Cutaneous T‐cell lymphomas (CTCLs) are telomerase‐positive tumors expressing hTERT, although neither gene rearrangement/amplification nor promoter hotspot mutations could explain the hTERT ...re‐expression. As the hTERT promoter is rich in CpG, we investigated the contribution of epigenetic mechanisms in its re‐expression. We analyzed hTERT promoter methylation status in CTCL cells compared with healthy cells. Gene‐specific methylation analyses revealed a common methylation pattern exclusively in tumor cells. This methylation pattern encompassed a hypermethylated distal region from −650 to −150 bp and a hypomethylated proximal region from −150 to +150 bp. Interestingly, the hypermethylated region matches with the recently named TERT hypermethylated oncogenic region (THOR). THOR has been associated with telomerase reactivation in many cancers, but it has so far not been reported in cutaneous lymphomas. Additionally, we assessed the effect of THOR on two histone deacetylase inhibitors (HDACi), romidepsin and vorinostat, both approved for CTCL treatment and a DNA methyltransferase inhibitor (DNMTi) 5‐azacytidine, unapproved for CTCL. Contrary to our expectations, the findings reported herein revealed that THOR methylation is relatively stable under these epigenetic drugs' pressure, whereas these drugs reduced the hTERT gene expression.
Cutaneous T‐cell lymphomas (CTCLs) represent a group of lymphoproliferative disorders arising from the skin. CTCLs are characterized by hTERT gene expression despite the lack of hTERT amplifications or rearrangements. Here, we investigated hTERT promoter methylation and associated TERT hypermethylated oncogenic region (THOR) with hTERT reactivation in CTCL. Additionally, we evaluated THOR methylation and hTERT expression after treatment with epigenetic drugs. Altogether, our study offers a better understanding of the response to epigenetic drugs in patients with CTCL.
Telomeric Repeat-containing RNA (TERRA) are long non-coding RNAs transcribed from telomeric DNA sequences from multiple chromosome ends. Major research efforts have been made to understand TERRA ...roles and functions in several physiological and pathological processes. We summarize herein available data regarding TERRA's roles in human cells and we report the first investigation in cutaneous T-cells lymphomas (CTCL) using real-time PCR. Among the TERRA analysed, our data suggest a particular role for TERRA 16p downregulation and TERRA 11q upregulation in CTCL lymphomagenesis.
Telomerase expression and telomere maintenance are critical for cell proliferation and survival, and they play important roles in development and cancer, including hematological malignancies. ...Transcriptional regulation of the rate-limiting subunit of human telomerase reverse transcriptase gen (hTERT) is a complex process, and unveiling the mechanisms behind its reactivation is an important step for the development of diagnostic and therapeutic applications. Here, we review the main mechanisms of telomerase activation and the associated hematologic malignancies.
As a major cancer hallmark, there is a sustained interest in understanding the telomerase contribution to carcinogenesis in order to therapeutically target this enzyme. This is particularly relevant ...in primary cutaneous T-cell lymphomas (CTCL), a malignancy showing telomerase dysregulation with few investigative data available. In CTCL, we examined the mechanisms involved in telomerase transcriptional activation and activity regulation. We analyzed 94 CTCL patients from a Franco-Portuguese cohort, as well as 8 cell lines, in comparison to 101 healthy controls. Our results showed that not only polymorphisms (SNPs) located at the promoter of human telomerase reverse transcriptase (
gene (rs2735940 and rs2853672) but also an SNP located within the coding region (rs2853676) could influence CTCL occurrence. Furthermore, our results sustained that the post-transcriptional regulation of
contributes to CTCL lymphomagenesis. Indeed, CTCL cells present a different pattern of
spliced transcripts distribution from the controls, mostly marked by an increase in the
β+ variants proportion. This increase seems to be associated with CTCL development and progression. Through
splicing transcriptome modulation with shRNAs, we observed that the decrease in the α-β+ transcript induced a decrease in the cell proliferation and tumorigenic capacities of T-MF cells in vitro. Taken together, our data highlight the major role of post-transcriptional mechanisms regulating telomerase non canonical functions in CTCL and suggest a new potential role for the α-β+
transcript variant.
Background
Telomere shortening is linked to a range of different human diseases, hence reliable measurement methods are needed to uncover such associations. Among the plethora of telomere length ...measurement methods, qPCR is reported as easy to conduct and a cost‐effective approach to study samples with low DNA amounts.
