Glial-specific retrovirally mediated gas1 gene expression induces glioma cell apoptosis and inhibits tumor growth in vivo Absalom Zamorano, a Britt Mellstro ¨m, b Paula Vergara, a Jose ´ R. Naranjo, b and Jose ´ Segovia a, * a Departamento de Fisiologı ´a, Biofı ´sica y Neurociencias, Centro de Investigacio ´n y de Estudios Avanzados del IPN, Me ´xico, 07300, D.F., Mexico b Departamento de Biologı ´a Molecular y Celular, Centro Nacional de Biotecnologı ´a, CSIC, Cantoblanco, 28049 Madrid, Spain Received 1 July 2003; revised 28 October 2003; accepted 21 November 2003 We recently reported that the targeted expression of growth arrest specific 1 (Gas1) induces apoptosis in glioma cells. Because the vast majority of gliomas present genetic alterations that reduce their ability to undergo apoptosis, a gene therapy strategy aimed at reinstating apoptotic processes in glioma cells is an interesting approach for the treatment of these tumors. We used a retroviral gene transfer system to transduce C6 glioma cells with a transgene in which the expression of a full-length human gas1 cDNA is under the transcriptional control of a human promoter of the glial fibrillary acidic protein (gfa2). In vitro experiments showed that the retroviral transfer of gas1 significantly reduces the number of viable cells, and induces apoptosis in C6 cells, through the activation of caspase-3. Furthermore, retroviral-mediated transfer of gas1 to gliomas implanted in nude mice induces a significant inhibition of tumor growth, accompanied by increased caspase-3 activation. In the present experiments, we have taken advantage of the property of retrovirus to transfer transgenes exclusively to proliferat- ing cells, together with the use of a glial specific promoter, to selectively target the expression of gas1, a pro-apoptotic gene, to glioma cells. D 2004 Elsevier Inc. All rights reserved. Keywords: Tumor; C6 cells; p53; Gene therapy; Gene transfer; Astrocytes; Cell death Introduction Tumor development occurs by means of a series of dynamic changes in the genome that confer growth advantages to trans- formed cells. This leads to disruption of cellular regulatory circuits that impact on cell cycle control and death signals. Therefore, therapies aimed at blocking the inappropriate proliferation of cells, or inducing cell death, represent attractive strategies for tumor treatment (Bamford et al., 2000). In particular, approximately 75% of glioblastomas present a reduced capacity to undergo apoptosis, due to p53-associated lesions (Wechsler-Reya and Scott, 2001). This indicates that decreased sensitivity to apoptosis signals is a crucial factor in the malignancy of gliomas. Thus, therapies based on the restoration of apoptotic pathways, or the ectopic expression of molecules that induce apoptosis, are attractive options for the treatment of gliomas. Growth arrest specific-1 (gas1) was isolated by differential screening of NIH-3T3 fibroblasts after serum withdrawal (Schneider et al., 1988). Gas1 is a gene that codes for a protein inserted into the cell membrane through a glycosyl-phosphatidyli- nositol (GPI) anchor at its carboxy terminus (Del Sal et al., 1992; Stebel et al., 2000). This protein has been directly related to cell arrest and/or apoptosis in different cell systems (Del Sal et al., 1992; Mellstro ¨m et al., 2002), and is also a tumor suppressor that prevents DNA synthesis by blocking entry of cells into the S phase (Del Sal et al., 1992, 1995; Evdokiou and Cowled, 1998). The growth arrest function of Gas1 requires appropriate levels of p53, although it is independent of its transactivating function (Del Sal et al., 1995). Interestingly, Gas1-induced apoptosis is p53 indepen- dent and it has been observed in interdigital cell death during development (Lee et al., 2001). Moreover, gas1 induction has been described in apoptotic secretory epithelial cells during the physi- ological involution of the mammary gland, the ventral prostate and the ovarian corpus luteum (Bielke et al., 1997; Guo et al., 1998). Gas1 transcription is repressed by c-Myc, and this regulation may contribute to the oncogenic potential of c-Myc (Gartel and Shchors, 2003). On the other hand, Gas1 has also been identified as a positive growth regulator in cerebellum, where it is required to promote normal proliferation of precursor cells in the external germinal layer and Bergman glia, but not for their differentiation (Liu et al., 2001). Taken together, these results suggest that Gas1 may mediate different responses depending on the specific devel- opmental context and cellular type. Gas1 induction has been reported in neurons and neuroblastoma cells committed to die, both in culture and in vivo, and gas1- induced neuronal death was attenuated by co-expression of the human Bcl-2 protein or the baculoviral caspase inhibitor OpIAP2 (Mellstro ¨m et al., 2002). Furthermore, we have shown that the transcriptionally mediated targeting of gas1 markedly reduces the number of viable cells and induces a caspase-3-mediated apoptotic process in cultured C6 glioma cells (Zamorano et al., 2003). These 0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2003.11.022 * Corresponding author. Departamento de Fisiologı ´a, Biofı ´sica y Neurociencias, Centro de Investigacio ´n y de Estudios Avanzados del IPN, Av. IPN #2508, Me ´xico, 07300, D.F., Mexico. Fax: +52-55-5061- 3754. E-mail address: jsegovia@fisio.cinvestav.mx (J. Segovia). Available online on ScienceDirect (www.sciencedirect.com.) www.elsevier.com/locate/ynbdi Neurobiology of Disease 15 (2004) 483 – 491