Extra-telomeric functions of telomerase in the pathogenesis of Epstein-Barr virus-driven B-cell malignancies and potential therapeutic implications

Abstract The Epstein-Barr virus (EBV) is a ubiquitous human \u3b3-herpesvirus causally linked to a broad spectrum of both lymphoid and epithelial malignancies. In order to maintain its persistence in host cells and promote tumorigenesis, EBV must restrict its lytic cycle, which would ultimately lead to cell death, selectively express latent viral proteins, and establish an unlimited proliferative potential. The latter step depends on the maintenance of telomere length provided by telomerase. The viral oncoprotein LMP-1 activates TERT, the catalytic component of telomerase. In addition to its canonical role in stabilizing telomeres, TERT may promote EBV-driven tumorigenesis through extra-telomeric functions. TERT contributes toward preserving EBV latency; in fact, through the NOTCH2/BATF pathway, TERT negatively affects the expression of BZLF1, the master regulator of the EBV lytic cycle. In contrast, TERT inhibition triggers a complete EBV lytic cycle, leading to the death of EBV-infected cells. Interestingly, short-term TERT inhibition causes cell cycle arrest and apoptosis, partly by inducing telomere-independent activation of the ATM/ATR/TP53 pathway. Importantly, TERT inhibition also sensitizes EBV-positive tumor cells to antiviral therapy and enhances the pro-apoptotic effects of chemotherapeutic agents. We provide here an overview on how the extra-telomeric functions of TERT contribute to EBV-driven tumorigenesis. We also discuss the potential therapeutic approach of TERT inhibition in EBV-driven malignancies

Short-term inhibition of TERT induces telomere length-independent cell cycle arrest and apoptotic response in EBV-immortalized and transformed B cells

open7siBesides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumorigenesis through extra-telomeric functions. The possible therapeutic effects of BIBR1532 (BIBR), a powerful TERT inhibitor, have been evaluated in different cellular backgrounds, but no data are currently available regarding Epstein-Barr virus (EBV)-driven B-cell malignancies. Our aim was to characterize the biological effects of TERT inhibition by BIBR on EBV-immortalized lymphoblastoid cell lines (LCLs) and fully transformed Burkitt's lymphoma (BL) cell lines. We found that BIBR selectively inhibits telomerase activity in TERT-positive 4134/Late and 4134/TERT+ LCLs and EBV-negative BL41 and EBV-positive BL41/B95.8 BL cell lines. TERT inhibition led to decreased cell proliferation, accumulation of cells in the S-phase and ultimately to increased apoptosis, compared with mock-treated control cells. All these effects occurred within 72 h and were not observed in BIBR-treated TERT-negative 4134/TERT- and U2OS cells. The cell cycle arrest and apoptosis, consequent upon short-term TERT inhibition, were associated with and likely dependent on the activation of the DNA damage response (DDR), highlighted by the increased levels of γH2AX and activation of ATM and ATR pathways. Analyses of the mean and range of telomere lengths and telomere dysfunction-induced foci indicated that DDR after short-term TERT inhibition was not related to telomere dysfunction, thus suggesting that TERT, besides stabilizing telomere, may protect DNA via telomere-independent mechanisms. Notably, TERT-positive LCLs treated with BIBR in combination with fludarabine or cyclophosphamide showed a significant increase in the number of apoptotic cells with respect to those treated with chemotherapeutic agents alone. In conclusion, TERT inhibition impairs cell cycle progression and enhances the pro-apoptotic effects of chemotherapeutic agents in TERT-positive cells. These results support new therapeutic applications of TERT inhibitors in EBV-driven B-cell malignancies.openCeleghin, Andrea; Giunco, Silvia; Freguja, Riccardo; Zangrossi, Manuela; Nalio, Silvia; Dolcetti, Riccardo; De Rossi, AnitaCeleghin, Andrea; Giunco, Silvia; Freguja, Riccardo; Zangrossi, Manuela; Nalio, Silvia; Dolcetti, Riccardo; DE ROSSI, Anit

