Telomerase (hTERT) Overexpression Reveals a Promising Prognostic Biomarker and Therapeutical Target in Different Clinical Subtypes of Pediatric Acute Lymphoblastic Leukaemia

Acute Lymphoblastic Leukemia (ALL) is a neoplasm of the hematopoietic system defined as a clonal expansion of an abnormal lymphoid precursor cell. It mostly affects children under five years of age and is the most common tumor to afflict pediatric patients. The expression of the human telomerase gene (hTERT) in patients with ALL has been studied as a biomarker and could become a new therapeutic target. We evaluate the role of hTERT gene expression in ALL pediatric patients, through quantitative real-time PCR technique, and the possible correlation between hTERT expression and clinical variables: gender, age, white blood cells (WBC), gene fusions, and immunophenotyping. The analysis between healthy controls and ALL patients (N = 244) was statistically significant (p < 0.001), demonstrating hTERT overexpression in these patients. In comparison with the usual set of clinical variables, the data were not statistically significant (p > 0.05), indicating that hTERT is equally overexpressed among patients regardless of gender, age, gene fusions, and immunophenotyping. Moreover, patients who presented a higher hTERT expression level had a significant (p < 0.0001) lower overall survival rate. In summary, hTERT expression emerges as an important molecular pathway in leukemogenesis regardless patient’s clinical variables, thus, the data here presented pointed it as a valuable biomarker in pediatric acute lymphoblastic leukemia and a promising target for new therapeutic and prognostic measures.

Mesenchymal stem cells immortalization's and state-of-art: a systematic review and meta-analysis

Background The immortalization of mesenchymal stem cells (MSCs) allows them to avoid senescence and be cultured through limitless cell passages. Thus, several experimental strategies, such as retrovirus-mediated gene transfer or viral oncogenesis, have been applied for the immortalization of MSCs. The aim was to identifier the most commonly used methodologies and their particularities for the immortalization of human and animal MSCs. Methods The search was conducted in June 2019 and developed in SCOPUS, PUBMED, and SCIENCE DIRECT. Statistical analysis was performed, obtaining the values of total n, mean and standard deviation, confidence interval (CI), and percentage (frequency) for all the predictors. Results The most used immortalization methodology was viral transfection, being the most common immortalized cell type was the bone marrow-derived MSC, and the most used gene for immortalizing both human and animal MSCs was hTERT (39.3%) and SV40T (54.5%). Among the articles analyzed in this review, only 39.3% and 36.4% of human and animal MSCs immortalization protocols, respectively, underwent the tumorigenicity test. Conclusions The virus-mediated gene transfection was observed as the most used and established technique. The insertion of the hTERT gene is still the most used gene for cell immortalization, suggesting that the maintenance of telomerase is efficient for maintaining cell proliferation and bypassing cell senescence. The review concluded that the tumorigenicity tests should become mandatory in order to safely use the immortalized MSCs for translation.

IMPLICATIONS AND POTENTIAL ROLE OF HUMAN TELOMERASE REVERSE TRANSCRIPTASE (HTERT) IN BLADDER CARCINOGENESIS

Bladder cancer is a heterogeneous disease and ranks as 10th most common cancer worldwide. Urothelial carcinoma (UC) is the most common histologic type of BC and majority constitute of papillary tumors that are well-differentiated (low-grade). Several genetic changes may occur in bladder cancer, but hTERT promoter mutations and its expression has been detected in most cases of transitional cell carcinoma. Numerous researches have led to the findings which suggest that the hTERT promoter mutations in conjunction with the common polymorphism and hTERT expression have potential of being used as clinical biomarkers in bladder cancer. Further studies need to explore the potential use of hTERT gene in bladder cancer detection, diagnosis and prognosis. This review focuses on the role of hTERT in bladder tumors in the backdrop of various studies published.

Design and development of nanostructured co delivery of artemisinin and chrysin for targeting hTERT gene expression in breast cancer cell line: possible clinical application in cancer treatment

Background: Breast cancer is one of the most significant causes of female cancer death worldwide. To explore the possibility of a novel chemo-preventive strategy for improving breast cancer treatment, the anticancer effects of a combination two natural compounds, Artemisinin (Art) and Chrysin (Chr), against T47D breast cancer cells were investigated.Methods: For this purpose, Art and Chr were co-encapsulated in PEGylated PLGA nanoparticles (NPs) and evaluated for their therapeutic efficacy. The morphology and dynamic light scattering (DLS) analyses were carried out to optimize the Nano formulations. Drug release study was performed using the dialysis method and then the cytotoxic and inhibitory effect of individual and combined drugs on the expression level of hTERT in the T47D breast cell line was evaluated using MTT assay and qPCR, respectively. Results: The results showed that pure drugs and formulations exhibited dose-dependent cytotoxicity against T47D cells and especially, Art/Chr–PLGA/PEG NPs had a more synergistic anti-proliferative effect and significantly arrested the growth of cancer cells than the other groups. Real-time PCR results revealed that Art, Chr and combination of Art–Chr in pure and encapsulated forms inhibited hTERT gene expression. Conclusions: It was found that Art–Chr–PLGA/PEG NPs relative to pure combination could further decline hTERT expression in all concentrations. Our study demonstrated that Art–Chr–PLGA/PEG NPs based combinational therapy holds promising potential for the treatment of breast cancer.

