hTERT Expression Research Articles

The Effect of Circulating Exosomes Obtained from Young and Old Individuals on the Aging related hTERT and P16 Expression in Hematopoietic Stem Cells

Introduction: Functional reduction of telomeres can induce DNA damage response through cell cycle checkpoints and contribute to the senescence of stem cells. The effect of exosomes on the aging and rejuvenation of hematopoietic stem cells (HSCs) is not well known. Therefore, the present study is designed to examine the impact of plasma exosomes derived from young and old individuals on hTERT and P16 expression involved in the cellular aging process. Methods: Exosomes isolated from four young (Y-Exo) and four old (O-Exo) men were evaluated for CD63 protein expression, morphology, size and zeta potential. HSCs were treated with exosomes, and then, the cell viability and the mRNA expression (hTERT and P16) were evaluated using MTT and qRT-PCR methods, respectively. To measure the hTERT protein level, a western blot technique was performed. Results: The gene expression of hTERT was significantly decreased in HSCs treated with 5 μg/ml (O5-Exo) and 10 μg/ml (O10-Exo) doses of exosomes obtained from elderly individuals compared to the cells treated with young exosomes and the untreated HSCs (p < 0.05). In addition, there was a profound elevation of hTERT protein in the HSCs treated with both doses of young exosomes in comparison with the cells treated with both doses of old exosomes (p < 0.05). Moreover, P16 expression was markedly upregulated in the O5-Exo and O10-Exo groups compared to the untreated group (p < 0.05). Conclusion: Our findings reinforce the concept that depending on the age of individuals, circulating exosomes may acquire properties that affect the pathways involved in the aging process in HSCs.

Transduction of Human Fetal Liver Hematopoietic CD34+ Stem and Progenitor Cells into a Cell Line by Enhancing Telomerase Activity

Background: Human fetal liver hematopoietic stem cells have proven potential as therapeutics but lack extensive research due to their limited supply. Even in vitro expanded fetal liver hematopoietic stem cells enter senescence or lose their self-renewal capacity after a few days in culture. The present study aimed to obtain a homogeneous and persistent supply of hematopoietic stem cells from the fetal liver by establishing a cell line through immortalization of cells by enhancing telomerase activity. Materials and Methods: Human fetal liver hematopoietic CD34+ stem and progenitor cells were transformed and immortalized using retroviruses carrying the human telomerase (hTERT) gene. Following transduction, telomerase activity was assessed using the TRAP assay and telomere length was examined by Southern blotting in transduced cells. Their characterization was conducted using flowcytometry to analyze the CD34+ population of hematopoietic stem cells and their colony forming potential using colony forming unit (CFU) assay. Results: After transduction with hTERT, the life span of human fetal liver hematopoietic CD34+ stem and progenitor cells were extended to 80 population doublings, without any change in cell morphology or population doubling times. Constitutive hTERT expression enhanced the replicative capacity and prevented terminal differentiation of CD34+ fetal liver hematopoietic stem and progenitor cells (FLHSPCs). Moreover, hTERT-transduced stem cells maintained their telomere length and telomerase activity. Conclusion: By introducing telomerase activity into hematopoietic stem and progenitor cells, their lifespan can be extended while maintaining stemness. These modified cells hold promise for in vitro research focused on studying hematopoietic stem cells derived from fetal liver.

Annexin A1 protects periodontal ligament cells against lipopolysaccharide-induced inflammatory response and cellular senescence: An implication in periodontitis.

Periodontitis is an inflammatory condition that affects the tooth-supporting structures, triggered by the host's immune response toward the bacterial deposits around the teeth. Annexin A1 (AnxA1), a vital member of the annexin superfamily, is known for its diverse physiological functions, particularly its anti-inflammatory and anti-senescence properties. We hypothesized that AnxA1 has a protective effect against lipopolysaccharide (LPS)-induced inflammatory responses and cellular damage in periodontal ligament cells (PDLCs). In this study, we demonstrate that LPS stimulation significantly reduced telomerase activity in PDLCs, a decline that was dose-dependently reversed by AnxA1. Importantly, AnxA1 protected the cells from LPS-induced cellular senescence and the downregulation of human telomerase reverse transcriptase (hTERT) expression. In line with this, AnxA1 suppressed the LPS-induced expression of p21 and p16 at both the mRNA and protein levels. Furthermore, AnxA1 demonstrated potent anti-inflammatory effects by inhibiting the secretion of interleukin 6 (IL-6), interleukin 8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1). It also mitigated LPS-induced oxidative stress by reducing the levels of phosphorylated Foxo3a (Ser253) and restored sirtuin 1 (SIRT1) expression. Notably, SIRT1 silencing abolished AnxA1's protective effects on Foxo3a phosphorylation and cellular senescence, suggesting that SIRT1 mediates AnxA1's actions. In conclusion, AnxA1 protected PDLCs against LPS-triggered inflammation and cell senescence by activating SIRT1 signal pathway. These findings indicate that AnxA1 could serve as a promising therapeutic strategy for the treatment of periodontitis.

