The knockdown of the maternal estrogen receptor 2a (esr2a) 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 17ß-estradiol stored in the oocyte from enveloping granulosa cells, in the epigenetic programming of zebrafish development.

Anti-Proliferative and Pro-Apoptotic Effects of Short-Term Inhibition of Telomerase In Vivo and in Human Malignant B Cells Xenografted in Zebrafish.

Besides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumor growth/progression through extra-telomeric functions. Our previous in vitro studies demonstrated that short-term TERT inhibition by BIBR1532 (BIBR), an inhibitor of TERT catalytic activity, negatively impacts cell proliferation and viability via telomeres' length-independent mechanism. Here we evaluate the anti-proliferative and pro-apoptotic effects of short-term telomerase inhibition in vivo in wild-type (wt) and tert mutant (terthu3430/hu3430; tert-/-) zebrafish embryos, and in malignant human B cells xenografted in casper zebrafish embryos. Short-term Tert inhibition by BIBR in wt embryos reduced cell proliferation, induced an accumulation of cells in S-phase and ultimately led to apoptosis associated with the activation of DNA damage response; all these effects were unrelated to telomere shortening/dysfunction. BIBR treatment showed no effects in tert-/- embryos. Xenografted untreated malignant B cells proliferated in zebrafish embryos, while BIBR pretreated cells constantly decreased and were significantly less than those in the controls from 24 to up to 72 h after xenotransplantation. Additionally, xenografted tumor cells, treated with BIBR prior- or post-transplantation, displayed a significant higher apoptotic rate compared to untreated control cells. In conclusion, our data demonstrate that short-term telomerase inhibition impairs proliferation and viability in vivo and in human malignant B cells xenografted in zebrafish, thus supporting therapeutic applications of TERT inhibitors in human malignancies.

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

The Epstein-Barr virus (EBV) is a ubiquitous human γ-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.

Besides 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.

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.

hTERT inhibition triggers Epstein-Barr virus lytic cycle and apoptosis in immortalized and transformed B cells: a basis for new therapies.

PURPOSE: Induction of viral lytic cycle, which induces death of host cells, may constitute a useful adjunct to current therapeutic regimens for Epstein-Barr virus (EBV)-driven malignancies. Human telomerase reverse transcriptase (hTERT), essential for the oncogenic process, may modulate the switch from latent to lytic infection. The possible therapeutic role of hTERT inhibition combined with antiviral drugs was investigated. EXPERIMENTAL DESIGN: EBV-negative BL41 and convertant EBV-positive BL41/B95.8 Burkitt's lymphoma cell lines and lymphoblastoid cell lines (LCL) were infected with retroviral vector encoding short hairpin RNA (shRNA) anti-hTERT and cultured with or without the prodrug ganciclovir. The effects on EBV lytic replication, cell proliferation, and apoptosis were characterized. RESULTS: hTERT silencing by shRNA induced the expression of BZLF1, EA-D, and gp350 EBV lytic proteins and triggered a complete lytic cycle. This effect was associated with downregulation of BATF, a negative regulator of BZLF1 transcription. hTERT silencing also resulted in antiproliferative and proapoptotic effects. In particular, hTERT inhibition induced an accumulation of cells in the S-phase, an effect likely due to the dephosphorylation of 4E-BP1, an AKT1-dependent substrate, which results in a decreased availability of proteins needed for cell-cycle progression. Besides inducing cell death through activation of complete EBV lytic replication, hTERT inhibition triggered AKT1/FOXO3/NOXA-dependent apoptosis in EBV-positive and -negative Burkitt's lymphoma cells. Finally, ganciclovir enhanced the apoptotic effect induced by hTERT inhibition in EBV-positive Burkitt's lymphomas and LCLs. CONCLUSIONS: These results suggest that combination of antiviral drugs with strategies able to inhibit hTERT expression may result in therapeutically relevant effects in patients with EBV-related malignancies.

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 1-6 days 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.

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.