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 activation is a critical step for human carcinogenesis through the maintenance of telomeres, but the activation mechanism during carcinogenesis remains unclear. Transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene is the major mechanism for cancer-specific activation of telomerase, and a number of

Human cytochrome P450 (CYP) 1B1 is a key enzyme in the metabolism of 17␤-estradiol (E2). CYP1B1 is mainly expressed in endocrine-regulated tissues, such as mammary, uterus, and ovary. Because many CYP enzymes are likely to be induced by the substrates themselves, we examined whether the human CYP1B1 expression is regulated by E2 in the present study. Real-time reverse transcription-PCR analysis revealed that treatment with 10 nM E2 for 12 h induced CYP1B1 mRNA expression in estrogen receptor (ER)-positive MCF-7 cells. Luciferase reporter assays using MCF-7 cells showed a significant transactivation up to 7-fold by E2 with a reporter plasmid containing a region from ؊152 to ؉25 of the human CYP1B1 gene. A computer-assisted homology search indicated a putative estrogen response element (ERE) between ؊63 and ؊49 in the CYP1B1 promoter region. Specific binding of ER␣ to the putative ERE was demonstrated by chromatin immunoprecipitation assays and gel shift analyses. With reporter plasmids containing the wild or mutated putative ERE on the CYP1B1 gene and the wild or mutated ER␣ expression vectors, luciferase assays using Ishikawa cells demonstrated that the putative ERE and ER␣ are essential for the transactivation by E2. Because endometrial tissue is highly regulated by estrogens, the expression pattern of CYP1B1 protein in human endometrial specimens was examined by immunohistochemistry. The staining of CYP1B1 was stronger in glandular epithelial cells during a proliferative phase than those during a secretory phase, consistent with the pattern of estrogen secretion. These findings clearly indicated that the human CYP1B1 is regulated by estrogen via ER␣. Because 4-hydroxylation of estrogen by CYP1B1 leads to decrease of the estrogenic activity but the produced metabolite is toxicologically active, our findings suggest a clinical significance in the estrogenregulated CYP1B1 expression for the homeostasis of estrogens as well as estrogen-dependent carcinogenesis.

Hypoxia occurs during the development of the placenta in the first trimester and correlates with both trophoblast differentiation and the induction of telomerase activity through hTERT expression. We sought to determine the mechanism of regulation of hTERT expression during hypoxia. We show that hypoxia-inducible factor 1␣ (HIF-1␣) and hTERT expression in the human placenta decrease with gestational age and that these are overexpressed in preeclamptic placenta, a major complication of pregnancy. Hypoxia not only transactivates the hTERT promoter activity but also enhances endogenous hTERT expression. The hTERT promoter region between ؊165 and ؉51 contains two HIF-1 consensus motifs, and in vitro reporter assays show that these are essential for hTERT transactivation by HIF-1. Introduction of an antisense oligonucleotide for HIF-1 diminishes hTERT expression during hypoxia, indicating that upregulation of hTERT by hypoxia is directly mediated through HIF-1. Our results provide persuasive evidence that the regulation of hTERT promoter activity by HIF-1 represents a mechanism for trophoblast growth during hypoxia and suggests that this may be a generalized response to hypoxia in various human disorders including resistance to cancer therapeutics by upregulating telomerase.

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...

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