The Impact of Ten Days of Periodic Fasting on the Modulation of the Longevity Gene in Overweight and Obese Individuals: A Quasi-Experimental Study.

Fasting potentially alters the aging process induced by obesity by regulating telomere integrity, which is related to longevity genes. However, the impact of periodic fasting (PF) on the expression of longevity genes, particularly Forkhead Box O Transcription Factors (FOXO3a) and the Human Telomerase Reverse Transcriptase (hTERT), is not fully understood. This study aimed to analyze the effects of PF, specifically on FOXO3a, hTERT expression, and other associated factors. A quasi-experimental 10-day study was conducted in Surabaya, East Java, Indonesia. This study consisted of an intervention group (PFG), which carried out PF for ten days using a daily 12 h time-restricted eating protocol, and a control group (CG), which had daily meals as usual. FOXO3a and hTERT expression were analyzed by quantitative real-time qPCR. A paired t-test/Wilcoxon test, independent t-test/Mann-Whitney U-test, and Spearman's correlation test were used for statistical analysis. Thirty-six young men participated in this study. During the post-test period, FOXO3a expression in the PFG increased 28.56 (±114.05) times compared to the pre-test, but the difference was not significant. hTERT expression was significantly higher in both the CG and PFG. The hTERT expression in the PFG was 10.26 (±8.46) times higher than in the CG, which was only 4.73 (±4.81) times higher. There was also a positive relationship between FOXO and hTERT in the CG. PF significantly increased hTERT expression in the PFG; however, no significant increase was found in FOXO3a expression. PF regimens using the 12 h time-restricted eating approach may become a potential strategy for preventing obesity-induced premature aging by regulating longevity gene expression.

Effect of aging and exercise on hTERT expression in thymus tissue of hTERT transgenic bacterial artificial chromosome mice.

Telomere shortening occurs with aging in immune cells and may be related to immunosenescence. Exercise can upregulate telomerase activity and attenuate telomere shortening in immune cells, but it is unknown if exercise impacts other immune tissues such as the thymus. This study aimed to examine human telomerase reverse transcriptase (hTERT) alternative splicing (AS) in response to aging and exercise in thymus tissue. Transgenic mice with a human TERT bacterial artificial chromosome integrated into its genome (hTERT-BAC) were utilized in two different exercise models. Mice of different ages were assigned to an exercise cage (running wheel) or not for 3weeks prior to thymus tissue excision. Middle-aged mice (16months) were exposed or not to treadmill running (30min at 60% maximum speed) prior to thymus collection. hTERT transcript variants were measured by RT-PCR. hTERT transcripts decreased with aging (r = - 0.7511, p < 0.0001) and 3weeks of wheel running did not counteract this reduction. The ratio of exons 7/8 containing hTERT to total hTERT transcripts increased with aging (r = 0.3669, p = 0.0423) but 3weeks of voluntary wheel running attenuated this aging-driven effect (r = 0.2013, p = 0.4719). Aging increased the expression of senescence marker p16 with no impact of wheel running. Thymus regeneration transcription factor, Foxn1, went down with age with no impact of wheel running exercise. Acute treadmill exercise did not induce any significant changes in thymus hTERT expression or AS variant ratio (p > 0.05). In summary, thymic hTERT expression is reduced with aging. Exercise counteracted a shift in hTERT AS ratio with age. Our data demonstrate that aging impacts telomerase expression and that exercise impacts dysregulated splicing that occurs with aging.

