BMC Cancer 2006, 6:66 http://www.biomedcentral.com/1471-2407/6/66 MFHiSCgVuFtk-r7/eTce4et-lOlsnwere infected with 105 vp/cell of rAAV/TRE/ MCF-7 cells were infected with 105 vp/cell of rAAV/ TRE/HSVtk/Tet-On. Twenty-four h after transduction, one group was exposed to 10 µg/ml of Dox and the other group without Dox administration. After 3 days, RNAs were extracted from the 2 groups and HSVtk gene expression was detected by RT-PCR with nested primers TK3/TK4 (a DNA fragment of 420 bp). For internal control, β-actin was co- amplified with HSVtk. The amplification product of HSVtk gene was observed in Dox inducement group but not in without inducement group. M: 100 bp DNA ladder; 1:group without induction; 2:group inducted with 1 g/L Dox capable of directing expression of the HSVtk gene in breast cancer and results in suppression of tumor growth rate, when delivered by direct intratumoral injection and induced by appropriate Dox with conjunction of GCV. Our results also suggest that direct intratumoral injection is not only a simple and practical gene delivery method in local cancer gene therapy, but that it also avoids systemic toxicity [25]. All the cancerous nude mice survived longer after the treatment, and toxicity of the treatment in mice was acceptable. In cancer gene therapy, viral vectors are the most efficient means of delivering a corrective gene into human cells, such as retroviral vectors and adenoviral vectors. How- ever, the applications of retroviral vectors for gene therapy face a number of challenges. Retroviral vectors has some disadvantage of infection only dividing cells, randomly integration of therapeutic gene, risk of insertional muta- genesis and activation of oncogenes, which correlates with tumor in human being [24]. The adenovirus vector is potentially of a strong immunogenic character and has a low transduction efficiency in solid tumor [26,27]. AAV can integrate into the specific site of human chromosome 19 and is not pathogenic for human being, especially without the risk of tumorigenesis. MCF-7 cells were highly infected and expressed by recombinant AAV-2 vector in nFHuiSgdVuetkrmegiec5neeaenxdpdretsesciotinonlevbeyl RinTi-mPCplRanted tumors of BALB/c HSVtk gene expression level in implanted tumors of BALB/c nude mice and detection by RT-PCR. Total RNA was isolated from implanted tumors of nude mice.HSVtk gene expression level was detected by RT-PCR with the nested primers TK3/TK4 (a DNA fragment of 420 bp). For internal control, β-actin was amplified together with HSVtk. The amplification product of HSVtk gene was observed in the Dox+GCV+virus group and the weak signal was detected in GCV+virus group. M: 100 bp Marker; 1: DMEM group; 2: GCV+Dox group; 3: GCV+virus group; 4: Dox+GCV+virus group. study of Veldwijk [28]. Due to the small fragment of the Tet-On regulatory units, AAV-2 vector was allowed to introduce the HSVtk gene although it possesses about 5 Kb capability. The tetracycline-responsive system is an ideal system for tightly regulation gene expression medi- ated by rAAV [29], because it is the tet responsive cassette inducible unit whose activity is modified in response to Dox. In rAAV vector we constructed, TRE and rtTA units of Tet gene regulation system were inserted inversely each other, to delete the downstream activity of the HSVtk gene by rtTA gene promoter through antisense suppression and subtraction. Tumor growth in nude mice given our treatment was inhibited but not eradicated. It is quite difficult to eradi- cate the tumors treated by one kind of a single gene since tumorigenesis and development is a complicated progress involving multigenes [30]. In order to enhance the effici- acy of the treatment, some approaches were adopted, such as combination of two suicide genes or combination with other therapies [30,31]. Suicide gene therapy in combina- tion with other methods, such as adjuvant radiotherapy or chemotherapy, may have some promising use in the clinic because it does not produce any elevation of the toxic side-effects in breast cancer treatment as it reaches drug concentration high enough to cure local tumors [32,33]. For some long period and progressive diseases, such as diabetes or cancer, the control of therapeutic genes expres- sion is of great importance in this gene therapy. However, intrinsic to lengthy gene therapy treatment is the need to Page 6 of 8 (page number not for citation purposes)