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1.
J Obstet Gynaecol Res ; 47(11): 3813-3820, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34490692

ABSTRACT

AIM: This study aimed to evaluate changes in prenatal testing among women with twin pregnancies before and after the introduction of noninvasive prenatal testing (NIPT). To date, no consensus on prenatal testing for twin pregnancies has been reached in Japan. METHODS: Women pregnant with twins who requested prenatal testing at Kyushu Medical Center from 2005 to 2018 were included in this study. Genetic counseling was provided to all participants. Their chosen methods of testing were collected and classified as invasive diagnosis (ID), noninvasive screening (NIS), and no test requested (NR). Parity, chorionicity, and methods of conception were assessed as attributes. The study period was divided into three terms according to testing availability in our center. RESULTS: After NIPT was introduced in our center, the use of ID methods decreased and eventually disappeared while NIS came to the forefront. NR was also the preferred choice of women with twin pregnancies before the introduction of NIPT and decreased but did not disappear after introducing NIPT. Women with twin pregnancies who underwent assisted reproduction initially showed hesitation to undergo testing but showed a strong preference for NIS after the introduction of NIPT. Differences in choice according to parity, chorionicity, and methods of conception were found before the introduction of NIPT but disappeared after introducing NIPT. CONCLUSION: Increasing information about NIPT has apparently influenced the attitudes of women with twin pregnancies to prenatal testing in Japan. In particular, those who conceive through assisted reproductive technologies exhibited a strong preference for NIPT.


Subject(s)
Genetic Counseling , Pregnancy, Twin , Aneuploidy , Attitude , Chorion , Female , Genetic Testing , Humans , Japan , Pregnancy , Prenatal Diagnosis
2.
Gynecol Minim Invasive Ther ; 8(2): 76-79, 2019.
Article in English | MEDLINE | ID: mdl-31143628

ABSTRACT

We present two cases of congenital vaginal agenesis with functional uterine corpus, manifesting with periodic lower abdominal pain and hematometra in adolescence. Both patients were successfully treated with the creation of neovagina and neocanal structures to discharge menstrual blood; this may also facilitate the preservation of fertility. Both cases were characterized by degrees of congenital vaginal agenesis, whether short or completely absent, with no communication between the uterine cavity and external genitalia, as confirmed by physical examination and imaging. We surgically reconstructed a neovagina with the modified McIndoe's procedure, using an artificial skin graft, and canalized to the caudal portion of the uterine cavity. Although redilatation of the neocanal was required, no patient suffered severe infection in postoperative course and both now exhibit regular menstruation. Although hysterectomy has classically been the preferred treatment for such cases, recent technical progression enables treatment of such diseases with conservative and minimally invasive surgery, in a safe manner.

3.
Cancer Sci ; 107(10): 1399-1405, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27486687

ABSTRACT

FBXW7 is a ubiquitin ligase that mediates ubiquitylation of oncoproteins, such as c-Myc, cyclin E, Notch and c-Jun. FBXW7 is a known tumor-suppressor gene, and mutations in FBXW7 have been reported in various human malignancies. In this study, we examined the sequences of the FBXW7 and p53 genes in 57 ovarian cancer clinical samples. Interestingly, we found no FBXW7 mutations associated with amino acid changes. We also investigated FBXW7 expression levels in 126 epithelial ovarian tumors. FBXW7 expression was negatively correlated with the malignant potential of ovarian tumors. That is to say, FBXW7 expression levels in ovarian cancer samples were significantly lower than those in borderline and benign tumors (P < 0.01). FBXW7 expression levels in serous carcinoma samples were the lowest among four major histological subtypes. In addition, p53-mutated ovarian cancer samples showed significantly lower levels of FBXW7 expression compared with p53 wild-type cancer samples (P < 0.001). DNA methylation arrays and bisulfite PCR sequencing experiments revealed that 5'-upstream regions of FBXW7 gene in p53-mutated samples were significantly higher methylated compared with those in p53 wild-type samples (P < 0.01). This data indicates that p53 mutations might suppress FBXW7 expression through DNA hypermethylation of FBXW7 5'-upstream regions. Thus, FBXW7 expression was downregulated in ovarian cancers, and was associated with p53 mutations and the DNA methylation status of the 5'-upstream regions of FBXW7.


