Socio-economic inequalities in physical activity among Japanese adults during the COVID-19 pandemic.

OBJECTIVES: This study aimed to explore the socio-economic inequalities in physical activity (PA) based on domains of daily life, such as work, transport, recreation and sedentary life, among Japanese adults during the COVID-19 pandemic. STUDY DESIGN: This was a cross-sectional study. METHODS: This study used data from the 2020 National Sport and Lifestyle Survey, conducted by the Sasakawa Sports Foundation. Data of 2,296 (1,103 women) participants were analysed. PAs were assessed using the Global Physical Activity Questionnaire. Educational level and household income were used as indicators of socio-economic status. We calculated the slope index of inequality (SII) and relative index of inequality (RII). RESULTS: We detected absolute and relative inequalities for household income in all PA domains, except for work-related PA. The higher the participants' income, the longer they engaged in transport- and recreation-related PA and sedentary behaviour. Recreation-related PA had a larger disparity than other domains, with SII at 20.8% (95% confidence interval [CI] -28.4 to -13.1) and RII at 0.58 (95% CI 0.47-0.71). At the educational level, each inequality was observed in work- and recreation-related PA and sedentary behaviour. The higher the participants' educational level, the longer they engaged in recreation-related PA and sedentary behaviour. However, work-related PA was longer at lower educational levels, with RII at 1.90 (95% CI 1.48-2.44). The inequality in recreation-related PA was also relatively large (SII 23.3%, 95% CI -30.9 to -15.7; RII 0.54, 95% CI 0.45-0.66). CONCLUSION: Our study revealed significant socio-economic disparities in each PA domain, particularly in recreational PA. These results suggest a widening gap because of the COVID-19 pandemic.

Associations between 24-h movement behaviors and self-rated health: a representative sample of school-aged children and adolescents in Okinawa, Japan.

OBJECTIVES: This study aimed to determine the associations between adherence to 24-h movement behavior guidelines and self-rated health (SRH) among Japanese adolescents according to their age group. STUDY DESIGN: This was a cross-sectional study. METHODS: Probability proportional sampling data, which were collected from six regions of Okinawa Prefecture, Japan, considering the number of schools, included 2408 fifth-grade students (aged 10-11 years) in 31 elementary schools and 4360 eighth-grade students (aged 13-14 years) in 30 junior high schools. SRH, moderate-to-vigorous physical activity (MVPA), screen time (ST), sleep duration, and confounding factors (sex, weight status, family affluence, parental support, school satisfaction, and school demands) were self-reported. RESULTS: The logistic regression models showed that adherence to ST and sleep recommendations in elementary school students was associated with a high prevalence of good health only, whereas adherence to only MVPA, only sleep, ST and sleep, MVPA and sleep, and all three recommendations were associated with a high prevalence of good health among junior high school students. All combinations that included achievement of the recommended sleep duration were associated with SRH. CONCLUSIONS: Achieving 24-h movement behavior guidelines, particularly sleep recommendations, is associated with better perceived health in school-aged children, especially in adolescents.

Circulating tumour cells detected by a novel adenovirus-mediated system may be a potent therapeutic marker in gynaecological cancers.

BACKGROUND: Recently developed detection system for circulating tumour cells (CTCs) using a telomerase-specific replicative adenovirus generated nonspecific green fluorescent protein (GFP) signals because of the co-presence of white blood cells (WBCs) nonspecifically infected by viruses. Here, we established a unique detection system for CTCs that completely excludes nonspecific signals. METHODS: Blood obtained from the patients was subjected to haemolytic processes to eliminate red blood cells. The cell pellets were then infected with OBP-401, fixed, incubated with fluorescence-labelled anti-CD45 antibody to mark white blood WBCs, and examined on slides under a microscope. RESULTS: Preparatory experiments with cancer cells artificially added to healthy donor samples confirmed that CD45 labelling could distinguish GFP-positive cancer cells from WBCs. In 53 patients with gynaecological cancers, CTCs were detected in 21 patients (39.6%) when CD45-positive cells were excluded as WBCs among GFP-positive cells. No CTCs were detected in samples from healthy volunteers. There was no significant correlation between CTC counts and known clinicopathological factors. The CTCs rapidly vanished after surgery or chemotherapy in most patients whose treatments were effective. In contrast, the persistence of CTCs even after treatments was tightly associated with poor response to the treatments (P<0.005). CONCLUSION: The presence of CTCs in our system may potentially be a novel therapeutic marker in gynaecological cancers.