Gene Expression Profile in Immortalized Human Periodontal Ligament Fibroblasts Through hTERT Ectopic Expression: Transcriptome and Bioinformatic Analysis

Human periodontal ligament fibroblast (hPLF) cells play an important role in maintaining oral cavity homeostasis with special function in tissue regeneration and maintenance of dental alveoli. Although their primary cell cultures are considered a good experimental model with no genetic changes, the finite life span may limit some experimental designs. The immortalization process increases cell life span but may cause genetic changes and chromosomal instability, resulting in direct effects on physiological cell responses. In this way, we aimed to investigate the global gene expression of hPLFs after the immortalization process by the ectopic expression of the catalytic subunit of the enzyme telomerase reverse transcriptase (hTERT) through transcriptome analysis. The embryonic origin of the primary culture of hPLF cells and immortalized hPLF-hTERT was also tested by vimentin staining, hTERT synthesis evaluated by indirect immunocytochemistry, analysis of cell proliferation, and morphology. The results indicated that hPLFs and hPLF-hTERT were positive for vimentin. On the 20th cell passage, hPLFs were in senescence, while hPLF-hTERT maintained their proliferation and morphology characteristics. At the same passage, hPLF-hTERT presented a significant increase in hTERT synthesis, but transcriptome did not reveal overexpression of the hTERT gene. Fifty-eight genes had their expression altered (11 upregulated and 47 downregulated) with the absence of changes in the key genes related to these cell types and in the main cancer-associated genes. In addition, the increase in hTERT protein expression without the overexpression of its gene indicates posttranscriptional level regulation. Successful immortalization of hPLFs through the ectopic expression of hTERT encourages further studies to design experimental protocols to investigate clinical questions from a translational perspective.

Taurine Augments Telomerase Activity and Promotes Chondrogenesis in Dental Pulp Stem Cells

Background: Stem cell therapy has become an advanced and state-of-the-art procedure to regenerate lost tissues of the human body. Cartilage repair is a challenging task in which stem cells find potential application. One of the important biologic modifiers that can cause chondrogenic differentiation of stem cells is taurine. However, taurine has not been investigated for its effects on dental pulp derived stem cell (DPSC) chondrogenic differentiation. Objective: The objective of the study was to investigate if taurine administration to DPSCs heralds chondrogenic differentiation as ascertained by expression of SOX9, COL2A1, ACAN, ELN, and COMP. The study also investigated if the differentiated cells synthesized glycosaminoglycans, a marker of cartilage formation. The study also aimed to assess proliferative activity of the cells after taurine administration by measuring the hTERT gene and protein expression. Materials and methods: DPSCs were obtained from a molecular biology laboratory and characterization of stem cell markers was done by flow cytometry. The cells were subjected to a MTT assay using various concentrations of taurine. Following this, hTERT gene and protein estimation was done in the control, telomerase inhibitor treated DPSC (TI-III), 10 μM taurine treated DPSC, and TI-III + 10 μM taurine treated DPSCs. A polymerase chain reaction was done to assess gene expression of SOX9, COL2A1, ACAN, ELN, and COMP genes and glycosaminoglycans were estimated in control cells, Induced DPSCs, induced and TI-III treated DPSCs, and 10 μM taurine treated DPSCs. Results: DPSCs expressed CD73, CD90, and CD105 and did not express CD34, CD45, and HLA-DR, which demonstrated that they were mesenchymal stem cells. The MTT assay revealed that various concentrations of taurine did not affect the cell viability of DPSCs. A concentration of 10 μM of taurine was used for further assays. With regard to the hTERT gene and protein expression, the taurine treated cells expressed the highest levels that were statistically significant compared to the other groups. Taurine was also found to restore hTERT expression in telomerase inhibitor treated cells. With regard to chondrogenesis related genes, taurine administration significantly increased the expression of SOX9, COL2A1, ACAN, and ELN genes in DPSCs and caused a significant increase in glycosaminoglycan production by the cells. Conclusions: Taurine can be regarded a biologic modifier that can significantly augment chondrogenic differentiation of DPSCs and can find potential applications in regenerative medicine in the area of cartilage regeneration.

Azacitidine, as a DNMT Inhibitor Decreases hTERT Gene Expression and Telomerase Activity More Effective Compared with HDAC Inhibitor in Human Head and Neck Squamous Cell Carcinoma Cell Lines

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the most fatal malignancies worldwide and despite using various therapeutic strategies for the treatment of HNSCC, the surveillance rate is low. Telomerase has been remarked as the primary targets in cancer therapy. Considering the key regulatory role of epigenetic mechanisms in controlling genome expression, the present study aimed to investigate the effects of two epigenetic modulators, a DNA methylation inhibitor and a histone deacetylase inhibitor on cell migration, proliferation, hTERT gene expression, and telomerase activity in HNSCC cell lines. Methods: Human HNSCC cell lines were treated with Azacitidine and Trichostatin A to investigate their effects on telomerase gene expression and activity. Cell viability, migration, hTERT gene expression, and telomerase activity were studied using MTT colorimetric assay, scratch wound assay, qRT-PCR, and TRAP assay, respectively. Results: Azacitidine at concentrations of ≤1μM and Trichostatin A at 0.1 to 0.3nM concentrations significantly decreased FaDu and Cal-27 cells migration. The results showed that Azacitidine significantly decreased hTERT gene expression and telomerase activity in FaDu and Cal-27 cell lines. However, there were no significant changes in hTERT gene expression at different concentrations of Trichostatin A in both cell lines. Trichostatin A treatment affected telomerase activity at the high dose of 0.3 nM Trichostatin A. Conclusion: The findings revealed that unlike histone deacetylase inhibitor, Azacitidine as an inhibitor of DNA methylation decreases telomerase expression in HNSCC cells. This might suggest the potential role of DNA methyltransferase inhibitors in telomerase-based therapeutic approaches in squamous cell carcinoma.