Derivation of human primary prostate epithelial cell lines by differentially targeting the CDKN2A locus along with expression of hTERT

Prostate cancer (PCa) is the most common cancer diagnosed in men worldwide and was the second leading cause of cancer-related deaths in US males in 2022. Prostate cancer also represents the second highest cancer mortality disparity between non-Hispanic blacks and whites. However, there is a relatively small number of prostate normal and cancer cell lines compared to other cancers. To identify the molecular basis of PCa progression, it is important to have prostate epithelial cell (PrEC) lines as karyotypically normal as possible. Our lab recently developed a novel methodology for the rapid and efficient immortalization of normal human PrEC that combines simultaneous CRISPR-directed inactivation of CDKN2A exon 2 (which directs expression of p16INK4A and p14ARF) and ectopic expression of an hTERT transgene. To optimize this methodology to generate immortalized lines with minimal genetic alterations, we sought to target exon 1α of the CDKN2A locus so that p16INK4A expression is ablated while the exons encoding p14ARF remains unaltered. Here we describe the establishment of two cell lines: one with the above-mentioned p16INK4A only loss, and a second line targeting both products in the CDKN2A locus. We characterize the potential lineage origin of these new cell lines along with our previously obtained clones, revealing distinct gene expression signatures. Based on the analyses of protein markers and RNA expression signatures, these cell lines are most closely related to a subpopulation of basal prostatic cells. Given the simplicity of this one-step methodology and the fact that it uses only the minimal genetic alterations necessary for immortalization, it should also be suitable for the establishment of cell lines from primary prostate tumor samples, an urgent need given the limited number of available prostate cancer cell lines.

Modulation of dynamic DNA G-quadruplex structures in the hTERT promoter region by ligands.

Small molecules can inhibit cellular processes such as replication and transcription by binding to the promoter regions that are prone to form G-quadruplexes. However, since G-quadruplexes exist throughout the human genome, the G-quadruplex binders suffer from specificity issues. To tackle this problem, a G-quadruplex binder (Pyridostatin, or PDS) is conjugated with a ligand (Polyamide, or PA) that can specifically recognize DNA sequences flanking the G-quadruplex forming region. The binding mechanism of this hybrid ligand to the hTERT promoter region (hTERT 5-12) is then elucidated using optical tweezers. During mechanical unfolding processes, different intermediate structures of hTERT 5-12 in presence of PDS, PA, or PA-PDS conjugateare observed. These intermediate structures are consistent with two folding patterns of G-quadruplexes in the hTERT 5-12 fragment. While the duplex DNA binder PA facilitates the folding of a hairpin-G-quadruplex structure, the PDS assists the formation of two tandem G-quadruplexes. Both replication stop assay in vitro and dual luciferase assay in vivo established the effectiveness of the PA-PDS conjugate for hTERT 5-12 targeting. We expect such a ligand dependent folding dynamics will provide guidelines to the development of drugs that not only target hTERT expressions, but also other oncogenes via interactions with specific G-quadruplex structures formed in their promotor regions.

Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq.

Telomerase is a specialized reverse transcriptase that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Biogenesis of human telomerase requires its RNA subunit (hTR) to fold into a multi-domain architecture that includes the template-containing pseudoknot (t/PK) and the three-way junction (CR4/5). These two hTR domains bind the telomerase reverse transcriptase (hTERT) protein and are thus essential for telomerase catalytic activity. Here, we probe the structure of hTR in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) and ensemble deconvolution analysis. Unexpectedly, approximately 15% of the steady state population of hTR has a CR4/5 conformation lacking features thought to be required for hTERT binding. The proportion of hTR CR4/5 that is folded into the primary functional conformation does not require hTERT expression and the fraction of hTR that assumes a misfolded CR4/5 domain is not refolded by overexpression of its hTERT binding partner. This result suggests a functional role for an RNA folding cofactor other than hTERT during telomerase biogenesis. Mutagenesis demonstrates that stabilization of the alternative CR4/5 conformation is detrimental to telomerase assembly and activity. Moreover, the alternative CR4/5 conformation is not found in telomerase RNP complexes purified from cells via an epitope tag on hTERT, supporting the hypothesis that only the major CR4/5 conformer is active. We propose that this misfolded portion of the cellular hTR pool is either slowly refolded or degraded. Thus, kinetic traps for RNA folding that have been so well-studied in vitro may also present barriers for assembly of ribonucleoprotein complexes in vivo.

Association of hTERT expression, Her2Neu, estrogen receptors, progesterone receptors, with telomere length before and at the end of treatment in breast cancer patients.