Methods
Cancer cells’ telomere length was evaluated by relative and absolute qPCR methods.
Results
Robust and reproducible telomere length measurements were optimized taking into account a careful reference gene selection and by knowing the cancer cells ploidy. qPCR data were compared to “gold standard” measurement from terminal restriction fragment (TRF).
Conclusions
Our study provides guidance and recommendations for accurate telomere length measurement by qPCR in cancer cells, taking advantage of our expertise in telomere homeostasis investigation in primary cutaneous T‐cell lymphomas. Furthermore, our data emphasize the requirement of samples with both, high DNA quality and high tumor cells representation.
In this original article we aimed to validate the applicability of qPCR when assessing telomere length in cancer cells, taking advantage of our expertise on telomere homeostasis investigation in primary cutaneous T‐cell lymphomas
Lymphomas represent a heterogeneous group of cancers characterized by clonal lymphoproliferation. Over the past decades, frequent epigenetic dysregulations have been identified in hematologic ...malignancies including lymphomas. Many of these impairments occur in genes with established roles and well-known functions in the regulation and maintenance of the epigenome. In hematopoietic cells, these dysfunctions can result in abnormal DNA methylation, erroneous chromatin state and/or altered miRNA expression, affecting many different cellular functions. Nowadays, it is evident that epigenetic dysregulations in lymphoid neoplasms are mainly caused by genetic alterations in genes encoding for enzymes responsible for histone or chromatin modifications. We summarize herein the recent epigenetic modifiers findings in lymphomas. We focus also on the most commonly mutated epigenetic regulators and emphasize on actual epigenetic therapies.
The ends of linear genomes are comprised of a unique and genetically stable structure termed telomere. Mammalian telomeres are composed of tandem repeats (TTAGGG)n that terminates in a 3’ ...single-stranded G-rich overhang that have a central role in sustaining a diverse array of telomeric functions. Indeed, the G-rich overhang that has approximately 30-500 nucleotides, folds back and invades the double-stranded telomeric helix, forming a lariat-like structure called telomeric loop or T-loop. The overhang pairs with the opposite strand, giving rise to a smaller displacement loop, the D-loop (Figure 1). This whole secondary structure is stabilized by the shelterin complex (Griffith, Comeau et al. 1999; di Fagagna, Reaper et al. 2003).The T-loop is also stabilized by the G-rich character of telomere 3’ single-stranded overhang. Indeed, the 3’ overhang takes on a secondary structure formed from the hydrogen bonding of guanine residues in tetrad formations, called G-quadruplex structure. This structure blocks telomerase physical access to telomeres, by preventing telomeric DNA linearization (Biffi, Tannahill et al. ; Sfeir ; Oganesian and Karlseder 2009).Mammalian telomeric DNA is assembled into evenly spaced nucleosomes that are enriched with repressive epigenetic marks that are characteristic of constitutive heterochromatin (Benetti, Garcia-Cao et al. 2007). The heterochromatic state of telomeres is important for proper telomere function, as it modulates telomere length and telomere ability to undergo homologous recombination (Schoeftner and Blasco).Telomeres solve two basic problems that are inherent to linear genomes. First, thanks to its specialized structure, they distinguish chromosome ends from DNA double-strand breaks, thereby preventing unwanted DNA-damage signaling and genome instability. Second, they prevent loss of essential genetic information (O'sullivan and Karlseder ; Gümüs-Akay and Tükün 2012). Telomere functions are mainly regulated by shelterin protein complex and telomerase enzyme.Shelterin complex Telomeric DNA is bound by the shelterin complex, composed of six proteins: TRF1 (telomeric repeat binding factor 1, also known as TERF1), TRF2 (telomeric repeat binding factor 2, also known as TERF2), RAP1 (TERF2 interacting protein, also known as TERF2IP), TIN2 (TRF1 interacting nuclear factor 2, also known as TINF2), TPP1 (adrenocortical dysplasia protein homolog, also known as ACD) and POT1 (protection of telomeres 1) (De Lange 2005). The exquisite specificity with which shelterin binds to the telomeric DNA is conferred by three of its components: TRF1 and TRF2 bind to the double stranded region of the DNA, whereas POT1 coats the single stranded overhang. The other three shelterin components bind to the telomeres through protein-protein interactions. RAP1 binds TRF2; TPP1 binds POT1; and TIN2 binds TRF1, TRF2 and TPP1 simultaneously (Sfeir ; Palm and de Lange 2008). Most shelterin components are essential to survival of mammalian cells, as its depletion either drives cells into cellular senescence or results in early embryonic lethality (Martinez and Blasco ; Patel, Vasan et al.). Together, shelterin complex protects chromosome ends from activating a DNA damage response, inhibits inappropriate repair mechanisms and maintains telomeric length and structure. However, each protein plays a unique role in telomere homeostasis.
As a major cancer hallmark, there is a sustained interest in understanding the telomerase contribution to carcinogenesis in order to therapeutically target this enzyme. This is particularly relevant ...in primary cutaneous T-cell lymphomas (CTCL), a malignancy showing telomerase dysregulation with few investigative data available. In CTCL, we examined the mechanisms involved in telomerase transcriptional activation and activity regulation. We analyzed 94 CTCL patients from a Franco-Portuguese cohort, as well as 8 cell lines, in comparison to 101 healthy controls. Our results showed that not only polymorphisms (SNPs) located at the promoter of human telomerase reverse transcriptase (hTERT) gene (rs2735940 and rs2853672) but also an SNP located within the coding region (rs2853676) could influence CTCL occurrence. Furthermore, our results sustained that the post-transcriptional regulation of hTERT contributes to CTCL lymphomagenesis. Indeed, CTCL cells present a different pattern of hTERT spliced transcripts distribution from the controls, mostly marked by an increase in the hTERT β+ variants proportion. This increase seems to be associated with CTCL development and progression. Through hTERT splicing transcriptome modulation with shRNAs, we observed that the decrease in the α-β+ transcript induced a decrease in the cell proliferation and tumorigenic capacities of T-MF cells in vitro. Taken together, our data highlight the major role of post-transcriptional mechanisms regulating telomerase non canonical functions in CTCL and suggest a new potential role for the α-β+ hTERT transcript variant.
Os compostos de arsénio são substâncias altamente tóxicas e carcinogénicas, libertadas na natureza através de fontes naturais e antropomórficas. Contudo, alguns compostos de arsénio como o trióxido ...de arsénio e a trypasamide são utilizados terapeuticamente no tratamento da leucemia promielocítica aguda e de doenças causadas por protozoários, respectivamente. Devido ao elevado grau de conservação nos processos celulares eucariotas, a levedura Saccharomyces cerevisiaeconstitui um excelente modelo para estudar como os organismos superiores, incluindo o homem, respondem as diversas condições de stress. Na maioria dos organismos, os mecanismos adaptativos relacionados com mudanças ambientais ocorrem sobretudo através da reprogramação da expressão genética que leva à indução de genes que conferem actividades de protecção. Estes processos são controlados por factores de transcrição específicos, que depois de serem activados, medeiam a montagem do complexo de préiniciação da transcrição na região promotora dos seus genes alvos. . O complexo Mediador, necessário para a activação de vários genes em leveduras e mamíferos, interage com factores de transcrição específicos através do domínio cauda e assim actua como co-activador.Neste trabalho investigamos o envolvimento do complexo Mediador na resposta da levedura Saccharomyces cerevisiae ao stress induzido pelos compostos de arsénio. Evidências sugerem que as subunidades Med2, Med3, Med14, Med15 e Med16 da cauda do complexo Mediador são importantes nessa resposta. A actividade do Yap8, o principal regulador da adaptação celular, depende destas subunidades. A sua ausência não afecta os níveis intracelulares do Yap8, contudo a função de transactivação deste factor de transcrição é severamente comprometida nos respectivos mutantes. Utilizando o sistema two-hybrid, fomos capazes de demonstrar que o Yap8 interage com a subunidade Med2 estabelecendo assim uma ligação funcional entre as duas proteínas. Estudos de interacção genética demonstraram que os duplos mutantes yap8med3 e yap8med16 são capazes de tolerar concentrações mais elevadas de arsénio do que o mutante simples yap8,surgindo assim a hipótese da activação de vias adaptativas alternativas. Os nossos resultados fornecem fortes evidências de que o Yap1 é o regulador desta via.
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