Satoru Kyo

Novel Cardiac Precursor‐Like Cells from Human Menstrual Blood‐Derived Mesenchymal Cells

Stem cell therapy can help repair damaged heart tissue. Yet many of the suitable cells currently identified for human use are difficult to obtain and involve invasive procedures. In our search for novel stem cells with a higher cardiomyogenic potential than those available from bone marrow, we discovered that potent cardiac precursor‐like cells can be harvested from human menstrual blood. This represents a new, noninvasive, and potent source of cardiac stem cell therapeutic material. We demonstrate that menstrual blood‐derived mesenchymal cells (MMCs) began beating spontaneously after induction, exhibiting cardiomyocyte‐specific action potentials. Cardiac troponin‐I‐positive cardiomyocytes accounted for 27%–32% of the MMCs in vitro. The MMCs proliferated, on average, 28 generations without affecting cardiomyogenic transdifferentiation ability, and expressed mRNA of GATA‐4 before cardiomyogenic induction. Hypothesizing that the majority of cardiomyogenic cells in MMCs originated from detached uterine endometrial glands, we established monoclonal endometrial gland‐derived mesenchymal cells (EMCs), 76%–97% of which transdifferentiated into cardiac cells in vitro. Both EMCs and MMCs were positive for CD29, CD105 and negative for CD34, CD45. EMCs engrafted onto a recipients heart using a novel 3‐dimensional EMC cell sheet manipulation transdifferentiated into cardiac tissue layer in vivo. Transplanted MMCs also significantly restored impaired cardiac function, decreasing the myocardial infarction (MI) area in the nude rat model, with tissue of MMC‐derived cardiomyocytes observed in the MI area in vivo. Thus, MMCs appear to be a potential novel, easily accessible source of material for cardiac stem cell‐based therapy.

Human CYP1B1 Is Regulated by Estradiol via Estrogen Receptor

Human cytochrome P450 (CYP) 1B1 is a key enzyme in the metabolism of 17beta-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 ERalpha 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 ERalpha expression vectors, luciferase assays using Ishikawa cells demonstrated that the putative ERE and ERalpha 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 ERalpha. 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 estrogen-regulated CYP1B1 expression for the homeostasis of estrogens as well as estrogen-dependent carcinogenesis.

Telomerase-Specific Replication-Selective Virotherapy for Human Cancer

Purpose: Replication-selective tumor-specific viruses present a novel approach for treating neoplastic disease. These vectors are designed to induce virus-mediated lysis of tumor cells after selective viral propagation within the tumor. Telomerase activation is considered to be a critical step in carcinogenesis, and its activity is closely correlated with human telomerase reverse transcriptase (hTERT) expression. We investigated the antitumor effect of the hTERT-specific replication-competent adenovirus on human cancer cells. Experimental Design: We constructed an adenovirus 5 vector [tumor- or telomerase-specific replication-competent adenovirus (TRAD)], in which the hTERT promoter element drives expression of E1A and E1B genes linked with an internal ribosome entry site, and we examined the selective replication and antitumor effect in human cancer cells in vitro and in vivo. Results: TRAD induced selective E1A and E1B expression in human cancer cells, but not in normal cells such as human fibroblasts. TRAD replicated efficiently and induced marked cell killing in a panel of human cancer cell lines, whereas replication as well as cytotoxicity was highly attenuated in normal human fibroblasts lacking telomerase activity. In nu/nu mice carrying s.c. human lung tumor xenografts, intratumoral injection of TRAD resulted in a significant inhibition of tumor growth. No evidence of TRAD was identified in tissues outside of the tumors, despite the presence of TRAD in the circulation. Moreover, TRAD replication in the distant, noninjected tumors was demonstrated. Conclusions: Our results suggest that the hTERT promoter confers competence for selective replication of TRAD in human cancer cells, an outcome that has important implications for the treatment of human cancers.

