BACKGROUND: It has been proposed that the human endometrium may contain a population of adult stem cells that are responsible for its remarkable regenerative capability. Recently, a subset of stem cells or progenitor cells in adult tissue has been identified as side-population cells (SP cells) displaying low staining with Hoechst 33342 by fluorescenceactivated cell sorter (FACS) analysis. In this study, we isolated SP cells from the human endometrium and analysed their properties. METHOD: Endometrial cells were obtained using enzymatic digestion from uterine hysterectomy for the treatment of uterine myoma and stained with Hoechst 33342 dye either alone or in combination with verapamil. The cells were then analysed using FACS. RESULTS: SP cells were present among normal human endometrial cells. Most SP cells were enriched in the CD9 2 CD13 2 fraction. These SP cells showed long-term repopulating properties and produced gland (CD9 CD132 cells isolated from the endometrium also generated gland-or stroma-like cells. CONCLUSIONS: SP cells in the human endometrium can function as progenitor cells. This is the first report of the phenotype of SP cells from normal human endometrial cells.

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Background: Trophoblast cells, the functional components of the placenta, are derived from multipotent trophoblast stem (TS) cells. Results: SATB homeobox proteins regulate the TS cell stem state through up-regulation of a stem-specific transcription factor, EOMES, and inhibition of trophoblast differentiation. Conclusion: SATB proteins regulate TS cell development. Significance: Understanding TS cell biology is crucial to determining processes underlying placental development.

Differentiated trophoblast cell lineages arise from trophoblast stem (TS) cells. To date such a stem cell population has only been established in the mouse. The objective of this investigation was to establish TS cell populations from rat blastocysts. Blastocysts were cultured individually on a feeder layer of rat embryonic fibroblasts (REFs) in fibroblast growth factor-4 (FGF4) and heparin supplemented culture medium. Once cell colonies were established REF feeder layers could be replaced with REF conditioned medium. The blastocyst-derived cell lines, in either proliferative or differentiated states, did not express genes indicative of ICM-derived tissues. In the proliferative state the cells expressed established stem cell-associated markers of TS cells. Cells ceased proliferation and differentiated when FGF4, heparin, and REF conditioned medium were removed. Differentiation was characterized by a decline of stem cell-associated marker gene expression, the appearance of large polyploid cells (trophoblast giant cells), and the expression of trophoblast differentiation-associated genes. Collectively, the data indicate that the rat blastocyst-derived cell lines possess many features characteristic of mouse TS cells but also possess some distinct properties. These rat TS cell lines represent valuable new in vitro models for analyses of mechanisms controlling TS cell renewal and differentiation.

Recently, numerous studies have identified that immature cell populations including stem cells and progenitor cells can be found among “side-population” (SP) cells. Although SP cells isolated from some adult tissues have been reported elsewhere, isolation and characterization of human trophoblast SP remained to be reported. In this study, HTR-8/SVneo cells and human primary villous cytotrophoblasts (vCTBs) were stained with Hoechst 33342 and SP and non-SP (NSP) fractions were isolated using a cell sorter. A small population of SP cells was identified in HTR-8/SVneo cells and in vCTBs. SP cells expressed several vCTB-specific markers and failed to express syncytiotrophoblast (STB) or extravillous cytotrophopblast (EVT)-specific differentiation markers. SP cells formed colonies and proliferated on mouse embryonic fibroblast (MEF) feeder cells or in MEF conditioned medium supplemented with heparin/FGF2, and they also showed long-term repopulating property. SP cells could differentiate into both STB and EVT cell lineages and expressed several differentiation markers. Microarray analysis revealed that IL7R and IL1R2 were exclusively expressed in SP cells and not in NSP cells. vCTB cells sorted as positive for both IL7R and IL1R2 failed to express trophoblast differentiation markers and spontaneously differentiated into both STB and EVT in basal medium. These features shown by the SP cells suggested that IL7R and IL1R2 are available as markers to detect the SP cells and that vCTB progenitor cells and trophoblast stem cells were involved in the SP cell population.