Subject(s)
Cell Cycle Proteins/genetics , F-Box Proteins/genetics , Neoplasms, Glandular and Epithelial/genetics , Neoplasms, Glandular and Epithelial/pathology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Phenotype , Ubiquitin-Protein Ligases/genetics , Adult , Aged , Carcinoma, Ovarian Epithelial , Cell Cycle Proteins/metabolism , Cell Line, Tumor , DNA Methylation , F-Box Proteins/metabolism , F-Box-WD Repeat-Containing Protein 7 , Female , Gene Expression Regulation, Neoplastic , Humans , Middle Aged , Mutation , Promoter Regions, Genetic , Tumor Suppressor Protein p53/genetics , Ubiquitin-Protein Ligases/metabolism , Young Adult
4.
BMC Med Genet ; 16: 67, 2015 Aug 21.
Article in English | MEDLINE | ID: mdl-26293665

ABSTRACT

BACKGROUND: The functional single nucleotide polymorphism (SNP) in the MDM2 promoter region, SNP309, is known to be associated with various diseases, particularly cancer. Although many studies have been performed to demonstrate the mechanism of allele-specific expression (ASE) on SNP309, they have only utilized in vitro techniques. It is unknown whether ASE of MDM2 is ascribed solely to SNP309, in vivo. METHODS: We attempted to evaluate ASE of MDM2 in vivo using post-labeling followed by automated capillary electrophoresis under single-strand conformation polymorphism conditions. For measuring a quantitative difference, we utilized the SNPs on the exons of MDM2 as markers, the status of which was heterozygous in a large population. To address the cause of ASE beyond 20%, we confirmed sequences of both MDM2-3'UTR and promoter regions. We assessed the SNP which might be the cause of ASE using biomolecular interaction analysis and luciferase assay. RESULTS: ASE beyond 20% was detected in endometrial cancers, but not in cancer-free endometria samples only when an SNP rs1690916 was used as a marker. We suspected that this ASE in endometrial cancer was caused by the sequence heterogeneity in the MDM2-P2 promoter, and found a new functional polymorphism, which we labelled SNP55. There was no difference between cancer-free endometria and endometrial cancer samples neither for SNP55 genotype frequencies nor allele frequencies, and so, SNP55 alone does not affect endometrial cancer risk. The SNP55 status affected the DNA binding affinity of transcription factor Sp1 and nuclear factor kappa-B (NFκB). Transcriptional activity of the P2 promoter containing SNP55C was suppressed by NFκB p50 homodimers, but that of SNP55T was not. Only ASE-positive endometrial cancer samples displayed nuclear localization of NFκB p50. CONCLUSIONS: Our findings suggest that both the SNP55 status and the NFκB p50 activity are important in the transcriptional regulation of MDM2 in endometrial cancers.


Subject(s)
Endometrial Neoplasms/genetics , Polymorphism, Single Nucleotide/genetics , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-mdm2/genetics , Binding Sites/genetics , Blotting, Western , DNA Primers/genetics , Endometrial Neoplasms/metabolism , Female , Gene Frequency , Genotyping Techniques , Humans , Immunohistochemistry , Luciferases , NF-kappa B p50 Subunit/metabolism , Plasmids/genetics
5.
Gynecol Obstet Invest ; 76(3): 188-92, 2013.
Article in English | MEDLINE | ID: mdl-23969319

ABSTRACT

Abdominal pregnancy is a rare condition that accounts for only 1% of all ectopic pregnancies but results in high maternal morbidity and mortality. We present a case of abdominal pregnancy with massive peritoneal bleeding successfully treated using systemic methotrexate (MTX). A 34-year-old woman with amenorrhea for 8 weeks and a positive pregnancy test was referred for evaluation of ectopic pregnancy. Transvaginal ultrasonographic scan showed a gestational sac measuring 25 mm in diameter containing a viable embryo in the cul-de-sac and a considerable amount of free fluid in the patient's lower abdomen and pelvis. Laboratory parameters showed that her hemoglobin concentration was 5.8 g/dl and serum human chorionic gonadotropin concentration was 13,195 mIU/ml. Emergency surgery revealed an abdominal pregnancy in the cul-de-sac and a massive intra-abdominal hemorrhage. After a hemostasis procedure, the patient was successfully treated using systemic MTX. We also present the review of abdominal pregnancy cases treated using systemic MTX at our institution over 10 years. Systemic MTX treatment for abdominal pregnancy is safe and effective and makes it possible to avoid the risk of excessive bleeding by surgical resection of the implantation site.


Subject(s)
Abortifacient Agents, Nonsteroidal/therapeutic use , Hemoperitoneum/drug therapy , Hemoperitoneum/surgery , Laparotomy/methods , Methotrexate/therapeutic use , Pregnancy, Abdominal/drug therapy , Pregnancy, Abdominal/surgery , Adult , Chorionic Gonadotropin/blood , Female , Hemoperitoneum/etiology , Humans , Pregnancy , Pregnancy, Abdominal/blood , Pregnancy, Abdominal/pathology
6.
Cell ; 152(5): 1106-18, 2013 Feb 28.
Article in English | MEDLINE | ID: mdl-23452856

ABSTRACT

In the mammalian circadian clockwork, CRY1 and CRY2 repressor proteins are regulated by posttranslational modifications for temporally coordinated transcription of clock genes. Previous studies revealed that FBXL3, an F-box-type E3 ligase, ubiquitinates CRYs and mediates their degradation. Here, we found that FBXL21 also ubiquitinates CRYs but counteracts FBXL3. Fbxl21(-/-) mice exhibited normal periodicity of wheel-running rhythms with compromised organization of daily activities, while an extremely long-period phenotype of Fbxl3(-/-) mice was attenuated in Fbxl3/Fbxl21 double-knockout mice. The double knockout destabilized the behavioral rhythms progressively and sometimes elicited arrhythmicity. Surprisingly, FBXL21 stabilized CRYs and antagonized the destabilizing action by FBXL3. Predominantly cytosolic distribution of FBXL21 contrasts with nuclear localization of FBXL3. These results emphasize the physiological importance of antagonizing actions between FBXL21 and FBXL3 on CRYs, and their combined actions at different subcellular locations stabilize oscillation of the circadian clock.


Subject(s)
Circadian Clocks , Cryptochromes/metabolism , F-Box Proteins/metabolism , Amino Acid Sequence , Animals , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , F-Box Proteins/genetics , Fibroblasts , Mice , Mice, Knockout , Molecular Sequence Data , Multiprotein Complexes , Sequence Alignment , Ubiquitination
7.
J Biol Chem ; 287(51): 42685-94, 2012 Dec 14.
Article in English | MEDLINE | ID: mdl-23071113

ABSTRACT

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.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Gene Expression Regulation, Developmental , Nuclear Proteins/genetics , Signal Transduction/genetics , Stem Cells/cytology , Stem Cells/metabolism , Trophoblasts/cytology , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/genetics , Cell Line , Female , Gene Expression Profiling , Mice , Mice, Inbred ICR , Nuclear Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Pregnancy , Proto-Oncogene Proteins c-akt/metabolism , Sp1 Transcription Factor/metabolism , Stem Cells/enzymology , Trophoblasts/metabolism , Up-Regulation/genetics
8.
Mol Cell Biol ; 32(3): 590-605, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22124152

ABSTRACT

D-type cyclins play a pivotal role in G(1)-S progression of the cell cycle, and their expression is frequently deregulated in cancer. Cyclin D1 has a half-life of only ~30 min as a result of its ubiquitylation and proteasomal degradation, with various F-box proteins, including Fbxo4, Fbxw8, Skp2, and Fbxo31, having been found to contribute to its ubiquitylation. We have now generated Fbxo4-deficient mice and found no abnormalities in these animals. Cyclin D1 accumulation was thus not observed in Fbxo4(-/-) mouse tissues. The half-life of cyclin D1 in mouse embryonic fibroblasts (MEFs) prepared from Fbxo4(-/-), Fbxw8(-/-), and Fbxo4(-/-); Fbxw8(-/-) mice also did not differ from that in wild-type MEFs. Additional depletion of Skp2 and Fbxo31 in Fbxo4(-/-); Fbxw8(-/-) MEFs by RNA interference did not affect cyclin D1 stability. Although Fbxo31 depletion in MEFs increased cyclin D1 abundance, this effect appeared attributable to upregulation of cyclin D1 mRNA. Furthermore, abrogation of the function of the Skp1-Cul1-F-box protein (SCF) complex or the anaphase-promoting complex/cyclosome (APC/C) complexes did not alter the half-life of cyclin D1, whereas cyclin D1 degradation was dependent largely on proteasome activity. Our genetic analyses thus do not support a role for any of the four F-box proteins examined in cyclin D1 degradation during normal cell cycle progression. They suggest the existence of other ubiquitin ligases that target cyclin D1 for proteolysis.


Subject(s)
Cyclin D1/metabolism , F-Box Proteins/genetics , Anaphase-Promoting Complex-Cyclosome , Animals , Cell Cycle , Cullin Proteins/metabolism , Female , Fibroblasts/metabolism , Male , Mice , Mice, Inbred C57BL , Proteasome Endopeptidase Complex/metabolism , Protein Stability , Proteolysis , RNA Interference , S-Phase Kinase-Associated Proteins/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism
9.
PLoS One ; 6(7): e21990, 2011.
Article in English | MEDLINE | ID: mdl-21760941

ABSTRACT

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.


Subject(s)
Cell Separation/methods , Side-Population Cells/cytology , Trophoblasts/cytology , Animals , Biomarkers/metabolism , Cell Line , Cell Proliferation/drug effects , Culture Media, Conditioned/pharmacology , Down-Regulation/drug effects , Embryo, Mammalian/cytology , Female , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Mice , Pregnancy , Pregnancy Trimester, First/drug effects , Receptors, Interleukin-1/metabolism , Receptors, Interleukin-7/metabolism , Side-Population Cells/drug effects , Side-Population Cells/metabolism , Trophoblasts/drug effects , Trophoblasts/metabolism , Up-Regulation/drug effects
10.
Fetal Diagn Ther ; 29(3): 248-52, 2011.
Article in English | MEDLINE | ID: mdl-21160169

ABSTRACT

An abdominal aortic aneurysm is a rare disease in the paediatric population and is mainly caused by intrauterine infection, connective tissue diseases, such as Ehlers-Danlos syndrome and Marfan's syndrome, and iatrogenic trauma due to umbilical artery catheterization. Although several cases have been reported in the English literature, they were rarely diagnosed prenatally. Vascular obstruction in utero is also believed to be the major cause of porencephaly. Recently, gene mutations have been reported as the cause of both the above-mentioned diseases. We present a prenatally diagnosed case of congenital abdominal aortic aneurysm with porencephaly.


Subject(s)
Aortic Aneurysm, Abdominal/diagnosis , Cerebrum/abnormalities , Fetal Diseases/diagnostic imaging , Adult , Aortic Aneurysm, Abdominal/complications , Aortic Aneurysm, Abdominal/congenital , Cerebrum/diagnostic imaging , Cerebrum/pathology , Female , Fetal Diseases/pathology , Humans , Male , Pregnancy , Radiography , Ultrasonography
12.
Lab Invest ; 89(6): 645-56, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19333234

ABSTRACT

SYT-SSX protein, resulted from chromosomal translocation, causes synovial sarcoma, which is a malignant tumor accounting for 10% of soft tissue sarcoma. However, biological functions of SYT (synovial sarcoma translocation), also known as SS18, are largely unclear, whereas it has been proven that Syt-null mice die at early stages of embryonic development. Here, we generated Syt-deficient mice and confirmed the reported phenotypes, including growth retardation, open neural tube and haplo-insufficient lethality, and therefore, there is no doubt that Syt is essential for embryonic development. However, placental defects, described in the earlier report, were rarely seen in our mice and we frequently observed cardiac defect in Syt-deficient mice. As the mechanisms responsible for embryonic lethality seem to be complicate, we performed additional experiments. By using primary cultured embryonic fibroblasts, we showed that Syt(-/-) MEFs deregulate actin organization and suppressed cell migration. These observations suggest that Syt may contribute to the signaling pathway important for various cellular functions in vivo and in vitro, and we propose that Syt-deficient MEFs would be a powerful means to understand the biological roles of SYT in vitro.


Subject(s)
Cell Movement , Proto-Oncogene Proteins/physiology , Repressor Proteins/physiology , Actins/metabolism , Animals , Animals, Newborn , Cells, Cultured , Embryo Loss , Embryo, Mammalian/cytology , Embryonic Development , Female , Fibroblasts/physiology , Heart Defects, Congenital/genetics , Humans , Mice , Mice, Knockout , Neural Tube/abnormalities , Placenta/abnormalities , Placenta/metabolism , Pregnancy , Proto-Oncogene Mas , Proto-Oncogene Proteins/genetics , Repressor Proteins/genetics , Signal Transduction
13.
J Exp Med ; 204(12): 2875-88, 2007 Nov 26.
Article in English | MEDLINE | ID: mdl-17984302

ABSTRACT

Cell proliferation is strictly controlled during differentiation. In T cell development, the cell cycle is normally arrested at the CD4(+)CD8(+) stage, but the mechanism underlying such differentiation-specific exit from the cell cycle has been unclear. Fbxw7 (also known as Fbw7, Sel-10, hCdc4, or hAgo), an F-box protein subunit of an SCF-type ubiquitin ligase complex, induces the degradation of positive regulators of the cell cycle, such as c-Myc, c-Jun, cyclin E, and Notch. FBXW7 is often mutated in a subset of human cancers. We have now achieved conditional inactivation of Fbxw7 in the T cell lineage of mice and found that the cell cycle is not arrested at the CD4(+)CD8(+) stage in the homozygous mutant animals. The mutant mice manifested thymic hyperplasia as a result of c-Myc accumulation and eventually developed thymic lymphoma. In contrast, mature T cells of the mutant mice failed to proliferate in response to mitogenic stimulation and underwent apoptosis in association with accumulation of c-Myc and p53. These latter abnormalities were corrected by deletion of p53. Our results suggest that Fbxw7 regulates the cell cycle in a differentiation-dependent manner, with its loss resulting in c-Myc accumulation that leads to hyperproliferation in immature T cells but to p53-dependent cell-cycle arrest and apoptosis in mature T cells.


Subject(s)
Cell Cycle , Cell Differentiation , F-Box Proteins/genetics , F-Box Proteins/metabolism , Lymphoma/pathology , Mutation/genetics , T-Lymphocytes/cytology , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Animals , Antigens/pharmacology , Cell Cycle/drug effects , Cell Differentiation/drug effects , Cell Lineage/drug effects , Cell Proliferation/drug effects , Disease Susceptibility , F-Box-WD Repeat-Containing Protein 7 , Gene Expression Regulation/drug effects , Integrases/metabolism , Mice , Mice, Nude , Phenotype , Proto-Oncogene Proteins c-myc/metabolism , Receptors, Notch/metabolism , Spleen/cytology , Spleen/drug effects , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Thymus Gland/drug effects , Thymus Gland/pathology , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/deficiency
14.
Mol Cell Biol ; 26(16): 6157-69, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16880526

ABSTRACT

Cullin-based ubiquitin ligases (E3s) constitute one of the largest E3 families. Fbxw8 (also known as Fbw6 or Fbx29) is an F-box protein that is assembled with Cul7 in an SCF-like E3 complex. Here we show that Cul7 forms a heterodimeric complex with Cul1 in a manner dependent on Fbxw8. We generated mice deficient in Fbxw8 and found that Cul7 did not associate with Cul1 in cells of these mice. Two-thirds of Fbxw8-/- embryos die in utero, whereas the remaining one-third are born alive and grow to adulthood. Fbxw8-/- embryos show intrauterine growth retardation and abnormal development of the placenta, characterized by both a reduced thickness of the spongiotrophoblast layer and abnormal vessel structure in the labyrinth layer. Although the placental phenotype of Fbxw8-/- mice resembles that of Cul7-/- mice, other abnormalities of Cul7-/- mice are not apparent in Fbxw8-/- mice. These results suggest that the Cul7-based SCF-like E3 complex has both Fbxw8-dependent and Fbxw8-independent functions.


Subject(s)
Cell Cycle Proteins/metabolism , Cullin Proteins/metabolism , F-Box Proteins/metabolism , Placenta/embryology , Animals , Crosses, Genetic , Embryo, Mammalian/abnormalities , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/pathology , Exons/genetics , Female , Fetal Growth Retardation , Gene Targeting , Genotype , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Genetic , Phenotype , Placenta/abnormalities , Placenta/cytology , Placenta/pathology , Pregnancy , Protein Binding
16.
Cancer Sci ; 97(8): 729-36, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16863506

