Telomerase Reverse Transcriptase Gene Research Articles

Metformin and p-coumaric acid downregulate the expression of hTERT in gastric cancer cell line AGS

Gastric cancer is among the most common and fetal cancers worldwide. Its treatments are expensive and cause side effects. Therefore, treating gastric cancer with natural compounds and known therapeutics, or combinations of them can be interesting alternatives. p-Coumaric acid (pCA) and metformin (Met) are among such anticancer agents. Gastric cancer cells express a high level of human telomerase reverse transcriptase (hTERT) gene, which enhances their invasion and metastasis. As a result, down-regulation of the hTERT gene may be an effective anticancer therapy. Since the effects of treatments with pCA, Met, and their combination on h-TERT expression have not been investigated, the present study was carried out to assess these effects. MTT assay was used to measure the cytotoxic effects of pCA and/or Met on the AGS cells during 48 h. Real-time PCR was used to assess the changes in the expression levels of hTERT gene after 48 h. Wound healing assay, transwell migration and invasion assays, and colony formation assay were performed to determine the metastatic potentials of the AGS cells treated with pCA and/or Met. pCA and Met had dose-dependent cytotoxic effects on the AGS cell line. Treatments with pCA, Met, and their combination significantly suppressed hTERT expression, significantly decreased cellular migration and invasion, and also significantly lowered colony numbers. pCA and Met decrease the expression of hTERT at very low, non-cytotoxic concentrations and inhibit migration, invasion, and colony formation in the AGS gastric cancer cell line. Therefore they may be potential candidates for further investigations in fighting against gastric cancer and may pave the way for the emergence on new anti-gastric cancer therapeutics.

Genetic Polymorphisms of the Telomerase Reverse Transcriptase Gene in Relation to Prostate Tumorigenesis, Aggressiveness and Mortality: A Cross-Ancestry Analysis.

Telomerase reverse transcriptase (TERT) has been consistently associated with prostate cancer (PCa) risk. However, few studies have explored the association between TERT variants and PCa aggressiveness. Individual and genetic data were obtained from UK Biobank and a Chinese PCa cohort (Chinese Consortium for Prostate Cancer Genetics). A total of 209,694 Europeans (14,550 PCa cases/195,144 controls) and 8873 Chinese (4438 cases/4435 controls) were involved. Nineteen susceptibility loci with five novel ones (rs144704378, rs35311994, rs34194491, rs144020096, and rs7710703) were detected in Europeans, whereas seven loci with two novel ones (rs7710703 and rs11291391) were discovered in the Chinese cohort. The index SNP for the two ancestries was rs2242652 (odds ratio [OR] = 1.16, 95% confidence interval [CI]:1.12-1.20, p = 4.12 × 10-16) and rs11291391 (OR = 1.73, 95%CI:1.34-2.25, p = 3.04 × 10-5), respectively. SNPs rs2736100 (OR = 1.49, 95%CI:1.31-1.71, p = 2.91 × 10-9) and rs2853677 (OR = 1.74, 95%CI:1.52-1.98, p = 3.52 × 10-16) were found significantly associated with aggressive PCa, while rs35812074 was marginally related to PCa death (hazard ratio [HR] = 1.61, 95%CI:1.04-2.49, p = 0.034). Gene-based analysis showed a significant association of TERT with PCa (European: p = 3.66 × 10-15, Chinese: p = 0.043) and PCa severity (p = 0.006) but not with PCa death (p = 0.171). TERT polymorphisms were associated with prostate tumorigenesis and severity, and the genetic architectures of PCa susceptibility loci were heterogeneous among distinct ancestries.

Immortalized cell lines derived from dental/odontogenic tissue.

