Zhang et al. Journal of Experimental & Clinical Cancer Research 2010, 29:52 http://www.jeccr.com/content/29/1/52 Page 2 of 7 studies NSCLC is a good target for HSV-TK gene therapy [6]. How to efficiently and selectively deliver HSV-TK gene into tumor cells? It has been reported that the non-repli- cative adenoviruses were able to infect and mediated gene transfer into NSCLC [7]. The replication-competent adenoviruses, also called oncolytic adenoviruses, are thereby a natural extension from the success of non-repli- cative adenoviruses mediated gene delivery. The advan- tage of using the replication-competent adenoviruses for therapeutic gene delivery is that it can selectively repli- cate and spread in malignant tumor tissues, and finally lead to remarkably increased therapeutic gene expression in tumor cells accompanying adenoviral replication and spread. The current strategy to generate tumor-selective replication-competent adenovirus is to replace the aden- ovirus E1 gene promoter with other tumor or tissue-spe- cific promoter [8,9]. Since human telomerase reverse transcriptase (hTERT) is over-expressed in all types of NSCLC, but is inactivated in normal cells [10], in the present study, we chose the hTERT promoter to drive adenoviral E1A gene expression and generated a tumor- selective replication-competent adenoviral vector. In order to further enhance therapeutic efficacy, we inserted a constitutive expression HSV-TK gene into this vector to develop a novel armed oncolytic adenovirus (Ad.hTERT- E1A-TK). We subsequently evaluated whether Ad.hTERT-E1A-TK could preferentially replicate in NSCLC and HSV-TK/GCV system could effectively kill NSCLC both in vitro and in vivo. Materials and methods Cells and cell culture HEK293 (human embryonic kidney 293) cells were pur- chased from Invitrogen (San Diego, CA, USA). NCIH460 (human large cell lung cancer), A549 (human lung adeno- carcinoma), SW1990 (human pancreas cancer), Hela (human cervical carcinoma), and SMMC-7721 (human hepatoma) were obtained from the Cells Bank of the Chi- nese Academy of Science (Shanghai, China). Primary human dermal fibroblasts were provided by our labora- tory, and which was derived from bioptic tissue for der- matoplasty (a written informed consent was obtained from patients). Cells were cultured in DMEM or RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS).All of the tumor cells had activated telomerase activities, while primary human dermal fibroblasts showed lower telomerase activity according to our previ- ous study. Adenoviral vectors Ad.hTERT-E1A-TK is an oncolytic adenoviral vector that is able to replicate in hTERT activated tumor cells and carries a constitutive expression HSV-TK-HAtag cassette. The construction of Ad.hTERT-E1A-TK has been described previously [11]. Ad.GFP is a replication-defec- tive Ad that lacks adenoviral E1A gene and expresses the green fluorescent protein gene (GFP). Ad.null is also a replication-defective Ad but expresses no extraneous gene. The dl309 is a wild-type Ad that contains intact adenoviral E1 gene. Ad.hTERT-E1A and Ad.hTERT-E1A- CD had similar structure with Ad.hTERT-E1A-TK and were used as positive control for oncolytic adenovirus. The former contains no therapeutic gene while the later has Escherichia colicytosine deaminase gene (E coli CD) instead of Herpes Simplex Virus Thymidine Kinase (HSV-TK). Purified, high titer viral stocks were generated according to the protocol described by Huang et al [12]. The schematic diagram of Ad.hTERT-E1A-CD or Ad.hTERT-E1A-TK was shown in Additional file 1. Western blot analysis The adenoviral HSV-TK and E1A expression was con- firmed by Western blot as described below. The cells were plated in 6-well plates and infected with the Ad.hTERT-E1A-TK at a multiplicity of infection (MOI) of 10 plaque forming units (PFU) per cell. Cells were har- vested and lysed 48 h later after infection in SDS sample buffer (containing 10 mM β-mercaptoethanol, 100 mM Tris-CL [pH 6.8], 2% SDS, and 0.1% bromophenol blue). Protein samples were separated by 10% SDS polyacrylam- ide gel and transferred to a polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The blots were probed with anti-HA (Sigma, St. Louis, MO, USA) monoclonal antibody which detected HSV-TK and anti-Ad2 E1A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) polyclonal antibody, followed by a secondary horseradish peroxidase-conjugated antibody. The anti- gen-antibody complexes were visualized using the enhanced chemiluminescence kit (Roche, New York, NY, USA) as recommended by the manufacturer. Cytopathic effect assays The cytopathic effect (CPE) was determined by three dif- ferent methods. At first, tumor cells such as NCIH460, SW1990, SMMC-7721 and Hela were plated into 24-well plates and either infected with different dose of Ad.hTERT-E1A-TK, Ad.hTERT-E1A-CD, dl309, Ad.GFP or treated with prodrug gancyclovir (GCV) or 5-fluoro- cytosine (5-FC) or untreated on the next day respectively. Five days later the plates were stained with crystal violet and the remaining living cells were determined by inten- sity of blue color. The 2nd method was Cell Counting Kit-8 assay (CCK-8, Dojindo Molecular Technologies Inc., Gaithersburg, MD, USA) which could quantitatively determine living cells by measuring optic intensity. The