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1.  Association of MDM2 SNP309 and TP53 Arg72Pro polymorphisms with risk of endometrial cancer 
Oncology Reports  2013;30(1):25-34.
The incidence of endometrial cancer, a common gynecological malignancy, is increasing in Japan. We have previously shown that the ER/MDM2/p53/p21 pathway plays an important role in endometrial carcinogenesis. In the present study, we investigated the effects of germline single nucleotide polymorphisms in murine double minute 2 (MDM2) SNP309, TP53 Arg72Pro, ESR1 PvuII and XbaI, and p21 codon 31 on endometrial cancer risk. We evaluated these polymorphisms in DNA samples from 125 endometrial cancer cases and 200 controls using polymerase chain reaction-based restriction fragment length polymorphism. The association of each genetic polymorphism with endometrial cancer was examined by the odds ratio and 95% confidence interval, which were obtained using logistic regression analysis. The SNP309 GG genotype non-significantly increased the risk of endometrial cancer. The 95% confidence interval for the GG genotype vs. the TT genotype of MDM2 SNP309 was 1.76 (0.93–3.30). Endometrial cancer was not associated with tested SNP genotypes for TP53, ESR1 and p21. The combination of SNP309 GG + TG and TP53 codon 72 Arg/Arg significantly increased endometrial cancer risk. The adjusted OR was 2.53 (95% confidence interval, 1.03–6.21) and P for the interaction was 0.04. This result was supported by in vitro data showing that endometrial cancer cell lines with the SNP309 G allele failed to show growth inhibition by treatment with RITA, which reduces p53-MDM2 binding. The presence of the SNP309 G allele and TP53 codon 72 Arg/Arg genotype is associated with an increased risk of endometrial cancer in Japanese women.
doi:10.3892/or.2013.2433
PMCID: PMC3729233  PMID: 23624782
MDM2 SNP309; TP53 Arg72Pro; ESR1 PvuII XbaI; p21 codon 31; endometrial cancer
2.  Oxidative Stress Produced by Xanthine Oxidase Induces Apoptosis in Human Extravillous Trophoblast Cells 
Abstract
Oxidative stress has been recognized as an important factor in the pathophysiology of preeclampsia. It has been reported that the expression of xanthine oxidase (XO) in the cytotrophoblast and plasma hydrogen peroxide (H2O2) level are significantly higher in preeclamptics than in control women. The aim of this study was to clarify the biological influence of reactive oxygen species (ROS) produced by XO on extravillous trophoblast (EVT) cells. TCL1 cells, a human immortalized EVT cell line, were incubated with xanthine and XO (X/XO). We then measured the cell number, urate level of the culture media and the apoptotic cell ratio. Similar experiments were performed with additional administration of allopurinol, catalase, L-NAME or D-NAME, and with administration of H2O2 in substitution for X/XO. We assessed the effects of H2O2 on invasion ability, tube-like formation and protein expression of HIF1A and ITGAV of TCL1. Finally, the apoptotic cell ratio using primary cultured trophoblasts was measured following exposure to H2O2. X/XO decreased the relative cell number and increased the urate level and apoptotic cell ratio significantly. Elevation of the urate level and apoptotic cell ratio was attenuated by allopurinol and catalase, respectively. L-NAME and D-NAME had no influence on these effects. H2O2 also decreased the relative cell number. Pretreatment with H2O2 significantly inhibited the invasion ability, tube-like formation and HIF1A and ITGAV of TCL1. H2O2 also induced apoptosis in primary cultured trophoblasts. In conclusion, ROS produced by XO induced apoptosis and affected EVT function including invasion and differentiation.
doi:10.1262/jrd.2012-053
PMCID: PMC3943235  PMID: 22986926
Apoptosis; Extravillous trophoblast; Oxidative stress; Xanthine oxidase
3.  The Maternally Expressed Gene Tssc3 Regulates the Expression of MASH2 Transcription Factor in Mouse Trophoblast Stem Cells through the AKT-Sp1 Signaling Pathway* 
The Journal of Biological Chemistry  2012;287(51):42685-42694.