Effetti del knockdown del recettore degli estrogeni β2 (esr2a) sullo sviluppo di Danio rerio

ABSTRACT This thesis work is focused on the study of the role of the maternal mRNA for the estrogen receptor erβ2 (esr2a) on the long-term epigenetic modulation of development in zebrafish (Danio rerio). Experimental evidence indicates that steroid hormones may participate in the maternal programming of the development in fish (Auperin and Geslin, 2008) and mammals (Darnaudéry and Maccari, 2007). In particular, estrogens secreted by the granulosa cells during vitellogenesis may be incorporated in fish oocytes (Hines et al, 1999) with possible significant effects on the subsequent ontogeny of the offspring. In the present work on zebrafish, the expression levels of the mRNA coding for the Esr2a receptor were found to be high in ovulated oocytes and early embryos. The concentration of these transcripts decreased along the 8 hours post-fertilization (hpf) and then returned to rise with the onset of embryonic transcription. The gene knockdown by morpholino antisense technology was applied to investigate the functional role of the esr2a in zebrafish development. Two different morpholinos were used: the first, designed on the ATG start translation codon, determines the knockdown by blocking the translation of both maternal and zygotic transcripts (esr2aATGMO); the second interferes with the correct splicing process to block only the post-transcriptional maturation of the zygotic messenger (esr2aSPLICMO). The embryos microinjected with esr2aATGMO died between 12 and 14 days after fertilization (dpf) and displayed several morphological alterations compared to non-injected embryos (WT) or to controls microinjected with a standard unrelated morpholino (StdMO). The morphant phenotype of fish treated with esr2aATGMO was characterized by delayed body growth, curved shape, abnormal development of brain and splanchnocranium, enlarged and hemorrhagic pericardial cavity, uninflated swim bladder, rudimental caudal fin and abnormal circular swimming. The specificity of the effects was verified by co-injecting the esr2aATGMO with a morpholino inactivating the p53 protein. The phenotype obtained with the co-injection corresponded to that of fish injected with esr2aATGMO alone. This shows that the effects of the morpholino for esr2a are specific and not due to the hyperactivation of p53 caused by the microinjection. Resumption of the WT phenotype was achieved by co-injecting esr2aATGMO with the transcript for zebrafish Ers2a bearing 8 silent mutations in the region complementary to the morpholino. This demonstrates that the binding of the oligonucleotide to its target was very specific. To further investigate the role of the gene esr2a, an experiment of gain of function was performed by increasing the concentration of the receptor transcript. Developmental abnormalities were also monitored during first days after fertilization following the microinjection of the mRNA for esr2a into eggs permeated with a solution of 17β-estradiol (E2). The embryos treated with esr2aSPLICMO were comparable to controls. This morpholino interferes in the process of maturation of the zygotic transcript, leading to the loss of the third exon and to a translated protein that doesn't work. Significantly, this result suggests that the observed phenotypic changes after treatment with esr2aATGMO are mainly due to the inactivation of the maternal transcript. The in situ hybridization analysis using developmental markers highlighted a possible up-regulation of empty spiracles homeobox 1 (emx1) and of sine oculis homeobox homolog 3a (six3a) at 24 and 48 hpf and of sonic hedgehog (shha) at 48 hpf. Staining with alcian blue revealed an altered pattern of cranial cartilage organization at 6 dpf in fish treated with esr2aATGMO. Subsequent hybridization with cranial markers showed a slightly different expression profiles between WT and morphants of the gene distal-less homeobox gene 2a (dlx2a) at 48 hpf and neurogenic differentiation (NeuroD) at 