Hypoxia-Inducible Factor 1 Mediates Upregulation of Telomerase (hTERT)

ABSTRACT 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 1α (HIF-1α) 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.

Induction of hTERT expression and phosphorylation by estrogen via Akt cascade in human ovarian cancer cell lines

We examined the mechanism by which estrogen regulates telomerase activity in Caov-3 human ovarian cancer cell lines, which express ER, to determine whether the regulation affects the expression and/or phosphorylation of the telomerase catalytic subunit (hTERT). 17β-Estradiol (E2) induced telomerase activity and hTERT expression. Transient expression assays using luciferase reporter plasmids containing various fragments of hTERT promoter showed that the estrogen-responsive element appeared to be partially responsible for the E2-induced activation of the hTERT promoter. Either pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, or transfection with a dominant-negative Akt attenuated the E2-induced activation of the hTERT promoter. In addition, estrogen induced the phosphorylation of IκB inhibitor protein via the Akt cascade, and cotransfection with a dominant-negative subunit of NFκB attenuated the response of the ERE-deleted hTERT promoter to E2. Moreover, E2 induced the phosphorylation of hTERT, the association of 14-3-3 protein and NFκB with hTERT, and nuclear accumulation of hTERT in an Akt-dependent manner. These results indicate that E2 induces telomerase activity not only by transcriptional regulation of hTERT via an ERE-dependent mechanism and a PI3K/Akt/NFκB cascade, but also by post-transcriptional regulation via Akt-dependent phosphorylation of hTERT. Thus, the phosphorylation of Akt is a key event in the induction of telomerase activity by E2 in human ovarian cancer cells.

In vivo imaging of lymph node metastasis with telomerase-specific replication-selective adenovirus

Currently available methods for detection of tumors in vivo such as computed tomography and magnetic resonance imaging are not specific for tumors. Here we describe a new approach for visualizing tumors whose fluorescence can be detected using telomerase-specific replication-competent adenovirus expressing green fluorescent protein (GFP) (OBP-401). OBP-401 contains the replication cassette, in which the human telomerase reverse transcriptase (hTERT) promoter drives expression of E1 genes, and the GFP gene for monitoring viral replication. When OBP-401 was intratumorally injected into HT29 tumors orthotopically implanted into the rectum in BALB/c nu/nu mice, para-aortic lymph node metastasis could be visualized at laparotomy under a three-chip color cooled charged-coupled device camera. Our results indicate that OBP-401 causes viral spread into the regional lymphatic area and selectively replicates in neoplastic lesions, resulting in GFP expression in metastatic lymph nodes. This technology is adaptable to detect lymph node metastasis in vivo as a preclinical model of surgical navigation.

Successful Immortalization of Endometrial Glandular Cells with Normal Structural and Functional Characteristics

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. These findings support the notion that both Rb inactivation/p53 inactivation and telomerase activation are necessary to immortalize endometrial epithelial cells, but additional factors are required for endometrial carcinogenesis. Our established cell lines show great promise for investigation of hormone functions, endometrial biology, and endometrial carcinogenesis.

A novel telomerase-specific gene therapy : gene transfer of caspase-8 utilizing the human telomerase catalytic subunit gene promoter

Apoptosis is a genetically encoded cell death process and is a pathway that may be disrupted in tumor cells. Therefore, therapies that restore the ability to undergo apoptosis are promising for the treatment of tumor cells. We have demonstrated that the transfer of apoptosis-inducible genes inhibits the growth of tumors in vitro and in vivo through induction of apoptosis. However, to restrict induction of apoptosis to tumor cells, we need to explore a tumor-specific expression system of these genes. In the present study, we developed the telomerase-specific transfer system of apoptosis-inducible genes, utilizing the promoter of the human telomerase catalytic subunit (hTERT) gene. Approximately 90% of tumors have telomerase activity whereas most normal cells do not express the activity. These observations indicate that telomerase is a particularly attractive target for the tumor-specific expression system of vectors. We demonstrate here that by using the hTERT promoter-driven caspase-8 expression vector (hTERT/caspase-8), apoptosis is restricted to telomerase-positive tumor cells of wide range, and is not seen in normal fibroblast cells without telomerase activity. Furthermore, treatment of subcutaneous tumors in nude mice with the hTERT/caspase-8 construct inhibited tumor growth significantly because of induction of apoptosis (p < 0.01). The telomerase-specific expression of apoptosis-inducible genes afforded by the hTERT promoter, therefore, may be a novel and promising targeting approach for the treatment of tumors with telomerase activity.