Breast cancer shows significant clinical, morphologic, and molecular variation. Telomeres are nucleoprotein complexes composed of hexanucleotide repeat DNA sequence, TTAGGG, and numerous telomere-associated proteins. The maintenance of telomere length is carried out by a ribonucleoprotein called telomerase, which consists of two main components: a catalytic subunit called hTERT (human telomerase reverse transcriptase) and an RNA template called hTR (human telomerase RNA). The importance of evaluating hTERT expression lies in its potential therapeutic application, being an attractive target due to its almost non-existent expression in normal somatic cells. It is also expected that the anti-neoplastic effect would appear earlier in neoplastic cells with shorter telomeres. Additionally, a significant relationship has been observed between Her2-Neu overexpression and Her2-Neu positivity, which could suggest new combined therapies.The aim of this study was to detect the expression of hTERT, estrogen receptor (ER), progesterone receptor (PR), and HER2-Neu in neoplastic breast tissue embedded in paraffin before treatment and to investigate the relationship between them and with baseline and post-treatment telomere length, as well as with various clinicopathological parameters. A cross-sectional-correlational, 21 women diagnosed with breast cancer at the Oncology Service of the High Specialty Medical Unit No. 1 of Bajio of the Mexican Institute of Social Security. The study complies with the Helsinki Declaration and was approved by the Institutional Ethical Committee of the Mexican Institute of Social Security (R-2019-1001-127). A peripheral blood sample was obtained before oncological treatment and at the end of oncological treatment for the measurement of telomere length by extracting DNA from leukocytes, was performed by the quantitative polymerase chain reaction (PCR) method described by Cawthon. Tumor samples were collected from each patient at the oncology department for immunohistochemical determination of biomarker expression (ER, PR, Her2/neu) and hTERT. Of the 21 cases included in the study, the median age was 57.57 years. Eighteen cases were classified as invasive ductal carcinoma NOS (85.71%), 10 were histologic grade 2 (47.61%), 16 cases were hormone receptor positive (76.19%), 7 were Her2Neu positive (33.33%), and only 2 cases were triple negative (9.52%). Positive hTERT expression was detected in 11 cases (52.38%) and was negative in the remaining cases. A significant association was identified between hTERT-positive cases and Her2-Neu positive cases (p = 0.04). Baseline and post-treatment telomere lengths showed a significant difference using the non-parametric Wilcoxon t-test (p = 0.002). In hTERT-positive cases, there was significant telomere shortening at the end of oncological treatment (6.14 ± 1.54 vs. 4.75 ± 1.96 Kb, p = 0.007). Positive hTERT immunostaining cases were associated with poor prognostic factors, such as Her2-Neu overexpression and post-treatment telomere shortening. In the future, hTERT immunostaining could be used to select patients for therapies with antagonistic effects on hTERT, as well as in the selection of more appropriate chemotherapy regimens for patients who express it.

Telomere Length, Telomerase Activity, and Vaginal Microbiome in Patients with HPV-Related Precancerous Lesions.

Persistent high-risk human papillomaviruses (HR HPVs) infection leads to the development of squamous intraepithelial lesions in cervical cells that may lead to cancer. The telomere length, telomerase activity, and species composition of the vaginal microbiome may influence the dynamic of changes and the process of carcinogenesis. In the present study, we analyze relative telomere length (RTL), relative hTERT expression (gene for the telomerase component-reverse transcriptase) in cervical smear cells and vaginal microbiomes. Total RNA and DNA were isolated from tissue samples of 109 patients from the following groups: control, carrier, low-grade or high-grade squamous intraepithelial lesion (L SIL and H SIL, respectively), and cancer. The quantitative PCR method was used to measure telomere length and telomerase expression. Vaginal microbiome bacteria were divided into community state types using morphotype criteria. Significant differences between histopathology groups were confirmed for both relative telomere length and relative hTERT expression (p < 0.001 and p = 0.001, respectively). A significant difference in RTL was identified between carriers and H SIL (p adj < 0.001) groups, as well as between carriers and L SIL groups (p adj = 0.048). In both cases, RTL was lower among carriers. The highest relative hTERT expression level was recorded in the H SIL group, and the highest relative hTERT expression level was recorded between carriers and the H SIL group (p adj < 0.001). A correlation between genotype and biocenosis was identified for genotype 16+A (p < 0.001). The results suggest that identification of HPV infection, telomere length assessment, and hTERT expression measurement together may be more predictive than each of these analyses performed separately.