Stem cells derived from dental/odontogenic tissue have the property of multiple differentiation and are prospective in tooth regenerative medicine and cellular and molecular studies. However, in the face of cellular senescence soon in vitro, the proliferation ability of the cells is limited, so studies are hindered to some extent. Fortunately, immortalization strategies are expected to solve the above issues. Cellular immortalization is that cells are immortalized by introducing oncogenes, human telomerase reverse transcriptase genes (hTERT), or miscellaneous immortalization genes to get unlimited proliferation. At present, a variety of immortalized stem cells from dental/odontogenic tissue has been successfully generated, such as dental pulp stem cells (DPSCs), periodontal ligament cells (PDLs), stem cells from human exfoliated deciduous teeth (SHEDs), dental papilla cells (DPCs), and tooth germ mesenchymal cells (TGMCs). This review summarized establishment and applications of immortalized stem cells from dental/odontogenic tissues and then discussed the advantages and challenges of immortalization.

Inhibition of Posterior Capsule Opacification by Adenovirus-Mediated Delivery of Short Hairpin RNAs Targeting TERT in a Rabbit Model

Purpose Posterior capsule opacification (PCO) is the most common postoperative complication after cataract surgery and cannot yet be eliminated. Here, we investigated the inhibitory effects of telomerase reverse transcriptase (TERT) gene silencing on PCO in a rabbit model. Methods After rabbit lens epithelial cells (LECs) were treated with adenovirus containing short hairpin RNAs (shRNA) targeting TERT (shTERT group), adenovirus containing scramble nonsense control shRNA (shNC group) or PBS (control group), quantitative real-time polymerase chain reaction and Western blotting were used to measure the expression levels of TERT, and a scratch assay was performed to assess the LEC migration. New Zealand white rabbits underwent sham cataract surgery followed by an injection of adenovirus carrying shTERT into their capsule bag. The intraocular pressure and anterior segment inflammation were evaluated on certain days, and EMT markers (α-SMA and E-cadherin) were evaluated by Western blotting and immunofluorescence. The telomerase activity of the capsule bag was detected by ELISA. At 28 d postoperatively, hematoxylin and eosin staining of the cornea and iris and electron microscopy of the posterior capsule were performed. Results Application of shTERT to LECs downregulated the expression levels of TERT mRNA and protein. The scratch assay results showed a decrease in the migration of LECs in the shTERT group. In vivo, shTERT decreased PCO formation after cataract surgery in rabbits and downregulated the expression of EMT markers, as determined by Western blotting and immunofluorescence. In addition, telomerase activity was suppressed in the capsule bag. Despite slight inflammation in the iris, histologic results revealed no toxic effects in the cornea and iris. Conclusion TERT silencing effectively reduces the migration and proliferation of LECs and the formation of PCO. Our findings suggest that TERT silencing may be a potential preventive strategy for PCO.

Overexpression of the telomerase holoenzyme induces EMT and tumorigenesis of HPV-immortalized keratinocytes.

Cervical cancer is the most frequent malignancy of the female genital tract and is associated with persistent infection of the uterine cervix with high-risk human papillomaviruses (HPV). The two HPV oncoproteins, E6 and E7, cooperatively immortalize cervical cells and are essential but insufficient for inducing tumorigenicity. During the progression of HPV-associated cervical dysplasia to carcinoma, the cellular telomerase reverse transcriptase (TERT) gene is activated and the TERC gene amplified. We questioned whether these increases in telomerase components might mediate the acquisition of the tumorigenic phenotype. We therefore transduced the TERT and TERC genes into E6/E7 immortalized keratinocytes that were anchorage-dependent and nontumorigenic. The resultant cells showed a profound morphological change characteristic of epithelial-mesenchymal transitionas well as a corresponding increase in expression of vimentin, N-cadherin, Zinc finger E-Box binding homeobox 1, snail family transcriptional repressor 1and matrix Metallopeptidase 2 and decrease in keratin and E-cadherin. More important, the transduced cells were now anchorage-independent and formed tumors in immunodeficient mice. Our findings indicate that overexpression of the telomerase holoenzyme in HPV-immortalized cells is sufficient to induce the complete transformed phenotype.

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 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.