Creation of immortalised epithelial cells from ovarian endometrioma.

BACKGROUND: Epithelial cells of endometriotic tissues are difficult to propagate in vitro as experimental material is scarce owing to their limited life span. However, there is an increasing concern regarding their malignant transformation in ovaries. The present study sought to generate their stable culture system. METHODS AND RESULTS: Purified epithelial cells isolated from ovarian endometriomas using microscopic manipulation were successfully immortalised by combinatorial transfection of human cyclinD1, cdk4 and human telomerase reverse transcriptase (hTERT) genes, whereas the introduction of hTERT alone, or together with cdk4, was insufficient for immortalisation, leading to cellular senescence. We confirmed stable cytokeratin expression in the immortalised cells, proving their epithelial origin. These cells expressed progesterone receptor B and showed significant growth inhibition by various progestins. Oestrogen receptor (ER) expression was detected in these cells, albeit at low levels. Additional overexpression of ERα generated stable cells with oestrogen-dependent growth activation. Soft-agar colony formation assay and nude mice xenograft experiments demonstrated that these cells, even those with additional inactivation of p53, did not have transformed phenotypes. CONCLUSION: We for the first time generated immortalised epithelial cells from ovarian endometrioma that retained sex steroid responsiveness. These cells are invaluable tools not only for the consistent in vitro work but also for the study of molecular pathogenesis or carcinogenesis of endometriosis.

Socioeconomic inequalities in toothbrushing behaviours in young children: a children's lifestyle survey in a representative population of A city, Okinawa prefecture, Japan.

PURPOSE: We have less understanding of which socioeconomic status (SES) indicators may be reflective of latent socioeconomic inequalities in toothbrushing behaviours, especially finishing-toothbrushing by parents in young children. The aim of this study was to reveal the socioeconomic inequalities in children's toothbrushing and finishing-toothbrushing by parents and if it varies by SES indicators. METHODS: We used data from 'Survey on Children's Life' conducted by A city of Okinawa Prefecture, Japan. The multiple imputed data of 902 (boys, 453) included self-reported children's toothbrushing behaviour and finishing-toothbrushing by parents in three-to six-year-old children. SES was assessed using self-reported household income and parental educational attainment. Absolute and relative inequalities in toothbrushing behaviours were quantified using the slope index of inequality (SII) and relative index of inequality (RII), respectively. RESULTS: There were significant absolute and relative inequalities of children's toothbrushing for household income (SII and RII were 0.241 and 2.73, respectively), of finishing-toothbrushing by parents for household income (SII and RII were 0.133 and 3.28, respectively), and educational attainment (SII and RII were 0.166 and 5.55, respectively). The same inequality trends were observed after adjusting for covariates (child's age and sex, family structure, breakfast and dinner frequency, and sleep duration). CONCLUSION: Socioeconomic inequalities in children's toothbrushing and finishing-toothbrushing by parents varied according to SES indicators.

Loss of Ftsj1 perturbs codon-specific translation efficiency in the brain and is associated with X-linked intellectual disability.

FtsJ RNA 2'-O-methyltransferase 1 (FTSJ1) gene has been implicated in X-linked intellectual disability (XLID), but the molecular pathogenesis is unknown. We show that Ftsj1 is responsible for 2'-O-methylation of 11 species of cytosolic transfer RNAs (tRNAs) at the anticodon region, and these modifications are abolished in Ftsj1 knockout (KO) mice and XLID patient-derived cells. Loss of 2'-O-methylation in Ftsj1 KO mouse selectively reduced the steady-state level of tRNAPhe in the brain, resulting in a slow decoding at Phe codons. Ribosome profiling showed that translation efficiency is significantly reduced in a subset of genes that need to be efficiently translated to support synaptic organization and functions. Ftsj1 KO mice display immature synaptic morphology and aberrant synaptic plasticity, which are associated with anxiety-like and memory deficits. The data illuminate a fundamental role of tRNA modification in the brain through regulation of translation efficiency and provide mechanistic insights into FTSJ1-related XLID.

