Zhang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:52 http://www.jeccr.com/content/29/1/52 Page 3 of 7 tumor cells, NCIH460, A549 and Hela grown in 96-well plates were treated with 10 MOI of Ad.hTERT-E1A-TK, Ad.hTERT-E1A-TK plus GCV or GCV alone. Five days later the remaining living cells were determined by CCK- 8 assay. The cytopathic effect was also observed by microscopy for morphologic changes. NCIH460 cells and primary human fibroblasts were plated into 6-well plates and infected with 10 MOI of Ad.hTERT-E1A-TK, dl309, or Ad.GFP respectively on the next day. CPE was moni- tored and photographed by light microscopy at the differ- ent time points. Viral replication To determine viral progeny production, NCIH460 cells (4 × 105cells/well) and primary fibroblasts (4 × 105cells/well) were plated into 6-well plates and infected with Ad.hTERT-E1A-TK at 10 MOI for 4 h. The medium con- taining extra virus was removed and the cells were washed once with PBS and cultured with fresh mediun. 24 h and 5 days later after infection, the cells were col- lected and lysed by three rounds of freezing and thawing, and then centrifuged to collect the supernatant. The ade- noviral particles in the infected tumor cells or fibroblasts supernatant were determined by plaque assay in HEK293 cells. Animal experiments Specific pathogen-free male athymic BALB/c nude mice, 4-6 weeks old (20-30 g), were obtained from the Institute of Animal Center (Chinese Academy of Sciences, Shang- hai, China). Mice were housed five per cage and allowed free access to food and water. All animal procedures were performed according to principles of laboratory animal care (NIH publication No. 85-23, revised 1985) and the current Chinese regulations and standards on the use of laboratory animals. For tumor cell implantation, NCIH460 cells (5 × 106) were subcutaneously injected into the right dorsal lumbar region in 100 μl of phosphate buffered saline (PBS). When the tumors grew up to approximately 100 mm3, the animals were randomly divided into 4 groups i.e. Ad.null, Ad.hTERT-E1A-TK, Ad.hTERT-E1A-TK plus GCV and PBS plus GCV, and each group contained at least 7 animals. About 1 × 109 PFU of Ad.null orAd.hTERT-E1A-TK in 100 μl PBS or 100 μl PBS alone were injected into tumors respectively. On the 3rd day post virus injection, GCV (100 mg/kg/ day) was intraperitoneally administered for 14 consecu- tive days. The tumor growth was assessed by measuring bi-dimensional diameters twice a week with calipers. The tumor volumes (V) were calculated according to the for- mula V = 1/2ab2 (a represents the largest diameter and b represents the smallest diameter). All animals were killed 4 weeks later after treatment and then the tumors were removed and weighed. Histopathologic examination of tumors The resected tumors were fixed with 10% formalin and embedded in paraffin. The tumor sections were stained with hematoxylin-eosin and evaluated by two individual pathologists. Statistical analysis All numerical data were expressed as mean ± SD. A com- parison of means among two or more groups was per- formed using one-way analysis of variance or nonparametric test, and further confirmed by post-hoc analyses with S-N-K or Games-Howell test. All statistical analyses were conducted using SPSS 11.5 software (SPSS, Chicago, IL). Differences with p < 0.05 were considered as significant. Results and discussion Tumor specific replication and killing effect of Ad.hTERT- E1A-TK In the present study we generated a novel oncolytic aden- oviral vector, Ad. hTERT-E1A-TK, in which tumor selec- tive replication was mediated by the hTERT promoter and HSV-TK gene expression was controlled by CMV promoter. Given Ad.hTERT-E1A-TK contained a suicide gene HSV-TK, we first examined TK expression in Ad.hTERT-E1A-TK infected cells by Western blot. Our results showed that TK expression could be detected in Ad.hTERT-E1A-TK-infected tumor cells but not in con- trol cells (Additional file 2). We next examined Ad.hTERT-E1A-TK/GCV induced cytopathic effect. As shown as crystal violet staining in Fig. 1A and Additional file 3, Ad.hTERT-E1A-TK/GCV was able to kill different type of tumor cells including NCIH460, SW1990, SMMC-7721 and Hela. Its tumor killing effect was com- parable with other oncolytic adenoviral vector such as Ad.hTERT-E1A-CD/5-FC, and even superior to Ad.hTERT-E1A as well as wild type adenovirus dl309 in most tested cell lines. Furthermore, Ad.hTERT-E1A-TK killed tumor cell in dose dependent manner. Ad.hTERT- E1A-TK induced tumor cell killing effect was further con- firmed by CCK-8 assay. As shown in Fig. 1B, two NSCLC cell lines, NCIH460 and A549, and one human cervical carcinoma cell line Hela showed significant reduction in surviving cells after Ad.hTERT-E1A-TK infection, and GCV could further enhance Ad.hTERT-E1A-TK induced tumor cell killing effect. 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-replica- tive 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,