Targeting hTERT Promoter G-Quadruplex DNA Structures with Small-Molecule Ligand to Downregulate hTERT Expression for Triple-Negative Breast Cancer Therapy.

Human telomerase reverse transcriptase (hTERT) may have noncanonical functions in transcriptional regulation and metabolic reprogramming in cancer cells, but it is a challenging target. We thus developed small-molecule ligands targeting hTERT promoter G-quadruplex DNA structures (hTERT G4) to downregulate hTERT expression. Ligand 5 showed high affinity toward hTERT G4 (Kd = 1.1 μM) and potent activity against triple-negative breast cancer cells (MDA-MB-231, IC50 = 1 μM). In cell-based assays, 5 not only exerts markedly inhibitory activity on classical telomere functions including decreased telomerase activity, shortened telomere length, and cellular senescence but also induces DNA damage, acute cellular senescence, and apoptosis. This study reveals that hTERT G4-targeting ligand may cause mitochondrial dysfunction, disrupt iron metabolism and activate ferroptosis in cancer cells. The in vivo antitumor efficacy of 5 was also evaluated in an MDA-MB-231 xenograft mouse model and approximately 78.7% tumor weight reduction was achieved. No observable toxicity against the major organs was observed.

DDB2 promotes melanoma cell growth by transcriptionally regulating the expression of KMT2A and predicts a poor prognosis.

Identification of potential key targets of melanoma, a fatal skin malignancy, is critical to the development of new cancer therapies. Lysine methyltransferase 2A (KMT2A) promotes melanoma growth by activating the human telomerase reverse transcriptase (hTERT) signaling pathway; however, the exact mechanism remains elusive. This study aimed to reveal new molecular targets that regulate KMT2A expression and melanoma growth. Using biotin-streptavidin-agarose pull-down and proteomics, we identified Damage-specific DNA-binding protein 2 (DDB2) as a KMT2A promoter-binding protein in melanoma cells and validated its role as a regulator of KMT2A/hTERT signaling. DDB2 knockdown inhibited the expression of KMT2A and hTERT and inhibited the growth of melanoma cells invitro. Conversely, overexpression of DDB2 activated the expression of KMT2A and promoted the growth of melanoma cells. Additionally, we demonstrated that DDB2 expression was higher in tumor tissues of patients with melanoma than in corresponding normal tissues and was positively correlated with KMT2A expression. Kaplan-Meier analysis showed a poor prognosis in patients with high levels of DDB2 and KMT2A. Overall, our data suggest that DDB2 promotes melanoma cell growth through the transcriptional regulation of KMT2A expression and predicts poor prognosis. Therefore, targeting DDB2 may regulate the effects of KMT2A on melanoma growth and progression, providing a new potential therapeutic strategy for melanoma.

Development and validation of novel keloid-derived immortalized fibroblast cell lines.

Keloids are a common connective tissue disorder with an ill-understood etiopathogenesis and no effective treatment. This is exacerbated because of the absence of an animal model. Patient-derived primary keloid cells are insufficient as they age through passaging and have a limited supply. Therefore, there is an unmet need for development of a cellular model that can consistently and faithfully represent keloid's pathognomic features. In view of this, we developed keloid-derived immortalized fibroblast (KDIF) cell lines from primary keloid fibroblasts (PKF) by transfecting the human telomerase reverse transcriptase (hTERT) gene. The TERT gene encodes the catalytic subunit of the telomerase enzyme, which is responsible for maintaining the cellular replicative potential (cellular immortalization). Primary fibroblasts from keloid-specific lesional (peripheral, middle, and top) as well as extralesional sites were isolated and evaluated for cell line development and comparative cellular characteristics by employing qRT-PCR and immunofluorescence staining. Moreover, the immortalized behavior of KDIF cell lines was evaluated by comparing with cutaneous fibrosarcoma and dermatofibrosarcoma protuberans cell lines. Stable KDIF cell lines with elevated expression of hTERT exhibited the cellular characteristics of site-specific keloid fibroblasts. Histochemical staining for β-galactosidase revealed a significantly lower number of β-gal-positive cells in all three KDIF cell lines compared with that in PKFs. The cell growth curve pattern was studied over 10 passages for all three KDIF cell lines and was compared with the control groups. The results showed that all three KDIF cell lines grew significantly faster and obtained a fast growing characteristic as compared to primary keloid and normal fibroblasts. Phenotypic behavior in growth potential is an indication of hTERT-mediated immortalized transformation. Cell migration analysis revealed that the top and middle KDIF cell lines exhibited similar migration trend as site-specific PKFs. Notably, peripheral KDIF cell line showed significantly enhanced cell migration in comparison to the primary peripheral fibroblasts. All KDIF cell lines expressed Collagen I protein as a keloid-associated fibrotic marker. Functional testing with triamcinolone inhibited cell migration in KDIF. ATCC short tandem repeat profiling validated the KDIF as keloid representative cell line. In summary, we provide the first novel KDIF cell lines. These cell lines overcome the limitations related to primary cell passaging and tissue supply due to immortalized features and present an accessible and consistent experimental model for keloid research.