Expression of HER-2 affects patient survival and paclitaxel sensitivity in endometrial cancer.

BACKGROUND: Disabled phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase/extracellular signal-regulated kinase signalling is involved in endometrial carcinogenesis, and there is evidence that expression of epidermal growth factor receptor (EGFR) family members has a role in such intracellular signalling pathways. This study analysed the prognostic impact of EGFR family expression in endometrial cancer in relation to PI3K-AKT and MAPK-ERK signalling, as well as drug sensitivity. METHODS AND RESULTS: Immunohistochemical analysis using 63 surgical specimens of endometrioid-type endometrial cancers revealed that EGFR, human epidermal growth factor receptor (HER)-2 and HER-4 were expressed in 25 (39.7%) of 63, 26 (41.3%) of 63 and 31 (49.2%) of 63 tumours, respectively. Gene amplification of HER-2 was observed in 2 of 26 patients with high HER-2 expression. Kaplan-Meier analysis revealed that high HER-2 expression was a factor that negatively influenced the progression-free and overall survival rate (P<0.05), and multivariate analysis showed high HER-2 expression to be an independent prognostic factor. Subsequently, we performed in vitro knockdown analysis to investigate the linkage between HER-2 expression and PI3K-AKT pathways. Short interfering RNA (siRNA)-based knockdown of HER-2 in endometrial cancer cells led to a significant reduction in phosphorylated AKT (p-AKT) expression, indicating the existence of a HER-2/PI3K-AKT axis. As the PI3K-AKT pathway is known to have crucial roles in anticancer drug sensitivity, we examined the involvement of HER-2 in sensitivity to paclitaxel. Short interfering RNA-based knockdown of HER-2 conferred increased sensitivity to paclitaxel in endometrial cancer cells, attenuating the induction of p-AKT on paclitaxel stimulation, which was cancelled by inactivating AKT by the introduction of a dominant-negative form. CONCLUSION: HER-2 is a significant prognostic factor of endometrioid-type endometrial cancer, as well as a key molecule that affects paclitaxel sensitivity by HER-2 interaction with the PI3K-AKT pathway.

Creation of tumorigenic human endometrial epithelial cells with intact chromosomes by introducing defined genetic elements.

Several genetic mutations have been identified in human endometrial cancers, but the specific combinations of mutations required to form endometrial cancer cells remain unknown. In the present study, we established an in vitro model of endometrial carcinogenesis, in which defined genetic elements were introduced into endometrial epithelial cells to create transformed endometrial cells at different stages. Introduction of the human papillomavirus type 16 E6/E7 gene and the human telomerase reverse transcriptase (hTERT) gene into human primary endometrial epithelial cells was sufficient to generate immortalized cells. Introduction of hTERT in early passages stabilized telomeres and created immortalized cells with normal karyotype, whereas introduction of hTERT in later passages generated immortalized cells but with widespread chromosome abnormalities. However, neither of those two immortalized cell lines exhibited tumorigenic phenotypes. Tumorigenic endometrial epithelial cells with invasive capacity were created by introducing a mutant K-ras allele into immortalized cells, keeping their chromosomes intact. Inhibiting the PTEN gene and activating Akt pathways did not create tumorigenic phenotypes, although the latter conferred anchorage-independent growth capacity. These findings suggest that neoplastic transformation of human endometrial cells can occur in the absence of widespread chromosomal abnormality, and that the combination of Rb inactivation, telomerase activation and altered K-ras signaling is sufficient for in vitro neoplastic transformation. The present experimental model can help clarify the genetic requirements for endometrial carcinogenesis, and it is useful for testing and developing specific inhibitors of specific oncogenic pathways.

Analysis of outcome of Stage I-III endometrial cancer treated with systematic operation omitting paraaortic lymphadenectomy.

