The Tax protein of human T-cell lymphotropic virus type 1 (HTLV-1) as multifunctional oncoprotein [Elektronische Ressource] = Das Tax-Protein des humanen T-Zell-lymphotropen Virus Typ 1 (HTLV-1) als multifunktionelles Onkoprotein / vorgelegt von Karin Andrea Kreß

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• Biologie

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• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2011
• Nombre de lectures: 18
• Poids de l'ouvrage: 3 Mo

Extrait

THE TAX PROTEIN OF HUMAN T CELL
LYMPHOTROPIC VIRUS TYPE 1 (HTLV-1) AS
A MULTIFUNCTIONAL ONCOPROTEIN

DAS TAX PROTEIN DES HUMANEN T-ZELL-
LYMPHOTROPEN VIRUS TYP 1 (HTLV-1)
ALS MULTIFUNKTIONELLES ONKOPROTEIN






Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität
Erlangen-Nürnberg
zur Erlangung des
Doktorgrades Dr. rer. nat.










vorgelegt von
Karin Andrea Kreß
aus Neustadt/Aisch























Als Dissertation genehmigt
von der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg

















Tag der mündlichen Prüfung: 31. Januar 2011

Vorsitzender der
Promotionskommission: Prof. Dr. Rainer Fink
Erstberichterstatter: Prof. Dr. Bernhard Fleckenstein
Zweitberichterstatter: Prof. Dr. Robert Slany





















Jeder Augenblick ist
von unendlichem Wert.
Seneca

















Im Gedenken an meinen ersten Doktorvater
Prof. Dr. Ralph Grassmann †
1957-20084
THE TAX PROTEIN OF HUMAN T CELL LYMPHOTROPIC VIRUS TYPE 1
(HTLV-1) AS A MULTIFUNCTIONAL ONCOPROTEIN

Contents 

I. Summary ........................................................................................................................... 6

I. Zusammenfassung ........................................................................................................... 7

II. Introduction ...................................................................................................................... 8
1. Human T cell lymphotropic virus type 1 (HTLV-1) ............................................................. 8
2. Differentiation of human T lymphocytes and HTLV-1 persistence................................... 11
3. Cyclic AMP metabolism ........................................................................................................ 13
4. The tumour marker and actin-bundling protein Fascin..................................................... 14

III. Project Rationale............................................................................................................ 16

IV. Material and Methods.................................................................................................... 17
1. Cell culture.............................................................................................................................. 17
2. Nucleic acid detection............................................................................................................. 20
3. manipulation ..................................................................................................... 22
4. Transfection and transduction techniques........................................................................... 25
5. Protein detection..................................................................................................................... 26
6. Cell based assays and functional assays ............................................................................... 29
7. Bioinformatics and statistics.................................................................................................. 33

V. Results ............................................................................................................................. 34
1. The costimulatory receptors GITR, 4-1BB and 4-1BB ligand (4-1BBL) are overexpressed
on HTLV-1-transformed cells ............................................................................................... 34
2. HTLV-1-transformed cells exhibit a long-lived phenotype ................................................ 39
3. The transactivating capacity of Tax varies during T cell activation ................................. 43
4. cAMP levels are elevated in HTLV-1-transformed cells .................................................... 46
5. Tax is necessary for the maintenance of intracellular cAMP............................................. 48
6. Phosphodiesterase PDE3B is downregulated in HTLV-1-transformed cells.................... 51
7. PDE3B and cAMP concentrations inversely correlate in HTLV-1-transformed cells..... 55
8. Inhibitory histone modifications are increased at the PDE3B promoter.......................... 57
9. cAMP and PDE3B are deregulated in other lymphomas ................................................... 58
10. HTLV-1-transformed cells communicate with other lymphocytes via gap junctions, but
transfer of ectopic cAMP is not mediated by gap junctions............................................... 60 5
11. The tumour marker Fascin is specifically overexpressed in virus-transformed cells.. 63
12. Tax is necessary and sufficient to induce Fascin............................................................. 65
13. Tax-mediated induction of Fascin requires NF- κB signalling ....................................... 67
14. Fascin is induced by other viral oncogenes...................................................................... 69
15. Knockdown of Fascin reduces the invasive capacity of ATLL-derived cells................ 70

VI. Discussion ....................................................................................................................... 73
1. HTLV-1-transformed cells have a long-lived phenotype with potential relevance to
proliferation and survival.................................................................................................... 73
2. Cyclic AMP and PDE3B are deregulated in HTLV-1-transformed cells.................... 75
3. The tumour marker Fascin is strongly induced by HTLV-1/Tax through NF- κB
signals in T cells .................................................................................................................... 79

VII. References....................................................................................................................... 83

VIII. Publications....... 93

IX. Appendix ........................................................................................................................... 97

6 I. SUMMARY
I. SUMMARY
+Oncogenic transformation of CD4 T cells by human T cell lymphotropic virus type 1
(HTLV-1) is understood as the initial step to adult T cell leukaemia/lymphoma (ATLL), a
+severe neoplasm of CD4 T cells. This process is mainly initiated by perturbation of
cellular signalling by the viral Tax oncoprotein, a potent transcriptional regulator. In this
study, novel Tax functions (1) on the phenotype of HTLV-1-transformed cells, (2) on
cyclic AMP (cAMP) regulation, and (3) on expression of the tumour marker Fascin, were
identified. First, characterisation of the phenotype of HTLV-1-transformed cells revealed
that these cells share phenotypic properties with long-lived T cell populations such as
memory and regulatory T cells (T reg) with regard to the expression of characteristic
surface markers. Given the fact that murine T reg-mediated suppression employs elevated
cAMP levels as a key regulator, cAMP levels were investigated in HTLV-1-transformed
cells next. Elevated cAMP concentrations were a consistent feature of all HTLV-1-
transformed cell lines, including HTLV-1 in vitro-transformed, Tax-transformed, and
patient-derived cells. In transformed cells with conditional Tax expression, high cAMP
coincided with the presence of Tax, but was lost without it. This work shows that the
cAMP-degrading phosphodiesterase 3B (PDE3B) was specifically downregulated in
HTLV-1-transformed cells, which was independent of Tax. PDE3B transcripts and cAMP
levels were inversely correlated in HTLV-1-transformed cells and overexpression of
PDE3B led to a decrease of cAMP. Decreased expression of PDE3B was associated with
inhibitory histone modifications at the PDE3B promoter and the PDE3B locus. In search
of novel biomarkers with relevance to oncogenesis, the tumour marker and actin-
bundling protein Fascin (FSCN1) was identified and found to be specifically and strongly
+upregulated in both HTLV-1-transformed and ATLL-patient-derived CD4 T cells.
Fascin is important for migration and metastasis in various types of cancer. In this study,
a direct link between a viral oncoprotein and Fascin expression was identified, as Tax
was sufficient to induce high levels of Fascin. NF- κB signals were important for Tax-
mediated induction of Fascin in T cells providing a new mode of transcriptional
regulation of Fascin. Functionally, knockdown of Fascin reduced the invasive capacity of
ATLL-derived cells into extracellular matrix. In summary, novel functions of Tax were
identified in this work. The data show that HTLV-1-transformed cells assume biological
features of long-lived T cell pop