Targeted-simultaneous expression of Gas1 and p53 using a bicistronic adenoviral vector in gliomas

Abstract

The targeted expression of transgenes is one of the principal goals of gene therapy, and it is particularly relevant for the treatment of brain tumors. In this study, we examined the effect of the overexpression of human gas1 (growth arrest specific 1) and human p53 cDNAs, both under the transcriptional control of a promoter of the human glial fibrillary acidic protein (gfa2), employing adenoviral expression vectors, in glioma cells. We showed that the targeted overexpression of gas1 and p53 (AdSGas1 and AdSp53, respectively) in rat glioma cells (C6) reduced the number of viable cells and induced apoptosis. Moreover, the adenovirally targeted expression of these genes also reduced tumor growth in vivo. Unexpectedly, there was no additive effect when both gas1 and p53 were simultaneously expressed in the same cells using a bicistronic adenoviral vector. We suggest that Gas1 does not act in combination with p53 in the C6 and U373 glioma cell lines, inducing apoptosis and cell cycle arrest. Our results indicate that the targeted expression of tumor suppressor genes (gas1 and p53) regulated by the gfa2 promoter, together with adenoviral vectors may provide an interesting approach for adjuvant selective glioma gene therapy.

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.…”

“…As a positive control, we used an anti-bactin antibody, 17 as previously described. 9,10 Proteins were revealed using a Western Lightning Plus-ECL Kit (PerkinElmer, Waltham, MA). Images were digitally captured with a BioDoc-It Imaging System (UVP, Upland, CA).…”

“…SH-SY5Y were cultured and assayed as previously described. 12 Immunoprecipitation Cells were seeded as previously described for immunocytochemistry, culture media were collected and incubated with the antibody against Gas1 (Proscience) 9,12 and then immunoprecipitated with Tachizorb (Roche) following the manufacturer's instructions; as a control, medium was incubated with an unrelated polyclonal antibody, in this case, against glial fibrillary acidic protein (Dako, Carpinteria, CA). After immunoprecipitation, western blot assays to detect Gas1 were performed as previously described, including the control medium.…”

“…In all, 1 Â 10 6 C6 cells were subcutaneously inoculated into the dorsal flank of 6-8-week-old nude (nu/nu) mice, as previously described. 8,9 When the tumors reached 4-5 mm (large axis), animals were divided into three groups; one received three doses of 5 Â 10 5 viral particles (v.p.) expressing tgas1, another received the lacZ-expressing virus (5 Â 10 5 v.p.)…”

“…[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.…”