Zhang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:52 http://www.jeccr.com/content/29/1/52 Page 6 of 7 GCV started to grow slowly on the 8th day after treatment when compared with that treated with Ad.null or PBS plus GCV. On 16th day, the differences became more sig- nificant. At the end of observation, the average tumor sizes were 2440.00 mm3, 2287.00 mm3, 1274.50 mm3 and 435.01 mm3 in group of Ad.null, PBS plus GCV, Ad.hTERT-E1A-TK alone, and Ad.hTERT-E1A-TK plus GCV, respectively. Since Ad.null and PBS plus GCV showed no difference in tumor growth or tumor size (p > 0.05), we took both Ad.null and PBS plus GCV together as control. The tumor growth curve and tumor size between control and Ad.hTERT-E1A-TK or Ad.hTERT- E1A-TK plus GCV was significantly different (p = 0.025 or p = 0.008) and by 54.39% and 74.34% reduction in tumor weight in Ad.hTERT-E1A-TK or Ad.hTERT-E1A- TK plus GCV treated groups compared with controls. More importantly, the tumor growth curve and tumor size between Ad.hTERT-E1A-TK and Ad.hTERT-E1A-TK plus GCV also showed different (p = 0.040), it was about 43.75% reduction in tumor size in Ad.hTERT-E1A-TK plus GCV treated group (Fig. 3C). It is necessary to mention that the timing for prodrug giving was on the 3rd day in this study. The reason was mainly dependent on our previous study in which the transgene expression reached the peak on the 3rd day after intratumoral injection of either replication-competent or replication-deficient adenoviral vectors. In that study the transgene (red fluorescent protein, RFP) expression was visualized by in vivo imaging (data not shown), it reached the peak on the 3rd day which might reflect full replica- tion and distribution of adenoviral vectors in tumor tis- sue. Whether the advanced administration of GCV would result in the suppression on adenoviral replication in tumor tissues or interferer with therapeutic efficacy of Ad.hTERT-E1A-TK has not been investigated in this study. The tumor sections from different groups also showed differential histopathologic features. The most obvious difference was the numbers of apoptotic cells and the range of necrosis area. As shown in Fig. 4, tumors treated with Ad.hTERT-E1A-TK plus GCV showed wider necro- sis and more dark-stained and condensed nuclei. Conclusion Taking together, we have generated a novel oncolytic ade- noviral vector in which the main difference with cur- rently used oncolytic adenoviral vector ONYX-015 is hTERT controlled replication and armed with HSV-TK. The hTERT promoter used in this study is high strin- gency and provide the base for tumor-specific replication. Ad.hTERT-E1A-TK itself was able to inhibit tumor growth thanks to its replicative ability and oncolytic effect. Moreover, its tumor killing effect could be further enhanced by prodrug GCV. Our study showed that Figure 4 Histological examinations of NCIH460 tumors treated by different conditions. The hematoxylin-eosin stain of NCIH460 tu- mors treated by different conditions, PBS plus GCV treated (A); Ad.null treated (B); Ad.hTERT-E1A-TK alone treated (C). Ad.hTERT-E1A-TK plus GCV treated (D). The necrosis is barely seen in control groups (A and B), while there are obvious necrotic areas and numerous apoptotic bodies in the tumor tissues treated by Ad.hTERT-E1A-TK alone or Ad.hTERT- E1A-TK plus GCV treated (C and D). Original magnification ×400. Ad.hTERT-E1A-TK/GCV could efficiently kill NSCLC tumor cells both in vitro and in vivo. Therefore, we con- cluded that Ad.hTERT-E1A-TK, as a potent and safe anti- tumor strategy, could provide a potential new option for NSCLC biotherapy. Additional material Additional file 1 Schematic diagram of Ad.hTERT-E1A-CD or Ad.hTERT-E1A-TK adenoviral construct. Ad.hTERT-E1A-CD or Ad.hTERT- E1A-TK adenoviral vector had been constructed in the way described in this figure. ITR, inverted repeats of the adenovirus genome; ΔE1 and ΔE3, E1 and E3 region deleted. Additional file 2 Western blotting analysis of TK gene expression. NCIH460 Cells were infected with Ad-hTERT-E1A-TK at a MOI of 10. Cell lysates were harvested 48 h later, and immunobloted by anti HA-tag anti- body. NCIH460 Cells which had been transfected with plasmid containing TK gene were used as positive control, and uninfected NCIH460 cells were used as negative control. Additional file 3 Tumor cell killing effect of Ad.hTERT-E1A-TK on dif- ferent tumor cells. Crystal violet staining of tumor cells after infection with different adenoviral vectors. SW1990, SMMC-7721 and HeLa cells were plated into 24-well plates and treated with different dose of adenoviral vec- tors or prodrug or untreated as indicated in figure. 5 days later the plates were stained with crystal violet. Abbreviations NSCLC: Non-small cell lung cancer; HSV: Herpes Simplex Virus; TK: Thymidine Kinase; CGV: gancyclovir; hTERT: human telomerase reverse transcriptase; MOI: multiplicity of infection; PFU: plaque forming units; CPE: cytopathic effect; CCK- 8: Cell Counting Kit-8. Competing interests The authors declare that they have no competing interests.