Zhang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:52 http://www.jeccr.com/content/29/1/52 Page 5 of 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. The expression of E1A gene can also be regarded as an indirect evidence for adenoviral replication, hence we performed Western blot to detect E1A expression in Ad.hTERT-E1A-TK infected NCIH460 cells and primary fibroblasts. 48 h after infection E1A expression was only detected in NCIH460 cells but not in primary fibroblasts which supported Ad.hTERT-E1A-TK selective-replica- tion in tumor cells (Fig. 2C). GCV enhanced Ad.hTERT-E1A-TK tumor killing effect in vitro The advantage of using suicide gene as therapeutic gene is that it can convert non-toxic prodrug into toxic thera- peutic agent. Since this converting process occurs in tumor site, it will save normal tissues from potential dam- age by systemic administration of toxic therapeutic agent. Next we investigated whether GCV could enhance Ad.hTERT-E1A-TK mediated tumor cell killing effect in vitro. To do this, NCIH460 tumor cells were infected with 10 MOI of Ad.hTERT-E1A-TK and then exposed to dif- ferent concentration of GCV for 5 days. According to our previous data, 10 MOI of Ad.GFP infection resulted in approximately 80% GFP positive expression cells in NCIH460 that suggested NCIH460 cells could be effi- ciently transduced by Ad, therefore, we applied 10 MOI of Ad.hTERT-E1A-TK to NCIH460 cells. The cells, infected by Ad.hTERT-E1A-TK alone for 5 days, showed about 60% death while the addition of GCV resulted in significantly more cell death. For example, about 85% or 95% cell death were observed when GCV was o.4 μg/ml or 0.8 μg/ml respectively. Therefore, GCV synergistically- enhanced Ad.hTERT-E1A-TK induced tumor cell killing effect in dose-dependent manner (Fig. 3A). Ad.hTERT-E1A-TK/GCV suppressed tumor growth in vivo The therapeutic effect of Ad.hTERT-E1A-TK alone or in combination with GCV was evaluated using human NSCLC nude mice models. The mice models were estab- lished by subcutaneous injection of NCIH460 cells. When the tumors grew up to approximately 100 mm3, about 1 × 109 PFU of Ad.null orAd.hTERT-E1A-TK in 100 μl PBS or 100 μl PBS alone was injected into tumors respectively. On the 3rd day post virus injection, GCV (100 mg/kg/day) was intraperitoneally administered for 14 consecutive days. The tumor growth was assessed by measuring bi-dimensional diameters twice a week with calipers. As shown in Fig. 3B, the tumors treated by Ad.hTERT-E1A-TK alone or Ad.hTERT-E1A-TK plus Figure 3 GCV enhanced inhibition on tumor growth in vitro and in vivo. A. GCV enhanced Ad.hTERT-E1A-TK tumor killing effect in vitro. NCIH460 tumor cells were infected with 10 MOI of Ad.hTERT- E1A-TK and then exposed to different concentration of GCV for 5 days. The surviving cells were quantified with CCK-8 assay and plotted. B. Ad.hTERT-E1A-TK/GCV suppressed tumor growth in vivo. NCIH460 xenograft tumors in nude mice were treated by Ad.null, PBS plus GCV, Ad.hTERT-E1A-TK alone or Ad.hTERT-E1A-TK plus GCV. Tu- mor sizes were measured twice a week using calipers and tumor vol- umes were plotted. C. Tumor weight at the end of the study. On day 28 post treatment, all animals were sacrificed and the tumors were re- moved and weighted. The data represent the mean ± SD from at least 7 animals per group.