Telomerase inhibition is an effective therapeutic strategy in TERT promoter-mutant glioblastoma models with low tumor volume.

Glioblastoma is one of the most lethal forms of cancer, with 5-year survival rates of only 6%. Glioblastoma-targeted therapeutics have been challenging to develop due to significant inter- and intra-tumoral heterogeneity. Telomerase reverse transcriptase gene (TERT) promoter mutations are the most common known clonal oncogenic mutations in glioblastoma. Telomerase is therefore considered to be a promising therapeutic target against this tumor. However, an important limitation of this strategy is that cell death does not occur immediately after telomerase ablation, but rather after several cell divisions required to reach critically short telomeres. We, therefore, hypothesize that telomerase inhibition would only be effective in glioblastomas with low tumor burden. We used CRISPR interference to knock down TERT expression in TERT promoter-mutant glioblastoma cell lines and patient-derived models. We then measured viability using serial proliferation assays. We also assessed for features of telomere crisis by measuring telomere length and chromatin bridge formation. Finally, we used a doxycycline-inducible CRISPR interference system to knock down TERT expression in vivo early and late in tumor development. Upon TERT inactivation, glioblastoma cells lose their proliferative ability over time and exhibit telomere shortening and chromatin bridge formation. In vivo, survival is only prolonged when TERT knockdown is induced shortly after tumor implantation, but not when the tumor burden is high. Our results support the idea that telomerase inhibition would be most effective at treating glioblastomas with low tumor burden, for example in the adjuvant setting after surgical debulking and chemoradiation.

Epigenetic changes of Arabidopsis MET1 cytosine methyltransferase mutants under salt stress

In this study, we investigated the morphological and molecular responses of Arabidopsis met1-7 and met1-3 mutants under salinity stress. Global DNA methylation changes of mutants were compared and genes known to be involved in DNA methylation in plants were analyzed. Also, the expression of the telomerase reverse transcriptase (TERT) gene as a stress response indicator was analyzed in order to examine the effects of stress on the telomerase enzyme in the mutants. We found that mutants have a higher rate of hypomethylation than Col-0, under all conditions. According to the qPCR analysis, expression levels of Domains Rearranged Methylase 2 (DRM2), Polymerase IV (Pol IV) and Polymerase V (Pol V) genes were higher in mutants than Col-0 plants. Contrary to expectations for the TERT gene, there was an increase in expression in the stress-applied mutant plants. Taken together, results suggest that the RNA-directed DNA methylation (RdDM) pathway is enhanced in mutants in order to deal with the lack of DNA methylation in CG sites. In conclusion, we believe that the morphological and molecular data obtained regarding the effects of NaCl application to met1 mutants will help us to understand the epigenetic basis of stress mechanisms.

Dva ko-nasleđena SNPs gena telomeraza reverzne transkriptaze (TERT) koja se dovode u vezu sa pacijentima od raka pluća u Iraku

Background: The telomerase reverse transcriptase (TERT) gene is essential polymorphic loci linked to most malignant tumors. This study assessed the association between the TERT gene and non-small cell lung carcinoma (NSCLC) in Iraq. Methods: Genomic DNA samples were extracted from a total of 200 samples of blood. Four specific PCR fragments were designed to amplify four high-frequency rs2735940, rs2736098, rs2736100, and rs10069690 SNPs within the TERT gene. Single-strand conformation polymorphism (SSCP) followed by sequencing reactions were used for genotyping and validating the amplified fragments. Results: Individuals with the genotype rs2735940: A/G were at a significantly greater risk of developing NSCLC (P=0.0299; OD 2.3158; Cl95% 1.0853 to 4.9414). Individuals with the genotype rs2736098: C/T were also significantly associated with the increased likelihood of developing NSCLC (P=0.0363; OD 2.1583; Cl95% 1.0503 to 4.4351). Linkage disequilibrium analysis showed that both SNPs showed a very high level of patient coinheritance. The LD plot showed that allele T of rs2736098 had collaborated with allele G of rs2735940 to generate TG haplotype in patients. According to our findings, both TERTrs2735940: A/G and TERT-rs2736098: C/T SNPs were found to be significant associations with the elevated risk of NSCLC. Both SNPs showed the highest values of co-inheritance in patients. This co-inheritance is mainly represented by alleles rs2735940: A and rs2736098: C. Both pathogenic T and G alleles have generated TG haplotype that is only available in patients' samples. Conclusion: This study suggests employing the haplotype TG as a promising biomarker for the early diagnosis of NSCLC. These findings need further validation by largescale investigation with a larger size of samples in the study population.

