EXPERIMENTAL CELL RESEARCH 228, 306–312 (1996) ARTICLE NO. 0330 FGF-2 Inhibits Apoptosis in Human Teratocarcinoma Cells during Differentiation on Collagen Substratum TUOMO ALANKO,* ,1 JUKKA TIENARI,† EERO LEHTONEN,† AND OLLI SAKSELA* , ‡ Departments of *Virology and †Pathology, Haartman Institute, and ‡Department of Dermatology, University of Helsinki, FIN-00014 Helsinki, Finland bers found in adult tissues, and probably all FGFs play Tera-2 is a human teratocarcinoma cell line, which significant roles during embryogenesis. FGFs have the is induced to differentiate into neuronal direction by common feature of binding to heparin, and both cell retinoic acid. Once differentiated, the cells form an surface and extracellular matrix heparan sulfate pro- almost nondividing population that can be maintained teoglycans (HSPG) serve as low-affinity binding sites for weeks under conventional culture conditions. If for the growth factors [4, 5]. The extracellular HSPGs differentiation by retinoic acid is induced while the function as a growth factor reservoir and protect FGF- cells are growing on type I collagen or if the already- 2 from denaturation and proteolytic degradation [6 – differentiated cells are transferred onto collagen, they 8]. Heparan sulfates also promote the binding of FGF- survive only a few days unless the cultures are repeat- 2 to and signal transduction via the cell surface high- edly supplied with FGF-2. Lack of this growth factor affinity tyrosine kinase receptors [9 – 12]. induces programmed cell death (apoptosis) detectable FGF-2, the most abundant member of the fibroblast after 24 – 48 h, as marked by DNA cleavage and nuclear growth factor family, regulates growth of practically fragmentation. The undifferentiated stem cells sur- all mesodermal and neuroectodermal cell types and vive and proliferate readily on collagen without addi- participates in mesodermal induction during develop- tion of FGF-2. Tera-2 cells express two members of the ment [13, 14]. It also supports the growth and survival FGF family, FGF-2 and FGF-4. The expression of both of neural cells from both the central and the peripheral FGFs is turned off during differentiation on collagen nervous systems [15, 16]. FGF-2 probably plays an im- substratum, whereas when cultivated on plain tissue portant role in the determination of cell fate in the culture dish, the expression of only FGF-4 becomes un- peripheral nervous system in vivo [17], as do certain detectable. The results indicate that signaling through other members of the FGF family. The neurotrophic cell surface FGF receptors is vital for the cells, and differentiation on collagen substratum results in com- potential of FGF-9, the most recent member of the FGF plete extinction of the autocrine stimulatory loop. In family secreted by glial cells [3], is not known yet. vivo, such induction of growth factor dependency upon We have used the human embryonal carcinoma cell differentiation would result in apoptotic death of line Tera-2 as a model for neuronal differentiation. Em- those cells which fail to find adequate conditions for bryonal carcinoma cell lines originate from germ cell continuing FGF stimulation.  1996 Academic Press, Inc. tumors and are poorly differentiated cells that often retain their ability to differentiate into several cell types in vitro. Tera-2 is tumorigenic when injected into INTRODUCTION athymic mice. Retinoic acid (RA) induces the cells to differentiate in vitro, which leads to the expression of The fibroblast growth factor family consists of at neuronal marker peptides and loss of tumorigenicity least nine growth-promoting peptides [see 1, 2 for re- [18, 19]. Simultaneously, the expression of several view, 3]. FGF-1, 2 FGF-2, and FGF-7 are the main mem- growth factors is altered. FGF-2 and FGF-4 are ex- pressed in the undifferentiated cells, and especially 1 To whom correspondence and reprint requests should be ad- FGF-4 is down regulated during differentiation, becom- dressed at the Department of Virology, Haartman Institute, P.O. ing practically undetectable in the fully differentiated Box 21, FIN-00014 Helsinki University, Finland. Fax: -358-0-434 cells [20]. The possible autoregulatory roles of these 6491. 2 Abbreviations used: ECM, extracellular matrix; FGF-1, acidic fi- and other growth factors are not yet fully understood. broblast growth factor; FGF-2, basic FGF; FGF-4, Kaposi-FGF; FGF- The great majority of developing cells in the nervous 7, keratinocyte growth factor; FGF-9, glia-activating factor; HSPG, system is eliminated by programmed cell death during heparan sulfate proteoglycan; MEM, Eagle’s minimum essential me- the formation of the functional neuropil that still has dium; PBS, phosphate-buffered saline; RA, all-trans-retinoic acid; SDS, sodium dodecyl sulfate. substantial plasticity [see 21 for review]. Programmed 306 0014-4827/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.