Background: Tssc3 is a maternally expressed imprinted gene.
Results: TSSC3 regulates Mash2 transcription in TS cells through phosphorylation of AKT and Sp1 translocation from cytoplasm to nucleus.
Conclusion: TSSC3 determines the fate of TS cells in terms of development into trophoblast progenitors and/or labyrinth trophoblasts.
Significance: TSSC3 regulates TS cell differentiation through the AKT/Sp1/MASH2 signaling pathway.
Tssc3 is a maternally expressed/paternally silenced imprinted gene. Recent evidence suggests that the loss of TSSC3 results in placental overgrowth in mice. These findings showed that the TSSC3 gene functions as a negative regulator of placental growth. In this study, we describe the function of TSSC3 and its signaling pathway in mouse trophoblast stem (TS) cell differentiation. First of all, we tested Tssc3 expression levels in TS cells. TS cells expressed Tssc3, and its expression level was the highest from day 1 to 4 but was down-regulated at day 5 after the induction of differentiation. Overexpression of TSSC3 in TS cells up-regulated Gcm1 and Mash2, which are marker genes of mouse trophoblast differentiation. Down-regulation of TSSC3 by siRNA enhanced Pl1 and Tpbpa expression in TS cells cultured under stem cell conditions, suggesting the contribution of TSSC3 to the differentiation from TS to trophoblast progenitors and/or labyrinth trophoblasts. TSSC3 activated the PI3K/AKT pathway through binding with phosphatidylinositol phosphate lipids and enhanced the activity of a promoter containing an E-box structure, which is the binding sequence of the Mash2 downstream target gene promoter. PI3K inhibitor suppressed the promoter activity induced by TSSC3. TSSC3 induced Sp1 translocation from cytoplasm to nucleus through the PI3K/AKT pathway. Nuclear Sp1 activated the Mash2 transcription by Sp1 binding with a consensus Sp1-binding motif. This is the first report describing that TSSC3 plays an important role in the differentiation from TS to trophoblast progenitors and/or labyrinth trophoblasts through the TSSC3/PI3K/AKT/MASH2 signaling pathway.
doi:10.1074/jbc.M112.388777
PMCID: PMC3522269  PMID: 23071113
Molecular Cell Biology; Placenta; Stem Cells; Transcription Factors; Trophoblast
4.  FGF4-DEPENDENT STEM CELLS DERIVED FROM RAT BLASTOCYSTS DIFFERENTIATE ALONG THE TROPHOBLAST LINEAGE 
Developmental biology  2011;351(1):110-119.
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.
doi:10.1016/j.ydbio.2010.12.038
PMCID: PMC3039089  PMID: 21215265
trophoblast stem cells; trophoblast differentiation; placenta; rat
5.  Successful Management of Pregnancy Complicated by Klippel-Trenaunay Syndrome Using MR Angiography-Based Evaluation 
Klippel-Trenaunay syndrome (KTS) is a rare congenital disease, and extensive cutaneous hemangiomas and abnormal venous vessels are characteristic. In our case, to manage her pregnancy with KTS, whole-body MRA was performed before delivery. A 29-year-old woman was referred at 28 weeks because of prominent vulvovaginal varicosities due to KTS. At 35 weeks, hypertrophy and multiple venous varicosities of her leg as well as massive vulvovaginal varicosities became prominent with a normal coagulation profile. Systematic MRAs revealed hemangiomas and varicosities in the right leg, the lower abdomen, and the pubic region, while no obvious AVM was detected around the bronchial tube and spine. We decided to deliver her baby by cesarean section at 37 weeks under general anesthesia, and a healthy baby was delivered. No blood transfusion was required. Prophylaxis against thrombosis was performed after the operation. She was discharged with her baby. Her vulvovaginal varicosities shrunk considerably one month later.
doi:10.1155/2011/723467
PMCID: PMC3335629  PMID: 22567516
6.  Isolation and Characterization of Human Trophoblast Side-Population (SP) Cells in Primary Villous Cytotrophoblasts and HTR-8/SVneo Cell Line 
PLoS ONE  2011;6(7):e21990.