Establishing an hTERT-driven immortalized umbilical cord-derived mesenchymal stem cell line and its therapeutic application in mice with liver failure

Acute liver failure (ALF) is characterized by rapid liver cell destruction. It is a multi-etiological and fulminant complication with a clinical mortality of over 80%. Therapy using mesenchymal stem cells (MSCs) or MSCs-derived exosomes can alleviate acute liver injury, which has been demonstrated in animal experiments and clinical application. However, similar to other stem cells, different cell sources, poor stability, cell senescence and other factors limit the clinical application of MSCs. To achieve mass production and quality control on stem cells and their exosomes, transfecting umbilical cord mesenchymal stem cell (UCMSC) with lentivirus overexpressing human telomerase reverse transcriptase (hTERT) gene, the hTERT-UCMSC was constructed as an immortalized MSC cell line. Compared with the primary UCMSC (P3) and immortalized cell line hTERT-UCMSC at early passage (P10), the hTERT-UCMSC retained the key morphological and physiological characteristics of UCMSC at the 35th passage (P35), and showed no signs of carcinogenicity and toxic effect in mice. There was no difference in either exosome production or characteristics of exosomes among cultures from P3 primary cells, P10 and P35 immortalized hTERT-UCMSCs. Inoculation of either hTERT-UCMSC (P35) or its exosomes improved the survival rate and liver function of ALF mice induced by thioacetamide (TAA). Our findings suggest that this immortalized cell line can maintain its characteristics in long-term culture. Inoculation of hTERT-UCMSC and its exosomes could potentially be used in clinics for the treatment of liver failure in the future.

TERT Mutations in Non-Small Cell Lung Cancer: Clinicopathologic Features and Prognostic Implications.

The associations between the clinical characteristics of non-small cell lung cancer (NSCLC) and mutations in telomerase reverse transcriptase (TERT) gene remain unclear. In this study, we used next-generation sequencing (NGS) to investigate the incidence rate and clinical correlates of TERT mutations in patients with NSCLC. In total, 283 tumor samples from patients with NSCLC were tested using an NGS panel from September 2017 to May 2020. The genetic testing results and clinical data of all patients were collected. TERT mutations were found in 30 patients, which were significantly associated with age, smoking history, sex, and metastasis (P < 0.05). Survival analyses showed that patients who carried TERT mutations had a poorer prognosis. Of the 30 TERT-mutation carriers, 17 harbored epidermal growth factor receptor (EGFR) mutations, which were significantly associated with sex, histopathology type, and metastasis (P < 0.05; overall survival [OS], 21 months; 95% confidence interval [CI], 8.153-33.847 months). Three TERT mutation patients harbored Kirsten rat sarcoma virus (KRAS) mutations, which were significantly associated with metastasis risk (P < 0.05), KRAS mutations carriers had a worse prognosis, with an OS of 10 months (95% CI, 8.153-33.847 months). Multivariate Cox regression analyses showed that age, cancer stage, and TERT mutation carrier status were independent risk factors for NSCLC, and the TERT mutation was 2.731 times higher than that without TERT mutation (95% CI, 1.689-4.418, P < 0.001). TERT mutations were present in 11% of patients with NSCLC. TERT mutations were associated with age, smoking history, sex, and distant metastasis. Co-mutations in TERT and EGFR/KRAS indicated a poor prognosis. The co-mutations of TERT and EGFR differed according to sex, histopathology type, and metastasis, whereas TERT and KRAS co-mutations were only associated with patient metastasis. Age, cancer stage, and TERT mutation carrier status were independent risk factors for poor prognosis in patients with NSCLC.

