRNAs were investigated in HTLV-transformed T cells. Four out of those, miRs21,24,146a and 155, were significantly upregulated and one, miR-223, significantly downregulated. Sub-sequent testing for sensitivity of microRNA expression to viral proteins returned one – miR-146a – that was affected by Tax. The effect of Tax turned out to be transactivation of the promoter ofMIRN146Agene via the NF-κB pathway. This constituted the first description of a direct influence of HTLV-1on cellular microRNA expression. Anin silico target gene analysis singled out genes involved in regulation of transcription and cell survival, which might be used by the virus to maintain persistence after infection.
1
z u samm e n fassung
Das Humane T-lymphotrope Virus Typ1(HTLV-1) kann eine aggressive Neoplasie ver-ursachen, die adulte T-Zell-Leukämie, und stimuliert sowohl in Zellkultur als auch in asymptomatisch Infizierten das Zellwachstum. In letzteren findet sich HTLV-1in T-Zell-klonen, die über lange Zeit persistieren. Das Überleben von T-Zellen wird unter anderem
durch kostimulatorische Rezeptoren reguliert, die wachstumsstimulierende und antia-poptotische Funktionen ausüben. In HTLV-infizierten Zellen werden diese Funktionen
vom Virus beeinflusst, hauptsächlich vermittelt durch dessen Onkoprotein Tax. Ein Ver-treter der kostimulatorischen Rezeptoren aus der Superfamilie der Tumor-Nekrosefaktor-Rezeptoren,4-1BB (TNFRSF9/CD137/ILA), war in allen betrachteten HTLV-transformie-rten Zelllinien stark exprimiert, unabhängig von deren Herkunft aus Patienten oder Im-mortalisierungin vitroreichte aus, die endogene Expression von. Tax 4-1BB in uninfi-zierten T-Zellen zu steigern, und es aktivierte den4-1BBPromotor über eine einzelne NF-κB Bindestelle. Da auch der passende Ligand zum4-1BB Rezeptor,4-1BBL, auf den transformierten Zelllinien vorhanden war, besteht die Möglichkeit einer autostimulatori-schen Schleife.Ex vivoUntersuchungen von Zellen, die aus HTLV-1-infizierten Patienten gewonnen worden waren, bestätigten die aus der Zellkultur gewonnenen Ergebnisse: die Tax-Expression korrelierte mit der von4-1BB, was eine Stimulation des4-1BBGens durch Tax auchin vivonahe legt. Eine parallele Analyse der Expression von4-1BB in HTLV-transformierten Zellen und ihrer Zellzyklusphase zeigte ein vermehrtes Vorkom-men von Zellen mit hohem4-1BB Niveau in einem prämitotischen Zustand in der G2-bzw. M-Phase. Dies kann als Hinweis auf eine beschleunigte Proliferation der betreffen-den Zellen gedeutet werden. Auf diese Weise könnte HTLV-1Tax zum Wachstum, zum Überleben und zur klonalen Expansion infizierter Zellen während der Persistenz und HTLV-assoziierter Krankheit beitragen. MicroRNAs sind beteiligt an der Regulation von Genexpression und beeinflussen in dieser Funktion auch das Wachstum und die Apoptose von Zellen. Da HTLV-1selbst keine microRNAs kodiert, muss das Virus das regulatorische Potential zellulärer mi-croRNAs nutzen. Ausgehend von phänotypischen Gemeinsamkeiten zwischen HTLV-transformierten und regulatorischen T-Zellen sowie Überlegungen zur Onkogenität von microRNAs wurden sieben microRNAs in HTLV-transformierten Zellen untersucht. Vier der sieben – miRs21,24,146a und155– waren signifikant überexprimiert und eine, miR-223, signifikant reprimiert. In einem HTLV-freien Zellsystem stieg das Niveau endo-gener microRNA miR-146a in Gegenwart von ektopisch exprimiertem Tax an. Nachfol-gende Analysen des betreffendenMIRN146APromotors ergaben eine Transaktivierung durch Tax über den NF-κB Signalweg. Dies stellt die erste Beschreibung eines direkten Einflusses von HTLV-1auf die Expression zellulärer microRNAs dar und legt somit ei-ne Funktion in der Transformation von T-Zellen durch HTLV-1nahe. Die Anwendung bioinformatischer Methoden bei der Suche nach Genen, die möglicherweise durch die erwähnten, deregulierten microRNAs beeinflusst werden, erbrachte eine Liste von Ziel-genen, die an der Regulierung von Zellproliferation und Apoptose beteiligt sind.
