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1.
Nat Biomed Eng ; 5(8): 926-940, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34373601

RESUMO

Current protocols for the differentiation of human pluripotent stem cells (hPSCs) into chondrocytes do not allow for the expansion of intermediate progenitors so as to prospectively assess their chondrogenic potential. Here we report a protocol that leverages PRRX1-tdTomato reporter hPSCs for the selective induction of expandable and ontogenetically defined PRRX1+ limb-bud-like mesenchymal cells under defined xeno-free conditions, and the prospective assessment of the cells' chondrogenic potential via the cell-surface markers CD90, CD140B and CD82. The cells, which proliferated stably and exhibited the potential to undergo chondrogenic differentiation, formed hyaline cartilaginous-like tissue commensurate to their PRRX1-expression levels. Moreover, we show that limb-bud-like mesenchymal cells derived from patient-derived induced hPSCs can be used to identify therapeutic candidates for type II collagenopathy and we developed a method to generate uniformly sized hyaline cartilaginous-like particles by plating the cells on culture dishes coated with spots of a zwitterionic polymer. PRRX1+ limb-bud-like mesenchymal cells could facilitate the mass production of chondrocytes and cartilaginous tissues for applications in drug screening and tissue engineering.


Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/transplante , Condrogênese , Doenças do Colágeno/terapia , Meios de Cultura/química , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Pluripotentes/metabolismo , Polímeros/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Antígenos Thy-1/metabolismo , Engenharia Tecidual
2.
Int J Cancer ; 149(8): 1593-1604, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34152598

RESUMO

Lung adenocarcinoma (LUAD) is the most common types among lung cancers generally arising from terminal airway and understanding of multistep carcinogenesis is crucial to develop novel therapeutic strategy for LUAD. Here we used human induced pluripotent stem cells (hiPSCs) to establish iHER2-hiPSCs in which doxycycline induced the expression of the oncoprotein human epidermal growth factor receptor 2 (HER2)/ERBB2. Lung progenitors that differentiated from iHER2-hiPSCs, which expressed NKX2-1/TTF-1 known as a lung lineage maker, were cocultured with human fetal fibroblast and formed human lung organoids (HLOs) comprising alveolar type 2-like cells. HLOs that overexpressed HER2 transformed to tumor-like structures similar to atypical adenomatous hyperplasia, which is known for lung precancerous lesion and upregulated the activities of oncogenic signaling cascades such as RAS/RAF/MAPK and PI3K/AKT/mTOR. The degree of morphological irregularity and proliferation capacity were significantly higher in HLOs from iHER2-hiPSCs. Moreover, the transcriptome profile of the HLOs shifted from a normal lung tissue-like state to one characteristic of clinical LUAD with HER2 amplification. Our results suggest that hiPSC-derived HLOs may serve as a model to recapitulate the early tumorigenesis of LUAD and would provide new insights into the molecular basis of tumor initiation and progression.


Assuntos
Adenocarcinoma de Pulmão/patologia , Carcinogênese , Regulação Neoplásica da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/patologia , Neoplasias Pulmonares/patologia , Organoides/patologia , Receptor ErbB-2/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Organoides/metabolismo , Receptor ErbB-2/genética , Transcriptoma , Células Tumorais Cultivadas
3.
Transl Oncol ; 14(1): 100960, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33395745

RESUMO

Paired related homeobox 1 (PRRX1) is a marker of limb bud mesenchymal cells, and deficiency of p53 or Rb in Prrx1-positive cells induces osteosarcoma in several mouse models. However, the regulatory roles of PRRX1 in human osteosarcoma have not been defined. In this study, we performed PRRX1 immunostaining on 35 human osteosarcoma specimens to assess the correlation between PRRX1 level and overall survival. In patients with osteosarcoma, the expression level of PRRX1 positively correlated with poor prognosis or the ratio of lung metastasis. Additionally, we found PRRX1 expression on in 143B cells, a human osteosarcoma line with a high metastatic capacity. Downregulation of PRRX1 not only suppressed proliferation and invasion but also increased the sensitivity to cisplatin and doxorubicin. When 143B cells were subcutaneously transplanted into nude mice, PRRX1 knockdown decreased tumor sizes and rates of lung metastasis. Interestingly, forskolin, a chemical compound identified by Connectivity Map analysis using RNA expression signatures during PRRX1 knockdown, decreased tumor proliferation and cell migration to the same degree as PRRX1 knockdown. These results demonstrate that PRRX1 promotes tumor malignancy in human osteosarcoma.

