E-type Cells Research Articles

Human Amniocytes: a Comprehensive Study on Morphology, Frequency and Growth Properties of Subpopulations from a Single Clone to the Senescence

Objective. Amniotic fluid contains a broad range of cells called amniocytes. These cells exhibit various morphologies, activities and features, depending on gestational age and fetal development. Here we present comprehensive data on growth properties of different subpopulations from single clone to senescence under different culture media. Materials and methods. Amniotic fluid samples were obtained from high risk pregnant women, who were candidates for amniocentesis. We investigated samples from 68 cases after completion of their diagnosis procedure. Accordingly, amniotic fluid cells were cultured until cells became senescent and culture growth ceased. Cells were cultured by three protocols: (1) AmnioMAX-II supplemented with 20 mM HEPES and 1% PenStrep; (2) DMEM supplemented with 4 mM L-glutamine, 10 mM HEPES, 15% FBS and 1% PenStrep; (3) a modified medium composed of 2 : 1 v/v DMEM : AmnioMAX-II which were supplemented as indicated in the second and first protocols, respectively. Culture characteristics and growth kinetics of different cell populations were further investigated. Results. All amniocytes had the ability to form colonies due to their fetal origin. Here, we show that life span of various cell types in amniotic fluid differs significantly: F-type cells have longer life span (on average 15 passages) compared to AF-type cells (6.6 on average) and E-type cells (4.6 on average). Student’s t-test analysis revealed a non-significant difference between the life span of amniocytes in AmnioMAX-II and DMEM-AmnioMAX-II (2 : 1 v/v), nearly 7 passages on average, which was significantly higher than their life span in DMEM (4 passages on average). Conclusion. Our study showed that all three cell types are clonogenic with distinguishable characteristics. Considering the effects of medium on growth properties of these subpopulations, we have found that morphological features and growth characteristics of amniocytes subjected to in vitro expansion can be modulated by the medium formulation used for cultivation.

Abstract 2401: Epigenetics-regulated microRNAs related with epithelial-mesenchymal transition of breast cancer cells

Abstract Background: Epithelial-mesenchymal transition (EMT) is a program in which biological cells change morphologically and functionally from an epithelial phenotype to a mesenchymal phenotype. The EMT is involved in the process of cancer metastasis. On the other hand, accumulated evidence showed epigenetics and microRNA plays important roles in breast cancer. However, to date, biological networks between epigenetics and microRNAs regarding EMT remains largely unclear. Thus, the aim of this study is to identify epigenetics-regulated microRNAs related with EMT of breast cancer. Materials / Methods and Results: MicroRNA expression profiles of 11 breast cancer cell lines (8 epithelial-phenotype (E-type) cells and 3 mesenchymal-phenotype (M-type) cells) were obtained by microarray method. Unsupervised clustering analysis showed that E- and M-type breast cancer cells had different microRNA expression profiles. On the other hand, we obtained a genomewide methylation status of these breast cancer cell lines by a MeDIP-seq method. An integrated in silico analysis identified microRNAs which microRNA and DNA methylation were inversely correlated. All of miR-200b/a/429 cluster members were listed in top5 differentially expressed miRs between E- and M-type cells, and also in top 5 epigenetics-regulated microRNAs. In the further study, we focused upon the miR-200b/a/429 cluster. A COBRA assay revealed that promoter regions of miR-200b/a/429 cluster in M-type breast cancer cells were more frequently methylated than that in E-type cells (65.1% vs 6.8%, p<0.0001, respectively). The methylation levels were inversely associated with miR-200b/a/429 cluster expression (Taqman assay, p<0.01). In addition, demethylating treatment using 5-aza-dC unmasked miR-200b/a/429 expression in M-typed breast cancer cell lines. Taken together, the finding indicated that the expression of miR-200b/a/429 cluster is epigenetically regulated. Next, we investigated an effect of the miR-200b/a/429 cluster upon cell motility as a function of the EMT. A transfection of exogenous miR-200b/429 inhibited 24% of cell migration ability (Transwell assay). Utilizing microRNA target prediction algorithm, we identified fibronectin as a target gene of miR-200b/429. Utilizing several prediction algorithms for microRNA target genes, we identified fibronectin as a target gene of miR-200b/429. A luciferase-based reporter assay demonstrated that miR-200b/429 were directly associated with fibronectin-3′UTR and repressed 21% (p<0.0001) of the reporter gene expression post-transcriptionally. Conclusion: The promoter hypermethylation of miR-200b cluster is associated with epithelial-mesenchymal transition, and stimulates cell motility by upregulating fibronectin expression in mesenchymal-phenotype breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2401. doi:1538-7445.AM2012-2401

