Transcriptionally mediated gene targeting of gas1 to glioma cells elicits growth arrest and apoptosis

Abstract

Induction of growth arrest-specific genes (gas1) prevents cell proliferation and/or leads to apoptosis in different cell types. In neurons, it has been recently reported that mild excitotoxic neuronal death is associated with gas1 induction, and that overexpression of Gas1 induces apoptosis in terminally differentiated neurons or in proliferating neuroblastoma cells. In the present study, we have analysed the effects of the transcriptionally mediated targeting of gas1 to C6 rat glioma cells. Expression of Gas1 decreased glial proliferation and induced C6 cell apoptosis. While the identity of the caspase(s) responsible for Gas1-induced apoptosis in neurons has remained elusive, in C6 glioma cells, overexpression of Gas1 reproducibly activated caspase-3. Our results support the concept of targeted expression of gas1 as a potentially useful gene therapy strategy in the treatment of human gliomas.

Lentiviral transfer of an inducible transgene expressing a soluble form of Gas1 causes glioma cell arrest, apoptosis and inhibits tumor growth

“…tGas1 from infected cells. These results are in agreement with our previous data, [7][8][9][10] and further support the concept of the apoptotic effect induced by the secretable form of Gas1 (tGas1) that acts on the cells that express the transgene, as well as in a soluble form in the medium of transfected and infected cells expressing this truncated Gas1 protein.…”

“…Gas1 is a molecule that can induce cell arrest and apoptosis of different tumor cells, [34][35][36] including glioma cell lines and human primary gliomas. [7][8][9][10] Bioinformatics analysis uncovered significant structural homology between Gas1 and GFRas. 11 Based on this Soluble Gas1 inhibits glioma growthinformation, we showed that the effect of Gas1 arresting cell cycle and inducing apoptosis is caused by its ability inhibiting the GDNF-mediated intracellular signaling cascade, a mechanism consistent with the effect of GDNF inducing glioma proliferation.…”

“…[7][8][9][10] We also showed that Gas1 exhibits significant structural homology with Glial-cell-line-derived neurotrophic factor (GDNF) receptors (GFRas) and demonstrated that Gas1 inhibits GDNF-mediated intracellular signaling cascades, therefore, shutting down cellular survival pathways. [10][11][12] As the effect of Gas1 interfering with the function of the GFRa-GDNF-Ret complex is due to its structural similarity to GFRas, and it is known that GFRa1 can be released from cells and in this soluble form binds and interacts with GDNF, at the level of the first cysteine-rich domain of the GFRa1, 13,14 we hypothesized that a secreted, soluble Gas1 protein, could exert its inhibitory effect both in the cells that produce soluble Gas1 and in neighbor cells, thus extending its therapeutic capacity to treat tumors.…”

“…7 Four fragments were generated: two starting from the site of the initiation of translation up to the codon for Arginine 315, thus obtaining the truncated form, lacking the GPI consensus signal, and two full-length fragments that included up to phenylalanine 345, the last amino acid of the Gas1 sequence. However, one of each type of constructs (full length and truncated) had a stop codon in the 3 0 oligonucleotide to detain translation, and the other pair of constructs had no stop codons, thus when subcloned into the pENTRTOPO expression vector (Invitrogen, Carlsbad, CA), the V5 epitope tag would also be expressed as a fusion protein (Figure 1).…”

Hypoxia Responsive Vectors Targeting Astrocytes in Glioma

“…Another important strategy may be to induce cell cycle arrest and prevent the proliferation of the tumor cells. Soluble form of Gas1 protein is being used by lenti-viral gene transfer methods to arrest the cell cycle (Lopez-Ornelas et al, 2011), (Zamorano et al, 2003), (Zamorano et al, 2004) and preventing the proliferation of glioma cells in nude mice. Hypoxia regulated expression of these proteins has been proposed as a viable approach that can be applied to tumor killing.…”