NSCLC NCIH460 cells were plated into 24-well plates and treated with different doses of adenoviral vectors or prodrug or untreated as indicated in figure. 5 days later the plates were stained with crystal violet. B. CCK-8 assay for surviving cells after infection with Ad.hTERT-E1A-TK. NSCLC NCIH460 and A549 cells, and cervical carcinoma Hela cellswere

plated into 96-well plates and infected by 10 MOI of Ad.hTERT-E1A-TK with or without 0.5 μg/ml GCV. 5 days later the surviving cells were quantified with CCK-8 assay and normalized by untreated cells. In order to demonstrate that Ad.hTERT-E1A-TK induced tumor cell killing effect was tumor specific, we compared the cytopathic effect between NCIH460 tumor cells and primary fibroblasts after 10 MOI of Ad.GFP, Ad.hTERT-E1A-TK or dl309 infection. The non-replicative adenovirus Ad.GFP caused no CPE in either tumor or normal cells, while wild type adenovirus dl309 caused similar CPE in both tumor and normal cells. Interestingly, PLX3397 supplier Ad.hTERT-E1A-TK did not cause CPE in primary fibroblasts but caused CPE in tumor cells which is similar with that in dl309 infected tumor

cells (Fig. 2A). In order to confirm OICR-9429 cost that Ad.hTERT-E1A-TK induced tumor specific killing effect was associated with its tumor specific replication, we performed plaque assay to quantify viral progeny production. As shown in Fig. 2B, Ad.hTERT-E1A-TK progenies detected in NCIH460 cells were approximately 7000 times more than that detected in primary fibroblasts. In more detail, about 2 × 107 and 2 × 105 of plaques were detected in supernatant from Ad.hTERT-E1A-TK infected NCIH460 cells and primary fibroblasts at 24 h after Cell Penetrating Peptide infection, whereas on day 5 the plaques were 7 × 1010 and 1 × 105 in supernatant from NCIH460 cells and primary fibroblasts respectively.

The plaques detected at 24 h post infection might derive from left vital adenovirus in the infected cells, however, the plaques detected on day 5 faithfully reflected the differential replication between tumor and normal cells. Figure 2 Selective replication and oncolysis of Ad.hTERT-E1A-TK. A. Comparison of cytopathic effects between NSCLC NCIH460 and primary fibroblasts. NSCLC NCIH460 and primary fibroblasts were plated into 6-well plates and infected with 10 MOI of Ad.hTERT-E1A-TK, dl309 or Ad.GFP. 5 days later cytopathic effects were observed and photographed by light microscopy. B. The virus progeny production in NCIH460 cells and primary fibroblasts. NCIH460 and primary fibroblasts were infected with 10 MOI of Ad.hTERT-E1A-TK for 4 h then washed once with PBS and then cultured with fresh medium. On 24 h and day 5 post infection, the cells were harvested for plaque assay. The plaques on HEK293 cells were counted and plotted. C. Western blotting analysis of E1A gene expression. NCIH460 and SW1990 Cells were infected with Ad-hTERT-E1A-TK at a MOI of 10. Cell lysates were harvested 48 h later, and immunobloted by anti E1A antibody.