Gas1 inhibits cell proliferation and induces apoptosis of human primary gliomas in the absence of Shh Gabriela Domı ´nguez-Monzo ´n a , Jorge A. Benı ´tez b,1 , Paula Vergara b , Rodrigo Lorenzana c , Jose ´ Segovia b, * a Seccio ´n Externa de Farmacologı´a, Centro de Investigacio ´n y de Estudios Avanzados del IPN, Me ´xico, 07360, D.F., Mexico b Departamento de Fisiologı´a, Biofı´sica y Neurociencias, Centro de Investigacio ´n y de Estudios Avanzados del IPN, Me ´xico, 07360, D.F., Mexico c Hospital Regional 1 o de Octubre, Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado, Mexico 1. Introduction Growth arrest specific1 (Gas1) is a protein expressed during development, that is also associated with cell arrest and capable of inducing apoptosis in different cellular systems. During embryonic development, Gas1 is differentially expressed and its presence has been linked with cell death, however, in cerebellum Gas1 acts as a positive growth regulator (K.K. Lee et al., 2001; Liu et al., 2001); Gas1 also affects the formation of the head and face (Seppala et al., 2007). Furthermore, although many reports show that Gas1 is related with cell growth arrest, acts as a tumor suppressor and as an inducer of cell death and apoptosis (Schneider et al., 1988; Del Sal et al., 1992; Evdokiou and Cowled, 1998; Mellstro ¨ m et al., 2002; Zamorano et al., 2003, 2004; Benitez et al., 2007), in other systems it has been indicated that it acts by suppressing apoptosis (Spagnuolo et al., 2004). All these data suggest that Gas1 is a pleiotropic protein that may exert different functions depending on the cellular context, and may interact with distinct molecular effectors. The role of Gas1 in development is also marked by its expression in both invertebrates and vertebrates (Airaksinen et al., 2006; Hatinen et al., 2007). The Gas1 protein is glycosylphosphatidylinositol (GPI)-linked to the outer cell membrane (Stebel et al., 2000), and several possible mechanisms explaining its effects have been proposed (Del Sal et al., 1995; C.S. Lee et al., 2001; Baladron et al., 2002; Cabrera et al., 2006; Schueler-Furman et al., 2006; Allen et al., 2007; Martinelli and Fan, 2007a; Lo ´ pez-Ramı ´rez et al., 2008). Recently, we showed the structural relationship between Gas1 and the a receptors (GFRas) for the Glial-cell line-Derived Neuro- trophic Factor (GDNF) family of ligands (Schueler-Furman et al., 2006). Based on this information, we proposed that the effects of Gas1 arresting cell cycle and inducing apoptosis are caused by its Int. J. Devl Neuroscience 27 (2009) 305–313 ARTICLE INFO Article history: Received 5 February 2009 Received in revised form 16 March 2009 Accepted 26 March 2009 Keywords: Gas1 GDNF Sonic Hedgehog Glioma Akt Ret ABSTRACT Growth arrest specific1 (Gas1) is a protein expressed during development and when cells arrest their growth. The potential of Gas1 as an adjuvant in the treatment of cancer, and its role as a tumor suppressor have also been proposed. In this work we are addressing the molecular mechanisms by which Gas1 induces cell arrest and apoptosis of cancer cells, using primary cultures of human gliomas as a model. We had previously demonstrated the structural relationship between Gas1 and the a receptors for the Glial-cell line-Derived Neurotrophic Factor (GDNF) family of ligands, and showed that Gas1 acts by inhibiting the intracellular signaling induced by GDNF. There are also reports indicating that Gas1 positively cooperates with Sonic Hedgehog (Shh) during embryonic development and in this paper we analyzed the potential interactions between Gas1 and Shh. We show that human gliomas do not express Shh, whereas GDNF and the molecular components necessary to transduce its signaling are present in human gliomas. Furthermore, the over-expression of Gas1 induces cell arrest, apoptosis and prevents the activation of Akt, a crucial mediator of survival and cellular proliferation pathways. In the present work, we present evidence demonstrating that Gas1 exerts its effects inhibiting cell growth and inducing apoptosis of glioma cells in the absence of Shh. ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved. Abbreviations: Akt, protein kinase B; DAPI, 4 0 ,6-diamino-2-phenyldole; Gas1, Growth arrest specific 1; GDNF, Glial-cell line-Derived Neurotrophic Factor; GFRa1, receptor a1 for the Glial-cell line-Derived Neurotrophic Factor family; Gli1, glioma-associated oncogene homolog 1; GPI, glycosylphosphatidylinositol; ISSSTE, Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado; PCNA, Proliferating Cell Nuclear Antigen; p-AKT, phosphorylated-AKT; ret, rearranged during transfection; Shh, Sonic Hedgehog; t-AKT, total-AKT; TUNEL, TdT-mediated dUTP nick ending labeling; WHO, World Health Organization. * Corresponding author at: Departamento de Fisiologı ´a, Biofı ´sica y Neurociencias, Centro de Investigacio ´ n y de Estudios Avanzados del IPN, Av. Instituto Polite ´ cnico Nacional # 2508, Me ´ xico, 07360, D.F., Mexico. Tel.: +52 55 5747 3958; fax: +52 55 5747 3754. E-mail address: jsegovia@fisio.cinvestav.mx (J. Segovia). 1 Present address: Ludwig Institute for Cancer Research, University of California- San Diego, La Jolla, CA 92093-0660, USA. Contents lists available at ScienceDirect International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu 0736-5748/$36.00 ß 2009 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2009.03.009