The Effects of Smoking on Telomere Length, Induction of Oncogenic Stress, and Chronic Inflammatory Responses Leading to Aging.

Telomeres, potential biomarkers of aging, are known to shorten with continued cigarette smoke exposure. In order to further investigate this process and its impact on cellular stress and inflammation, we used an in vitro model with cigarette smoke extract (CSE) and observed the downregulation of telomere stabilizing TRF2 and POT1 genes after CSE treatment. hTERT is a subunit of telomerase and a well-known oncogenic marker, which is overexpressed in over 85% of cancers and may contribute to lung cancer development in smokers. We also observed an increase in hTERT and ISG15 expression levels after CSE treatment, as well as increased protein levels revealed by immunohistochemical staining in smokers' lung tissue samples compared to non-smokers. The effects of ISG15 overexpression were further studied by quantifying IFN-γ, an inflammatory protein induced by ISG15, which showed greater upregulation in smokers compared to non-smokers. Similar changes in gene expression patterns for TRF2, POT1, hTERT, and ISG15 were observed in blood and buccal swab samples from smokers compared to non-smokers. The results from this study provide insight into the mechanisms behind smoking causing telomere shortening and how this may contribute to the induction of inflammation and/or tumorigenesis, which may lead to comorbidities in smokers.

TPP1 is associated with risk of advanced precursors and cervical cancer survival.

It is unclear how telomere-binding protein TPP1 interacts with human telomerase reverse transcriptase (hTERT) and influences cervical cancer development and progression. This study included all eligible 156 cervical cancers diagnosed during 2003-2008 and followed up through 2014, 102 cervical intraepithelial neoplasia (CIN) patients, and 16 participants with normal cervix identified at the same period. Correlation of expression of TPP1 and hTERT in these lesions was assessed using Kappa statistics. TPP1 was knocked down by siRNA in three cervical cancer cell lines. We assessed mRNA expression using quantitative real-time polymerase chain reaction and protein expression using tissue microarray-based immunohistochemical staining. We further analyzed the impact of TPP1 expression on the overall survival of cervical cancer patients by calculating the hazard ratio (HR) with 95% confidence intervals (CIs) using the multivariable-adjusted Cox regression model. Compared to the normal cervix, high TPP1expression was significantly associated with CIN 3 and cervical cancers (P<0.001 for both). Expressions of TPP1 and hTERT were highly correlated in CIN 3 (Kappa statistics = 0.50, P = 0.005), squamous cell carcinoma (Kappa statistics = 0.22, P = 0.011), and adenocarcinoma/adenosquamous carcinoma (Kappa statistics = 0.77, P = 0.001). Mechanistically, knockdown of TPP1 inhibited the expression of hTERT in both mRNA and protein levels. High expression of TPP1 (HR = 2.61, 95% CI 1.23-5.51) and co-high expression of TPP1 and hTERT (HR = 2.38, 95% CI 1.28-4.43) were independently associated with worse survival in cervical cancer patients. TPP1 and hTERT expression was correlated and high expression of TPP1 was associated with high risk of CIN 3 and cervical cancer and could predict a worse survival in cervical cancer.

Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine.

