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Manicone, Mariangela (2013) Functional interactions of the Tax and p13 proteins of Human T-cell Leukemia Virus Type I. [Ph.D. thesis]

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Abstract (english)

Human T-cell leukemia virus type 1 (HTLV-1) establishes a lifelong persistent infection in humans. Approximately 3% of the infected individuals will develop adult T-cell leukemia/lymphoma (ATLL), an aggressive malignancy of mature CD4+ T-cells. The viral protein Tax plays a major role in HTLV-1 pathogenicity by activating the NF-κB pathway. Tax activates both the canonical and non-canonical NF-κB pathways, promoting NF-κB translocation to the nucleus and transcription of genes that favour T-cell proliferation and survival. Our previous studies showed that the p13 protein of HTLV-1 enhances mitochondrial ROS production, resulting in activation of normal T-cells. ROS constitute a homeostatic rheostat that controls the activity of several key pathways, including the NF-κB pathway.Thus, we hypothesized that the effects of p13 on ROS production could affect the activation of the NF-κB pathway by Tax in primary T-cells.
The work described in the present thesis was aimed at testing the hypothesis that Tax and p13 might act in concert to activate the NF-κB signal transduction pathway in primary T-cells. To this end, we optimized a transfection protocol for primary T-cells using an innovative approach based on the electroporation of in vitro-transcribed RNA. Activation of the NF-κB pathway was then analysed by measuring expression of the NF-κB target genes CD25 and 4-1BB.
Results showed that the co-transfection of Tax and p13 resulted in a synergistic activation of the NF-κB pathway in primary T-cells measured as an increase in the expression levels of both CD25 and 4-1BB. In addition to being a transcriptional target of NF-κB, CD25 is also an early marker of T-cell activation. To further test the effects of Tax and p13 on cell activation, we measured CD38 expression by flow cytometry. Jurkat T-cells, which exhibit a constitutively activated CD38 positive phenotype, were used as a control. Results of this analysis confirmed the synergy of Tax and p13, although the effect was not so prominent as that observed for the CD25 marker, suggesting that, within the time frame of our experiments, Tax and p13 drove T-cells to an early-intermediate stage of activation.
Taken together, these findings suggest that, in contrast to the well-established role of Tax as an activator of the NF-κB pathway in tumor cell lines, in the context of normal T-cells, the induction of NF-κB target genes requires the concerted action of Tax and p13.
Current studies are aimed at verifying the ROS-dependence of this effect and testing the functional interaction of Tax and p13 in the context of the complete HTLV-1 genome using wild type HTLV-1 and a p13-knock-out HTLV-1 molecular clone. These experiments will be carried out in primary T-cells as well as in dendritic cells, which have recently emerged as an important target of the virus in vivo.

Abstract (italian)

Il virus T-linfotropico umano di tipo 1 (HTLV-1) stabilisce un'infezione persistente negli uomini. Circa il 3% degli individui infettati sviluppa la leucemia/linfoma a cellule T dell'adulto (ATLL), un'aggressiva neoplasia a carico dei linfociti T CD4+ maturi. L'attivazione della via di segnale di NF-κB mediata dalla proteina virale Tax è un evento cruciale nella patogenesi dell’infezione da HTLV-1. Tax attiva entrambe le vie di segnale di NF-κB, canonica e non-canonica, promuovendo la traslocazione nucleare di NF-κB e la trascrizione di geni che favoriscono la proliferazione e la sopravvivenza delle cellule T. I nostri studi precedenti hanno rivelato che la proteina virale p13 favorisce la produzione di specie reattive dell'ossigeno (ROS) a livello mitocondriale, causando l'attivazione di cellule T normali. I ROS possono essere paragonati ad un reostato che controlla l'attività di diverse vie di trasduzione del segnale, inclusa la via di NF-κB.
Lo scopo primario di questa tesi è stato quindi di verificare l'ipotesi che Tax e p13 potessero attivare sinergicamente la via di trasduzione del segnale di NF-κB in cellule T normali. A tal fine, è stato ottimizzato un protocollo di trasfezione di cellule T primarie utilizzando un approccio innovativo basato sull'elettroporazione di RNA trascritto in vitro. L'attivazione della via di NF-κB è stata analizzata misurando l'espressione dei geni target di NF-κB CD25, mediante analisi citofluorimetrica, e 4-1BB, mediante RT-PCR quantitativa.
I risultati ottenuti hanno mostrato che in cellule T normali, la co-trasfezione di Tax e p13 causa l'attivazione sinergica della via di NF-κB misurata come incremento dei livelli di espressione di entrambi i geni target. Oltre ad essere un target trascrizionale di NF-κB, il CD25 è anche un marcatore precoce di attivazione di cellule T. Per verificare il possibile effetto di Tax e p13 sull'attivazione cellulare, abbiamo misurato mediante analisi citofluorimetrica l'espressione del CD38, un marcatore intermedio-tardivo di attivazione. La linea T-cellulare leucemica Jurkat, caratterizzata da un fenotipo costitutivamente CD38 positivo, è stata utilizzata come controllo. I risultati di questa analisi hanno confermato la sinergia di Tax e p13, nonostante l'effetto sull’espressione del CD38 non fosse così prominente come quello osservato per il CD25, suggerendo che, nel nostro contesto sperimentale, Tax e p13 spingano le
cellule T in uno stadio precoce-intermedio di attivazione.
In complesso questi risultati suggeriscono che, in contrasto con il ruolo ben stabilito di Tax nell'attivazione della via di NF-κB in linee cellulari tumorali, nel contesto delle cellule T normali, l'induzione dei geni target di NF-κB necessita l'azione sinergica di Tax e p13.
Gli studi attualmente in corso sono volti a verificare la ROS-dipendenza dell'effetto sinergico di Tax e p13 sulla via di segnale di NF-κB. Inoltre, verificheremo la validità dell'interazione funzionale di Tax e p13 nel contesto del intero genoma di HTLV-1. A tal fine, paragoneremo l'attivazione della via di NF-κB indotta da un clone molecolare di HTLV-1 wild type, con quella indotta da un clone molecolare di HTLV-1 p13-knock-out. Questi esperimenti verranno condotti in cellule T primarie ed in cellule dendritiche, che rappresentano il principale target infezione da HTLV-1 in vivo.

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EPrint type:Ph.D. thesis
Tutor:Ciminale, Vincenzo
Ph.D. course:Ciclo 25 > Scuole 25 > ONCOLOGIA E ONCOLOGIA CHIRURGICA
Data di deposito della tesi:24 January 2013
Anno di Pubblicazione:23 January 2013
Key Words:Leukemia, Virus, T-cell, NF-κB
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/06 Oncologia medica
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Chirurgiche Oncologiche e Gastroenterologiche
Codice ID:5445
Depositato il:15 Oct 2013 09:54
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