The TERT Promoter: A Key Player in the Fight for Cancer Cell Immortality

The review describes the role of telomeres and telomerase in tumor progression, as well as various mechanisms of the activation of telomerase reverse transcriptase (TERT) expression in CNS tumors and other cancers. The main mechanism of TERT activation involves acquisition of somatic mutations by the TERT gene promoter (TERTp). The article presents information on the TERTp structure and transcription factors directly interacting with TERTp and regulating its transcription. The prospects of using the mutational status of TERTp as a prognostic marker of CNS malignancies and other tumors with a common profile of TERTp mutations are discussed.

DNA TYPING BY TELOMERASE REVERSE GENE TRANSCRIPTASE (TERT)

The length of the telomeric DNA and polymorphism of the TERT (Telomerase Reverse Transcriptase) gene can be the basis for the development of molecular genetic markers for the productive traits of agricultural animals, in particular pigs.&#x0D; The purpose of the work was to analyze the databases of the primary structure of the pig TERT gene, to determine single nucleotide polymorphisms (SNPs), to develop a DNA system for animal typing based on the TERT gene.&#x0D; Samples of biomaterials (blood, bristles) for DNA typing were selected from the leading farms of Ukraine in groups of 4 breeds of pigs and hybrid animals. Isolation of DNA from biomaterial was carried out using the Chelex 100 reagent. Genotyping of animals by the telomerase locus was carried out on the basis of standard methods of polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). The alignment of nucleotide sequences during the analysis of the primary structure of the TERT gene was carried out using the MegaX software and the Blast service. Design of the structure of oligonucleotide primers for PCR was carried out using the computer program Primer3Plus.&#x0D; During the development of the method of pigs genotyping according to TERT gene, a database analysis was carried out regarding the primary structure of the gene, a region of the gene with a significant number of SNPs was identified, and oligonucleotide primers were designed for PCR amplification of this region of the gene. The study presents the conditions of amplification of the TERT gene fragment, its cleavage by restriction endonuclease RsaI at the location of SNP rs698799571, electrophoresis of the obtained restriction DNA fragments in an 8% polyacrylamide gel, and the genotypes of the animals are also determined. The developed DNA typing technique for the TERT gene was used to analyze its polymorphism in groups of 4 purebred pigs and a group of hybrid anjmals. Monomorphic homozygous TERTAA genotype was present in purebred pigs. The polymorphism of the TERT gene by SNP rs698799571 was detected in a group of pigs of the final Irish hybrid (LW x L) x Maxgro (TERTAT genotype).&#x0D; Considering the fact that studies on the telomerase gene and determination of the TERT polymorphism for SNP rs698799571 in pigs have not yet been conducted in Ukraine, the developed technique of DNA typing by the telomerase gene has a perspective for further determination of the distribution of alleles and genotypes in domestic and imported breeds, as well as in marker-associated selection.

Genetic Variants in Telomerase Reverse Transcriptase Contribute to Solar Lentigines

Solar lentigines (SLs) are a hallmark of human skin aging. They result from chronic exposure to sunlight and other environmental stressors. Recent studies also imply genetic factors, but findings are partially conflicting and lack of replication. Through a multi-trait based analysis strategy, we discovered that genetic variants in telomerase reverse transcriptase were significantly associated with non-facial SL in two EastAsian (Taizhou longitudinal cohort, n= 2,964 and National Survey of Physical Traits, n= 2,954) and one Caucasian population (SALIA, n= 462), top SNP rs2853672 (P-value for Taizhou longitudinal cohort= 1.32× 10‒28 and P-value for National Survey of Physical Traits= 3.66× 10‒17 and P-value for SALIA= 0.0007 and Pmeta= 4.93× 10‒44). The same variants were nominally associated with facial SL but not with other skin aging or skin pigmentation traits. The SL-enhanced allele/haplotype upregulated the transcription of the telomerase reverse transcriptase gene. Of note, well-known telomerase reverse transcriptase‒related aging markers such as leukocyte telomere length and intrinsic epigenetic age acceleration were not associated with SL. Our results indicate a previously unrecognized role of telomerase reverse transcriptase in skin aging‒related lentigines formation.