P1-02-09: Epigenetics-Regulated microRNAs Related with Epithelial-Mesenchymal Transition of Breast Cancer Cells.

Abstract Background: Epithelial-mesenchymal transition (EMT) is a program in which biological cells change morphologically and functionally from an epithelial phenotype to a mesenchymal phenotype. The EMT is involved in the process of cancer metastasis. On the other hand, accumulated evidence showed epigenetics and microRNA plays important roles in breast cancer. However, to date, biological networks between epigenetics and microRNAs regarding EMT remains largely unclear. Thus, the aim of this study is to identify epigenetics-regulated microRNAs related with EMT of breast cancer. Materials / Methods and Results: MicroRNA expression profiles of 11 breast cancer cell lines (8 epithelial-phenotype (E-type) cells and 3 mesenchymal-phenotype (M-type) cells) were obtained by microarray method. Unsupervised clustering analysis showed that E- and M-type breast cancer cells had different microRNA expression profiles. On the other hand, we obtained a genomewide methylation status of these breast cancer cell lines by a MeDIP-seq method. An integrated in silico analysis identified microRNAs which microRNA and DNA methylation were inversely correlated. All of miR-200b/a/429 cluster members were listed in top5 differentially expressed miRs between E-and M-type cells, and also in top 5 epigenetics-regulated microRNAs. In the further study, we focused upon the miR-200b/a/429 cluster. A COBRA assay revealed that promoter regions of miR-200b/a/429 cluster in M-type breast cancer cells were more frequently methylated than that in E-type cells (65.1% vs 6.8%, p<0.0001, respectively). The methylation levels were inversely associated with miR-200b/a/429 cluster expression (Taqman assay, p<0.01). In addition, demethylating treatment using 5-aza-dC unmasked miR-200b/a/429 expression in M-typed breast cancer cell lines. Taken together, the finding indicated that the expression of miR-200b/a/429 cluster is epigenetically regulated. Next, we investigated an effect of the miR-200b/a/429 cluster upon cell motility as a function of the EMT. A transfection of exogenous miR-200b/429 inhibited 24% of cell migration ability (Transwell assay). Utilizing microRNA target prediction algorithm, we identified fibronectin as a target gene of miR-200b/429. Utilizing several prediction algorithms for microRNA target genes, we identified fibronectin as a target gene of miR-200b/429. A luciferase-based reporter assay demonstrated that miR-200b/429 were directly associated with fibronectin-3'UTR and repressed 21% (p<0.0001) of the reporter gene expression post-transcriptionally. Conclusion: The promoter hypermethylation of miR-200b cluster is associated with epithelial-mesenchymal transition, and stimulates cell motility by upregulating fibronectin expression in mesenchymal-phenotype breast cancer cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-09.