24 hpf. Two-color microarray analysis of the total genome of zebrafish was used to study gene expression in embryos at 8 and 48 hpf after treatment with esr2aATGMO compared to controls injected with StdMO. Analysis at 8 hpf highlights changes in gene expression due mainly to the maternal receptor knockdown. The analysis at 48 hpf allows to detect morpholino effects due to the inactivation of embryonic transcripts as well. At 8 hpf, 237 transcripts were significantly up-regulated, while 219 were down-regulated as a result of the absence of the Esr2a receptor in embryos injected with esr2aATGMO. At 48 hpf, 165 and 124 transcripts, presumably of zygotic origin, were up- and down-regulated, respectively. Among these, only 8 were in common with those up-regulated at 8 hpf and 17 with the down-regulated ones. Interestingly, transcripts involved in the negative regulation of cell proliferation were up-regulated. It can be assumed that the absence of Esr2a leads to a decrease in cell proliferation, perhaps due to an increased apoptosis. This would be in keeping with the TUNEL analysis on the rate of cell death, which showed a greater number of apoptotic cells in the brain of morphant fish at 24 and 48 hpf as compared to controls. The analysis of embryonic vascularization in the transgenic line TG (fli1:EGFP) at 3 and 6 dpf, based on the vascular activity of endogenous alkaline phosphatase, evidenced a marked decrease in subintestinal vessel formation and derangement in the distribution patterns of trunk and tail vessels in fish treated with esr2aATGMO. These results suggest that the development of zebrafish is influenced by epigenetic control dependent on maternal esr2a mRNA and interaction with the inheritance of maternal estrogens.RIASSUNTO Il mio lavoro di tesi si è focalizzato sullo studio del ruolo svolto dagli mRNA ovocitari di origine materna codificanti la forma erβ2 (esr2a) del recettore degli estrogeni nella modulazione epigenetica a lungo termine dello sviluppo in zebrafish (Danio rerio). Crescenti evidenze indicano che gli ormoni steroidei possono partecipare alla programmazione materna nei processi di sviluppo dei pesci (Auperin e Geslin, 2008) e dei mammiferi (Darnaudéry e Maccari, 2007). In particolare, gli estrogeni secreti dalle cellule della granulosa durante la vitellogenesi possono essere accumulati negli ovociti dei pesci (Hines et al., 1999) con un possibile ruolo di programming iniziale avente effetti profondi sulla ontogenesi successiva. Nel presente lavoro sullo zebrafish, è stato osservato mediante ibridazione in situ che i livelli di espressione degli mRNA che codificano per il recettore Esr2a sono alti negli oociti ovulati e nell'embrione subito dopo la fecondazione. La concentrazione di questi trascritti diminuisce nelle 8 ore che seguono la fecondazione (hpf), per poi riprendere a crescere dopo l'inizio della trascrizione embrionale, confermando precedenti dati di qRT-PCR (Pikulkaew et al., 2010). Il ruolo funzionale del gene esr2a è stato studiato mediante silenziamento genico ottenuto con la tecnologia del morfolino antisenso utilizzando due diversi morfolini microiniettati nelle uova appena deposte: il primo, progettato sull'ATG d'inizio traduzione, determina il knockdown mediante blocco della traduzione dei trascritti di origine materna e zigotica (esr2aATGMO), mentre il secondo interferisce nel corretto processo di splicing per bloccare la maturazione post-trascrizionale del solo messaggero zigotico (esr2aSPLICMO). Gli embrioni ottenuti dopo microiniezione di esr2aATGMO presentano numerose alterazioni morfologiche rispetto agli embrioni non iniettati (WT) o microiniettati con morfolino standard di controllo (StdMO) e muoiono tra i 12 e i 14 giorni dopo la fecondazione (dpf). Il fenotipo morfante è caratterizzato da: ritardo nella crescita corporea, forma ricurva, anormale sviluppo di cervello e splancnocranio, cavità pericardica ampia ed emorragica, vescica natatoria non insufflata, pinna caudale rudimentale e anomala natazione circolare. La