Prenatal stress modulates HPA axis homeostasis of offspring through dentate TERT independently of glucocorticoids receptor

In response to stressful events, the hypothalamic-pituitary-adrenal (HPA) axis is activated, and consequently glucocorticoids are released by the adrenal gland into the blood circulation. A large body of research has illustrated that excessive glucocorticoids in the hippocampus exerts negative feedback regulation of the HPA axis through glucocorticoid receptor (GR), which is critical for the homeostasis of the HPA axis. Maternal prenatal stress causes dysfunction of the HPA axis feedback mechanism in their offspring in adulthood. Here we report that telomerase reverse transcriptase (TERT) gene knockout causes hyperactivity of the HPA axis without hippocampal GR deficiency. We found that the level of TERT in the dentate gyrus (DG) of the hippocampus during the developmental stage determines the responses of the HPA axis to stressful events in adulthood through modulating the excitability of the dentate granular cells (DGCs) rather than the expression of GR. Our study also suggests that the prenatal high level of glucocorticoids exposure-induced hypomethylation at Chr13:73764526 in the first exon of mouse Tert gene accounted for TERT deficiency in the DG and HPA axis abnormality in the adult offspring. This study reveals a novel GR-independent mechanism underlying prenatal stress-associated HPA axis impairment, providing a new angle for understanding the mechanisms for maintaining HPA axis homeostasis.

Midazolam alleviates cellular senescence in SH-SY5Y neuronal cells in Alzheimer's disease.

Alzheimer's disease (AD) impacts the daily life of aging people. Oligomerized amyloid β (Aβ)-associated neuronal senescence is involved in the pathological mechanism of AD. Blockage of neuronal senescence has been considered an important strategy for the treatment of AD. Midazolam is a hypnotic-sedative drug with pleiotropic properties. However, the effects of Midazolam in oligomerized Aβ1.42 -induced neurotoxicity have not been reported previously. Here, we investigate whether Midazolam possesses a beneficial effect against oligomerized Aβ1.42 in SH-SY5Y neuronal cells. Cellular senescence was assessed using senescence-associated β-galactosidase staining. Telomerase activity was measured using the TeloTAGGG Telomerase PCR ELISA. First, the lactate dehydrogenase release assay demonstrates that 10 and 20 µM are the optimal concentrations of Midazolam used for cell cultures. Senescence-associated β-galactosidase staining results indicate that exposure to oligomerized Aβ1.42 significantly increased cellular senescence of SH-SY5Y cells, but it was significantly alleviated by Midazolam. Additionally, Midazolam restored the oligomerized Aβ1.42 -induced reduction of telomerase activity. Interestingly, we found that oligomerized Aβ1.42 remarkably reduced human telomerase reverse transcriptase (hTERT) gene expression but increased the telomeric repeat-binding factor 2 (TERF2) expression. However, treatment with Midazolam reversed the effects of oligomerized Aβ1.42 on the hTERT and TERF2 gene expressions. Importantly, the presence of Midazolam attenuated Aβ1.42 -induced p53 and p21 expressions. Mechanistically, Midazolam repressed the level of cyclooxygenase-2 (COX-2) and the release of prostaglandin E2. Importantly, overexpression of COX-2 abolished the impact of Midazolam against oligomerized Aβ1.42 in neuronal senescence. We conclude that the usage of Midazolam is a potential treatment strategy for AD.

TERT Promoter Mutations as Simple and Non-Invasive Urinary Biomarkers for the Detection of Urothelial Bladder Cancer in a High-Risk Region.