ABSTRACT

Fbxw7 (also known as Sel-10, hCdc4 or hAgo) is the F-box protein component of a Skp1-Cul1-F-box protein (SCF) ubiquitin ligase. Fbxw7 contributes to the ubiquitin-mediated degradation of cyclin E, c-Myc, Aurora-A, Notch and c-Jun, all of which appear to function as cell-cycle promoters and oncogenic proteins. Loss of Fbxw7 results in elevated expression of its substrates, which may lead to oncogenesis. However, it remains largely unclear which accumulating substrate is most related to cancer development in Fbxw7-mutant cancer cells. In the present study, we examined the abundance of cyclin E, c-Myc and Aurora-A in seven cancer cell lines, which harbor wild-type (three lines) or mutant (four lines) Fbxw7. Although these three substrates accumulated in the Fbxw7-mutant cells, the extent of increase in the expression of these proteins varied in each line. Forced expression of Fbxw7 reduced the levels of cyclin E, c-Myc and Aurora-A in the Fbxw7-mutant cells. In contrast, a decrease in the expression of cyclin E, c-Myc or Aurora-A by RNA interference significantly suppressed the rate of proliferation and anchorage-independent growth of the Fbxw7-mutant cells. These findings thus suggest that the loss of Fbxw7 results in accumulation of cyclin E, c-Myc and Aurora-A, all of which appear to be required for growth promotion of cancer cells. Fbxw7 seems to regulate the levels of multiple targets to suppress cancer development.


Subject(s)
Breast Neoplasms/metabolism , Cell Cycle Proteins/physiology , F-Box Proteins/physiology , Neoplasm Proteins/metabolism , Tumor Suppressor Proteins/physiology , Ubiquitin-Protein Ligases/physiology , Ubiquitin/metabolism , Aurora Kinases , Breast Neoplasms/genetics , Cell Cycle Proteins/genetics , Cell Proliferation , Cyclin E/genetics , Cyclin E/metabolism , F-Box Proteins/genetics , F-Box-WD Repeat-Containing Protein 7 , Humans , Mutation , Neoplasm Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA Interference , Tumor Suppressor Proteins/genetics , Ubiquitin-Protein Ligases/genetics
17.
Nature ; 432(7018): 775-9, 2004 Dec 09.
Article in English | MEDLINE | ID: mdl-15592418

ABSTRACT

The FBXW7/hCDC4 gene encodes a ubiquitin ligase implicated in the control of chromosome stability. Here we identify the mouse Fbxw7 gene as a p53-dependent tumour suppressor gene by using a mammalian genetic screen for p53-dependent genes involved in tumorigenesis. Radiation-induced lymphomas from p53+/- mice, but not those from p53-/- mice, show frequent loss of heterozygosity and a 10% mutation rate of the Fbxw7 gene. Fbxw7+/- mice have greater susceptibility to radiation-induced tumorigenesis, but most tumours retain and express the wild-type allele, indicating that Fbxw7 is a haploinsufficient tumour suppressor gene. Loss of Fbxw7 alters the spectrum of tumours that develop in p53 deficient mice to include a range of tumours in epithelial tissues such as the lung, liver and ovary. Mouse embryo fibroblasts from Fbxw7-deficient mice, or wild-type mouse cells expressing Fbxw7 small interfering RNA, have higher levels of Aurora-A kinase, c-Jun and Notch4, but not of cyclin E. We propose that p53-dependent loss of Fbxw7 leads to genetic instability by mechanisms that might involve the activation of Aurora-A, providing a rationale for the early occurrence of these mutations in human cancers.


Subject(s)
Cell Cycle Proteins/metabolism , F-Box Proteins/metabolism , Genes, Tumor Suppressor/physiology , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Base Sequence , Cell Cycle Proteins/genetics , Cell Transformation, Neoplastic/pathology , Cell Transformation, Neoplastic/radiation effects , F-Box Proteins/genetics , F-Box-WD Repeat-Containing Protein 7 , Female , Fibroblasts , Gene Deletion , Loss of Heterozygosity/genetics , Male , Mice , Mice, Knockout , Mutation/genetics , Neoplasms/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Survival Rate , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics
18.
Mol Cell Biol ; 24(19): 8386-94, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15367660

ABSTRACT

The Wnt signaling pathway plays a pivotal role in vertebrate early development and morphogenesis. Duplin (axis duplication inhibitor) interacts with beta-catenin and prevents its binding to Tcf, thereby inhibiting downstream Wnt signaling. Here we show that Duplin is expressed predominantly from early- to mid-stage mouse embryogenesis, and we describe the generation of mice deficient in Duplin. Duplin(-/-) embryos manifest growth retardation from embryonic day 5.5 (E5.5) and developmental arrest accompanied by massive apoptosis at E7.5. The mutant embryos develop into an egg cylinder but do not form a primitive streak or mesoderm. Expression of beta-catenin target genes, including those for T (brachyury), Axin2, and cyclin D1, was not increased in Duplin(-/-) embryos, suggesting that the developmental defect is not simply attributable to upregulation of Wnt signaling caused by the lack of this inhibitor. These results suggest that Duplin plays an indispensable role, likely by a mechanism independent of inhibition of Wnt signaling, in mouse embryonic growth and differentiation at an early developmental stage.