4.
Proc Natl Acad Sci U S A ; 117(46): 28579-28581, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33139551

RESUMO

Embryo implantation is achieved upon successful interaction between a fertilized egg and receptive endometrium and is mediated by spatiotemporal expression of implantation-associated molecules including leukemia inhibitory factor (LIF). Here we demonstrate, in mice, that LIF knockdown via a photoactivatable CRISPR-Cas9 gene editing system and illumination with a light-emitting diode can spatiotemporally disrupt fertility. This system enables dissection of spatiotemporal molecular mechanisms associated with embryo implantation and provides a therapeutic strategy for temporal control of reproductive functions in vivo.


Assuntos
Implantação do Embrião , Fator Inibidor de Leucemia/metabolismo , Optogenética , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Fertilidade , Fator Inibidor de Leucemia/genética , Camundongos Endogâmicos ICR
5.
Placenta ; 101: 194-203, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33011563

RESUMO

INTRODUCTION: P2Y14, one of the P2Y purinergic G-protein coupled receptors, is expressed in a variety of cells and tissues. Its ligand, UDP-glucose (UDPG), is released from damaged and stress-stimulated cells and acts as a danger signal via P2Y14. Thus, P2Y14 plays an important role in immunological defense systems. Here, we aimed to elucidate the expression, localization, and role of P2Y14 in human trophoblasts and the placenta. METHODS: Human chorionic villus and placental tissues were subjected to immunostaining for P2Y14 protein and an extravillous trophoblast (EVT) marker, HLA-G. We examined the expression of P2Y14 and the effect of UDPG on cell proliferation and invasion in an EVT cell line, HTR-8/SVneo, using an MTS assay and a Transwell assay, respectively. We tested the effect of UDPG on cell invasion in P2Y14-underexpressing HTR-8/SVneo clones established by the lentiviral introduction of shRNA for P2RY14 mRNA. RESULTS: Immunostaining revealed that P2Y14 was exclusively expressed by EVTs. P2RY14 mRNA and P2Y14 protein were expressed in HTR-8/SVneo cells. UDPG did not affect cell proliferation but it did enhance invasion. Inhibition of P2Y14 and decreasing the expression of P2Y14 suppressed UDPG-mediated invasive activity. CONCLUSIONS: These results showed that EVT selectively expressed P2Y14 and that P2Y14 was positively involved in UDPG-enhanced EVT invasion. It suggests the possible existence of a danger signal-mediated physiological system at the fetomaternal interface where UDPG released from maternal tissues through destruction by EVT invasion may accelerate EVT invasion, allowing EVTs to undergo successful placentation and vascular remodeling.

6.
Biochem Biophys Res Commun ; 526(1): 213-217, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32204914

RESUMO

The Cre-loxP recombination system is widely used to generate genetically modified mice for biomedical research. Recently, a highly efficient photoactivatable Cre (PA-Cre) based on reassembly of split Cre fragments has been established. This technology enables efficient DNA recombination that is activated upon blue light illumination with spatiotemporal precision. In this study, we generated a tTA-dependent photoactivatable Cre-loxP recombinase knock-in mouse model (TRE-PA-Cre mice) using a CRISPR/Cas9 system. These mice were crossed with ROSA26-tdTomato mice (Cre reporter mouse) to visualize DNA recombination as marked by tdTomato expression. We demonstrated that external noninvasive LED blue light illumination allows efficient DNA recombination in the liver of TRE-PA-Cre:ROSA26-tdTomato mice transfected with tTA expression vectors using hydrodynamic tail vein injection. The TRE-PA-Cre mouse established here promises to be useful for optogenetic genome engineering in a noninvasive, spatiotemporal, and cell-type specific manner in vivo.


