Induction of human embryonal carcinoma cell differentiation using N,N′-hexamethylene bisacetamide in vitro

Sekiya, Souei; Kimura, Hiroaki; Yamazawa, Koji; Kera, Kota; Kawata, Makoto; Takamizawa, H; Oda, Kinichiro

doi:10.1016/0090-8258(90)90111-w

Cited by 7 publications

(6 citation statements)

References 11 publications

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“…The medium was changed every day and the cells were subcultivated by repeated pipetting at a 1 : 3 dilution when they had reached con¯uence. Dierentiation of NEC14 cells was induced by the addition of 10 72 M N,N'-hexamethylene-bis-acetamide (HMBA) (Sekiya et al, 1990;Hasegawa et al, 1991).…”

Section: Cell Culturementioning

confidence: 99%

A novel E2F binding protein with Myc-type HLH motif stimulates E2F-dependent transcription by forming a heterodimer

et al. 1998

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The human embryonal carcinoma cells NEC14 can be induced to dierentiate morphologically by the addition of 10 72 M N, N'-hexamethylene-bis-acetamide and cease to grow in several days. Transcription factors of the E2F/DP family have been shown to be closely related to the regulation of cell proliferation. To analyse cellular proteins which interact with E2F in NEC14 cells, cDNA clones encoding E2F binding proteins were isolated from a lZAP II NEC14 cell library with the 32 P-labeled GST (Glutathione S-transferase)-E2F-1 fusion protein as a probe. One of the clones encodes E2FBP1 which has the helix ± loop ± helix (HLH) motif, but lacks the basic domain and the zipper structure usually found at N-and C-terminal sides to the HLH motif, respectively. The arrangement of amino acids in the helix 1 and helix 2 regions is quite similar to those of Mxi and Mad, but dierent from those of E2F-1 and DP-1. Western blot analysis of the immunoprecipitates prepared with anti-E2FBP1 antibody showed that E2FBP1 associates with both E2F-1 and DP-1 in vivo. E2FBP1 alone has no DNA binding activity, but bind to the E2F site through heterodimerization with E2F-1 but not with DP-1. Although E2FBP1 lacks the transactivation domain, it stimulates E2F site-dependent transcription in cooperation with E2F-1.

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Section: Cell Culturementioning

confidence: 99%

A novel E2F binding protein with Myc-type HLH motif stimulates E2F-dependent transcription by forming a heterodimer

et al. 1998

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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 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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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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“…The deviation of amplified cDNA sequences from the original mRNA MATERIALS AND METHODS Cell culture The human embryonal carcinoma (EC) cell line NEC14, derived from a testicular germ cell tumor ( Fig.lA) was cultivated in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) (10). Differentiation of NEC14 cells was induced by the addition of 10-2M N,N'-hexamethylene-bis acetamide (HMBA) (11,12).…”

Section: Introductionmentioning

confidence: 99%

Subtractive cDNA cloning using oligo(dt)₃₀-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells

Hara¹,

Kato²,

Nakada

et al. 1991

Nucl Acids Res

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The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of 10(-2)M N,N'-hexamethylene-bis-acetamide (HMBA). A subtractive cDNA library specific to undifferentiated NEC14 cells was constructed using oligo(dT)30-Latex and polymerase chain reaction (PCR). The method was designed to improve the efficiency of subtraction and the enrichment of cDNA clones corresponding to low abundance mRNAs. The single strand of cDNA was made from mRNA prepared from the HMBA-treated NEC14 cells using an oligo(dT)30 primer covalently linked to Latex particles. After removal of the mRNA template by heat-denaturation and centrifugation, the subtractive hybridization was carried out between the cDNA-oligo(dT)30-Latex and mRNA from untreated NEC14 cells. Unhybridized mRNA collected by centrifugation was hybridized repeatedly to the cDNA-oligo(dT)30-Latex and subtractive mRNA was converted to cDNA. The subtractive cDNA was then amplified by PCR and cloned into pBluescript II KS-. The cDNA library thus constructed consisted of approximately 10,000 independent clones with cDNA inserts of 1.7 Kb on average. Differential hybridization of these transformants indicated that approximately 3% of them contained cDNA inserts specific to the undifferentiated EC cells, some of which were derived from low abundance mRNAs.

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Induction of human embryonal carcinoma cell differentiation using N,N′-hexamethylene bisacetamide in vitro

Cited by 7 publications

References 11 publications

A novel E2F binding protein with Myc-type HLH motif stimulates E2F-dependent transcription by forming a heterodimer

A novel E2F binding protein with Myc-type HLH motif stimulates E2F-dependent transcription by forming a heterodimer

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

Subtractive cDNA cloning using oligo(dt)₃₀-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells

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Induction of human embryonal carcinoma cell differentiation using N,N′-hexamethylene bisacetamide in vitro

Cited by 7 publications

References 11 publications

A novel E2F binding protein with Myc-type HLH motif stimulates E2F-dependent transcription by forming a heterodimer

A novel E2F binding protein with Myc-type HLH motif stimulates E2F-dependent transcription by forming a heterodimer

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

Subtractive cDNA cloning using oligo(dt)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells

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Subtractive cDNA cloning using oligo(dt)₃₀-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells