T-cell lymphocytes' aging clock: telomeres, telomerase and aging.

Aging is the decline of physiological capabilities required for life maintenance and reproduction over time. The human immune cells, including T-cells lymphocytes, undergo dramatic aging-related changes, including those related to telomeres and telomerase. It was demonstrated that telomeres and telomerase play crucial roles in T-cell differentiation, aging, and diseases, including a well-documented link between short telomeres and telomerase activation demonstrated in several T-cells malignancies. Herein, we provide a comprehensive review of the literature regarding T-cells' telomeres and telomerase in health and age related-diseases.

Design, synthesis, and antiproliferative effect of 2,9-bis[4-(pyridinylalkylaminomethyl)phenyl]-1,10-phenanthroline derivatives on human leukemic cells by targeting G-quadruplex.

Current multiagent chemotherapy regimens have improved the cure rate in acute leukemia patients, but they are highly toxic and poorly efficient in relapsed patients. To improve the treatment approaches, new specific molecules are needed. The G-quadruplexes (G4s), which are noncanonical nucleic acid structures found in specific guanine-rich DNA or RNA, are involved in many cellular events, including control of gene expression. G4s are considered as targets for the development of anticancer agents. Heterocyclic molecules are well known to target and stabilize G4 structures. Thus, a new series of 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline derivatives (1a-i) was designed, synthesized, and evaluated against five human myeloid leukemia cell lines (K562, KU812, MV4-11, HL60, and U937). Their ability to stabilize various oncogene promoter G4 structures (c-MYC, BCL-2, and K-RAS) as well as the telomeric G4 was also determined through the fluorescence resonance energy transfer melting assay and native mass spectrometry. In addition, the more bioactive ligands 1g-i were tested for telomerase activity in HuT78 and MV4-11 protein extracts.

Intrahepatic Xenograft of Cutaneous T-Cell Lymphoma Cell Lines: A Useful Model for Rapid Biological and Therapeutic Evaluation.

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of diseases primarily involving the skin that could have an aggressive course with circulating blood cells, especially in Sézary syndrome and transformed mycosis fungoides. So far, few CTCL cell lines have been adapted for in vivo experiments and their tumorigenicity has not been adequately assessed, hampering the use of a reproducible model for CTCL biological evaluation. In fact, both patient-derived xenografts and cell line xenografts at subcutaneous sites failed to provide a robust tool, because engraftment was dependent on mice strain and cell line subtype. Herein, we describe an original method of intrahepatic injection into adult NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice liver of both aggressive (My-La, HUT78, HH, MAC2A, and MAC2B) and indolent (FE-PD and MAC1) CTCL cell lines. Six of the seven CTCL cell lines were grafted with a high rate of success (80%). Moreover, this model provided a quick (15 days) and robust assay for in vivo evaluation of CTCL cell lines tumorigenicity and therapeutic response in preclinical studies. Such a reproducible model can be therefore used for further functional studies and in vivo drug testing.

Design, Synthesis and Biological Evaluation of New Substituted Diquinolinyl-Pyridine Ligands as Anticancer Agents by Targeting G-Quadruplex.

G-quadruplexes (G4) are stacked non-canonical nucleic acid structures found in specific G-rich DNA or RNA sequences in the human genome. G4 structures are liable for various biological functions; transcription, translation, cell aging as well as diseases such as cancer. These structures are therefore considered as important targets for the development of anticancer agents. Small organic heterocyclic molecules are well known to target and stabilize G4 structures. In this article, we have designed and synthesized 2,6-di-(4-carbamoyl-2-quinolyl)pyridine derivatives and their ability to stabilize G4-structures have been determined through the FRET melting assay. It has been established that these ligands are selective for G4 over duplexes and show a preference for the parallel conformation. Next, telomerase inhibition ability has been assessed using three cell lines (K562, MyLa and MV-4-11) and telomerase activity is no longer detected at 0.1 µM concentration for the most potent ligand 1c. The most promising G4 ligands were also tested for antiproliferative activity against the two human myeloid leukaemia cell lines, HL60 and K562.

Telomerase functions beyond telomere maintenance in primary cutaneous T-cell lymphoma.

Telomere erosion may be counteracted by telomerase. Here we explored telomere length (TL) and telomerase activity (TA) in primary cutaneous T-cell lymphoma (CTCL) by using quantitative polymerase chain reaction and interphase quantitative fluorescence in situ hybridization assays. Samples from patients with Sézary syndrome (SS), transformed mycosis fungoides (T-MF), and cutaneous anaplastic large cell lymphoma were studied in parallel with corresponding cell lines to evaluate the relevance of TL and TA as target candidates for diagnostic and therapeutic purposes. Compared with controls, short telomeres were observed in aggressive CTCL subtypes such as SS and T-MF and were restricted to neoplastic cells in SS. While no genomic alteration of the hTERT (human telomerase catalytic subunit) locus was observed in patients' tumor cells, TA was detected. To understand the role of telomerase in CTCL, we manipulated its expression in CTCL cell lines. Telomerase inhibition rapidly impeded in vitro cell proliferation and led to cell death, while telomerase overexpression stimulated in vitro proliferation and clonogenicity properties and favored tumor development in immunodeficient mice. Our data indicate that, besides maintenance of TL, telomerase exerts additional functions in CTCL. Therefore, targeting these functions might represent an attractive therapeutic strategy, especially in aggressive CTCL.

Evaluation of Quantitative Fluorescence in situ Hybridization for Relative Measurement of Telomere Length in Placental Mesenchymal Core Cells.

BACKGROUND: Reduced telomere length in placental mesenchymal core cells has been reported during pregnancies complicated by intrauterine growth restriction. To estimate telomere length, a precise, accurate and reproducible technique must be used. OBJECTIVE: We evaluated the characteristics of a quantitative fluorescence in situ hybridization (Q-FISH) technique for measuring relative telomere length in placental mesenchymal core cells. METHODS: From late chorionic villus samplings, telomere length in placental mesenchymal core cells was estimated by a Q-FISH technique using peptide nucleic acid telomere probes. The main characteristics of the Q-FISH technique, such as precision and reproducibility, were evaluated. RESULTS: The telomere length of the cultured placental mesenchymal cells did not follow a normal distribution. When the Q-FISH technique was performed on interphase nuclei of uncultured mesenchymal core cells, normal telomere length distribution was observed. The precision of the technique when applied to cultured placental mesenchymal core cells was estimated to be <6%, and its reproducibility ranged from to 92.9 to 104.7%. CONCLUSION: Our results showed that cell culture of placental villi produced a non-normal telomere length distribution, probably related to telomere DNA replication during the cell cycle. Despite the influence of cell culture, the Q-FISH technique reported herein showed good precision and reproducibility.

Delving into the Metabolism of Sézary Cells: A Brief Review.

Primary cutaneous lymphomas (PCLs) are a heterogeneous group of lymphoproliferative disorders caused by the accumulation of neoplastic T or B lymphocytes in the skin. Sézary syndrome (SS) is an aggressive and rare form of cutaneous T cell lymphoma (CTCL) characterized by an erythroderma and the presence of atypical cerebriform T cells named Sézary cells in skin and blood. Most of the available treatments for SS are not curative, which means there is an urgent need for the development of novel efficient therapies. Recently, targeting cancer metabolism has emerged as a promising strategy for cancer therapy. This is due to the accumulating evidence that metabolic reprogramming highly contributes to tumor progression. Genes play a pivotal role in regulating metabolic processes, and alterations in these genes can disrupt the delicate balance of metabolic pathways, potentially contributing to cancer development. In this review, we discuss the importance of targeting energy metabolism in tumors and the currently available data on the metabolism of Sézary cells, paving the way for potential new therapeutic approaches aiming to improve clinical outcomes for patients suffering from SS.

Spotlight on hTERT Complex Regulation in Cutaneous T-Cell Lymphomas.

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 ß+ 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-ß+ 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-ß+ hTERT transcript variant.

New 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazoline and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinoline Derivatives: Synthesis and Biological Evaluation as Novel Anticancer Agents by Targeting G-Quadruplex.

The syntheses of novel 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinazolines 12 and 2,4-bis[(substituted-aminomethyl)phenyl]phenylquinolines 13 are reported here in six steps starting from various halogeno-quinazoline-2,4-(1H,3H)-diones or substituted anilines. The antiproliferative activities of the products were determined in vitro against a panel of breast (MCF-7 and MDA-MB-231), human adherent cervical (HeLa and SiHa), and ovarian (A2780) cell lines. Disubstituted 6- and 7-phenyl-bis(3-dimethylaminopropyl)aminomethylphenyl-quinazolines 12b, 12f, and 12i displayed the most interesting antiproliferative activities against six human cancer cell lines. In the series of quinoline derivatives, 6-phenyl-bis(3-dimethylaminopropyl)aminomethylphenylquinoline 13a proved to be the most active. G-quadruplexes (G4) stacked non-canonical nucleic acid structures found in specific G-rich DNA, or RNA sequences in the human genome are considered as potential targets for the development of anticancer agents. Then, as small aza-organic heterocyclic derivatives are well known to target and stabilize G4 structures, their ability to bind G4 structures have been determined through FRET melting, circular dichroism, and native mass spectrometry assays. Finally, telomerase inhibition ability has been also assessed using the MCF-7 cell line.

[Building partnerships from the training of advanced practice nurses]. / Construction de partenariats dès la formation des infirmiers en pratique avancée.

The introduction of advanced practice nurses in France is recent. It requires the development of partnerships from the training stage onwards, in order to promote the integration of these new professionals in the field. In this sense, the university and the university hospital of Bordeaux, in Gironde, have undertaken joint actions.

Telomeric Repeat-Containing RNA (TERRA): A Review of the Literature and First Assessment in Cutaneous T-Cell Lymphomas.

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.

Silencing of RND3/RHOE inhibits the growth of human hepatocellular carcinoma and is associated with reversible senescence.

Rnd3/RhoE is an atypical Rho GTPase family member, known to be deregulated in many types of cancer. Previously, we showed that RND3 expression is downregulated in hepatocellular carcinoma (HCC) cell lines and tissues. In cancer cells, Rnd3 is involved in the regulation of cell proliferation and cell invasion. The implication of Rnd3 in HCC invasion was importantly studied whereas its role in cell growth needs further investigation. Thus, in this work, we aimed to better understand the impact of Rnd3 on tumor hepatocyte proliferation. Our results indicate that the silencing of RND3 induces a cell growth arrest both in vitro in 2D and 3D culture conditions and in vivo in tumor xenografts. The growth alteration after RND3 silencing in HCC cells is not due to an increase of cell death but to the induction of senescence. This RND3 knockdown-mediated phenomenon is dependent on the decrease of hTERT expression. Interestingly, after re-expression of RND3, these cells are able to bypass senescence and regain the ability to proliferate, with a re-expression of hTERT. Given that a low expression of Rnd3 is linked to the presence of satellite nodules in HCC, the transient senescence state observed might play a role in cancer progression.

The Journey of SCAPs (Stem Cells from Apical Papilla), from Their Native Tissue to Grafting: Impact of Oxygen Concentration.

Tissue engineering strategies aim at characterizing and at optimizing the cellular component that is combined with biomaterials, for improved tissue regeneration. Here, we present the immunoMap of apical papilla, the native tissue from which SCAPs are derived. We characterized stem cell niches that correspond to a minority population of cells expressing Mesenchymal stromal/Stem Cell (CD90, CD105, CD146) and stemness (SSEA4 and CD49f) markers as well as endothelial cell markers (VWF, CD31). Based on the colocalization of TKS5 and cortactin markers, we detected migration-associated organelles, podosomes-like structures, in specific regions and, for the first time, in association with stem cell niches in normal tissue. From six healthy teenager volunteers, each with two teeth, we derived twelve cell banks, isolated and amplified under 21 or 3% O2. We confirmed a proliferative advantage of all banks when cultured under 3% versus 21% O2. Interestingly, telomerase activity was similar to that of the highly proliferative hiPSC cell line, but unrelated to O2 concentration. Finally, SCAPs embedded in a thixotropic hydrogel and implanted subcutaneously in immunodeficient mice were protected from cell death with a slightly greater advantage for cells preconditioned at 3% O2.

Exploring hTERT promoter methylation in cutaneous T-cell lymphomas.

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.

Diagnosis and treatment of lymphomas in the era of epigenetics.

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.

Targeting Epigenetic Modifiers Can Reduce the Clonogenic Capacities of Sézary Cells.

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL) in which the human Telomerase Reverse Transcriptase (hTERT) gene is re-expressed. Current available treatments do not provide long-term response. We previously reported that Histone deacetylase inhibitors (HDACi, romidespin and vorinostat) and a DNA methyltransferase inhibitor (DNMTi, 5-azacytidine) can reduce hTERT expression without altering the methylation level of hTERT promoter. Romidepsin and vorinostat are approved for CTCL treatment, while 5-azacytidine is approved for the treatment of several hematological disorders, but not for CTCL. Here, using the soft agar assay, we analyzed the functional effect of the aforementioned epidrugs on the clonogenic capacities of Sézary cells. Our data revealed that, besides hTERT downregulation, epidrugs' pressure reduced the proliferative and the tumor formation capacities in Sézary cells in vitro.

C6 Ceramide (d18:1/6:0) as a Novel Treatment of Cutaneous T Cell Lymphoma.

Cutaneous T cell lymphomas (CTCLs) represent a heterogeneous group of T cell lymphomas that primarily affect the skin. The most frequent forms of CTCL are mycosis fungoides and Sézary syndrome. Both are characterized by frequent recurrence, developing chronic conditions and high mortality with a lack of a curative treatment. In this study, we evaluated the effect of short-chain, cell-permeable C6 Ceramide (C6Cer) on CTCL cell lines and keratinocytes. C6Cer significantly reduced cell viability of CTCL cell lines and induced cell death via apoptosis and necrosis. In contrast, primary human keratinocytes and HaCaT keratinocytes were less affected by C6Cer. Both keratinocyte cell lines showed higher expressions of ceramide catabolizing enzymes and HaCaT keratinocytes were able to metabolize C6Cer faster and more efficiently than CTCL cell lines, which might explain the observed protective effects. Along with other existing skin-directed therapies, C6Cer could be a novel well-tolerated drug for the topical treatment of CTCL.

CDKN2A-CDKN2B deletion defines an aggressive subset of cutaneous T-cell lymphoma.

Inactivation of the CDKN2A-CDKN2B locus has been reported in the most frequent subtypes of cutaneous T-cell lymphomas (CTCLs), mycosis fungoides, Sézary syndrome (SS) and CD30+ cutaneous anaplastic large cell lymphoma. To investigate whether genetic or epigenetic inactivation of CDKN2A-CDKN2B is more specifically observed in certain CTCL subtypes with clinical impact, we used array-comparative genomic hybridization, quantitative PCR, interphase fluorescent in situ hybridization and methylation analyses of p14(ARF) p16(INK4A) and p15(INK4B) promoters. We studied 67 samples from 58 patients with either transformed mycosis fungoides (n=24), SS (n=16) or CD30+ cutaneous anaplastic large cell lymphoma (n=18). We observed combined CDKN2A-CDKN2B deletion in both transformed mycosis fungoides (n=17, 71%) and SS patients (n=7, 44%), but, surprisingly, in only one CD30+ cutaneous anaplastic large cell lymphoma case. Interphase fluorescent in situ hybridization showed 9p21 loss in 17 out of 19 cases, with 9p21 deletion indicating either hemizygous (n=4) or homozygous (n=2) deletion, with mixed patterns in most patients (n=11). The limited size of 9p21 deletion was found to account for false-negative detection by either BAC arrays (n=9) or fluorescent in situ hybridization (n=2), especially in patients with Sézary syndrome (n=6). Methylation was found to be restricted to the p15(INK4B) gene promoter in patients with or without 9p21 deletion and did not correlate with prognosis. In contrast, CDKN2A-CDKN2B genetic loss was strongly associated with a shorter survival in CTCL patients (P=0.002) and more specifically at 24 months in transformed mycosis fungoides and SS patients (P=0.02). As immunohistochemistry for p16(INK4A) protein was not found to be informative, the genetic status of the CDKN2A-CDKN2B locus would be relevant in assessing patients with epidermotropic CTCLs in order to identify those cases where the disease was more aggressive.

Complex context relationships between DNA methylation and accessibility, histone marks, and hTERT gene expression in acute promyelocytic leukemia cells: perspectives for all-trans retinoic acid in cancer therapy.

Telomerase (hTERT) reactivation and sustained expression is a key event in the process of cellular transformation. Therefore, the identification of the mechanisms regulating hTERT expression is of great interest for the development of new anticancer therapies. Although the epigenetic state of hTERT gene promoter is important, we still lack a clear understanding of the mechanisms by which epigenetic changes affect hTERT expression. Retinoids are well-known inducers of granulocytic maturation in acute promyelocytic leukemia (APL). We have previously shown that retinoids repressed hTERT expression in the absence of maturation leading to growth arrest and cell death. Exploring the mechanisms of this repression, we showed that transcription factor binding was dependent on the epigenetic status of hTERT promoter. In the present study, we used APL cells lines and publicly available datasets from APL patients to further investigate the integrated epigenetic events that promote hTERT promoter transition from its silent to its active state, and inversely. We showed, in APL patients, that the methylation of the distal domain of hTERT core promoter was altered and correlated with the outcome of the disease. Further studies combining complementary approaches carried out on APL cell lines highlighted the significance of a domain outside the minimal promoter, localized around 5 kb upstream from the transcription start site, in activating hTERT. This domain is characterized by DNA hypomethylation and H3K4Me3 deposition. Our findings suggest a cooperative interplay between hTERT promoter methylation, chromatin accessibility, and histone modifications that force the revisiting of previously proposed concepts regarding hTERT epigenetic regulation. They represent, therefore, a major advance in predicting sensitivity to retinoid-induced hTERT repression and, more generally, in the potential development of therapies targeting hTERT expression in cancers.

Reliable blood cancer cells' telomere length evaluation by qPCR.

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.