Bladder cancer (BC) is the 10th most common cancer in the world. While there are FDA-approved urinary assays to detect BC, none have demonstrated sufficient sensitivity and specificity to be integrated into clinical practice. Telomerase Reverse Transcriptase (TERT) gene mutations have been identified as the most common BC mutations that could potentially be used as non-invasive urinary biomarkers to detect BC. This study aims to evaluate the validity of these tests to detect BC in the Kerman province of Iran, where BC is the most common cancer in men. Urine samples of 31 patients with primary (n = 11) or recurrent (n = 20) bladder tumor and 50 controls were prospectively collected. Total urinary DNA was screened for the TERT promoter mutations (uTERTpm) by Droplet Digital PCR (ddPCR) assays. The performance characteristics of uTERTpm and the influence by disease stage and grade were compared to urine cytology results. The uTERTpm was 100% sensitive and 88% specific to detect primary BC, while it was 50% sensitive and 88% specific in detecting recurrent BC. The overall sensitivity and specificity of uTERTpm to detect bladder cancer were 67.7% and 88.0%, respectively, which were consistent across different tumor stages and grades. The most frequent uTERTpm mutations among BC cases were C228T (18/31), C250T (4/31), and C158A (1/31) with mutant allelic frequency (MAF) ranging from 0.2% to 63.3%. Urine cytology demonstrated a similar sensitivity (67.7%), but lower specificity (62.0%) than uTERTpm in detecting BC. Combined uTERTpm and urine cytology increased the sensitivity to 83.8%, but decreased the specificity to 52.0%. Our study demonstrated promising diagnostic accuracy for the uTERTpm as a non-invasive urinary biomarker to detect, in particular, primary BC in this population.

Establishment and characterization of the immortalized porcine lung-derived mononuclear phagocyte cell line.

Mononuclear phagocytes (MNP), including monocytes, dendritic cells (DC), and macrophages, play critical roles in innate immunity. MNP are abundant in the lungs and contribute to host defense against airborne agents and pulmonary immune homeostasis. In this study, we isolated porcine lung-derived MNP (PLuM) from primary cultures of parenchymal lung cells and then immortalized them by transferring the SV40 large T antigen gene and porcine telomerase reverse transcriptase gene using lentiviral vectors. The established cell line, immortalized PLuM (IPLuM), expressed DC/macrophage markers; i.e., CD163, CD172a, and major histocompatibility complex class II, whereas they did not express a porcine monocyte-specific marker, CD52. The expression patterns of these cell surface markers indicate that IPLuM originate from the DC/macrophage lineage rather than the monocyte lineage. The bacterial cell wall components muramyl dipeptide and lipopolysaccharide induced the production of the interleukin-1 family of pro-inflammatory cytokines in IPLuM. Phagocytotic activity was also detected by time-lapse fluorescence imaging of live cells when IPLuM were cultured in the presence of pHrodo dye-conjugated E. coli BioParticles. It is worth noting that IPLuM are susceptible to African swine fever virus infection and support the virus' efficient replication in vitro. Taken together, the IPLuM cell line may be a useful model for investigating host-agent interactions in the respiratory microenvironments of the porcine lung.