c BHLHE40 and BHLHE41 (BHLHE40/41) are basic helix-loop-helix type transcription factors that play key roles in multiple cell behaviors. BHLHE40/41 were recently shown to be involved in an epithelial-to-mesenchymal transition (EMT). However, the precise mechanism of EMT control by BHLHE40/41 remains unclear. In the present study, we demonstrated that BHLHE40/41 expression was controlled in a pathological stage-dependent manner in human endometrial cancer (HEC). Our in vitro assays showed that BHLHE40/41 suppressed tumor cell invasion. BHLHE40/41 also suppressed the transcription of the EMT effectors SNAI1, SNAI2, and TWIST1. We identified the critical promoter regions of TWIST1 for its basal transcriptional activity. We elucidated that the transcription factor SP1 was involved in the basal transcriptional activity of TWIST1 and that BHLHE40/41 competed with SP1 for DNA binding to regulate gene transcription. This study is the first to report the detailed functions of BHLHE40 and BHLHE41 in the suppression of EMT effectors in vitro. Our results suggest that BHLHE40/41 suppress tumor cell invasion by inhibiting EMT in tumor cells. We propose that BHLHE40/41 are promising markers to predict the aggressiveness of each HEC case and that molecular targeting strategies involving BHLHE40/41 and SP1 may effectively regulate HEC progression. Basic helix-loop-helix (bHLH) type transcription factors play key roles in cell differentiation, proliferation, apoptosis, and metabolism. BHLHE40 (basic helix-loop-helix family member e40 gene) and BHLHE41 are members of the Hairy/E(spl)/HES family. BHLHE40 and BHLHE41 (BHLHE40/41) exhibit more than 90% similarity in the bHLH region and approximately 50% in total. BHLHE40/41 have been shown to function as transcriptional repressors by binding to the class B E-box. BHLHE40/41 interact with TF2B, TBP, or TF2D or recruit a histone deacetylase at the E-box site (1-5). On the other hand, BHLHE40/41 were previously reported to modulate the expression of some genes in an E-box-independent manner. BHLHE40 has been shown to associate with SP1 binding sites in the BIRC5 promoter to activate its transcription (6) and with STAT3 to regulate the transcription of STAT3-dependent target genes (7). BHLHE41 suppressed VEGF transcription by interacting with HIF1A (8). BHLHE40 and BHLHE41 were reported to associate with retinoid X receptor (RXR), MYOD1, or CEBP in order to regulate the transcription of their target genes (9-12).In diverse types of cancer species, such as colon, oral, and liver cancer or brain tumors, BHLHE40 expression levels were found to be higher in tumors than in adjacent normal tissues (13-15). On the other hand, in human endometrial cancer (HEC) and nonsmall-cell lung cancer, no changes in BHLHE40 expression were reported between cancer and normal tissues (16,17). Regarding expression profiles with the development of cancer, studies on oral, lung, liver, and esophageal cancer showed that BHLHE40 expression inversely correlated with the tumor stage or differentiation grade (18-21)...

HOPX (homeodomain only protein X) is a newly identified homeobox gene whose loss of expression has been reported for several types of neoplasm. Although we found most human uterine endometrial cancers (HEC) defective in HOPX expression, genetic mutations in the HOPX gene were undetectable. As is the case with several tumor suppressor genes, the promoter region of HOPX is densely methylated in HEC tissue samples obtained by laser capture microdissection. HOPX mRNA and protein levels were reduced in the majority of samples, and this correlated with hypermethylation of the HOPX promoter. Forced expression of HOPX resulted in a partial block in cell proliferation, in vivo tumorigenicity and c-fos gene expression in HEC and MCF7 cells in response to 17b-estradiol (E 2 ) stimulation. Analysis of the serum response element (SRE) of c-fos gene promoter showed that the effect of HOPX expression is associated with inhibition of E 2 -induced c-fos activation through the serum response factor (SRF) motif. Knockdown of HOPX in immortalized human endometrial cells resulted in accelerated proliferation. Our study indicates that transcriptional silencing of HOPX results from hypermethylation of the HOP promoter, which leads to HEC development. ' 2008 Wiley-Liss, Inc.Key words: human uterine endometrial cancer; 17b-estradiol; DNA methylation; HOPX; tumor suppressor gene; serum response factor Human endometrial cancer (HEC) is one of the most common gynecological malignancies in Japan, where its incidence has increased dramatically over the past decade. Previous genetic analysis has identified several genetic abnormalities involved in endometrial carcinogenesis, but the detailed mechanism that is responsible for endometrial carcinogenesis remains unclear.Although several typical tumor suppressor genes are inactivated by deletions and/or mutations in cancer cells, epigenetic mechanisms including aberrant methylation of CpG sites within the promoter region also contribute to gene silencing. Even though there is an overall decrease in DNA methylation during carcinogenesis, particular CpG island sequences are hypermethylated resulting in the transcriptional silencing of critical genes and contributing to development and progression of a variety of human cancers. 1,2 c-fos is a typical ''immediate early'' gene (IEG) that is rapidly induced in response to diverse extracellular stimuli including various mitogens and steroid hormones. c-fos proteins hetero-and homo-dimerize with other basic ZIP transcription factors to form activating protein 1 (AP1) transcription factor complexes that regulate the expression of multiple genes involved in cell growth, differentiation and transformation. It has been shown that 17b-estradiol (E 2 ) induces c-fos expression in human endometrial epithelial cells and endometrial cancer cells, and this induction has been implicated as a possible mechanism by which E 2 stimulates the growth of endometrial epithelia. 3,4 Cyclin D1 induced by AP1 is reported to regulate the growth of endometrial granular cells a...

