306. A Novel Thymidine Kinase in Enhanced Gap Junction Mediated Suicide Gene Therapy

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Top of pageAbstract Patients with malignant gliomas have a poor prognosis due to failure of conventional therapy, and new treatment paradigms are urgently needed. The extensive heterogeneity of glioblastoma cells calls for a treatment strategy with multiple (cellular) targets. Suicide gene therapy for malignant gliomas has showed promising results in experimental animal models, but has failed in clinical trials due to low transfection rate, insufficient expression of the transgene in tumor cells, and low bystander killing effect. Gap junction communication (GJC) - allowing intercellular transport of small water soluble molecules - is generally low in malignant cells, leading to insufficient bystander effect. We have previously shown that treatment of glioma cells with the histone deacetylase inhibitor 4-phenyl butyrate (PB) leads to upregulation of the gap junction protein connexin 43 expression in both its phosphorylated and non-phosphorylated forms 1. This correlates with enhanced intercellular gap junction communication and increased bystander killing effect in a herpes simplex thymidine kinase (HSVtk)/ganciclovir suicide gene therapy in vitro model 2. A novel plant thymidine kinase with high affinity for the nucleoside analogue AZT was employed in a suicide gene therapy in vitro model. We show that the plant-TK/AZT system is superior to the HSV-TK/GCV system (app. 450- vs 55-fold time of activation) in the glioblastoma cell line U-87 MG. In addition, U-87 MG cells recombinantly expressing this thymidine kinase, showed a synergistic sensitivity to an AZT/PB combination therapy. This effect was shown to be partially gap junction mediated. Similar to glioblastoma cells, we show that neural stem cells (NSC) are subject to enhanced gap junction communication by treatment with PB. This was shown by enhanced fluorescent dye transfer between NSCs and glioblastoma cells. Furthermore, the bystander killing of tumor cells was investigated using plant kinase expressing NSCs co-cultured with glioblastoma cells in the presence of an AZT/PB combination compared to AZT alone. The presence of PB also resulted in an upregulation of connexin 43 in NSCs, as was shown for glioma cells. The successful cell killing in vitro using the plant-TK/AZT system prompted us to test this in vivo. An intracranial glioblastoma xenograft model in immuno-suppressed rats was developed. Plant-TK expressing U-87 MG glioma cell xenografts were established. The sensitivity of the tumor cells to AZT was assessed by intracranial infusion by use of an osmotic minipump. Drug concentrations of 40, 80, and 160 mM, with a flow of 5 ml/hour were used. 80 mM AZT was well tolerated by the rats for a 2 week period. The anti-tumor effect was assessed by histological and immunohistochemical techniques after sacrifice of animals and sectioning of the brain. To explore the NSC migration in rat brains carrying glioma xenografts, BrdU labeled NSCs or NSCs recombinantly expressing green fluorescent protein have been injected at some distance from the tumor site and their migrational capacity is being studied.