Telomerase is a regulated enzyme and its activity is tightly associated with cell proliferation. The mechanisms of this association are unclear, but speci®c growth factors may regulate telomerase activity. The present study examines the eect of epidermal growth factor (EGF) on telomerase activity and identi®es the signal transduction pathway involved in this process. EGF upregulated telomerase activity in EGF receptor-positive cells after the activation of telomerase reverse transcriptase (TERT) mRNA expression. This activation was rapid, peaked after 6 or 12 h and was not blocked by the concurrent exposure to cycloheximide, suggesting a direct eect of EGF on TERT transcription. Transient expression assays revealed that EGF activates the hTERT promoter and that the proximal core promoter is responsible for this regulation. The activation of hTERT mRNA expression by EGF was speci®cally blocked by MEK inhibitor, and in vitro kinase assays demonstrated that ERK is activated in response to EGF. Transient expression assays using mutant reporter plasmids revealed that an ETS motif located in the core promoter of hTERT is required for the EGF-induced transactivation of hTERT. Overexpression of wild type Ets in cells enhanced the EGF eect on hTERT transcription, while that of dominant negative Ets signi®cantly repressed EGF action. These ®ndings suggest that EGF activates telomerase through the direct activation of TERT transcription, in which the Ras/ MEK/ERK pathway and Ets factor play major roles. Our data support the notion that growth factors directly regulate telomerase via speci®c signal transduction pathways.

The human endometrium is a dynamic tissue, the proliferative activity of which dramatically changes throughout the menstrual cycle, with exquisite regulation by sex-steroid hormones. Primary endometrial epithelial cells fall into senescence within 2 weeks when cultured on plastic dishes, and more complete understanding of endometrial biology has been delayed because of, in part, a lack of an in vitro culture model for endometrial epithelial cells. Our goal was to establish immortalized human endometrial glandular cells that retain the normal functions and characteristics of the primary cells. Because the Rb/p16 and p53 pathways are known to be critical elements of epithelial senescence in early passages, we used human papillomavirus E6/E7 to target these pathways. The combination of human papillomavirus-16 E6/E7 expression and telomerase activation by the introduction of human telomerase reverse transcriptase (hTERT) led to successful immortalization of the endometrial glandular cells. E6/E7 expression alone was sufficient to extend their life span more than 20 population doublings, but the telomerase activation was further required to enable the cells to pass through the subsequent replicative senescence at 40 population doublings. Isolated immortalized cells contained no chromosomal abnormalities or only nonclonal aberrations, retained responsiveness to sex-steroid hormones, exhibited glandular structure on three-dimensional culture, and lacked transformed phenotypes on soft agar or in nude mice. The human endometrium is a unique tissue characterized by constant and rapid cell proliferation, differentiation, and breakdown during a menstrual cycle. This cyclic change in proliferation is exquisitely regulated by the cooperative actions of estrogen and progesterone, indicating that human endometrium is highly susceptive to sexsteroid hormones and that endometrial glandular epithelial cells may provide a good model with which to study hormone function and regulation. However, in classical tissue culture using plastic or glass dishes, epithelial cells loose their proliferative capacity during ongoing cultivation throughout several days, whereas stromal cells are more easily cultured in the longer term. Most investigators in this field have tried to develop endometrial cell cultures with a mixture of stromal cells and/or organotypic cultures, but both for short-term experiments only. [1][2][3] The lack of a stable in vitro culture system of endometrial cells also renders studying the molecular carcinogenesis of the endometrium difficult. Most endometrial cancers arise from endometrial glandular cells via the multistep accumulation of abnormalities in oncogenes and tumor suppressor genes, including PTEN, Ras, and p53. 4 These factors are frequently mutated or deregulated in endometrial cancers or even in its precursors. However, in vitro experiments to investigate the role of these factors in endometrial carcinogenesis have been impossible because of the extremely short life span of primary cultured endometrial epith...

