Understanding apoptotic signaling pathways in cytosine deaminase-uracil phosphoribosyl transferase-mediated suicide gene therapy in vitro.
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.
Gopinath
P
P
Department of Biotechnology, Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039, India.
Ghosh
Siddhartha Sankar
SS
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D024201:Bystander Effect / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D006224:Cricetinae; D043525:Cytosine Deaminase / Q000235:genetics*; D041722:Genes, Transgenic, Suicide / Q000502:physiology*; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics; D010430:Pentosyltransferases / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology* / Q000627:therapeutic use; D015398:Signal Transduction / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D043525:Cytosine Deaminase