Assuntos
Técnicas de Introdução de Genes , Engenharia Genética , Genoma , Integrases/metabolismo , Optogenética , Animais , Sequência de Bases , DNA/genética , Feminino , Luz , Masculino , Camundongos Endogâmicos C57BL , Modelos Animais , Tetraciclina/farmacologia
7.
Biol Reprod ; 100(5): 1215-1227, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649202

RESUMO

A decellularized uterine scaffold (DUS) prepared from rats permits recellularization and regeneration of uterine tissues when placed onto a partially excised uterus and supports pregnancy in a fashion comparable to the intact uterus. The underlying extracellular matrix (ECM) together with an acellular, perfusable vascular architecture preserved in DUS is thought to be responsible for appropriate regeneration of the uterus. To investigate this concept, we examined the effect of the orientation of the DUS-preserving ECM and the vascular architecture on uterine regeneration through placement of a DUS onto a partially defective uterine area in the reversed orientation such that the luminal face of the DUS was outside and the serosal face was inside. We characterized the tissue structure and function of the regenerated uterus, comparing the outcome to that when the DUS was placed in the correct orientation. Histological analysis revealed that aberrant structures including ectopic location of glands and an abnormal lining of smooth muscle layers were observed significantly more frequently in the reversed group than in the correct group (70% vs. 30%, P < 0.05). Despite the changes in tissue topology, the uteri regenerated with an incorrectly oriented DUS could achieve pregnancy in a way similar to uteri regenerated with a correctly oriented DUS. These results suggest that DUS-driven ECM orientation determines the regenerated uterus structure. Using DUS in the correct orientation is preferable when clinically applied. The disoriented DUS may deteriorate the tissue topology leading to structural disease of the uterus even though the fertility potential is not immediately affected.


Assuntos
Técnicas de Cultura de Células/métodos , Polaridade Celular/fisiologia , Matriz Extracelular/fisiologia , Regeneração/fisiologia , Tecidos Suporte , Útero/citologia , Útero/fisiologia , Animais , Técnicas de Cultura de Células/veterinária , Células Cultivadas , Matriz Extracelular/química , Feminino , Intestino Delgado/citologia , Intestino Delgado/ultraestrutura , Gravidez , Ratos , Ratos Sprague-Dawley , Engenharia Tecidual/métodos , Engenharia Tecidual/veterinária , Tecidos Suporte/química , Útero/ultraestrutura
8.
Mol Cell Biol ; 35(24): 4096-109, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26391953

RESUMO

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.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias do Endométrio/genética , Proteínas de Homeodomínio/genética , Proteínas Nucleares/genética , Fator de Transcrição Sp1/genética , Proteína 1 Relacionada a Twist/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Sítios de Ligação/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Neoplasias do Endométrio/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Células HEK293 , Proteínas de Homeodomínio/biossíntese , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Transcrição Genética/genética
9.
FASEB J ; 29(1): 182-92, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25351988

RESUMO

The oral cavity provides an entrance to the alimentary tract to serve as a protective barrier against harmful environmental stimuli. The oral mucosa is susceptible to injury because of its location; nonetheless, it has faster wound healing than the skin and less scar formation. However, the molecular pathways regulating this wound healing are unclear. Here, we show that transient receptor potential vanilloid 3 (TRPV3), a thermosensitive Ca(2+)-permeable channel, is more highly expressed in murine oral epithelia than in the skin by quantitative RT-PCR. We found that temperatures above 33°C activated TRPV3 and promoted oral epithelial cell proliferation. The proliferation rate in the oral epithelia of TRPV3 knockout (TRPV3KO) mice was less than that of wild-type (WT) mice. We investigated the contribution of TRPV3 to wound healing using a molar tooth extraction model and found that oral wound closure was delayed in TRPV3KO mice compared with that in WT mice. TRPV3 mRNA was up-regulated in wounded tissues, suggesting that TRPV3 may contribute to oral wound repair. We identified TRPV3 as an essential receptor in heat-induced oral epithelia proliferation and wound healing. Our findings suggest that TRPV3 activation could be a potential therapeutic target for wound healing in skin and oral mucosa.