PURPOSE: The aim of this study was to assess the outcomes of endometrial cancer patients treated with systematic surgery omitting paraarotic lymphadenectomy. PATIENTS AND METHODS: We retrospectively analyzed a consecutive series of 84 endometrioid-type endometrial cancer patients at FIGO Stage I, II or III without grossly metastatic paraaortic lymphadenodes, who underwent surgery at our institute. RESULTS: Sixty-five patients (77%) underwent primary surgery with pelvic lymphadenectomy while the remaining 19 patients underwent surgery without lymphadenectomy due to severe medical complications or age greater than 70 years. The patients with high risk for recurrence were treated mainly by adjuvant irradiation therapy of the whole pelvis. The median follow-up period was 44 months. The 5-year overall survival (OS) rate was 92%, 92% and 65% for FIGO Stage I, II and III, respectively. Recurrence was detected in eight of the 82 optimally operated patients (9.8%). Out of the eight recurrent patients, five patients had a recurrent tumor at extra-pelvic sites (chest or abdomen), two patients had a recurrent tumor only in a paraaortic lymph node, and one patient had a recurrent tumor only in the vagina. Thus, the recurrence rate was relatively low, with 2.4% relapse at the paraarotic lymph nodes, and 5-year OS rate appeared to be favorable. However, all the six recurrent patients who underwent adjuvant radiation therapy had distant recurrence. CONCLUSIONS: These findings indicate that omission of paraarotic lymphadenectomy may be acceptable for endometrial cancer patients without gross metastasis at this site. However, the high rate of distant recurrence after whole pelvic irradiation strongly indicates an urgent need to develop potent systemic adjuvant therapy, potentially by chemotherapy or chemoradiation therapy.

Identification and characterization of negative regulatory elements of the human telomerase catalytic subunit (hTERT) gene promoter: possible role of MZF-2 in transcriptional repression of hTERT.

Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of human telomerase and is a critical determinant of the enzymatic activity of telomerase. Expression of hTERT is known to be regulated mainly at the transcriptional level. In the present study, using transient expression assays, we identified a 400 bp silencer of the hTERT promoter between -776 and -378 upstream of the proximal core promoter. The inhibitory effects of this silencer were enhanced with cellular differentiation. A computer-assisted homology search identified multiple binding motifs for myeloid-specific zinc finger protein 2 (MZF-2) within this region. Mutation introduced in these sites resulted in significant activation of hTERT transcription. Gel shift assays demonstrated that MZF-2 proteins specifically bound to these sites. Overexpression of MZF-2 in cells led to down-regulation of hTERT transcription as well as telomerase activity. These findings suggest that the 400 bp region upstream of the hTERT core promoter that we identified functions as a negative regulatory region and that MZF-2 may be an effector of negative regulation of hTERT.

Telomerase activation by histone deacetylase inhibitor in normal cells.

Although telomerase activity is known to be regulated mainly at the level of transcription of the human telomerase catalytic subunit (hTERT) gene, the molecular mechanism underlying tumor-specific expression of telomerase remains unclear. Emerging evidence suggests that reversible acetylation of nucleosomal histones and the resultant changes in the chromatin structure are important processes in gene transcription. In particular, histone deacetylase (HDAC) inhibitors activate the transcription of certain genes by altering the acetylation status of nucleosomal histones. The present study examines the effects of HDAC inhibitor on hTERT gene transcription. Treatment with tricostatin A (TSA) induced significant activation of hTERT mRNA expression and telomerase activity in normal cells, but not in cancer cells. Transient expression assays revealed that TSA activates the hTERT promoter. Furthermore, the proximal 181 bp core promoter of hTERT, which contains two c-Myc and five Sp1 sites, was determined to be the responsible element. Overexpression of Sp1 enhanced responsiveness to TSA, and mutation of Sp1 sites, but not c-Myc sites, of the core promoter of hTERT abrogated this activation. Introduction of the dominant-negative form of the Sp family inhibited TSA activation. These results indicate that HDAC inhibitor activates the hTERT promoter in normal cells, in which Sp1 plays a key role. This finding suggests one way whereby histone deacetylation may be involved in silencing the hTERT gene in normal cells.

Sp1 cooperates with c-Myc to activate transcription of the human telomerase reverse transcriptase gene (hTERT).

