Lab-on-Chip assay of tumour markers and Human Papilloma Virus for cervical cancer detection at point-of-care

Cervical cancer affects over half a million people worldwide each year, the majority of whom are in resource-limited settingswhere cytology screening is not available. As persistent human papilloma virus (HPV) infections are a key causative factor,detection of HPV strains now complements cytology where screening services exist. This work demonstrates the efficacy of ahandheld Lab-on-Chip (LoC) device in detecting cervical cancer from biopsy samples. The device is based on Ion-SensitiveField-Effect Transistor (ISFET) sensors used in combination with loop-mediated isothermal amplification (LAMP) assays, toamplify HPV DNA and human telomerase reverse transcriptase (hTERT) mRNA. These markers were selected because of theirhigh levels of expression in cervical cancer cells, but low to nil expression in normal cervical tissue. The achieved analyticalsensitivity for the molecular targets resolved down to a single copy per reaction for the mRNA markers, achieving a limit ofdetection of 102for hTERT. In the tissue samples, HPV-16 DNA was present in 4/5 malignant and 2/5 benign tissues, withHPV-18 DNA being present in 1/5 malignant and 1/5 benign tissues. hTERT mRNA was detected in all malignant and nobenign tissues, with the demonstrated pilot data to indicate the potential for using the LoC in cervical cancer screening inresource-limited settings on a large scale

GDF-15 as a biomarker of aging

Background: The aging process is accompanied by the gradual development of chronic systemic inflammation (inflamm-aging). Growth differentiation factor-15 (GDF-15) is associated with inflammation and known to be a stress-induced factor. The present study aimed to explore the association of GDF-15 with aging.Methods: In this cross-sectional study, serum GDF-15, hematological parameters, and biomedical parameters were determined in 120 healthy individuals (23-83 years old, males). Three telomere related parameters, including telomere length, telomerase activity, and the expression of human telomerase reverse transcriptase (hTERT) mRNA were also quantified.Results: The older group has a higher levels of GDF-15 and lower expression of hTERT mRNA, and PBMC telomerase activity (p<0.001). In individuals with high GDF-15 levels, they were older, and presented with the lower level of hTERT mRNA and T/S ratio (p<0.01). Spearman correlation analysis shows that GDF-15 positively correlated with age (r=0.664, p<0.001), and negatively correlated with telomere length (r=-0.434, p<0.001), telomerase activity (r=-0.231, p=0.012), and hTERT mRNA (r=-0.206, p=0.024). Furthermore, in multivariate regression analysis, GDF-15 levels showed a statistically significant linear and negative relationship with PBMC telomerase activity (β-coefficient=-0.583, 95% CI -1.044 to -0.122, p=0.014), telomere length (β-coefficient=-0.200, 95% CI -0.305 to -0.094, p<0.001), and hTERT mRNA (β-coefficient=-0.207, 95% CI -0.312 to -0.102, p<0.001) after adjusting for confounders.Conclusions: In conclusion, our results show that circulating GDF-15 is the potential biomarkers of aging that may influence the risk and progression of multiple aging conditions.

Imetelstat Sensitizes Hematopoietic Malignancy Cells to Genotoxic Agent Via Suppression of Telomerase Mediated DNA Repair Process