The tumorigenic, invasive and metastatic potential of epithelial and round subpopulations of the SW480 human colon cancer cell line

It has been reported that the SW480 human colon cancer cell line consists of E-type and R-type cells. The long-term tumorigenic potential, invasive and metastatic properties of these subclones have not been characterized. E-type and R-type cells were subcloned using limiting dilution methods from parental SW480 cells. The cell growth rate was determined by MTT colorimetric assay, and colony forming efficiency was analyzed using Matrigel-coated plates. The activity of matrix metalloproteinase (MMP) and of urokinase plasminogen activator (uPA) was assessed by zymography. Invasive and locomotive ability was analyzed using transwell chambers. In situ apoptosis detection of these subclones was also performed. In vivo long-term tumorigenicity and nodal metastasis were evaluated using nude mice. E-type cells produced spontaneously regressive tumors in spite of invasion and lymph node metastasis. In contrast, R-type cells revealed progressively growing tumors without invasion or metastasis. E-type cells exhibited increased apoptosis and invasive and motile ability, as well as strong MMP-9 and -2 activity. Although phorbol 12-myristate 13-acetate treatment induced MMP-9 activity in E-type cells, it had no effect on R-type cells. These findings suggest that E- and R-type cells may have different biological properties in terms of colon cancer progression, regression, invasion and nodal metastasis, and might serve as a useful model for these studies.

Receptive field organization and response properties of visual neurons in the pigeon nucleus semilunaris

The present study provides the first electrophysiological evidence that the nucleus semilunaris is a visual center in the pigeon midbrain. The receptive field of E-type cells is either an excitatory field alone or an excitatory center with an inhibitory periphery, which in most cases is surrounded by a disinhibitory region. Cells of I-type possess only an inhibitory receptive field. Semilunar cells are selective for fast (80–160 °/s), intermediate (40 °/s) and slow (10–20 °/s) velocities of motion, with directional cells mainly preferring forward and downward motion. About 40% of cells prefer a white stimulus moving against a black background, and 60% of cells prefer a black stimulus against a white background. The physiological significance of these properties is discussed.

Variation in RNA polymerase sigma subunit composition within different stocks of Escherichia coli W3110

The composition of RNA polymerase sigma subunits was analyzed for stock strains of Escherichia coli K-12 W3110 in Japan. Heterogeneity was discovered with respect to two sigma subunits, sigma28 (sigmaF, the rpoF gene product) and sigma38 (sigmaS, the rpoS gene product). Five different types of W3110 were identified: A-type lineages have both sigma subunits in intact forms; B-type lineages carry a truncated sigma38 subunit and an intact sigma28 subunit; C-type lineages carry an intact sigma28 subunit but lack a sigma38 subunit; D-type lineages have only a sigma38 subunit without a sigma28 subunit; and E-type stocks lack both sigma subunits. All the lineages examined, however, contain the intact forms of sigma70 (sigmaD, the rpoD gene product) and sigma54 (sigmaN, the rpoN gene product). As expected from the lack of a sigma28 subunit, cells of D- and E-type lineages are nonmotile. The truncated form of the sigma38 subunit in B-type stocks carries two mutations near its N terminus and lacks C-terminal proximal region 4 due to an amber mutation. The failure of C- and E-type W3110 cells to express sigma38 and that of D- and E-type cells to express sigma28 were found to be due to defects in transcription even though the respective sigma subunit genes remain intact. These findings emphasize the importance of paying attention to possible variations in the genetic background between laboratory stocks originating from the same strain.

Induction of mating-type change in a non-selfer mutant of Paramecium by microinjecting wild-type genomic DNA

Summary Mating-type change in the life cycle of Paramecium caudatum is based on intrinsic tem poral programming related to clonal aging. Approximately 80–120 fissions after conjugation, some of the E-type cells change their mating-type to O-type, resulting in an intraclonal mating reaction and in selfing-pair formation. We have found that a natural stock, C103, is a non-selfer mutant with the homozygote of a recessive allele. Microinjection of genomic DNA isolated from selfer strains induced a mating-type change in the non-selfer mutant, C103. These results were consistent with those of previous studies showing that a dominant modifier gene, Su(Mt), suppresses the stable expression of the Mt gene. The present report represents the first demonstration that microinjection of genomic DNA restores the genetic defect related to stable expression of mating type in Paramecium.