Small bowel obstruction caused by intrauterine device infection.

A 39-year-old previously healthy woman was referred to our emergency department by a primary care doctor on suspected to be acute enteritis, complaining of fever, anorexia, lower abdominal pain, and frequent diarrhea. The day after admission, although frequent diarrhea stopped, the abdominal distension worsened. An abdominal radiograph revealed several dilated loops of the small bowel, suggested that small bowel obstruction (SBO) had developed. White blood cell count and c-reactive protein were markedly increased, and abdominal contrast-enhanced computed tomography scan showed localized severely edematous bowel mucosa, increased adipose tissue concentration in the pelvis, and a beaded low absorption area in the uterus. Gynecological examination revealed the presence of a pus-filled plastic intrauterine device (IUD) in the uterus. The patient confided that she had sex with her husband 2 days before the onset of symptoms. A diagnosis of SBO due to pelvic peritonitis caused by IUD infection during sexual activity was made. The SBO was cleared in 12 days with fasting, peripheral parenteral nutrition, antibiotic treatment, and insertion of an ileus tube. This case reminds us that it needs to consider disorders associated with the uterine appendages, in women of reproductive age with lower abdominal pain.

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.

Frequent hypermethylation of MLH1 promoter in normal endometrium of patients with endometrial cancers.

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.

HIF-1-mediated activation of telomerase in cervical cancer cells.

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.

Direct activation of telomerase by EGF through Ets-mediated transactivation of TERT via MAP kinase signaling pathway.

Telomerase is a regulated enzyme and its activity is tightly associated with cell proliferation. The mechanisms of this association are unclear, but specific growth factors may regulate telomerase activity. The present study examines the effect of epidermal growth factor (EGF) on telomerase activity and identifies the signal transduction pathway involved in this process. EGF up-regulated 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 effect 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 specifically 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 effect on hTERT transcription, while that of dominant negative Ets significantly repressed EGF action. These findings 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 specific signal transduction pathways.

Tamoxifen regulates human telomerase reverse transcriptase (hTERT) gene expression differently in breast and endometrial cancer cells.

Tamoxifen is widely applied as an antiestrogenic agent for adjuvant therapy in the treatment of breast cancer, while its estrogen-agonistic activity occasionally causes proliferative disorders or carcinogenesis at other sites, such as the uterus. We reported that estrogen activates telomerase in breast and endometrial cancer cells. The present study examines the effects of tamoxifen on the gene expression of human telomerase reverse transcriptase (hTERT) in breast and endometrial cancer cells. Tamoxifen inhibited the cell growth of MCF-7 cells, as well as hTERT mRNA expression in the presence of estrogen (E2), antagonizing the E2 effects. In contrast, tamoxifen stimulated the growth of Ishikawa cells and activated hTERT mRNA expression in the absence or presence of E2, exhibiting estrogen-agonistic action. Transient expression assays revealed that these actions of tamoxifen are achieved by transcriptional regulation of the hTERT promoter. An estrogen responsive element (ERE) in the hTERT 5' regulatory region was partly responsible for both the E2-antagonistic and -agonistic actions of tamoxifen. Tamoxifen activated the MAP kinase cascade in Ishikawa cells, but not in MCF-7 cells, and the activation of hTERT mRNA expression was effectively blocked by MEK inhibitor, suggesting that the MAP kinase pathway is involved in the tamoxifen-induced activation of hTERT. These findings indicate that tamoxifen regulates hTERT expression in a cell-type specific manner. Tamoxifen-induced activation of hTERT may be one component of estrogen agonistic function of tamoxifen that is involved in endometrial carcinogenesis induced by this agent.

Efficient inhibition of human telomerase reverse transcriptase expression by RNA interference sensitizes cancer cells to ionizing radiation and chemotherapy.