2
I N T R O D U C T I O N
1 hu m a n t-l y mpho t r opic virus typ e 1
II
The human T-lymphotropic virus type1(HTLV-1), aδ-retrovirus, is the etiologic agent of a severe and fatal lymphoproliferative disorder of CD4+T cells, the adult T cell leukemia/lymphoma (ATLL), and of the neurodegenerative, inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Gessain et al.,1985, Osame et al.,1986,Poiesz et al.,1980,Yoshida et al.,1984 diseases develop as a). These consequence of prolonged viral persistence in T cells. As documented by many observations, HTLV-1has developed a unique strategy for lifelong persistence in the presence of an active immune system. It is characterized by replication of the virus mainly in its provirus form, stimulation of cell division by the virus, and, as a consequence, clonal amplification of infected cells. Evidence that HTLV-1 rarely replicates via reverse transcription but mostly by division of infected cells using cellular DNA polymerase is provided by the low mutation rate of the HTLV-1genome (Ina and Gojobori,1990) despite the poor fidelity of the HTLV-1reverse transcriptase, which is comparable to that of other retroviruses (Mansky and Bernard,2000). The stimulation of T cell proliferation in patients by viral gene expression was substantiated recently by cell dynamic studies (Asquith et al.,2007 revealed a correlation of the). These in vivoproliferation rate of CD4+CD45RO+cells with viral expression (ex vivo). Another indication that HTLV-1stimulates growth and survival of T lymphocytes is provided by their expansion to detectable clones, which can persist over many years even in non-leukemic individuals (Etoh et al.,1997,Gabet et al.,2000). Finally, the virus’s ability to stimulate permanent T lymphocyte growthin vitro, resulting in T cell immortalization (Grassmann et al.,2005), completes the arguments in favor of viral gene functions as cause of host cell proliferation and clonal expansion of patients’ lymphocytes. Besides structural proteins, HTLV-1the regulatory proteins Tax and Rex,encodes which are essential for viral replication (Kashanchi and Brady,2005), and the accessory proteins p12, p30, p13(Nicot et al.,2005,Lairmore and Franchini,2007) and HBZ (Mes-nard et al.,2006Tax strongly enhances viral mRNA synthesis by transactivating). While the HTLV-1terminal repeat promoter, Rex controls the synthesis of the structurallong proteins on a posttranscriptional level (Bogerd et al.,1992,Gröne et al.,1996). Although the accessory proteins are important for viral infectivity and replication (Nicot et al., 2005,Matsuoka and Jeang,2007), p12, p13, p30and HBZ are not required for lympho-cyte immortalization (Derse et al.,1997,Robek et al.,1998,Arnold et al.,2006).
3
Introduction
4
Tax is able to stimulate transcription by interacting with various signaling pathways. It activates nuclear factor kappa B (NF-κB) by two pathways: While the canonical pathway is induced by binding and stimulating IKKα, a component of the inhibitor of kappa B kinase (IKK) (Grassmann et al.,2005,Matsuoka and Jeang,2007), the activation of the non-canonical pathway is less well understood. Transactivation of various cellular promoters is mediated by Tax via direct contact with transcriptional activators CREB and SRF and with the coactivators p300/CBP (Kashanchi and Brady,2005,Fujii et al., 1995). Tax confers the transforming properties on HTLV-1(Grassmann et al.,2005). The pro-tein can immortalize T lymphocytes (Akagi et al.,1997,Schmitt et al.,1998) and induce leukemia in transgenic mice (Hasegawa et al.,2006). Biochemically, several Tax func-tions may contribute to its transforming capacity. At several check points, Tax interferes with normal cell cycle control (Grassmann et al.,2005,Marriott and Semmes,2005). In particular, it is capable of stimulating the G1to S phase transition through activation of cyclin-dependent kinase holoenzymes (Fraedrich et al.,2005,Haller et al.,2002), and, by disturbing cellular DNA repair and chromosomal segregation control pathways, it can induce chromosomal damage (Marriott and Semmes,2005 Tax interferes). Importantly, with tumor suppressor functions, for instance, it inactivates p53(Pise-Masison et al.,1998) and PDZ proteins (Ishioka et al.,2006,Xie et al.,2006). As a further means to promote cellular proliferation, Tax can stimulate the expression of cellular proteins controlling growth and survival (Silbermann and Grassmann,2007 particular, Tax-mediated). In modulation of cellular gene expression may explain the resistance of HTLV-1-positive cells to various proapoptotic stimuli (Tsukahara et al.,1999,Copeland et al.,1994,Yang et al.,2002,Wäldele et al.,2006,Hamasaki et al.,2001 the presence of Tax-). Moreover,
stimulated antiapoptotic proteins can be essential for cellular survival. For example, RNA interference(RNAi)-mediated knock-down of expression of the antiapoptotic pro-tein HIAP1/ciap2induces apoptosis in HTLV-1-transformed T cell lines (Wäldele et al., 2006).