Stem cells are undifferentiated cells that possess the potential for self-renewal with the capacity to differentiate into multiple lineages. In humans, their limited numbers pose a challenge in fulfilling the necessary demands for the regeneration and repair of damaged tissues or organs. Studies suggested that mesenchymal stem cells (MSCs), necessary for repair and regeneration via transplantation, require doses ranging from 10 to 400 million cells. Furthermore, the limited expansion of MSCs restricts their therapeutic application. To optimize a novel protocol to achieve qualitative and quantitative expansion of MSCs to reach the targeted number of cells for cellular transplantation and minimize the limitations in stem cell therapy protocols. Human umbilical cord (hUC) tissue derived MSCs were obtained and re-cultured. These cultured cells were subjected to the following evaluation procedures: Immunophenotyping, immunocytochemical staining, trilineage differentiation, population doubling time and number, gene expression markers for proliferation, cell cycle progression, senescence-associated β-galactosidase assay, human telomerase reverse transcriptase (hTERT) expression, mycoplasma, cytomegalovirus and endotoxin detection. Analysis of pluripotent gene markers Oct4, Sox2, and Nanog in recultured hUC-MSC revealed no significant differences. The immunophenotypic markers CD90, CD73, CD105, CD44, vimentin, CD29, Stro-1, and Lin28 were positively expressed by these recultured expanded MSCs, and were found negative for CD34, CD11b, CD19, CD45, and HLA-DR. The recultured hUC-MSC population continued to expand through passage 15. Proliferative gene expression of Pax6, BMP2, and TGFb1 showed no significant variation between recultured hUC-MSC groups. Nevertheless, a significant increase (P < 0.001) in the mitotic phase of the cell cycle was observed in recultured hUC-MSCs. Cellular senescence markers (hTERT expression and β-galactosidase activity) did not show any negative effect on recultured hUC-MSCs. Additionally, quality control assessments consistently confirmed the absence of mycoplasma, cytomegalovirus, and endotoxin contamination. This study proposes the development of a novel protocol for efficiently expanding stem cell population. This would address the growing demand for larger stem cell doses needed for cellular transplantation and will significantly improve the feasibility of stem cell based therapies.

CRL4DCAF4 E3 ligase-mediated degradation of MEN1 transcriptionally reactivates hTERT to sustain immortalization in colorectal cancer cells.

Telomerase reactivation is implicated in approximately 85% of human cancers, yet its underlying mechanism remains elusive. In this study, we elucidate that the cullin-RING ubiquitin ligase 4 (CRL4) complex drives the reactivation of human telomerase reverse transcriptase (hTERT) in colorectal cancer (CRC) by degrading the tumor suppressor, menin 1 (MEN1). Our data show that, in noncancerous intestinal epithelial cells, the transcription factor specificity protein 1 (Sp1) recruits both the histone acetyltransferase p300 and MEN1 to suppress hTERT expression, thus maintaining telomere shortness post-cell division. Inflammation-induced microenvironments trigger an activation of the CRL4DCAF4 E3 ligase, leading to MEN1 ubiquitination and degradation in CRC cells. This process nullifies MEN1's inhibitory action, reactivates hTERT expression at the transcriptional level, interrupts telomere shortening and spurs uncontrolled cellular proliferation. Notably, MEN1 overexpression in CRC cells partially counteracts these oncogenic phenotypes. NSC1517, an inhibitor of the CRL4DCAF4 complex identified through high-throughput screening from a plant-derived chemical pool, hinders MEN1 degradation, attenuates hTERT expression and suppresses tumor growth in mouse xenograft models. Collectively, our research elucidates the transcriptional mechanism driving hTERT reactivation in CRC. Targeting the CRL4DCAF4 E3 ligase emerges as a promising strategy to counteract cancer cell immortalization and curb tumor progression.

Pazopanib stimulates senescence of renal carcinoma cells through targeting nuclear factor E2-related factor 2 (Nrf2).

Renal cell carcinoma (RCC) is the most common kidney cancer with high mortality rate. Pazopanib has been approved for the treatment of RCC. However, the underlying mechanism is not clear. Here, we report a novel finding by showing that treatment with Pazopanib could promote cellular senescence of the human RCC cell line ACHN. Cells were stimulated with 5, 10, and 20 μM Pazopanib, respectively. Cellular senescence was measured using senescence-associated β-galactosidase (SA-β-Gal) staining. Western blot analysis and real-time polymerase chain reactionwere used to measure the mRNA and protein expression of nuclear factor E2-related factor 2 (Nrf2), γH2AX, human telomerase reverse transcriptase (hTERT), telomeric repeat binding factor 2 (TERF2), p53 and plasminogen activator inhibitor (PAI). First, we found that exposure to Pazopanib reduced the cell viability of ACHN cells. Additionally, Pazopanib induced oxidative stress by increasing the production of reactive oxygen species,reducing the levels of glutathione peroxidase,and promoting nuclear translocation of Nrf2. Interestingly, Pazopanib exposure resulted in DNA damage by increasing the expression of γH2AX. Importantly, Pazopanib increased cellular senescence and reduced telomerase activity. Pazopanib also reduced the gene expression of hTERT but increased the gene expression of TERF2. Correspondingly, we found that Pazopanib increased the expression of p53 and PAI at both the mRNA and protein levels. To elucidate the underlying mechanism, the expression of Nrf2 was knocked down by transduction with Ad- Nrf2 shRNA. Results indicate that silencing of Nrf2 in ACHN cells abolished the effects of Pazopanib in stimulating cellular senescence and reducing telomerase activity. Consistently, knockdown of Nrf2 restored the expression of p53 and PAI in ACHN cells. Based on these results, we explored a novel mechanism whereby which Pazopanib displays a cytotoxicity effect in RCC cells through promoting cellular senescence mediated by Nrf2.

