Expression of various viral and cellular enhancer-promoters during differentiation of human embryonal carcinoma cells

Hasegawa, Tomomi; Nakada, Susumu; Nakajima, Takuma; Oda, Kazuhiro; Kawata, Makoto; Kimura, Hiroaki; Sekiya, Souei

doi:10.1111/j.1432-0436.1990.tb00761.x

Cited by 8 publications

(13 citation statements)

References 44 publications

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“…Cloning of Human UTF1 and Its Sequence Analysis-A cDNA library was constructed according to the standard procedure (25) using poly(A) ϩ RNA from undifferentiated human teratocarcinoma cell line, NEC14 (26) and screened with the entire mouse UTF1 cDNA (24) as a probe. Sequencing of the obtained clones was done by the dideoxynucleotide procedure using M13 universal or reverse primer.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Functional Domains of an Embryonic Stem Cell Coactivator UTF1 Which Are Conserved and Essential for Potentiation of ATF-2 Activity

Fukushima¹,

Okuda²,

Nishimoto³

et al. 1998

Journal of Biological Chemistry

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We have recently cloned a cDNA encoding an embryonic stem cell transcriptional coactivator termed UTF1 from the mouse F9 teratocarcinoma cell line (Okuda, A., Fukushima, A., Nishimoto, M., Orimo, A., Yamagishi, T., Nabeshima, Y., Kuro-o, M., Nabeshima, Y., Boon, K., Keaveney, M., Stunnenberg, H.G., and Muramatsu, M. (1998) EMBO J. 17, 2019 -2032). Here we have cloned a cDNA for human UTF1 and identified two highly conserved domains termed conserved domain (CD)1 and CD2. Human UTF1, like that of mouse, binds to ATF-2 and the mutagenesis analyses reveal that the leucine zipper motif within the CD2 of the UTF1 and metal binding motif of ATF-2 are involved in this interaction. The factor also binds to TATA-binding protein containing complex. By means of immunoprecipitation analysis, we mapped two domains which are independently able to bind to the complex. Importantly, both domains are located within the conserved domains (one in CD1 and the other in CD2). Furthermore, transient transfection analyses point out the importance of these domains for activating ATF-2. Thus, these results suggest that these two conserved domains identified here play important roles in activating specific transcription at least in part by supporting physical interaction between the upstream factor, ATF-2, and basal transcription machinery.

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Section: Methodsmentioning

confidence: 99%

Characterization of Functional Domains of an Embryonic Stem Cell Coactivator UTF1 Which Are Conserved and Essential for Potentiation of ATF-2 Activity

Fukushima¹,

Okuda²,

Nishimoto³

et al. 1998

Journal of Biological Chemistry

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“…The medium was changed every day, and the cells were subcultivated by repeated pipetting at a 1:3 to 1:4 dilution when they had reached confluence. Differentiation of NEC14 cells was induced by the addition of 10 Ϫ2 M HMBA (20,48).…”

Section: Methodsmentioning

confidence: 99%

“…The human EC cell line NEC14, established from a testicular germ cell tumor, consists exclusively of stem cells that are small and polygonal and form densely packed clusters (20,46). The cells can be induced to differentiate in several days by the addition of 10 Ϫ2 M HMBA, and the differentiated cells undergo drastic morphological changes, becoming larger and flattened, and cease to proliferate (20,21,48).…”

Section: Induction Of Fn Gene Expression In Nec14 Cells After Inductimentioning

confidence: 99%

“…The cells can be induced to differentiate in several days by the addition of 10 Ϫ2 M HMBA, and the differentiated cells undergo drastic morphological changes, becoming larger and flattened, and cease to proliferate (20,21,48).…”

Section: Induction Of Fn Gene Expression In Nec14 Cells After Inductimentioning

confidence: 99%

“…Owing to the shortage of established human cell lines that change their growth properties in response to external signals, the positive and negative transcription factors interacting with the G-rich sequences in the human FN promoter and its relation to the growth potential of cells have not yet been clarified. The human embryonal carcinoma (EC) cell line NEC14, established from a testicular germ cell tumor (46,47), offers a good system for analysis of these factors, since it proliferates unlimitedly but can be induced to differentiate by the addition of 10 Ϫ2 M N, NЈ-hexamethylene bisacetamide (HMBA) in vitro (20,48). The cells tend to lose proliferative potential upon induction of differentiation, appearing larger and flattened, and finally cease their growth.…”

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confidence: 99%

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Induction of Sp1 in Differentiating Human Embryonal Carcinoma Cells Triggers Transcription of the Fibronectin Gene