specificità degli effetti ottenuti con la microiniezione è stata verificata anche mediante coiniezione, con l'esr2aATGMO, di un morfolino inattivante la proteina p53. Il fenotipo ottenuto silenziando anche p53 corrisponde a quello morfante; questo dimostra che gli effetti di esr2aATGMO sono specifici e non collegati ad un'iperattivazione di p53 causata dall'iniezione. E' stato possibile inoltre riottenere il fenotipo WT mediante la coiniezione con l'esr2aATGMO del trascritto di zebrafish per Esr2a mutato nella regione complementare al morfolino. Questo dimostra che il legame dell'oligonucleotide al target è estremamente specifico. Per approfondire in maniera più dettagliata la funzione del gene esr2a ho eseguito un esperimento di gain of function con il quale ho aumentato la concentrazione del trascritto del recettore. In un esperimento successivo ho iniettato l'mRNA per esr2a e trattato le uova con 17β-estradiolo (E2) per osservare eventuali anomalie nei primi giorni dopo la fecondazione. Dalla microiniezione di esr2aSPLICMO si ottengono embrioni con fenotipo paragonabile ai controlli. Questo morfolino interferisce nel processo di maturazione del messaggero zigotico, portando alla perdita del terzo esone e a una proteina tradotta non funzionante. Il risultato che ho ottenuto E' un dato importante, perchè suggerisce come le variazioni osservabili nel fenotipo esr2aATGMO siano imputabili principalmente all'inattivazione del trascritto materno. Un'analisi mediante ibridazione in situ utilizzando marcatori di sviluppo ha evidenziato nei morfanti esr2aATGMO una possibile up-regolazione di empty spiracles homeobox 1 (emx1) e sine oculis homeobox homolog 3a (six3a) a 24 e 48 hpf e di sonic hedgehog a (shha) a 48 hpf. La colorazione con alcian blu delle cartilagini craniche ha rivelato un pattern alterato a 6 dpf. Le successive analisi mediante in situ di alcuni marcatori della regione cranica hanno evidenziato un profilo di espressione lievemente differente tra WT e morfanti per il gene distal-less homeobox gene 2a (dlx2a) a 48 hpf e di neurogenic differentiation (neurod) a 24 hpf. Un'analisi di microarray two-color sul genoma totale di zebrafish è stata utilizzata per studiare l'espressione genica su embrioni di 8 e 48 hpf trattati con esr2aATGMO rispetto ai controlli iniettati con StdMO. Con l'analisi microarray a 8 hpf, si possono evidenziare le variazioni nell'espressione dovute principalmente al knockdown del recettore materno. L'analisi a 48 hpf permette invece di rilevare effetti del morfolino dovuti all'inattivazione anche dei trascritti embrionali. L'analisi pangenomica di microarray a 8 hpf, ha individuato 237 trascritti significativamente up-regolati e 219 down-regolati a causa dell'assenza del recettore Esr2a materno negli embrioni iniettati con esr2aATGMO. A 48 hpf, 165 e 124 trascritti, presumibilmente zigotici, sono rispettivamente up-e down-regolati. Tra questi solo 8 sono in comune con quelli up-regolati a 8 hpf e 17 con quelli down-regolati. E' interessante osservare la presenza di un'up-regolazione dei trascritti implicati nella regolazione negativa della proliferazione cellulare. Si può ipotizzare che l'assenza di Esr2a porti ad una diminuzione della proliferazione cellulare, forse dovuta ad un aumento dell'apoptosi. Il dato è in accordo con i risultati del saggio di morte cellulare, mediante TUNEL, che ha evidenziato un numero di cellule apoptotiche nei morfanti notevolmente aumentato e concentrato nell'area cerebrale a 24 e 48 hpf. L'analisi della vascolarizzazione embrionale sfruttando l'attività vasale della fosfatasi alcalina endogena e mediante l'utilizzo della linea transgenica TG(fli1:EGFP) a 3 e 6 dpf ha mostrato nei morfanti esr2aATGMO una netta diminuzione dei vasi subintestinali ed un pattern di distribuzione dei vasi lungo il tronco e la coda meno definito. I risultati ottenuti suggeriscono che nello sviluppo di zebrafish sia presente un controllo epigenetico dipendente dall'mRNA materno di Esr2a e da un'interazione con gli estrogeni di eredità materna