2 cost i m ulatory tnf r e c e p t o r s
The persistence of HTLV-1in T cell clones, which are detectable over many years, sug-gests that proteins mediating survival and growth of long-lived T cells could be crucial for HTLV-1persistence and, thus, could be potential targets of its oncoprotein Tax. Such properties are characteristic of a subgroup of the tumor necrosis factor receptor (TNFR) superfamily, the costimulatory TNF receptors. In analogy to their counterpart in the immunoglobulin family, CD28, they provide additional signals to those from the T cell receptor, which are necessary to initiate or sustain T cell activation. While some costim-ulatory TNFR can, in principle, substitute for CD28signaling, most deliver their signals after CD28(Watts,2005). Within the large TNFR superfamily, costimulatory molecules are numbered among the TNF receptor-associated factor (TRAF)-binding subgroup (Figure1) and include GITR, OX40, CD27and4-1BB. Two other subgroups are the death domain-containing recep-tors, which can trigger apoptosis via interaction with FADD, and decoy receptors, which compete with the former for ligands yet do not signal (Aggarwal,2003 of the cos-). All timulatory receptors interact with TRAF2and one or more of the remaining five TRAFs,
factor receptor1; DR3/5, death receptors3and5; DD, death domain; FADD, Fas-associated via death domain; TRADD, TNFR1-associated death domain protein; DCR13, decoy receptors1,2 and3; GITR, glucocorticoid-induced TNFR-related gene; TRAF, TNF receptor-associated factor; JNK, janus kinase; AP-1, transcription factor; IKK, inhibitor ofκB kinase; NF-κB, nuclear factor κB. Based onWatts(2005) andAggarwal(2003).
dr3 dr5
inactive DD
DD
surface
dcr1
dcr3
fas tnfr1
death domain containing
Introduction
5
decoy
dcr2
AP-1
caspase cascade
apoptosis
Figure1: ThreeThe TNF receptor superfamily. major subgroups are distinguished – receptors containing cytoplasmic death domains, decoy receptors and those featuring cytoplasmic binding sites forTNF receptor associated factors(TRAFs). Fas, (TNFRSF6, CD95); TNFR1, tumor necrosis
IKK
TRAFs
JNK
NF-κB
survival
traf-binding
gitr ox40 cd27 4-1bb
TRAF bind-ing sites
cytoplasm
TRADD
FADD
Casp8
Introduction
Figure2:Biogenesis and functions of microRNAs. See text for details.
6
which determines the signaling pathways that can be activated by them (Dempsey et al., 2003). TRAF2and5, for example, activate NF-κB, which in turn triggers expression of TRAF1, cIAP1and2(HIAP1), Bcl-XLor c-flip – proteins involved in antiapoptosis (Karin and Lin,2002); TRAF2can also use the mitogen-activated protein (MAP) kinase pathway (MAP3K and MAP4providing a link to JNK/SAPK and pK) 38MAPK cascades (Can-nons et al.,2000 being mostly absent from naive T cells, costimulatory TNFR). While superfamily members are present on long-lived T lymphocyte populations like memory and regulatory T cells, where they can augment proliferation and survival (Watts,2005).
3
micr o r nas
MicroRNAs (miRNAs) have been described as posttranscriptional regulators of gene ex-pression (Filipowicz et al.,2008 genes encoding them are transcribed by RNA). The polymerase II producing primary transcripts (pri-miR) which feature a stem-loop struc-ture that is excised by an RNase, Drosha, in conjunction with the DGCR8protein (Figure 2)(Lee et al.,2003 resulting hairpin, termed precursor microRNA (pre-miR), is). The exported to the cytoplasm where another RNase, Dicer, converts it to the mature single-stranded, about23nucleotides long microRNA (Kim and Nam,2006). The microRNAs are incorporated into protein complexes, the microRNA-induced silencing complexes (miRISC), which then exert the microRNA’s function. Within the miRISC, the microRNA probably acts as a guide that directs the complex to its target by binding to the3’ un-translated regions (3 Unlike small interfering RNAs (siRNA),’-UTRs) of target mRNAs. sequence complementarity between microRNAs and their targets is rather imperfect with
only a core of seven nucleotides at the5’-end at the microRNA, the seed sequence, usu-
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