Expression of hTERT and Lp-PLA(2) genes is increased in primary hypothyroidism.

ObjectiveSightly low serum free T4 levels are associated with longer life expectancy. However, hypothyroidism is associated with increased risk of inflammation and vascular complications. Therefore, relation of thyroid hormones (THs) with life expectancy seems to be complex. This study was carried out in an effort to understand the contribution of THs in inflammation and aging. Methodology15 cases of treatment naïve primary hypothyroidism and 15 age and sex matched euthyroid controls were recruited. mRNA expression of Telomerase (hTERT), leucocyte telomere length (LTL) and Lipoprotein associated phospholipase A2 (Lp-PLA2), a marker of vascular inflammation and risk predictor of cardiovascular events were measured by qPCR in whole blood. ResultExpression of hTERT was found to be 6.95 times higher in the cases as compared to the controls. mRNA expression of Lp-PLA(2) was also 3.3 times higher in cases. LTL in euthyroid was approximately 81% of the length in hypothyroid patients. ConclusionA higher expression of hTERT indicates that hypothyroidism confers better cell survival in peripheral leucocytes inspite of the presence of vascular inflammation. However causal relationship cannot be ascertained with these results.

Abstract 656: A novel dual-targeting peptide HS1002 enhances anti-tumor activity via GnRH/human telomerase reverse transcriptase (hTERT) interaction in human prostate cancer cells

Abstract Human telomerase reverse transcriptase (hTERT) is overexpressed in most human cancers. hTERT is considered as an important target for cancer therapy. In the present study, a new peptide HS1002 were synthesized with sequence of pGlu-His-Trp-Ser-Tyr-Arg-Leu-Arg-Phe-Ile-Pro-NHEt based on the GnRH agonist and hTERT. The aim of this study was to evaluate HS1002 for its anticancer activity by dual-targeting to GnRH/hTERT in prostate cancer cells. Upon comparison with different prostate cancer cell lines, HS1002 exhibited highest cytotoxicity against LNCaP cells. The hTERT expression correlated with telomerase activity in LNCaP cells, was significantly inhibited by HS1002, resulting in reduction of proliferation, metastasis and increase in apoptosis. Additionally, HS1002 suppressed c-Myc and AKT/ERK pathway in LNCaP cells. In the tumor-bearing nude mice model, HS1002 significantly inhibited tumor growth and downregulated the expression of hTERT and Ki-67 in tumor tissue as evidenced by western blot and immunohistochemistry analysis, respectively. Moreover, repeated subcutaneous injection of HS1002 resulted in the reduction of both serum testosterone levels and seminal vesicle weight. HS1002 also increased cytosolic calcium influx and CRE-luciferase activity in GnRHR-overexpressing HEK293 cells compared to mock-transfected transfected cells. Furthermore, a potent cytotoxicity in LNCaP cells was exhibited by HS1002-activated peripheral blood mononuclear cell (PBMC) with interleukin (IL)-2 using LDH release assay. In addition, a flow-cytometric analysis confirmed an elevation in the production of granzyme B and IFN-γ in CD8+ T cells in the MC38 syngeneic mouse model by HS1002. These data indicate that the inhibition of hTERT expression by HS1002 suppresses prostate cancer cell growth and induced anti-cancer immunity. Therefore, HS1002 might be used as a novel therapeutic drug for prostate cancer. Citation Format: Jae Hyeon Park, Ju Ri Kim, Hwa Young Cha, Hyung Sik Kim. A novel dual-targeting peptide HS1002 enhances anti-tumor activity via GnRH/human telomerase reverse transcriptase (hTERT) interaction in human prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 656.