Telomerase activation is thought to be a critical step in cellular immortalization and carcinogenesis. The human telomerase catalytic subunit (hTERT) is a rate limiting determinant of the enzymatic activity of human telomerase. In the previous study, we identified the proximal 181 bp core promoter responsible for transcriptional activity of the hTERT gene. To identify the regulatory factors of transcription, transient expression assays were performed using hTERT promoter reporter plasmids. Serial deletion assays of the core promoter revealed that the 5'-region containing the E-box, which binds Myc/Max, as well as the 3'-region containing the GC-box, which binds Sp1, are essential for transactivation. The mutations introduced in the E-box or GC-box significantly decreased transcriptional activity of the promoter. Overexpression of Myc/Max or Sp1 led to significant activation of transcription in a cell type-specific manner, while Mad/Max introduction repressed it. However, the effects of Myc/Max on transactivation were marginal when Sp1 sites were mutated. Western blot analysis using various cell lines revealed a positive correlation between c-Myc and Sp1 expression and transcriptional activity of hTERT. Using fibroblast lineages in different stages of transformation, we found that c-Myc and Sp1 were induced to a dramatic extent when cells overcame replicative senescence and obtained immortal characteristics, in association with telomerase activation. These findings suggest that c-Myc and Sp1 cooperatively function as the major determinants of hTERT expression, and that the switching functions of Myc/Max and Mad/Max might also play roles in telomerase regulation.

Telomerase activity in human endometrium.

Human uterine endometrium undergoes a complex pattern of changes in proliferation and secretory activity during the menstrual cycle. In the present study, telomerase activity in normal endometrium was examined using a non-radioisotope PCR-based telomeric repeat amplification protocol assay. Various levels of telomerase activity were detected in the 60 normal endometrial samples examined, depending on the phase of the menstrual cycle. Of 21 proliferative-phase endometrial samples, 20 (95%) expressed telomerase activity, whereas 8 of 19 (42%) secretory-phase or menstrual endometrial samples did (P = 0.002). Five of nine (56%) samples from atrophic endometrium from postmenopausal women also expressed telomerase activity. Eleven of 21 (52%) endometrial samples in the proliferative phase expressed high telomerase activity detectable after 100-fold dilution of extracts, whereas none of the 19 endometrial samples from the secretory phase or during menstruation and none of the 9 postmenopausal endometrial samples did (P < 0.001). The highest activity was observed in the late proliferative phase, but activity dramatically decreased with the progression of the secretory phase. Surprisingly, the levels of telomerase activity detected in the late proliferative phase were comparable to those detected in the endometrial cancers examined. Immunohistochemical analysis of the expression of proliferating cell nuclear antigen revealed that telomerase activity is closely correlated with endometrial cell proliferative activity. These findings indicate that normal endometrium expresses telomerase, the activity of which changes dramatically over the course of the menstrual cycle, suggesting in turn that telomerase is a regulated enzyme linked to cellular proliferation and that hormone functions may be involved in its regulation.

Expression of human telomerase subunits and correlation with telomerase activity in cervical cancer.

Activation of telomerase and stabilization of telomeres are thought to be required for both cellular immortality and oncogenesis. Three major components of human telomerase, human telomerase RNA (hTR), telomerase-associated protein (TP1/TLP1), and human telomerase catalytic subunit (hTRT/hEST2), have been identified recently. However, it remains unclear what roles these subunits play in the regulation of telomerase activity. In the present study, a total of 25 cervical cancers and 14 normal cervices as well as various cell lines derived from cervical cancer were examined for the expression of hTR, TP1 mRNA, and hTRT mRNA, and the correlations between expression of these and telomerase activity were evaluated in 23 cancers and 14 normal cervices. Reverse transcription-PCR analysis revealed that hTR and TP1 mRNA were commonly expressed in cancers and noncancerous tissues. However, hTRT mRNA was observed only in cervical cancers and cell lines, and more than 80% of cervical cancers expressed it, whereas neither normal cervical tissues nor normal primary fibroblast cells did. There was a strong correlation of telomerase activity with hTRT mRNA expression but not with TP1 or hTR expression. Cervical exfoliated cells were subjected to reverse transcription-PCR analysis for detection of hTRT mRNA, and approximately 70% of cervical cancers were positive for such expression. These findings provide strong evidence that expression of hTRT is a rate-limiting determinant of the enzymatic activity of human telomerase and that up-regulation of hTRT expression may play a critical role in human carcinogenesis. Our findings also indicate that detection of hTRT mRNA is useful for cytological screening for cervical cancer.