Assuntos
Mucosa Bucal/lesões , Canais de Cátion TRPV/fisiologia , Cicatrização/fisiologia , Animais , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Receptores ErbB/metabolismo , Temperatura Alta , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa Bucal/patologia , Mucosa Bucal/fisiopatologia , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genética , Extração Dentária , Cicatrização/genética
10.
Toxicol Lett ; 232(2): 384-92, 2015 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-25445724

RESUMO

Dioxins are persistent environmental pollutants that cause multiple adverse health effects in humans, mainly through binding to the ligand-activated transcription factor, aryl hydrocarbon receptor (AhR). Genetic variation in AhR may modulate the susceptibility to dioxins. In this study, we aimed to evaluate the effects of the single nucleotide polymorphism (SNP) -130 C/T in the AhR promoter on dioxin-inducible gene transcription, and to investigate interleukin-24 (IL-24) and interleukin-1ß (IL-1ß) as proxies for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. Using primary human chorionic stromal cells, we found that cells with the TT genotype showed higher AhR mRNA and protein levels than did those of the CC genotype. Microarray was carried out to analyze the gene expression profiles of cells (CC and TT genotype) after exposing the cells to TCDD. Several genes associated with human disorders were more highly up-regulated in cells of the TT genotype. Higher up-regulation of IL-24 and IL-1ß mRNA in cells with the TT genotype was observed. Furthermore, blood samples from 64 Yusho patients who were accidentally exposed to high concentrations of dioxins were analyzed for the genotype, dioxins concentrations and serum levels of IL-24 and IL-1ß. We observed higher serum IL-24 levels and lower serum IL-1ß levels in Yusho patients with the TT genotype than in those with the CC genotype. AhR SNP -130 C/T affects serum IL-24 and IL-1ß levels, independently of serum dioxins concentrations in Yusho patients. Our observations demonstrate that SNP -130 C/T modulates AhR expression and expression levels of IL-24 and IL-1ß, and suggest an association of AhR SNP -130 C/T with the susceptibility to dioxins.


Assuntos
Dioxinas/toxicidade , Interleucinas/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Porfirias/genética , Porfirias/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Adulto , Grupo com Ancestrais do Continente Asiático , Vilosidades Coriônicas/efeitos dos fármacos , Vilosidades Coriônicas/metabolismo , Dioxinas/envenenamento , Poluentes Ambientais/toxicidade , Feminino , Humanos , Interleucina-1beta/genética , Japão , Análise em Microsséries , Dibenzodioxinas Policloradas/toxicidade , Gravidez , Células Estromais/efeitos dos fármacos
11.
J Obstet Gynaecol Res ; 39(7): 1230-5, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23803005

RESUMO

A total of 297 samples of hydropic villi were classified according to DNA polymorphisms as androgenetic moles, dispermic triploids, or biparental diploids. A subset of 267 appropriate samples was included in the study. Most of the macroscopically diagnosed complete mole cases were genetically androgenetic in origin. The partial mole cases consisted of 30 androgenetic moles and 12 dispermic triploids. For the 59 cases macroscopically categorized as hydropic abortion, the genetic analysis revealed 38 androgenetic moles, seven dispermic triploids and 14 biparental diploids. These results showed that a new diagnostic method was required for the management of patients with hydropic villi. We identified the TSSC imprint gene of which expression was shown in normal and partial mole villi but was silenced in complete mole villi. Immunohistochemistry using the TSSC3 antibody demonstrated its efficacy as the differential diagnostic method. TSSC3 play an important role in the differentiation from trophoblast stem cells to progenitors and/or labyrinth trophoblast through the TSSC3/PI3K/Akt/Mash2 signaling pathway.