EXTH-59. IMMORTALIZATION OF MOUSE BONE MARROW-DERIVED MACROPHAGES FOR BRAIN TUMOR THERAPY DEVELOPMENT

Abstract BACKGROUND Macrophages can cross blood-brain-barrier (BBB) and infiltrate brain parenchyma with malignancies such as glioma. Recently, many efforts have been made to develop macrophage-based cancer therapy, including CAR–macrophages. However, lack of proliferation in differentiated macrophages hinders their utility. We sought to ameliorate this problem by immortalizing macrophages to assist potential therapy development. Bone marrow derived macrophages (BMDMs) were harvested from C57Bl/6 mice and transduced with mouse telomerase reverse transcriptase (mTert) gene via retrovirus. Following puromycin selection, morphology, proliferation, lineage marker expression and phagocytosis were measured by microscopy, flow cytometry and IncuCyte assays. RESULTS Through overexpression of mTert, we have successfully immortalized mouse BMDMs, termed as iBMDMs. Morphologically, iBMDMs largely maintained features of primary BMDMs. The established iBMDMs proliferated for over 20 subcultures without obvious reduction in growth. Proliferation of iBMDMs (doubling time, dt=52h) was similar to IC-21(dt=53h), an established mouse macrophage line immortalized by SV40-T antigen, and slower than Raw264.7 (dt=23h), an immortalized mouse macrophage line driven by murine leukemia virus. Furthermore, iBMDMs can grow without macrophage colony-stimulating factor (M-CSF) (dt=54h). Macrophage lineage markers CD11B and F4/80 expression levels were higher in primary BMDMs (CD11B+=62.8±0.53%, F4/80+=54.9±0.38%, CD11B+F4/80+=52.7±0.41%, n=2 for all flow results) and iBMDMs (CD11B+=39.1±5.29%, F4/80+=26.4±7.93%, CD11B+F4/80+=24.6±5.95%) than in IC-21 (CD11B+=10.7±0.37%, F4/80+=19.4±1.88%, CD11B+F4/80+=9.13±0.37%) and Raw264.7 (CD11B+=34.3±5.32%, F4/80+=14.1±4.40%, CD11B+F4/80+=8.33±4.19%) cells lines. Lastly, we evaluated the phagocytic ability of iBMDMs through IncuCyte assays. Similar to primary BMDMs, we observed a concentration dependent increase in red fluorescence, which indicates the phagocytosis of E. coli derived bioparticles, in iBMDMs with and without M-CSF. CONCLUSIONS We immortalized bone marrow-derived macrophages of C57Bl/6 mouse origin with competent immune function via mTert transduction. These cells can be used in preclinical immunotherapy development for glioma treatment. Ongoing work is focused on assessing their ability to penetrate BBB and infiltrate brain tumor in a mouse glioma model.

Thyroid carcinoma-featured telomerase activation and telomere maintenance: Biology and translational/clinical significance.

Telomerase is a ribonucleoprotein complex consisting of a catalytic component telomerase reverse transcriptase (TERT), internal RNA template and other co-factors, and its essential function is to synthesize telomeric DNA, repetitive TTAGGG sequences at the termini of linear chromosomes. Telomerase is silent in normal human follicular thyroid cells, primarily due to the TERT gene being tightly repressed. During the development and progression of thyroid carcinomas (TCs), TERT induction and telomerase activation is in general required to maintain telomere length, thereby conferring TC cells with immortal and aggressive phenotypes. The genomic alterations of the TERT loci including TERT promoter's gain-of-function mutations, copy number gain, fusion and rearrangements, have recently been identified in TCs as mechanisms to induce TERT expression and to activate telomerase. Importantly, numerous studies have consistently shown that TERT promoter mutations and TERT expression occur in all TC subtypes, and are robustly associated with TC malignancy, aggressiveness, treatment failure and poor outcomes. Therefore, the assessment of TERT promoter mutations and TERT expression is highly valuable in TC diagnostics, prognosis, treatment decision, and follow-up design. In addition, the TERT promoter is frequently hypermethylated in TC cells and tumors, which is required to activate TERT transcription and telomerase. Dysregulation of other components in the telomerase complex similarly upregulate telomerase. Moreover, shortened telomeres lead to altered gene expression and metabolism, thereby actively promoting TC aggressiveness. Here we summarize recent findings in TCs to provide the landscape of TC-featured telomere/telomerase biology and discuss underlying implications in TC precision medicine. Mechanistic insights into telomerase activation and TERT induction in TCs are important both biologically and clinically. The TERT gene aberration and expression-based molecular classification of TCs is proposed, and for such a purpose, the standardization of the assay and evaluation system is required. Moreover, the TERT-based system and 2022 WHO TC classification may be combined to improve TC care.