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.
doi:10.1371/journal.pone.0021990
PMCID: PMC3131303  PMID: 21760941
7.  Prognosis and long-term neurodevelopmental outcome in conservatively treated twin-to-twin transfusion syndrome 
Background
Amnioreduction remains a treatment option for pregnancies with twin-to-twin transfusion syndrome (TTTS) not meeting criteria for laser surgery or those in which it is not feasible. Amnioreduction is a relatively simple treatment which does not require sophisticated technical equipment. Previous reports of conservative management have indicated that major neurodevelopmental impairment occurs in 14.3-26% of survivors. The purpose of this study was to investigate long-term neurodevelopmental outcome in conservatively treated TTTS.
Methods
During the nine-year study period from January 1996 to December 2004, all pregnancies with TTTS who were admitted to our center were investigated. TTTS was diagnosed by using standard prenatal ultrasound criteria, and staged according to the criteria of Quintero et al. We reviewed gestational age at diagnosis, gestational age at delivery, the stage of TTTS at diagnosis, and diagnosis to delivery interval. Neonatal cranial ultrasound findings were reviewed and the neurodevelopmental outcomes were evaluated.
Results
Twenty-one pregnancies with TTTS were included. Thirteen pregnancies (62%) were treated with serial amnioreduction. The mean gestational age at delivery was 28 weeks (22 - 34 weeks). The perinatal mortality rate was 42.9%. Twenty survivors were followed up until at least 3 years of age. The mean age at follow-up was 6.3 years (3 - 12 years). Six children (30%) had neurodevelopmental impairment. Four children (20%) had major neurodevelopmental impairment and two children (10%) had minor neurodevelopmental impairment. Children with neurodevelopmental impairment were delivered before 29 weeks of gestation.
Conclusions
Our study showed a high rate of perinatal mortality and a high rate of major neurodevelopmental impairment in conservatively treated TTTS. The long-term outcomes for the survivors with TTTS were good when survivors were delivered after 29 weeks of gestation.
doi:10.1186/1471-2393-11-32
PMCID: PMC3125386  PMID: 21510908
fetus; TTTS; long-term outcome
8.  Abnormal fetal movements, micrognathia and pulmonary hypoplasia: a case report. Abnormal fetal movements 
Background
Micrognathia is a facial malformation characterized by mandibular hypoplasia and a small, receding chin that fails to maintain the tongue in a forward position. We previously reported a system of prenatal screening that we developed to identify fetuses with compromised central nervous system function by observing fetal behavior. In this paper we report the case of a preterm infant with micrognathia and pulmonary hypoplasia who presented abnormal fetal movements.
Case presentation
A 27-year-old Japanese primigravida at 33 weeks of gestation was referred to our hospital. Ultrasonographic examination revealed clinical polyhydramnios. Micrognathia was evident on midsagittal and 3 D scan. The lung area was less than the mean -2.0 standard deviations for the gestational age. The infant had mandibular hypoplasia and glossoptosis. After emergency cesarean delivery for non-reasuring fetal status, required immediate tracheostomy and cardiopulmonary resuscitation with mechanical ventilatory support. However, the infant's cardiopulmonary condition did not improve and she died 21 hours after birth.
Conclusions
The findings of our ultrasound exam are suggestive of brain dysfunction. The observation of fetal behavior appears to be effective for the prediction of prognosis of cases with micrognathia.
doi:10.1186/1471-2393-10-46
PMCID: PMC2931455  PMID: 20716376
9.  Evaluation of Haplotype Inference Using Definitive Haplotype Data Obtained from Complete Hydatidiform Moles, and Its Significance for the Analyses of Positively Selected Regions 
PLoS Genetics  2009;5(5):e1000468.