Wounding in lizards results in the release of beta-defensins at the wound site and formation of an antimicrobial barrier.

After tail loss in lizards no infections occur indicating the presence of an effective anti-microbial barrier in the exposed tissues of the tail stump. Previous molecular studies on the lizard Anolis carolinensis have identified some beta-defensin-like genes and the deduced peptides that may be involved in anti-infective protection. The present study has analyzed the tissues of wounded and normal tails in lizards in order to immune-localize one of the beta-defensins previously found (AcBD15) and to detect variation in its gene expression during wounding. No immunoreactivity for this beta-defensin is present in normal tissues or in the epidermis of lizards, except for some sparse granulocytes. The latter are seen during the first 1-6days after tail amputation and AcBD15 immunoreactivity is present in their granules. Degenerating granulocytes are incorporated, together with dead erythrocytes, platelets and keratinocytes into the scab. Real time RT-PCR and western blotting analysis indicates up-regulation of AcBD15 expression during wounding with respect to normal tissues, indicating that production, storage and release of this beta-defensin from granulocytes are active following wounding. The production of beta-defensins from granulocytes would allow protection of exposed tissues from microbial invasion avoiding a persistent inflammation, a process that leads to tissue regeneration

Telomere and Telomerase in Carcinogenesis: Their Role as Prognostic Biomarkers

Unlimited replicative potential is the hallmark of cancer cells. Telomere shortening, which occurs at each cell division, restricts cell proliferation in normal somatic cells. Maintenance of telomere length, required for the unlimited cell proliferation displayed by cancer cells, is provided by telomerase activity, expressed in the vast majority of tumors. Telomere/telomerase interplay has a critical role in tumor initiation and progression. Many tumor-based studies have demonstrated that neoplastic cells generally have shorter telomeres than their adjacent non-cancerous mucosa, strongly supporting the concept that telomere erosion is a critical event in carcinogenesis. Telomerase reverse transcriptase (TERT), the catalytic component of the telomerase complex, is usually absent in normal somatic cells but is expressed at variable levels in tumors. Specific mutations in its promoter may influence TERT levels. A body of data indicates that telomere length and levels of TERT/telomerase activity may be prognostic markers in cancers. Circulating cell-free TERT RNA may also be a promising marker for minimally invasive moni- toring of disease progression and response to therap

Transcriptional control of human organic anion transporting polypeptide 2B1 gene

Organic anion transporting polypeptides (OATPs) are a group of transmembrane carriers with a wide spectrum of amphipathic substrates. In particular, OATP2B1 (previously called OATP-B) can transport steroid hormone conjugates and is expressed in organs with steroidogenic activity, such as placenta, brain and skin. In this work, we have analyzed the transcription of the OATP2B1 gene (SLCO2B1) in 14 different human tissues by means of 5'-RACE analysis. Five promoters (only two of which were present in GenBank), associated with distinct first exons, were found to drive OATP2B1 expression, giving rise to transcripts with unique 5'-untranslated termini. Exon 1b is widely expressed and was found here in 10 tissues. It is partially coding, while the other four different first exons are untranslated. All exons are spliced to a common exon 2 that contains a putative ATG in frame with the following coding region. Sequence analysis of the 5'-flanking region of each first exon revealed a lack of TATA box, thus accounting for the use of multiple transcriptional start sites in nearly all first exons

Epstein-Barr virus and telomerase: from cell immortalization to therapy

Overcoming cellular senescence is strictly required for virus-driven tumors, including those associated with Epstein-Barr virus (EBV). This critical step is successfully accomplished by EBV through TERT expression and telomerase activation in infected cells. We herein review the complex interplay between EBV and TERT/telomerase in EBV-driven tumorigenesis. Evidence accumulated so far clearly indicates that elucidation of this issue may offer promising opportunities for the design of innovative treatment modalities for EBV-associated malignancies. Indeed, several therapeutic strategies for telomerase inhibition have been developed and are being investigated in clinical trials. In this respect, our recent finding that TERT inhibition sensitizes EBV+ lymphoma cells to antivirals through activation of EBV lytic replication is particularly promising and provides a rationale for the activation of clinical studies aimed at assessing the effects of combination therapies with TERT inhibitors and antivirals for the treatment of EBV-associated malignancies