Application of telomerase assay for the screening of cervical lesions.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends. The expression of telomerase is thought to be required for cellular immortality and oncogenesis. Telomerase activity has been detected not only in most cancers but also in some types of premalignant lesions, such as squamous intraepithelial lesions (SILs). In the present study, we used the telomerase assay to detect uterine cervical lesions in cervical scraping samples. A total of 82 cervical scraping samples were obtained from women with or without cervical lesions and examined by nonradioisotope telomeric repeat amplification protocol assay. Fifteen of 17 (88%) cervical cancer specimens exhibited telomerase activity, whereas 5 of 8 (63%) and 14 of 24 (58%) specimens from low-grade and high-grade SILs, respectively, also exhibited telomerase activity. In contrast, 3 of 33 (9%) specimens from normal cervices exhibited telomerase activity. Dilution telomeric repeat amplification protocol assay was performed to estimate telomerase activity; it revealed that high levels of activity were often expressed in cervical cancer. Cytological examination was also performed by Pap smear test, and 4 of 8 (50%) low-grade SILs, 21 of 24 (88%) high-grade SILs, and 16 of 17 (94%) cervical cancers were found to have cytological abnormalities. There were discordances in some cases between findings of smear abnormality and telomerase positivity. In particular, we found five cases of SILs without smear abnormality but with telomerase activity, suggesting that some lesions with false negative cytology can be detected by telomerase assay. These findings suggest that telomerase assay using cervical scrapings might be a useful screening method for cervical lesions especially when combined with a Pap smear test.

Tumor-specific transgene expression from the human telomerase reverse transcriptase promoter enables targeting of the therapeutic effects of the Bax gene to cancers.

Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, which is highly active in immortalized cells and >85% of human cancers but is quiescent in most normal somatic cells. To test the feasibility of using the hTERT promoter to induce tumor-specific transgene expression in cancer gene therapy, we constructed an adenoviral vector expressing a LacZ reporter gene driven by the hTERT core promoter and evaluated its activity in vitro and in vivo. The hTERT promoter could drive high-level expression of LacZ in tumor cells but not in normal cells and normal mouse tissues. Using a binary adenoviral system that can induce Bax gene expression, we showed that induction of the Bax gene expression via the hTERT promoter elicited tumor-specific apoptosis in vitro, suppressed tumor growth in nude mice, and prevented the toxicity of the Bax gene in vitro and in vivo. Thus, the hTERT promoter is apparently a strong and tumor-selective promoter with potential application in targeted cancer gene therapy.

Estrogen activates telomerase.

Telomerase activity is present in most malignant tumors and provides a mechanism for the unlimited potential for division of neoplastic cells. Although telomerase is known to be a regulated enzyme, the factors and mechanisms involved in telomerase regulation are not well understood. In the present study, we examined the effects of estrogen on telomerase activity. Telomerase activity in estrogen receptor (ER)-positive MCF-7 cells was up-regulated by the treatment with 17beta-estradiol. This activation accompanied up-regulation of the telomerase catalytic subunit, hTERT mRNA. Gel shift assays revealed that the imperfect palindromic estrogen-responsive element in the hTERT promoter specifically binds to ER. Transient expression assays using luciferase reporter plasmids containing various fragments of hTERT promoter showed that this imperfect palindromic estrogen-responsive element is responsible for transcriptional activation by ligand-activated ER. We also found that estrogen activates c-Myc expression in MCF-7 cells and that E-boxes in the hTERT promoter that bind c-Myc/Max play additional roles in estrogen-induced transactivation of hTERT. Estrogen thus activates telomerase via direct and indirect effects on the hTERT promoter. These findings may help elucidate the mechanisms of hormonal control of telomerase activity and aid understanding of the roles of sex steroids in cellular senescence and aging as well as estrogen-induced carcinogenesis.

Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells.

