BMC Cancer 2006, 6:66 http://www.biomedcentral.com/1471-2407/6/66 pEFfrifgesucetrnsecoe1f orAfGAVC/VTRfoEl/lHowSVetdk/bTyett-Ohen ionnduMctCioFn-7ocfeDllsoxin the Effects of rAAV/TRE/HSVtk/Tet-On on MCF-7 cells in the presence of GCV followed by the induction of Dox. MCF-7 cells were transduced with various doses of rAAV/TRE/HSVtk/Tet-On. Twenty-four hours after transduc- tion, the rAAV+Dox+GCV group (blank bar) cells were exposed to 3.0 µg/ml GCV and 1.0 µg/ml Dox while the rAAV+Dox group (black bar) cells with 1.0 µg/ml Dox and the rAAV+GCV group (hatched bar) cells with 3.0 µg/ml GCV. After 3 days, the survival rate was calculated as described in Materials and Methods. Data showed that the survival rate of the rAAV+Dox+GCV group cells was differ- ent from that of the other two groups cells. Values are expressed as means ± SD. of Medical Sciences of Xiangya Medical College granted approval for the experimental research on animals. BALB/ c nude mice were given a single priming dose of cyclo- phosphamide (100 mg/kg body weight, subcutaneously [s.c.]) 3 days prior to inoculation of cells to deplete the natural killer cells of mice. MCF-7 cells were trypsinized, washed with DMEM 3 times to remove enzyme, and har- vested by centrifugation. Cell pellets were resuspended in serum-free DMEM. After a short period of ether inhalation anesthesia by nude mice, 2 × 107 cells in 0.2 ml of suspen- sion were injected s.c. into the left flank regions to pro- duce tumors. Tumor size was measured every 3 days with vernier caliper. Thirty-two mice were randomly divided into 4 groups: GCV + Dox (8 mice), GCV + rAAV (8 mice), GCV + Dox + rAAV (8 mice), DMEM only (8 mice). When tumors had reached 7–8 mm in diameter, intratumoral injection of AAV-2/TRE/HSVtk/Tet-On (100 µl, 2 × 1011 vp/ml) or DMEM (mock group) was performed 4 times (twice a week). Tumors in mice were induced by Dox (20 mg/kg) for 1 week prior to intraperitoneal administration of GCV (100 mg/kg) twice daily for 14 days. The volumes of tumor were calculated using the following formula: volume V = L × W2 π/2, where L is the length and W is the width of the tumor (in millimeters). Histological analysis Subcutaneous tumors were excised from BALB/c nude mice with different treatments. Tumors were rinsed twice in normal saline (NS) then fixed in 4% polyparaformalde- hyde for 12 h, embedded in paraffin and sections were stained with hematoxylin and eosin for light microscopic observation. Statistical analysis Results are expressed as means ± standard deviation (S.D). The statistical difference between the groups was deter- mined by one-way analysis of variance test. The software for statistics is SPSS (10.0 version). Differences among groups were considered statistically different at P <0.05. Results Killing effect of HSVtk on MCF-7 cells under the induction of Dox MCF-7 cells in 96-well plate were infected with 104 vp/ cell, 105 vp/cell and 106 vp/cell of AAV-2/TRE/HSVtk/Tet- On respectively. After induction of 1.0 µg/ml Dox, the kill- ing rate of rAAV+Dox+GCV with 104 vp/cell to MCF-7 reached 36%, as detected by using the MTT assay in the presence of GCV (3.0 µg/ml). Increasing the viral titre to 106 vp/cell raised the cell death rate to 47% (Fig. 1). Almost no difference was found between MCF-7 cells sur- vival as rAAV infection reached a plateau when rAAV titres ranged from 104 to 106vp/cell. Although CsCl2 density gradient ultracentrifugation procedure allows for the enrichment of pure and packaged AAV-2 vector particles, it often produces vector preparations of variable quality, and may lead to a loss of particle infectivity [21]. We also indirect tested the transduction efficiency with pAAV-LacZ according to the Stratagene protocol. In our experiment, the efficiency of AAV infection to MCF-7 cells is 15–20%. In all kinds of rAAV titres above, the cell death rate of rAAV+Dox+GCV group was obviously higher than rAAV+GCV group and rAAV+Dox group (P < 0.05). GCV treatment for implanted human breast cancers in vivo During the first 14 days of GCV treatment, tumor volume in all groups increased at almost the same rate. However, in the GCV + Dox + virus treated group, tumor growth rate was retarded after 30 days compared with the control groups (Fig. 2). In the controls, tumors grew continuously up to 2 cm in diameter by the end of the 40th day when the mice were killed. For tumors injected with rAAV, GCV treatment after the induction of Dox resulted in suppres- sion of all tumors by day 30 (Fig. 2). The suppression rate of xenograft tumor growth reached up to 84%, and showed a strong inhibitory effect on tumor growth in vivo. These data suggested that HSVtk gene was expressed Page 4 of 8 (page number not for citation purposes)