The aim of this project is to elucidate the intracellular and extracellular mechanisms that regulate the initiation, and subsequently the direction, of human embryonal carcinoma (EC) cell differentiation, as a model of the mechanisms that regulate cell differentiation in human tumors and in the human embryo. Focusing on the cloned pluripotent human EC cell line NTERA-2 cl.D1 (NT2/D1), we will isolate mutants that do not differentiate in response to the inducers retinoic acid (RA; a natural morphogen) or hexamethylene bisacetamide (HMBA). We will then prepare cell hybrids between the mutants in order to conduct a complementation analysis of the genetic mechanisms by which these inducers are able to initiate differentiation. By retrovirus- mediated transfection, we will introduce various putative regulatory genes (especially homeobox genes, which are developmentally regulated in NT2/D1 cells, and oncogenes) into the parental NT2/D1 cells and into non-responsive mutants, to determine whether and modulate the differentation of these human EC cells and whether any are involved in the mechanisms by which RA and/or HMBA are able to initiate cellular differentiation. In addition, we will continue to analyze and characterize the pathways by which NT2/D1 EC cells differentiate into a variety of derivatives, notably neurons and tentatively identified permitive gut epithelial cells. Immunofluorescence techniques using the FACS and the new ACAS 470 anchored cell analysis and sorting system will be used to follow the progress of differentiation, and to isolate putative intermediate cell types identified operationally on the basis of cell surface antigen expression. Finally, we will use the serum- free medium that we have defined for the growth of human EC cells to analyze the differentiation in the absence of serum and to test the effects of specific growth factors and extracellular matrix molecules (e.g., EGF, FGF, TGF-alpha, TGF-beta, fibronectin, laminin, etc.) on the direction of differentiation of NT2/D1 EC cells after induction with RA or HMBA. We will also use a specific inhibitor of glycosphingolipid synthesis to determine whether any of the developmentally-regulated glycoplipid antigens of NT2/D1 cells play a role in controlling the course of differentiation.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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Wistar Institute
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Andrews, P W; Casper, J; Damjanov, I et al. (1996) Comparative analysis of cell surface antigens expressed by cell lines derived from human germ cell tumours. Int J Cancer 66:806-16
Wenk, J; Andrews, P W; Casper, J et al. (1994) Glycolipids of germ cell tumors: extended globo-series glycolipids are a hallmark of human embryonal carcinoma cells. Int J Cancer 58:108-15
Ackerman, S L; Knowles, B B; Andrews, P W (1994) Gene regulation during neuronal and non-neuronal differentiation of NTERA2 human teratocarcinoma-derived stem cells. Brain Res Mol Brain Res 25:157-62
Rideg, K; Hirka, G; Prakash, K et al. (1994) DNA-binding proteins that interact with the 19-base pair (CRE-like) element from the HCMV major immediate early promoter in differentiating human embryonal carcinoma cells. Differentiation 56:119-29
Andrews, P W; Damjanov, I; Berends, J et al. (1994) Inhibition of proliferation and induction of differentiation of pluripotent human embryonal carcinoma cells by osteogenic protein-1 (or bone morphogenetic protein-7). Lab Invest 71:243-51
Fenderson, B A; Radin, N; Andrews, P W (1993) Differentiation antigens of human germ cell tumours: distribution of carbohydrate epitopes on glycolipids and glycoproteins analyzed using PDMP, an inhibitor of glycolipid synthesis. Eur Urol 23:30-6;discussion 36-7
Giwercman, A; Andrews, P W; Jorgensen, N et al. (1993) Immunohistochemical expression of embryonal marker TRA-1-60 in carcinoma in situ and germ cell tumors of the testis. Cancer 72:1308-14
Fenderson, B A; Andrews, P W (1992) Carbohydrate antigens of embryonal carcinoma cells: changes upon differentiation. APMIS Suppl 27:109-18
Marrink, J; Andrews, P W; van Brummen, P J et al. (1991) TRA-1-60: a new serum marker in patients with germ-cell tumors. Int J Cancer 49:368-72
Andrews, P W; Marrink, J; Hirka, G et al. (1991) The surface antigen phenotype of human embryonal carcinoma cells: modulation upon differentiation and viral infection. Recent Results Cancer Res 123:63-83

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