The haplotype map constructed by the HapMap Project is a valuable resource in the genetic studies of disease genes, population structure, and evolution. In the Project, Caucasian and African haplotypes are fairly accurately inferred, based mainly on the rules of Mendelian inheritance using the genotypes of trios. However, the Asian haplotypes are inferred from the genotypes of unrelated individuals based on population genetics, and are less accurate. Thus, the effects of this inaccuracy on downstream analyses needs to be assessed. We determined true Japanese haplotypes by genotyping 100 complete hydatidiform moles (CHM), each carrying a genome derived from a single sperm, using Affymetrix 500 K Arrays. We then assessed how inferred haplotypes can differ from true haplotypes, by phasing pseudo-individualized true haplotypes using the programs PHASE, fastPHASE, and Beagle. We found that, at various genomic regions, especially the MHC locus, the expansion of extended haplotype homozygosity (EHH), which is a measure of positive selection, is obscured when inferred Asian haplotype data is used to detect the expansion. We then mapped the genome using a new statistic, XDiHH, which directly detects the difference between the true and inferred haplotypes, in the determination of EHH expansion. We also show that the true haplotype data presented here is useful to assess and improve the accuracy of phasing of Asian genotypes.
Author Summary
Precise haplotype maps are preferred for the performance of a variety of genetic studies including identification of disease-associated loci and dissection of evolutionary mechanisms such as selection and recombination. For diploid organisms, the haplotype information appears as the genotypes when we obtain the information using widely used high-throughput techniques. The process of extracting haplotype information from genotypes is called phasing, which can be accurately done if the genotypes are from related individuals, such as parent–child trios, by considering the constraints imposed by the rules of Mendelian inheritance. For the genotype data without family information, phasing is done by one of the methods that are based on haplotype clustering, and the inferred haplotypes are known to be less accurate. Here, we experimentally determined genome-wide definitive haplotypes using a collection of Japanese complete hydatidiform moles (CHM), each of which carries a genome derived from a single sperm. Using these resources, we asked if the definitive haplotype data can detect long-distance information that has been obscured when we rely solely on the haplotypes inferred by clustering. We also show that by introducing definitive haplotypes as references, inference of haplotypes of unrelated individuals is significantly improved.
doi:10.1371/journal.pgen.1000468
PMCID: PMC2670534  PMID: 19424418
10.  Long-Term Effects of Polychlorinated Biphenyls and Dioxins on Pregnancy Outcomes in Women Affected by the Yusho Incident 
Environmental Health Perspectives  2008;116(5):626-630.
Background
Maternal exposure to polychlorinated biphenyls (PCBs) is associated with increased proportions of spontaneous abortion and stillbirth in animal studies. In Japan in 1968, accidental human exposure to rice oil contaminated with PCBs and other dioxin-related compounds, such as polychlorinated dibenzofurans (PCDFs), led to the development of what was later referred to as Yusho oil disease.
Objective
The aim of this study was to investigate the association of maternal PCB and dioxin exposure with adverse pregnancy outcomes in Yusho women.
Methods
In 2004, we interviewed 214 Yusho women (512 pregnancies) about their pregnancy outcomes over the past 36 years. Pregnancy outcomes included induced abortion, spontaneous abortion, preterm delivery, and pregnancy loss.
Results
In pregnancy years 1968–1977 (within the first 10 years after exposure), the proportions of induced abortion [odds ratio adjusted for age at delivery (ORadj) = 5.93; 95% confidence interval (CI), 2.21–15.91; two-tailed p < 0.001) and preterm delivery (ORadj = 5.70; 95% CI, 1.17–27.79; p = 0.03) were significantly increased compared with the proportions in pregnancy years 1958–1967 (10 years before the incident). Spontaneous abortion (ORadj = 2.09; 95% CI, 0.84–5.18), and pregnancy loss (ORadj = 2.11; 95% CI, 0.92–4.87) were more frequent (OR = 2.18; 95% CI, 1.02–4.66), but these were not significant (p = 0.11 and p = 0.08, respectively) in pregnancy years 1968–1977. We found no significant increases in the proportions of these adverse pregnancy outcomes in pregnancies occurring during 1978–1987 or 1988–2003 compared with those in pregnancies before 1968.
Conclusion
High levels of PCB/PCDF exposure had some adverse effects on pregnancy outcome in Yusho women.
doi:10.1289/ehp.10686
PMCID: PMC2367658  PMID: 18470296
dioxin; environmental exposure; pregnancy loss; preterm delivery; spontaneous abortion; Yusho
11.  ZAC, LIT1 (KCNQ1OT1) and p57KIP2 (CDKN1C) are in an imprinted gene network that may play a role in Beckwith–Wiedemann syndrome 
Nucleic Acids Research  2005;33(8):2650-2660.
Loss of genomic imprinting is involved in a number of developmental abnormalities and cancers. ZAC is an imprinted gene expressed from the paternal allele of chromosome 6q24 within a region known to harbor a tumor suppressor gene for several types of neoplasia. p57KIP2 (CDKN1C) is a maternally expressed gene located on chromosome 11p15.5 which encodes a cyclin-dependent kinase inhibitor that may also act as a tumor suppressor gene. Mutations in ZAC and p57KIP2 have been implicated in transient neonatal diabetes mellitus (TNDB) and Beckwith–Wiedemann syndrome, respectively. Patients with these diseases share many characteristics. Here we show that mouse Zac1 and p57Kip2 have a strikingly similar expression pattern. ZAC, a sequence-specific DNA-binding protein, binds within the CpG island of LIT1 (KCNQ1OT1), a paternally expressed, anti-sense RNA thought to negatively regulate p57KIP2 in cis. ZAC induces LIT1 transcription in a methylation-dependent manner. Our data suggest that ZAC may regulate p57KIP2 through LIT1, forming part of a novel signaling pathway regulating cell growth. Mutations in ZAC may, therefore, contribute to Beckwith–Wiedemann syndrome. Furthermore, we find changes in DNA methylation at the LIT1 putative imprinting control region in two patients with TNDB.
doi:10.1093/nar/gki555
PMCID: PMC1097765  PMID: 15888726
12.  Identification and characterization of two forms of mouse MUTYH proteins encoded by alternatively spliced transcripts 
Nucleic Acids Research  2004;32(2):477-487.
There are three types of mouse Mutyh mRNAs (type a, b and c) generated by alternative splicing, and type b mRNA is a major form among the three in most of the tissues examined. The level of type c mRNA is relatively high in brain. Type a and b mRNAs were expected to encode 57.7 kDa protein (MUTYHα), while type c mRNA had a partly different open reading frame encoding a 50.2 kDa protein (MUTYHβ). An in vitro translation of type b and c mRNAs produced a 50 kDa MUTYHα and 47 kDa MUTYHβ, respectively. MUTYHα and MUTYHβ were detected in wild-type embryonic stem (ES) cells or thymocytes prepared from wild-type mice, but neither MUTYH-null ES cells nor thymocytes prepared from MUTYH-null mice. Both MUTYHα and MUTYHβ were mainly localized in the nuclei and some in mitochondria in wild-type ES cells. Recombinant MUTYHα and β were expressed as fusion proteins with thioredoxin in Escherichia coli, but only MUTYHα was partly soluble and thus could be purified. Recombinant MUTYHα possessed DNA glycosylase activities to excise adenine opposite 8-oxoguanine and guanine but not AP lyase activity.
doi:10.1093/nar/gkh214
PMCID: PMC373338  PMID: 14742662

Results 1-12 (12)