E2F-1 represses transcription of the human telomerase reverse transcriptase gene

The ends of human chromosomes (telomeres) lose up to 200 bp of DNA per cell division. Chromosomal shortening ultimately leads to senescence and death in normal cells. Many human carcinoma lines are immortal in vitro, suggesting that these cells have a mechanism for maintaining the ends of their chromosomes. Telomerase is a ribonucleoprotein complex that synthesizes telomeric DNA onto chromosomes using its RNA component as template. Telomerase activity is found in most tumor cells, but is absent from normal cells. Little is known about how normal human cells repress telomerase (hTERT) gene expression. Mice carrying an E2F-1 null mutation develop a variety of malignant tumors, suggesting that this transcription factor has a tumor suppressor function. To determine mechanisms by which E2F-1 suppresses tumor formation, we examined the role of this transcription factor in regulation of the hTERT promoter in human cells. We identified two putative E2F-1-binding sites proximal to the transcriptional start site of the hTERT promoter. Mutation of these sites produced dramatic increases in promoter activity. Overexpression of E2F-1 but not a mutant E2F-1 repressed hTERT promoter activity in reporter gene assays. This repression was abolished by mutation of the E2F-1-binding sites in the hTERT promoter. Human cancer cell lines stably overexpressing E2F-1 exhibited decreased hTERT mRNA expression and telomerase activity. We conclude that E2F-1 has an atypical function as a transcriptional repressor of the hTERT gene in human cells.

Human telomerase reverse transcriptase as a critical determinant of telomerase activity in normal and malignant endometrial tissues

Telomerase activation is thought to be essential for cellular immortality and oncogenesis. It is observed in most malignant tumors but not in most normal somatic tissues. Normal human endometrium is, however, known to express significant telomerase activity in a menstrual phase-dependent manner. The 3 major subunits composing telomerase have been identified. Using normal and malignant endometrial tissues, we studied how these components are involved in telomerase activation. A total of 23 endometrial cancers and 32 normal human endometria in various menstrual phases as well as cell lines derived from endometrial cancer were examined for the expression of each telomerase subunit using RT-PCR analysis. Telomerase activity in each sample was determined by the TRAP assay, and the correlation between subunit expression and telomerase activity was examined. RT-PCR analysis revealed that telomerase RNA (hTR) and telomerase-associated protein (TP1) mRNA were constitutively expressed in both normal and malignant endometrial tissues. Expression of human telomerase reverse transcriptase (hTERT) mRNA was observed in most endometrial cancers, while that in normal endometrium depended on the phases of menstrual cycles. Proliferative phase normal endometria expressed hTERT mRNA, while secretory phase endometria did not. There was a strong association between telomerase activity and hTERT expression but not TP1 or hTR expression in both normal and tumor tissues. Five telomerase-positive endometrial cancer cell lines expressed each of the telomerase subunits including hTERT, while 2 telomerase-negative normal primary fibroblast cells expressed TP1 mRNA and hTR, but not hTERT mRNA. Our findings suggest that hTERT is a rate-limiting determinant of enzymatic activity of human telomerase. Since some normal tissues with high regenerative potential can express hTERT, special attention should be paid to the clinical use of hTERT inhibitors as anti-cancer drugs.