Understanding apoptotic signaling pathways in cytosine deaminase-uracil phosphoribosyl transferase-mediated suicide gene therapy in vitro
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Cytosine deaminase-uracil phosphoribosyl transferase (CD-UPRT) fusion gene is known to exhibit therapeutic effect by inducing apoptosis in vitro. However, bystander effects of 5-flurocytosine (5-FC)/CD-UPRT and the molecular mechanism for apoptosis are yet to be established. In the present study, we have generated BHK21 cell line expressing both CD-UPRT and green fluorescent protein (GFP) from two separate transcripts, where GFP was used as a noninvasive probe to monitor the therapeutic effect of CD-UPRT. Enzyme activity of CD-UPRT in the stable cell line was measured by the reverse phase high-performance liquid chromatography analysis. Inhibition of cell growth and strong bystander effects of 5-FC/CD-UPRT were established, whereas characteristic surface morphology of apoptotic cell death was identified by AFM analysis. Involvement of various apoptotic signaling genes using semi-quantitative RT-PCR has been explored to substantiate the potential application of 5-FC/CD-UPRT suicide gene in therapy.
KeywordsSuicide gene therapy Apoptosis CD-UPRT Bystander effects Atomic force microscope
Cytosine deaminase-uracil phosphoribosyl transferase
Green fluorescence protein
Atomic force microscope
This research work was supported by the Department of Biotechnology (No. BT/PR9988/NNT/28/76/2007), Council of Scientific and Industrial Research [No. 37 (1248)/06/EMR-II] Government of India. Assistance from Central instruments Facility (CIF) IIT Guwahati, for confocal analysis is gratefully acknowledged. We are also thankful to Mr. Vijay Kumar Ravi (Department of Biotechnology, IIT Guwahati) for AFM imaging. Our sincere thanks to www.imagemet.com for allowing us to use the software SPIP 4.6.3.
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