Telomerase activation plays critical roles in tumor growth and progression in part through the maintenance of telomere structure. Indeed, the ubiquitous expression of telomerase in human cancers makes telomerase a promising target for cancer therapy. Genetic, pharmacologic, and antisense methods to inhibit telomerase have been described; however, in most cases, cancer cell death was observed only after many cell divisions. Here, using retroviral delivery of small interfering RNAs (siRNAs) specific for the human telomerase reverse transcriptase (hTERT), we successfully inhibited telomerase activity in cervical cancer cell lines. Cells lacking hTERT expression exhibited significantly decreased telomerase activity and showed shortened telomeres and telomeric 3' overhangs with passage. These cells entered replicative senescence after a considerable number of cell divisions. Notably, the proliferative rate of these cells was significantly impaired, compared with control cells with telomerase activity, even in low-passage cells (population doubling 5). Likewise, colony-forming ability and tumorigenicity in mice were attenuated in low-passage cells lacking hTERT. We further examined the effects of chemotherapy and ionizing radiation on cells in which hTERT expression was suppressed. Cells lacking hTERT showed a significantly increased sensitivity, compared with control cells, to ionizing radiation or chemotherapeutic agents that induce DNA double- strand breaks, such as topoisomerase inhibitors or bleomycin. These findings suggest that an siRNA-based strategy can be applied to the development of novel telomerase inhibitors, the antitumor effects of which may be enhanced in combination with ionizing radiation and chemotherapy.

Association of mismatch repair deficiency with PTEN frameshift mutations in endometrial cancers and the precursors in a Japanese population.

We studied mismatch repair deficiency and PTEN (phosphatase and tensin homologue deleted on chromosome 10) mutations in endometrial cancers and hyperplasias in a Japanese population. Methylation-sensitive restriction enzyme polymerase chain reaction revealed MLH1 hypermethylation in 21 (38%) of 56 endometrial cancers. Sequencing analysis revealed PTEN mutations in 22 patients with cancer (39%) in exons 5 and 8. A PTEN frameshift mutation was associated significantly with MLH1 hypermethylation (P = .01) and a highly positive phenotype with microsatellite instability (P < .001) but not with a PTEN missense mutation. In hyperplasia, MLH1 hypermethylation was similarly observed (11/27 [41%]), but the PTEN mutation was less frequent (5/27 [19%]), observed only in atypical hyperplasias; among the 5 patients with a PTEN mutation, the 2 patients with frameshift mutations had MLH1 hypermethylation, but the 3 patients with missense mutations had unmethylated MLH1. These findings indicate that MLH1 hypermethylation is an early event frequently occurring in hyperplasia without atypia, whereas the PTEN mutation occurs later, mostly in atypical hyperplasia, possibly caused by MLH1 hypermethylation.

Pharmacokinetics of paclitaxel in ovarian cancer patients and genetic polymorphisms of CYP2C8, CYP3A4, and MDR1.

Interindividual differences in the pharmacokinetics of paclitaxel and its metabolites in Japanese ovarian cancer patients were investigated in relation to genetic polymorphisms of the CYP2C8, CYP3A4, and MDR1 genes. The area under the concentration-time curve (AUC) ratios of paclitaxel/6alpha-hydroxypaclitaxel and paclitaxel/3 -p-hydroxypaclitaxel calculated as the metabolic index of CYP2C8 and CYP3A4 showed 13- and 12-fold interindividual variations, respectively. No patient had any CYP2C8 variants, while 2 patients were heterozygotes of CYP3A4*16. For the MDR1 gene, the frequencies of -129C, 1236C, 2677T, 2677A, and 3435T alleles were 2.2%, 8.7%, 56.5%, 4.4%, and 52.2%, respectively. Subjects possessing the 3435T allele had a significantly (P < .05) higher AUC of 3'- p-hydroxypaclitaxel compared to those possessing the 3435C allele. Leukocytopenia was significantly (P < .05) related to the AUC of paclitaxel. Genotyping of the CYP2C8, CYP3A4, and MDR1 genes might not be essential to predict adverse effects of paclitaxel in Japanese patients, although an allelic variant of MDR1 may functionally affect the pharmacokinetics of its metabolite.

hTERT-promoter-based tumor-specific expression of MCP-1 effectively sensitizes cervical cancer cells to a low dose of cisplatin.