Assuntos
Córion/metabolismo , Doença Trofoblástica Gestacional/diagnóstico , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Placenta/metabolismo , Neoplasias Uterinas/diagnóstico , Animais , Biomarcadores/metabolismo , Córion/patologia , Feminino , Doença Trofoblástica Gestacional/metabolismo , Doença Trofoblástica Gestacional/patologia , Humanos , Imuno-Histoquímica , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Placenta/patologia , Guias de Prática Clínica como Assunto , Gravidez , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia
12.
J Reprod Dev ; 59(1): 7-13, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-22986926

RESUMO

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 (H(2)O(2)) 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 H(2)O(2) in substitution for X/XO. We assessed the effects of H(2)O(2) 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 H(2)O(2). 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. H(2)O(2) also decreased the relative cell number. Pretreatment with H(2)O(2) significantly inhibited the invasion ability, tube-like formation and HIF1A and ITGAV of TCL1. H(2)O(2) also induced apoptosis in primary cultured trophoblasts. In conclusion, ROS produced by XO induced apoptosis and affected EVT function including invasion and differentiation.


Assuntos
Apoptose , Regulação da Expressão Gênica no Desenvolvimento , Estresse Oxidativo , Trofoblastos/metabolismo , Xantina Oxidase/metabolismo , Diferenciação Celular , Linhagem Celular , Movimento Celular , Relação Dose-Resposta a Droga , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Microscopia de Fluorescência , NG-Nitroarginina Metil Éster/farmacologia , Gravidez , Trofoblastos/citologia , Ácido Úrico/metabolismo
13.
J Biol Chem ; 287(51): 42685-94, 2012 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-23071113

RESUMO

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.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/genética , Transdução de Sinais/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Trofoblastos/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos ICR , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição Sp1/metabolismo , Células-Tronco/enzimologia , Trofoblastos/metabolismo , Regulação para Cima/genética
14.
PLoS One ; 6(7): e21990, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21760941

RESUMO

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.


Assuntos
Separação Celular/métodos , Células da Side Population/citologia , Trofoblastos/citologia , Animais , Biomarcadores/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Regulação para Baixo/efeitos dos fármacos , Embrião de Mamíferos/citologia , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Camundongos , Gravidez , Primeiro Trimestre da Gravidez/efeitos dos fármacos , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-7/metabolismo , Células da Side Population/efeitos dos fármacos , Células da Side Population/metabolismo , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Regulação para Cima/efeitos dos fármacos
15.
Mol Cancer Ther ; 10(8): 1430-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21632462

RESUMO

We previously isolated side-population (SP) cells from a human endometrial cancer cell line, Hec1, and determined that Hec1-SP cells have cancer stem-like cell features. In this study, we isolated SP cells and non-SP (NSP) cells derived from a rat endometrial cell line expressing human [(12)Val] KRAS (RK12V cells) and determined the SP phenotype. RK12V-SP cells showed self-renewal capacity, the potential to develop into stromal cells, reduced expression levels of differentiation markers, long-term proliferating capacity in cultures, and enhanced tumorigenicity, indicating that RK12V-SP cells have cancer stem-like cell features. RK12V-SP cells also display higher resistance to conventional chemotherapeutic drugs. In contrast, treatment with a histone deacetylases (HDAC) inhibitor, sodium butyrate (NaB), reduced self-renewal capacity and completely suppressed colony formation of RK12V-SP cells in a soft agar. The levels of intracellular reactive oxygen species (ROS) and the number of γH2AX foci were increased by NaB treatment of both RK12V-SP cells and RK12V-NSP cells. The expression levels of γH2AX, p21, p27, and phospho-p38 mitogen-activated protein kinase were enhanced in RK12V-SP cells compared with RK12V-NSP cells. These results imply that treatment with NaB induced production of intracellular ROS and DNA damage in both RK12V-SP and RK12V-NSP cells. Following NaB treatment, DNA damage response signals were enhanced more in RK12V-SP cells than in RK12V-NSP cells. This is the first article on an inhibitory effect of NaB on proliferation of endometrial cancer stem-like cells. HDAC inhibitors may represent an attractive antitumor therapy based upon their inhibitory effects on cancer stem-like cells.


