Objective: To investigate the in vitro effects of cytosine deaminase-thymidine kinase/5-fluorocytosine + ganciclovir (CD-TK/5-FC + GCV) on ovarian cancer cells and normal cells using a human telomerase reverse transcriptase (hTERT) promoter-driven vector system.
Methods: hTERT promoter activity was determined by luciferase assay after the plasmids of pBTdel-279 containing hTERT core promoter were transfected into ovarian carcinoma-derived cell line 3AO, human normal ovarian epithelial cell (NOEC) and human embryonic lung fibroblast (HELF). hTERT mRNA expression levels of these cells were determined by reverse transcription polymerase chain reaction (RT-PCR). Eukaryotic expression vectors containing CD-TK fusion disuicide genes under the control of hTERT promoter (pBTdel-279-CD-TK) and cytomegalovirus (CMV) promoter (pcDNA3-CD-TK), as well as vectors containing TK gene under hTERT promoter control (pBTdel-279-TK), were constructed and transfected into 3AO, NOEC and HELF cells by cationic liposome. Following the transfection with CD and TK, 5-FC and GCV of different doses were added and methyl thiazolyl tetrazolium (MTT) and flow cytometry methods were applied to investigate the antitumor effects of pBTdel-279-CD-TK/5-FC + GCV and pcDNA3-CD-TK/5-FC + GCV systems. pBTdel-279-TK/GCV was considered as a control. RT-PCR was used to detect CD and TK genes in ovarian cancer cells and normal cells before and after the transfection of pBTdel-279-CD-TK or pcDNA3-CD-TK. Scanning electron microscopy (SEM) was used to examine the morphology of 3AO after the action of pBTdel-279-CD-TK/5-FC + GCV.
Results: hTERT promoter activity was 24.1% and hTERT mRNA expression was positive in ovarian cancer cell 3AO, whereas in NOEC or HELF the activity was 0.3% or 0.7% and hTERT mRNA expression was negative. Expression vectors of pBTdel-279-CD-TK, pcDNA3-CD-TK and pBTdel-279-TK were successfully constructed. Compared with the effects of pBTdel-279-TK/GCV, the pBTdel-279-CD-TK/5-FC + GCV system was more efficient on hTERT positive ovarian cancer cells, but had no effects on hTERT-negative NOEC and HELF cells. The killing rate was 74.5% when 5-FC was at 2 mmol/L and GCV at 10 mg/L. The hTERT promoter system was nearly as efficient as the CMV promoter system in inducing cancer cell death (P > 0.05). CD and TK genes were expressed in all the three kinds of cells after pcDNA3-CD-TK transfection, while positive only in ovarian cancer cells after pBTdel-279-CD-TK transfection. Apoptosis was the major death form of 3AO following the effects of pBTdel-279-CD-TK/5-FC + GCV.
Conclusion: The telomerase-specific expression of CD and TK genes under the hTERT promoter control is a novel targeting approach for gene therapy of ovarian cancer.