Enhanced EJ Cell Killing of (125)I Radiation by Combining with Cytosine Deaminase Gene Therapy Regulated by Synthetic Radio-Responsive Promoter

Abstract

Aim: To investigate the enhancing effect of radionuclide therapy by the therapeutic gene placed under the control of radio-responsive promoter.

Methods: The recombinant lentivirus E8-codA-GFP, including a synthetic radiation-sensitive promoter E8, cytosine deaminase (CD) gene, and green fluorescent protein gene, was constructed. The gene expression activated by (125)I radiation was assessed by observation of green fluorescence. The ability of converting 5-fluorocytosine (5-FC) to 5-fluorourial (5-FU) by CD enzyme was assessed by high-performance liquid chromatography. The viability of the infected cells exposed to (125)I in the presence of 5-FC was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the infected cells exposed to (125)I alone served as negative control and 5-FU as positive control.

Results: The recombinant lentiviral vector was constructed successfully. On exposure of infected cells to (125)I, green fluorescence can be observed and 5-FU can be detected. MTT assay showed that the survival rate for infected cells treated with (125)I was lower compared with the (125)I control group, but higher than the positive control group.

Conclusion: The synthetic promoter E8 can induce the expression of downstream CD gene under (125)I radiation, and the tumor killing effect of (125)I can be enhanced by combining CD gene therapy with radiosensitive promoter.

Keywords: 125I; 5-fluorocytosine; cytosine deaminase; radiation-sensitive promoter; recombinant lentivirus.

FIG. 1.

Gel electrophoresis of cytosine deaminase (CD) gene in pGC-FU-E8-codA-GFP plasmid. The right lane: DNA marker, the left lane: CD gene (1327 bp).

FIG. 2.

Gel electrophoresis of E8 promoter in pGC-FU-E8-codA-GFP plasmid. The right lane: molecular marker, the left lane: E8 gene (96 bp).

FIG. 3.

Infected EJ cells green fluorescence expressions induced by different doses of ¹²⁵I GFP-positive cells were seen in dark green. Group (a–e) performed cells induced by 0, 18.5, 37.0, 55.5, and 74.0 kBq ¹²⁵I. The expression of fluorescence can be seen at the dose as low as 18.5 kBq and was clearer with the increased dose of ¹²⁵I.