Assuntos
Butiratos/farmacologia , Dano ao DNA/efeitos dos fármacos , Neoplasias do Endométrio/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células da Side Population/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Ratos , Células da Side Population/metabolismo , Células da Side Population/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética , Proteínas ras/metabolismo
16.
Am J Pathol ; 176(1): 381-92, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20008133

RESUMO

Cancer stem-like cell subpopulations, referred to as "side-population" (SP) cells, have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Although SP cells have been identified in the normal human endometrium and endometrial cancer, little is known about their characteristics. In this study, we isolated and characterized the SP cells in human endometrial cancer cells and in rat endometrial cells expressing oncogenic human K-Ras protein. These SP cells showed i) reduction in the expression levels of differentiation markers; ii) long-term proliferative capacity of the cell cultures; iii) self-renewal capacity in vitro; iv) enhancement of migration, lamellipodia, and uropodia formation; and v) enhanced tumorigenicity. In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor cells and stromal-like cells with enriched extracellular matrix. The expression levels of vimentin, alpha-smooth muscle actin, and collagen III were enhanced in SP tumors compared with the levels in non-SP tumors. In addition, analysis of microdissected samples and fluorescence in situ hybridization of Hec1-SP-tumors showed that the stromal-like cells with enriched extracellular matrix contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. Moreober, in a Matrigel assay, SP cells differentiated into alpha-smooth muscle actin-expressing cells. These findings demonstrate that SP cells have cancer stem-like cell features, including the potential to differentiate into the mesenchymal cell lineage.


Assuntos
Diferenciação Celular , Linhagem da Célula , Movimento Celular , Neoplasias do Endométrio/patologia , Mesoderma/patologia , Actinas/metabolismo , Adulto , Idoso , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Separação Celular , Neoplasias do Endométrio/genética , Feminino , Genes Neoplásicos/genética , Humanos , Mesoderma/metabolismo , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Dados de Sequência Molecular , Pseudópodes/metabolismo , Ratos , Células Estromais/patologia
17.
Histochem Cell Biol ; 132(4): 423-33, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19579031

RESUMO

The oral mucosa is a highly specialised, stratified epithelium that confers protection from infection and physical, chemical and thermal stimuli. The non-keratinised junctional epithelium surrounds each tooth like a collar and is easily attacked by foreign substances from the oral sulcus. We found that TRPV2, a temperature-gated channel, is highly expressed in junctional epithelial cells, but not in oral sulcular epithelial cells or oral epithelial cells. Dual or triple immunolabelling with immunocompetent cell markers also revealed TRPV2 expression in Langerhans cells and in dendritic cells and macrophages. Electron microscopy disclosed TRPV2 immunoreactivity in the unmyelinated and thinly myelinated axons within the connective tissue underlying the epithelium. TRPV2 labelling was also observed in venule endothelial cells. The electron-dense immunoreaction in junctional epithelial cells, macrophages and neural axons occurred on the plasma membrane, on invaginations of the plasma membrane and in vesicular structures. Because TRPV2 has been shown to respond to temperature, hypotonicity and mechanical stimuli, gingival cells expressing TRPV2 may act as sensor cells, detecting changes in the physical and chemical environment, and may play a role in subsequent defence mechanisms.


Assuntos
Tecido Conjuntivo/metabolismo , Inserção Epitelial/metabolismo , Mucosa Bucal/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Axônios/metabolismo , Tecido Conjuntivo/ultraestrutura , Células Dendríticas/metabolismo , Inserção Epitelial/ultraestrutura , Gengiva/metabolismo , Gengiva/ultraestrutura , Células de Langerhans/metabolismo , Macrófagos/metabolismo , Masculino , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Mucosa Bucal/ultraestrutura , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar
18.
Int J Cancer ; 124(11): 2577-88, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19173292

RESUMO

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 17beta-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 HOPpromoter, which leads to HEC development.


Assuntos
Neoplasias do Endométrio/genética , Epigênese Genética , Estradiol/farmacologia , Inativação Gênica , Proteínas de Homeodomínio/genética , Fator de Resposta Sérica/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Metilação de DNA , Neoplasias do Endométrio/patologia , Feminino , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/fisiologia , Humanos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/análise , RNA Mensageiro/análise , Fator de Resposta Sérica/fisiologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/fisiologia
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