The knockdown of the maternal estrogen receptor 2a (ers2a) mRNA affects embryo transcript contents and larval development in zebrafish

In zebrafish, ovulated oocytes are loaded with maternal estrogen receptor 2a (esr2a) mRNA which is spread as granular and filamentous structures throughout the central ooplasm and is promptly relocated inside the blastodisc area at the 1-cell stage (0.2h post-fertilization, hpf), as shown by in situ hybridization. This transcript is available for translation until its sharp decline from 4 to 8 hpf, being replaced by low levels of zygotic esr2a mRNA mainly localized in the head region and around the yolk sac from 24 hpf until hatching at 48 hpf. To test the functional role of the maternal esr2a mRNA, 1- or 2-cell embryos were injected with 10.3 ng each of morpholino (MO) to knockdown translation (MO2-esr2a) of both maternal and zygotic esr2a transcripts, with a missplicing MO (MO3-esr2a) to effectively block post-transcriptionally the zygotic transcript alone, and with a non-specific MO-control. Treatment with MO2-esr2a increased apoptosis in embryos, especially in the brain, and caused severe malformations in 63% of 1-5 dpf larvae, as compared to 10-11% in those treated with MO3-esr2a and MO-control. Defects included body growth delay with curved shape, persistent yolk sac with reduced sub-intestinal veins and swollen yolk extension, abnormal brain and splanchnocranium development, smaller eyes and otic vesicles, pericardial oedema, uninflated swim bladder and rudimentary caudal fin with aberrant circular swimming. Affected larvae could survive for only 12-14 days. The MO2-esr2a phenotype was rescued with co-injection of 30 pg/embryo of mutated zebrafish esr2a mRNA encoding the full length of Esr2a, but containing eight silent mutations in the region recognised by MO2-esr2a. A lower dosage (15 pg) failed to recover mortality and abnormality. Raising the dosage to 60 and 90 pg increased abnormality, but not mortality, whereas with 120 pg both mortality and abnormality worsened, indicating a strict quantitative requirement of Esr2a. Co-injection of an anti-p53 MO failed to rescue the MO2-esr2a phenotype, eliminating the possibility of off-target effects. Pangenomic microarray analysis revealed that 240 and 219 significantly expressed transcripts were up- and down-regulated, respectively, by maternal Esr2a protein deficiency in 8-hpf MO2-esr2a embryos. Also at 48 hpf, 162 and 120 presumably zygotic transcripts were up- and down-regulated, respectively, but only 18 were in common with each of the 8-hpf sets. In total, the transcripts from 705 genes were affected by Esr2a knockdown. These findings suggest the involvement of maternal esr2a mRNA, presumably transactivated by maternal 17\u3b2-estradiol stored in the oocyte from enveloping granulosa cells, in the epigenetic programming of zebrafish development

The knockdown of maternal glucocorticoid receptor mRNA alters embryo development in zebrafish

In zebrafish, ovulated oocytes contain both maternal cortisol and the mRNA for the glucocorticoid receptor (gr), which is spread as granular structures throughout the ooplasm. At 0.2 hpf, this transcript is relocated in the blastodisc area and partitioned among blastomeres. At 6-8 hpf, it is replaced by zygotic transcript. We used morpholinos to block translation of both maternal and zygotic gr transcripts, and a missplicing morpholino to block post-transcriptionally the zygotic transcript alone. Only knockdown of translation produced an increase of apoptosis and subsequent craniofacial and caudal deformities with severe malformations of neural, vascular, and visceral organs in embryos and 5-dpf larvae. Such defects were rescued with trout gr2 mRNA. Microarray analysis revealed that 114 and 37 highly expressed transcripts were up- and down-regulated, respectively, by maternal Gr protein deficiency in 5-hpf embryos. These results indicate that the maternal gr transcript and protein participate in the maternal programming of zebrafish development