BACKGROUND: Double-suicide gene therapy is a promising strategy for the 
treatment of advanced cancer. It has become an important research line in the 
development of gene therapy to overcome the drawbacks of single-gene therapy.
OBJECTIVE: The aim of this study was to investigate the usefulness of 
double-suicide gene therapy with the two suicide genes, gef and apoptin, in 
colon carcinoma.
METHODS: gef and apoptin genes were cloned into a doxycycline-regulated 
retrovirus-mediated gene expression system. Expression of both genes in the 
DLD-1 cell line was confirmed by reverse transcriptase polymerase chain reaction 
(RT-PCR). Cell viability was determined with the sulforhodamine B colorimetric 
assay, and the cell cycle was studied by propidium iodide (PI) staining. Annexin 
V-FITC and PI assays were used to evaluate apoptosis, and the results were 
confirmed by electron microscopy. The mitochondrial membrane potential was 
measured by JC-1 assay.
RESULTS: Our results showed that the combined expression of gef and apoptin 
genes was strikingly more effective than the expression of either gene alone. 
Co-expression of gef and apoptin synergistically enhanced the decrease in cell 
viability, increasing necrosis and inducing apoptosis in colon cancer cells via 
the mitochondrial pathway, which can be deficient in advanced or metastatic 
colon cancer.
CONCLUSIONS: Double-suicide gene therapy based on gef and apoptin genes may be a 
candidate for the development of new colon cancer strategies, and further 
studies are warranted to establish the usefulness of double-suicide gene therapy 
in vivo.