Cervical cancers at advanced stages or with recurrent status are mainly treated by platinum-based chemotherapy, such as cisplatin. However, a novel strategy to reduce the minimally effective dose is required to prevent severe adverse effects that limit the effectiveness of the treatment. Monocyte chemoattractant protein-1 (MCP-1) is a subtype of chemokines that can promote monocyte/macrophage infiltration and enhance their phagocytosis at not only sites of inflammatory lesions but also of tumors. The present study applies MCP-1-based gene therapy to treat cervical cancers. To achieve tumor-specific expression of MCP-1, retroviral expression vector was constructed using the human telomerase reverse transcriptase gene (hTERT) promoter. Retroviral expression of MCP-1 into cervical cancer ME180 cells did not affect their proliferation either in vitro or in vivo. However, when combined with a suboptimal low dose of cisplatin, tumor formation was obviously reduced in clones transduced with MCP-1, but not in control clones. Histological examination revealed that a substantial number of macrophages infiltrated the tumor sites of MCP-1-transduced cells, but not of controls. These findings suggest that MCP-1 expression sensitizes cervical cancer cells to an otherwise ineffective low dose of cisplatin, possibly by inducing the migration of macrophages to eradicate tumor cells. This system may be a novel strategy for chemotherapy combined with immunogene therapy against otherwise intractable cervical cancers.

2-5A antisense therapy directed against human telomerase RNA inhibits telomerase activity and induces apoptosis without telomere impairment in cervical cancer cells.

Human telomerase RNA (hTR), an important component of telomerase, is a possible target of telomerase-based cancer gene therapy. The present study was undertaken to assess the efficacy of antisense hTR therapy using newly developed 2-5A (5'-phosphorylated 2'-5'-linked oligoadenylate)-linked oligonucleotides against cervical cancer cells. ME180 and SiHa cells were treated with 2-5A-linked antisense hTR designed to complement the region of hTR between residues 76 and 94. The hTR expression, telomerase activity, cell viability, and apoptosis were then examined. The 2-5A anti-hTR effectively degraded hTR and inhibited telomerase activity. The 2-5A mutant anti-hTR and the anti-hTR without 2-5A were not capable of inhibiting telomerase activity. Inhibition of telomerase by 2-5A anti-hTR rapidly decreased cell viability only in telomerase-positive cells within 3-6 days after the treatment, when telomere length has not yet been shortened. This inhibition was associated with apoptosis, possibly through activation of caspase family members. These findings suggest that 2-5A-linked antisense-hTR therapy has a potent telomerase-inhibitory effect associated with a cytocidal effect from caspase-induced apoptosis, and may therefore be a potential tool in telomerase-based gene therapy against cervical cancers.

Aberrant expression and mutations of TGF-beta receptor type II gene in endometrial cancer.

