An Experimental Study Of Adenovirus-Mediated Transfer Lf KDR Promoter-Thymidine Kinase Gene For Selective Killing Of Vascular Endothelial Cells And Tumor Cells

Tumor modules of 1-2mm in diameter can grow by deriving nutrients via diffusion. For additional growth, neovascularization is obligatory to provide an adequate blood supply and is an important step in the progression of a tumor. Increased vascularity may allow for not only an increase in tumor growth but also a greater chance of hematogenous tumor metastasis. Thus, it is hypothesized that the damage to tumor neovascularization will lead to extensive tumor cell death and decrease metastatic potential.Experimental studies in tumor blood vessel research have demonstrated that tumor endothelial cells proliferates 500 times faster than any normal (except placental) tissue endothelial cells in the adult. One characteristic of tumor endothelial cells is the increased expression of KDR (Kinase-domaine insert containing receptor, KDR). KDR is one of the receptors for vascular endothelial growth factor (VEGF). Mainly through KDR, VEGF stimulates endothelial cell proliferation and angiogenesis. Expression of KDR in normal tissues is restricted to the vascular endothelium in vivo. There is recent evidence for the increased expression of KDR not only in the endothelial cells of tumor vasculature, but also in tumor cells. The detailed mechanism of the elevation has been studied using the 5 -flanking region of KDR gene and the results indicated that the 5' -flanking sequence is responsible for thecontrol of KDR gene expression. This sequence has been shown to direct a specific expression of reporter genes in KDR-producing cells. However, there has been no investigation of KDR directing suicide gene specific expression in endothelial cells.Using the 5' -flanking sequences as a promoter for the herpes simplex virus thymidine kinase (HSV-TK) gene in an adenoviral vector (AdKDR-TK), the therapeutic efficacy of adenovirus-mediated HSV-TK gene transduction, followed by ganciclovir (GCV) administration, was studied in four cell lines, HUVEC~ NIH3T3~ Hep-G2 ~fl A375. As a control, a recombinant adenoviral vector (AdCMV-TK) carrying HSV-TK, which was driven by the CMV promoter, was constructed. The percentage survival of cells is presented as a percentage of the survival cell in the GCV-treated cells divided by that in the cells without GCV treatment (mean±SD).The results show:1.In the KDR-producing cell lines HUVEC and A375 there appeared be a relationship between the percentage survival of cells and the number of AdKDR-TK PFU used (M0I). Both cell lines infected with AdKDR-TK exhibited GCV sensitivity as low as 1~.tg/ml of concentration at a M0I of 100. There was a significant decrease in the percent survival at 10 and 50j.tg/ml when infected at a M0I of 100.2.The KDR nonproducing cell lines NIH3T3 and HepG2 infected with AdKDR-TK did not markedly show GCV sensitivity, even at high concentrations of GCV at a M0I of 100.3.Transduction by an adenoviral vector harboring a CMV promoter and HSV-TK gene (AdCMV-TK) showed similar GCV killing in four cell lines. High GCV killing effect of the HSV-TK gene by a CMV promoter of AdCMV-TK was found in four cell lines.In conclusion, AdKDR-TK transduction of four cell lines demonstrated GCV killing only in the KDR-producing cells (HUVEC and A375) and not in the KDR nonproducing cells (NIH3T3 and HepG2). On the other hand, four cell lines infected with AdCMV-TK similarly became sensitive to GCV. These experiments indicate that AdKDR-TK- mediated transfer of the HSV-TK gene resulted in GCV killing of only KDR-producing cells. Thus, cell-specific killing was achieved by adenoviral vector containing KDR promoter for the HSV-TK gene and GCV treatment.