Subject(s)
Apoptosis/physiology , Carrier Proteins/metabolism , Embryo, Mammalian/metabolism , Nuclear Proteins/metabolism , Animals , Blastocyst/metabolism , Blastocyst/pathology , Embryo, Mammalian/pathology , Gene Targeting , Immunohistochemistry , Mice
19.
EMBO J ; 23(10): 2116-25, 2004 May 19.
Article in English | MEDLINE | ID: mdl-15103331

ABSTRACT

The F-box protein Skp2 mediates c-Myc ubiquitylation by binding to the MB2 domain. However, the turnover of c-Myc is largely dependent on phosphorylation of threonine-58 and serine-62 in MB1, residues that are often mutated in cancer. We now show that the F-box protein Fbw7 interacts with and thereby destabilizes c-Myc in a manner dependent on phosphorylation of MB1. Whereas wild-type Fbw7 promoted c-Myc turnover in cells, an Fbw7 mutant lacking the F-box domain delayed it. Furthermore, depletion of Fbw7 by RNA interference increased both the abundance and transactivation activity of c-Myc. Accumulation of c-Myc was also apparent in mouse Fbw7-/- embryonic stem cells. These observations suggest that two F-box proteins, Fbw7 and Skp2, differentially regulate c-Myc stability by targeting MB1 and MB2, respectively.


Subject(s)
Cell Cycle Proteins/metabolism , F-Box Proteins/metabolism , Ligases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Cycle Proteins/genetics , Cells, Cultured , Cyclin E/genetics , Cyclin E/metabolism , F-Box Proteins/genetics , F-Box-WD Repeat-Containing Protein 7 , Humans , Ligases/genetics , Mice , Mice, Knockout , Peptides/genetics , Peptides/metabolism , Phosphorylation , RNA Interference , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , S-Phase Kinase-Associated Proteins/metabolism , Serine/metabolism , Stem Cells/cytology , Stem Cells/physiology , Threonine/metabolism , Transcriptional Activation , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics
20.
J Biol Chem ; 279(10): 9417-23, 2004 Mar 05.
Article in English | MEDLINE | ID: mdl-14672936

ABSTRACT

Mammalian Fbw7 (also known as Sel-10, hCdc4, or hAgo) is the F-box protein component of an SCF (Skp1-Cul1-F-box protein-Rbx1)-type ubiquitin ligase, and the mouse Fbw7 is expressed prominently in the endothelial cell lineage of embryos. We generated mice deficient in Fbw7 and found that the embryos died in utero at embryonic day 10.5-11.5, manifesting marked abnormalities in vascular development. Vascular remodeling was impaired in the brain and yolk sac, and the major trunk veins were not formed. In vitro para-aortic splanchnopleural explant cultures from Fbw7(-/-) embryos also manifested an impairment of vascular network formation. Notch4, which is the product of the proto-oncogene Int3 and an endothelial cell-specific mammalian isoform of Notch, accumulated in Fbw7(-/-) embryos, resulting in an increased expression of Hey1, which encodes a transcriptional repressor that acts downstream of Notch signaling and is implicated in vascular development. Expression of Notch1, -2, or -3 or of cyclin E was unaffected in Fbw7(-/-) embryos. Mammalian Fbw7 thus appears to play an indispensable role in negative regulation of the Notch4-Hey1 pathway and is required for vascular development.


Subject(s)
Blood Vessels/metabolism , Gene Expression Regulation, Developmental , Proto-Oncogene Proteins/metabolism , Receptors, Cell Surface/metabolism , Animals , Blood Vessels/embryology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , F-Box Proteins/genetics , F-Box Proteins/metabolism , F-Box-WD Repeat-Containing Protein 7 , Female , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins/genetics , Receptor, Notch4 , Receptors, Cell Surface/genetics , Receptors, Notch , Signal Transduction , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
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