OBJECTIVE: Transforming growth factor beta (TGF-beta) is a multifunctional cytokine that strongly inhibits epithelial cell growth. Disabling of TGF-beta signaling is thought to be involved in development of a variety of tumors in which abnormal expression or function of TGF-beta receptor plays critical roles. In the present study, we examined aberrant expression and mutation of the gene TGF-beta receptor type II (TbetaRII) in endometrial cancers of endometrioid subtype. METHODS AND RESULTS: Real-time PCR analysis using surgical tissue specimens of 27 endometrial cancers and 24 normal endometria revealed that endometrial cancers had significantly decreased levels of TbetaRII mRNA expression (mean level 2.44 +/- 2.65), compared to normal endometria (mean level 7.23 +/- 6.07) (P < 0.001). Methylation status of TbetaRII promoter containing 30 CpGs was examined by bisulfite sequencing analysis, and 98% (51/52) of the patients were found to have unmethylated TbetaRII promoter, indicating that promoter hypermethylation is not the major cause of decreased expression of TbetaRII in endometrial cancers. Mutational analysis revealed that 15.1% (8/53) of endometrial cancers had frameshift mutations at polyadenine repeats in exon 3 of the TbetaRII gene. Notably, these mutations were preferentially accumulated in patients with MSI-H phenotype (7/19:37%) (P < 0.001) or with those with methylated MLH1 promoters (6/16:38%) (P < 0.01). Thus, it appears that the TbetaRII gene is a target of mismatch repair deficiency. CONCLUSION: Taken together, we found that the decreased expression of TbetaRII as well as frameshift mutation of TbetaRII via mismatch repair deficiency frequently occurs in this tumor type, possibly causing loss of receptor function and unresponsiveness of TGF-beta signaling that may lead to endometrial carcinogenesis.

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.

Is the telomerase assay useful for screening of endometrial lesions?

Telomerase activation is specifically observed in most cancers but not in normal tissues with some exceptions, such as germ cells or certain tissues with regenerative potential, suggesting a diagnostic opportunity for cancers involving measurement of telomerase activity. Cytologic screening for endometrial cancer has not been well established, due to the complexity of diagnostic criterion. In the present study, we investigated the utility of the telomerase assay for screening endometrial lesions. A total of 100 patients with or without endometrial lesions were examined for telomerase activity by the telomeric repeat amplification protocol (TRAP) assay using endometrial scraping samples and the correlation with cytology was investigated. The TRAP assay revealed frequent telomerase activation in normal endometria at reproductive age, particularly in 67% of proliferative-phase endometria, suggesting that the telomerase assay is not suitable for screening women of reproductive age. However, in postmenopausal women, telomerase activity was rarely detected (8%) in normal endometria, while it was observed in >80% of endometrial cancers or hyperplasias. Interestingly, some cases of endometrial cancer and hyperplasia were misdiagnosed by cytology but correctly detected by the TRAP assay. The sensitivity of the TRAP assay to screen endometrial lesions was 87%, equivalent to that of cytology. Combination of cytology and the TRAP assay increased sensitivity to 100%. We thus concluded that measuring telomerase activity in endometrial scrapings is a useful diagnostic tool for the screening of endometrial lesions in postmenopausal women, particularly when used with cytology to increase screening sensitivity.

Evidence of the monoclonal composition of human endometrial epithelial glands and mosaic pattern of clonal distribution in luminal epithelium.

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.

Significance of immunological detection of human telomerase reverse transcriptase: re-evaluation of expression and localization of human telomerase reverse transcriptase.

Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase and is a potentially useful diagnostic marker for cancers. There have been few studies in which immunological detection of hTERT has been attempted and its subcellular localization has not been precisely defined. In the present study, we re-evaluated expression and localization of hTERT in cancer and normal cells using a newly developed antibody. Immunohistochemistry revealed that hTERT is expressed in approximately 80% of gynecological cancers, but some premalignant lesions exhibited weak expression of hTERT. Interestingly, not only nuclei but also cytoplasm of cancer cells were positive for hTERT staining. This finding was supported by the results of Western blot analysis of cell lines, in which both nuclear and cytoplasmic extracts exhibited significant hTERT bands. Cytoplasmic hTERT in cancer cells may be functional because the telomeric repeat amplification protocol assay of cytoplasmic extracts showed high levels of telomerase activity. Unexpectedly, not all normal primary cells and telomerase-negative cancer cell lines lacked hTERT expression; some exhibited weak TERT signals. In Western analysis, hTERT signals did not always correlate with telomerase activity of the various cell types. These findings suggest that functional hTERT is expressed in both the nucleus and cytoplasm of cancer cells and that hTERT expression does not strictly reflect telomerase activity. Further analysis is needed to clarify the biological significance of cytoplasmic hTERT.