Suzuki

Oda

Nakajima

et al. 1998

Molecular and Cellular Biology

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Cells of the human embryonal carcinoma line NEC14 proliferate as densely packed clusters consisting of small, polygonal stem cells and do not express a detectable level of fibronectin (FN). Upon induction of differentiation by treatment with N,N-hexamethylene bisacetamide (HMBA), the level of FN mRNA increased steeply within 24 h and FN began to be accumulated, along with the organization of actin filaments in the cells. The FN promoter elements required for the activation were analyzed in reference to a cluster of GC boxes by using the chloramphenicol acetyltransferase (CAT) gene fused to 5 sequential-deletion derivatives of the promoter and promoters carrying base substitutions in the GC boxes. Among four GC boxes, GC boxes 2 and 3 had the greatest effect on promoter activation, and base substitutions in these GC boxes resulted in 80% reduction in promoter activity. The pattern of DNA-protein complex formation with these GC boxes changed drastically after induction of differentiation. The extract prepared from undifferentiated NEC14 cells formed fast-migrating complexes (UnD complexes), while the extract prepared from NEC14 cells treated with HMBA for 24 h formed slow-migrating complexes containing Sp1. Both complexes were formed predominantly with GC box 2. Base substitutions within the GC boxes completely abolished the formation of both UnD and Sp1 complexes. Consistent with these changes, the Sp1 level increased steeply within 24 h. Induction of Sp1 expression in NEC14 cells effectively stimulated the promoter activity of the transfected FN promoter-CAT constructs. These results indicate that activation of the FN promoter in differentiating NEC14 cells occurs by the steep induction of Sp1, which prevents an undifferentiated cell factor from binding to the Sp1 sites.Fibronectin (FN) is a family of large glycoproteins of the extracellular matrix and has multiple domains with specific binding sites for other matrix macromolecules, such as fibrin, glycosaminoglycans, and collagen, and for its receptor, the ␣5␤1-type integrin on the cell surface (24). FN binds to its receptor as a dimer of similar subunits of about 250 kDa (23,24). The cytoplasmic domain of the receptor interacts with actin filaments via several adhesion proteins, connecting the extracellular matrix to actin filaments at the receptor site called focal contacts or adhesion plaques (3,4,23,25,44). FN thus plays an important role in organizing the extracellular matrix and enabling cells to attach to it. Through these functions, FN regulates cell adhesion, migration, growth, wound healing, and tumor metastasis (12,23).Expression levels of FN in cells vary markedly depending on the environment and cellular capacity for proliferation. Cells transformed by various oncogenes usually express very low levels of FN (14,17,29), while senescent cells inevitably express high levels of FN, ceasing their proliferation (18,35,39). We previously showed that the level of FN gene expression in the rat derivative cell line 3Y1 transformed by adenovirus E1A and E1B g...

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Construction and characterization of multiple human colon cancer cell lines for inducibly regulated gene expression

Welman

Cawthorne

Barraclough

et al. 2005

J of Cellular Biochemistry

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Validation of targets for cancer drug discovery requires robust experimental models. Systems based on inducible gene expression are well suited to this purpose but are difficult to establish in several epithelial cell types. Using the recently discovered transcriptional transactivator (rtTA2S-M2), we developed a strategy for fast and efficient generation of Tet On cells. Multiple clones of HCT116, SW480, and HT29 human colon cancer cells for doxycycline-regulated gene expression were constructed that constitutively express green fluorescent protein (GFP) for selection/maintenance purposes. The cell lines displayed good fold inducibility (49-124xHCT116; 178-621xSW480; 261-787xHT29) and minimal leakiness after transient transfection with a luciferase reporter or with vectors driving inducible expression of red fluorescent protein (dsRed2), constitutively active c-Src or dominant negative K-Ras4B. The clones preserved their transformed phenotype as demonstrated by comparing their properties to respective wild type cells, in terms of growth in vitro and in vivo (as tumor xenografts), cell cycle traverse, and sensitivity to drugs used in chemotherapy. These engineered cell lines enabled tightly controlled inducible gene expression both in vitro and in vivo, and proved well suited for construction of double-stable cell lines inducibly expressing a protein of interest. As such they represent a useful research tool for example, to dissect oncogene function(s) in colon cancer. Supplementary material for this article be found at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_welman.doc.

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Expression of various viral and cellular enhancer-promoters during differentiation of human embryonal carcinoma cells

Cited by 8 publications

References 44 publications

Characterization of Functional Domains of an Embryonic Stem Cell Coactivator UTF1 Which Are Conserved and Essential for Potentiation of ATF-2 Activity

Characterization of Functional Domains of an Embryonic Stem Cell Coactivator UTF1 Which Are Conserved and Essential for Potentiation of ATF-2 Activity

Induction of Sp1 in Differentiating Human Embryonal Carcinoma Cells Triggers Transcription of the Fibronectin Gene

Construction and characterization of multiple human colon cancer cell lines for inducibly regulated gene expression

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