Telomerase activation is thought to be a critical step in cellular immortalization and carcinogenesis. Of the three major subunits comprising human telomerase, human telomerase catalytic subunit (hTERT) has been shown to be a rate-limiting determinant of the enzymatic activity of human telomerase. However, little is known concerning how expression of hTERT is regulated in human cells. To identify the regulatory elements controlling hTERT gene expression, approximately 3.5 kb of the 5'-flanking sequence of hTERT was cloned and characterized. The promoter of hTERT was GC rich and lacked both TATA and CAAT boxes. The CapSite Hunting method identified transcription start site 19 bp upstream of the first nucleotide of the published cDNA sequence. Transient expression assays revealed that transcription of hTERT was significantly activated in cancer cell lines but repressed in normal primary cells. Using the fibroblast lineage at various stages of transformation, we found that transcription occurred in strains that had overcome replicative senescence and expressed telomerase activity. Deletion analysis of hTERT promoter identified the 181-bp core promoter region upstream of the transcription start site. Gel shift analysis revealed two major factors binding to core promoter, an E box (CACGTG) binding factor and Sp1. Overexpression of c-Myc resulted in a significant increase in transcriptional activity of the core promoter. These findings suggest that hTERT expression is strictly regulated at the transcription machinery, and that the proximal core promoter containing an E box and Sp1 sites is required for transactivation of hTERT.

Progesterone regulates human telomerase reverse transcriptase gene expression via activation of mitogen-activated protein kinase signaling pathway.

Emerging evidence indicates that sex steroid hormones regulate telomerase in target tissues. We have reported that estrogen activates telomerase through transactivation of the telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT). Progesterone usually antagonizes estrogen action in reproductive organs, but the effect on telomerase remains unclear. In this study, we examine the effects of progesterone on the gene expression of hTERT in breast and endometrial cancer cell lines expressing progesterone receptor. Progesterone significantly induced hTERT mRNA expression within 3 h after exposure. This transient effect peaked at 12 h and then decreased. In contrast, exposure to progesterone for > 48 h antagonized estrogen effects and inhibited the estrogen-induced activation of hTERT expression; the cyclin-dependent kinase inhibitor p21/Waf1/Cip1 plays an integral role in this inhibition. Thus, progesterone exerts diverse effects on hTERT mRNA expression in a time-dependent manner. We also found that the mitogen-activated protein kinase signaling pathway mediates both the short-term and long-term effects of progesterone on hTERT gene expression. These findings support the notion that hTERT gene is a target of both estrogen and progesterone.

Treatment of malignant glioma cells with the transfer of constitutively active caspase-6 using the human telomerase catalytic subunit (human telomerase reverse transcriptase) gene promoter.

Because the apoptotic pathway is often disrupted in tumor cells, its genetic restoration is a very attractive approach for the treatment of tumors. To treat malignant gliomas with this approach, it would be preferred to restrict induction of apoptosis to tumor cells by establishing a tumor-specific expression system. Telomerase is an attractive target because the vast majority of malignant gliomas have telomerase activity whereas normal brain cells do not. Activation of telomerase is tightly regulated at the transcriptional level of the telomerase catalytic subunit [human telomerase reverse transcriptase, (hTERT)]. Therefore, we hypothesized that using a hTERT promoter-driven vector system, an apoptosis-inducible gene may be preferentially restricted to telomerase- or hTERT-positive tumor cells. In this study, we constructed an expression vector consisting of the constitutively active caspase-6 (rev-caspase-6) under the hTERT promoter (hTERT/rev-caspase-6) and then investigated its antitumor effect on malignant glioma cells. The rationale for using the rev-caspase-6 gene is because it induces apoptosis independent of the initiator caspases. We demonstrated that the hTERT/rev-caspase-6 construct induced apoptosis in hTERT-positive malignant glioma cells, but not in hTERT-negative astrocytes, fibroblasts, and alternative lengthening of telomeres cells. In addition, the growth of s.c. tumors in nude mice was significantly suppressed by the treatment with hTERT/rev-caspase-6 construct. The present results strongly suggest that the telomerase-specific transfer of the rev-caspase-6 gene under the hTERT promoter is a novel targeting approach for the treatment of malignant gliomas.