Hypoxia-inducible factor 1 (HIF-1) is a key regulator of O 2 homeostasis, which regulates the expression of several genes linked to angiogenesis and energy metabolism. Tumor hypoxia has been shown to be associated with poor prognosis in a variety of tumors, and HIF-1 induced by hypoxia plays pivotal roles in tumor progression. The presence of putative HIF-1-binding sites on the promoter of human telomerase reverse transcriptase gene (hTERT) prompted us to examine the involvement of HIF-1 in the regulation of hTERT and telomerase in tumor hypoxia. The telomeric repeat amplification protocol (TRAP) assay revealed that hypoxia activated telomerase in cervical cancer ME180 cells, with peak induction at 24-48 h of hypoxia. Notably, hTERT mRNA expression was upregulated at 6-12 h of hypoxia, concordant with the elevation of HIF-1 protein levels at 6 h. hTERT protein levels were subsequently upregulated at 24 h and later. Luciferase assays using reporter plasmids containing hTERT core promoter revealed that hTERT transcription was significantly activated in hypoxia and by HIF-1 overexpression, and that the two putative binding sites within the core promoter are responsible for this activation. Chromatin immunoprecipitation assay identified the specific binding of HIF-1 to these sites (competing with c-Myc), which was enhanced in hypoxia. The present findings suggest that hypoxia activates telomerase via transcriptional activation of hTERT, and that HIF-1 plays a critical role as a transcription factor. They also suggest the existence of novel mechanisms of telomerase activation in cancers, and have implications for the molecular basis of hypoxia-induced tumor progression and HIF-1-based cancer gene therapy.

The endometrium is a highly regenerative tissue that plays a crucial role in implantation. We examined the clonal constitution of glandular cells as well as the luminal epithelium of this unique tissue. Using collagenase-based digestion techniques with microscopic manipulation, we isolated individual human endometrial glands and examined their clonality using a polymerase chain reaction-based assay for nonrandom X chromosome inactivation with an X-linked androgen receptor gene. Most of the glands analyzed were composed of monoclonal populations of epithelial cells and one of the glands exhibited a loss of heterogeneity in the androgen receptor gene. In addition, adjacent glands within a 1-mm(2) area shared clonality, suggesting that clonality of the luminal epithelium is regionally defined. The clonality of endometrium was further confirmed in a study of female mice that harbor the green fluorescent protein gene on either the maternal or paternal X chromosome. Fluorescent microscopy of uterine sections revealed that individual endometrial glands consisted completely of either fluorescent or nonfluorescent cells and that the surface epithelium exhibited a clear boundary between these cell types. These findings suggest that single or multiple stem cells with uniform clonality exist on the bottom of each endometrial gland and genetic alterations occurring in such cells may play a critical role in endometrial carcinogenesis. The possible association between area-specific X inactivation of the endometrial surface and the endometrial receptivity of embryo implantation remains to be clarified.

Silencing of the MLH1 gene by promoter hypermethylation is the mechanism underlying the microsatellite instability (MSI) phenotype in endometrial cancers. However, the profile of CpG methylation in a wide range of MLH1 promoters in endometrial cancers and in the normal endometrium is largely unknown. The present study investigates the region 700 bp upstream of MLH1 covering 48 CpG sites using bisulfite sequencing. Methylation status was classified as full (over 80% of CpGs are methylated), partial (10-80%) or nonmethylation (less than 10%). Of 56 endometrioid endometrial cancers, 16 (29%) were fully methylated, 14 (25%) were partially methylated and 26 (46%) were not methylated. Analyses of MLH1 by immunohistochemical means and of MSI revealed that the degree, rather than region-specific methylation of CpG islands is critical for decreased MLH1 expression and the MSI phenotype. Among 12 patients with methylated cancers, five (42%) patients contained methylated promoters in their normal endometria with profiles similar to those of cancer lesions, and these were associated with the MSI phenotype. In contrast, only one of 31 (3%) normal endometria from patients without endometrial malignancies harbored methylated promoters. These findings suggest that hypermethylation of the MLH1 promoter is frequent in the histologically normal endometrium adjacent to cancers, supporting the notion that hypermethylation of mismatch repair genes is the initial step that triggers various genetic events in endometrial carcinogenesis.