Previous observations indicate that transfer of human chromosome (chr.) 1 induces senescence of endometrial cancer cells. To identify the gene(s) responsible for the senescence, we first analyzed the structural integrity of the introduced chr. 1 in immortal revertant from chr.1-transferred HHUA cells. The data demonstrated a correlation between nonrandom deletions within the 1q31-qter region and reversion to immortality. Next, by using a panel of 12 microsatellite markers, we found high frequencies of loss of heterozygosity in the particular 1q region (1q41-42), in surgically removed samples. Then, we screened the genetic mutation of the genes involved in this region, with endometrial cancer panel. Among them, EGLN1, that is a member of prolyl hydroxylase and can facilitate HIF-1 degradation by ubiquitination through the hydroxylation of HIF-1, was mutated at significantly higher frequencies (12/20, 60%). Introduction of wild-type EGLN1 into endometrial cancer cell lines (HHUA, Ishikawa and HWCA), that carry EGLN1 gene mutations induced senescence. This was invoked through the negative regulation of HIF-1 expression. In addition, alternative way of negative regulation of HIF-1 by Factor inhibiting HIF-1(FIH), SiRNA against HIF-1, and HIF-1 inhibitor, YC-1, could also induce senescence. Thus, EGLN1 can be considered as a candidate tumor suppressor on chr. 1q, and our observation could open the new aspect in exploring the machinery of senescence induction associated with HIF-1 signal transduction. These results also suggested the availability of negative regulation of HIF-1 signals for uterine cancer treatment, especially for uterine sarcomas that have worse prognosis and show a high frequency of EGLN1 gene abnormality. ' 2005 Wiley-Liss, Inc.Key words: HIF-1; EGLN1; endometrial cancer; prolyl hydroxylase; Chromosome 1Endometrial cancer is one of the most common gynecologic malignancies. Considerable evidence has suggested a number of genetic events associated with this tumor. However, the molecular pathogenesis of endometrial cancers remains poorly understood. Genetic alterations that represent gross chromosomal alterations are known to occur during endometrial carcinogenesis. Among them, nonrandom chromosome changes have been found in almost 80% of endometrial cancers and are consistent with rearrangements and deletion in a centromeric or paracentromeric area, leading to an excess of chromosome (chr.) 1q.1-3 Previously, we transferred a single human chromosome into endometrial cancer cells (HHUA and Ishikawa cells) via microcell fusion to investigate the role of chr.1 alterations in the carcinogenesis. As a result, we found that the tumorigenicity of microcell hybrid clones with an introduced chr.1, 6, or 9 is completely suppressed. However, the hybrids with an introduced chr.1 differed from those with an introduced chr. 6 or 9.4,5 A large proportion of the hybrids with chr. 1 had arrested cell growth and showed remarkable alterations in cell morphology. This finding is in contrast with the transfer of chr. 6 ...