Telomerase is the ribonucleoprotein enzyme that has the main function of extension of telomeric repeat. Telomerase consists of two constructions: telomerase reverse transcriptase protein (hTERT) and RNA template. Inhibition of telomerase activity is considered to be a therapeutic approach for malignant tumors since telomerase is dominantly expressed in germ cells, stem cells, and cancer cells but not in normal cells. We found that (TTAGGG)n repeats could be integrated and repair artificially induced DNA double strand break (DSB) sites (Onozawa M et al. PNAS, 2014). Therefore, we hypothesized that inhibition of telomerase activity may interfere with the DNA repair process and enhance the effects of DNA damaging agents. Imetelstat competitively suppresses telomerase activity. It has a binding site complementary to the RNA template of telomerase and induces specific inhibition. Imetelstat showed clinical benefits in patients with essential thrombocytosis or myelofibrosis used as monotherapy. However, the effectiveness of imetelstat combined with other cytotoxic agent for hematological malignancies is not clear. We therefore evaluated the synergistic anti-tumor effects of imetelstat and cytotoxic agents on hematological malignancies. First, the expression of hTERT mRNA, protein, and activity was confirmed in 8 human cell lines of hematological malignancies (U937, HL60, PALL2, CEM, IM9, RPMI8226, ST1, and SU9T01) but not in peripheral blood mononuclear cells (PBMCs) from a healthy individual. There was a significant correlation between mRNA expression and activity measured by TRAP assay (r=0.839, 95% confidence interval: 0.396-0.965, p=0.00467). The expression of hTERT mRNA in clinical samples was analyzed using primary acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) cells. The expression level of hTERT in ALL cells was significantly higher than that in AML cells (p=0.0093). The telomerase inhibitor, imetelstat had a concentration-dependent effect on telomerase inhibition, while a control oligo did not affect telomerase activity. The expression of hTERT protein was not suppressed by imetelstat treatment. Imetelstat also had a concentration-dependent suppressive effect on cell growth without affecting cell viability. Next, we evaluated the synergistic anti-tumor effects of etoposide and imetelstat at the concentration at which it did not affect cell growth by monotherapy. Cell proliferation at day 4 was inhibited by coadministration of etoposide (1 µM) and imetelstat (1 µM) compared to that with administration of etoposide alone (Figure A). Western blot analysis showed that gamma-H2AX expression level after radiation and imetelstat administration was significantly higher than that of after radiation alone (Figure B). Therefore, imetelstat enhanced DNA-DSB induced by a genotoxic agent. We determined whether the synergistic effect was mediated by shortening of telomere length. Telomere length was analyzed in long-term culture using the flow-FISH method. Imetelstat significantly shortened telomere length compared to a control oligo after day 14, while there was almost no change in telomere length at day 4. We also analyzed the changes of hTERT activity and gamma-H2AX expression after radiation in PBMCs from a healthy individual. Although hTERT activity was not detected in stable state in PBMC, hTERT activity appeared and gradually increased over time and gamma-H2AX expression peaked from 2 to 4 hours after radiation. Images of immunofluorescence staining revealed co-localization of hTERT and gamma-H2AX in the nucleus of PBMCs after radiation (Figure C). Therefore, hTERT induced by DNA damage might be directly involved in the DNA-DSB repair process. We clearly showed synergistic effects of imetelstat and a genotoxic agent in short-term treatment, in which shortening of telomeres was not observed. Potentially, telomerase inhibition could stand as a beneficial addition to other treatment methods when the inhibitor is administered at low non-toxic doses. Our hypothesis needs to be clarified in a clinical trial. Further studies are required to optimize these therapies in a variety of hematopoietic malignancies or other cancers. Figure Disclosures Nakagawa: akeda Pharmaceutical Company Limited: Research Funding. Teshima:Novartis: Honoraria, Research Funding.

Effect of arsenic exposure on human telomerase reverse transcriptase (hTERT) gene expression: Risk of cardiovascular diseases

Background: Exposure to inorganic arsenic (iAs) through drinking water is currently a serious threat to public health of millions of people worldwide including Bangladesh. Some recent studies have shown that telomere dysfunction is emerging as an important factor in the pathogenesis of different cardiovascular diseases. Arsenic plays significant role on telomere dysfunction by altering the expression of telomere-related genes. The study was aimed to investigate the effects of arsenic on hTERT mRNA levels and their combined role in increasing CVD susceptibility. Methods: In this cross sectional study, total of 50 CVD patients who underwent open heart surgery were recruited for this study. Urine, nail and cardiac tissue samples were collected and analyzed for As. Blood samples were quantified for hTERT expression analysis using real-time polymerase chain reaction. Results: The hTERT mRNA expression was found approx. 10 fold higher in the As-exposed patients than the As-unexposed patients (p<0.01). A strong positive correlation (p<0.01, R>0.3) was found between the hTERT mRNA levels and As contents in the cardiac tissue, nail and urine samples of the study subjects. The significant increase (approx. 4 fold) in the hTERT mRNA expression was found in the patients with coronary artery disease (CAD) than the non-CAD patients. Conclusions: The results of the study suggest that arsenic exposure increases hTERT mRNA expression which may in turn modify As-induced cardiovascular outcomes. The findings of this study will help to look deep into the association of As exposure in cardiovascular disease pathogenesis to open a new window in the diagnosis and treatment procedure of CVD. Bangladesh Med Res Counc Bull 2019; 45: 03-10

Visualizing the down-regulation of hTERT mRNA expression using gold-nanoflare probes and verifying the correlation with cancer cell apoptosis

A locked nucleic acid-functionalized gold nanoparticle flare probe was designed to achieve in situ monitoring and regulating intracellular hTERT mRNA which also induced the apoptosis of cancer cells.