BMC Cancer 2006, 6:66 http://www.biomedcentral.com/1471-2407/6/66 Background For enhancing gene therapeutic effects of breast cancer, there are several approaches including the inhibition of oncogene functioning [1], the rehabilitation of the tumor suppressor genes [2], enhancement of the immune response of tumor cells or immunological cells [3,4], increase in the immunological tolerance at chemotherapy of normal cells [5], suppression of tumor angiogenesis [6], and the transformation of some apoptotic genes into tumor cells [7], as well as suicide gene therapy [8]. The last approach, which can offer the prospect of selectively introducing genes into cancer cells, rendering them sus- ceptible to specific antitumor drugs, is especially appeal- ing for this bystander effect [9]. This is significant since the high therapeutic index that can be achieved with a high concentration of toxic drug will occur only at the tumor site. The more widely used suicide gene system is the her- pes simplex virus thymidine kinase/ganciclovir (HSVtK/ GCV). GCV, a kind of antiviral drug, can be phosphor- ylated by HSVtK protein finally into triphosphates, which are potent inhibitors of DNA polymerase, leading to the disruption of cellular DNA synthesis and ultimately cell death. These properties make the system attractive in can- cer therapy [9,10]. The progress was made in increasing the product of thera- peutic genes combined with suicide gene therapy using either tumor cell-specific or tissue-specific promoters. Both are capable of directing expression of a heterologous gene in tumor cells [11-13]. However, only a few workers have reported precise regulation of the HSVtk gene. Recent studies have also shown that a specific dose of the thera- peutic gene product is required for successful treatment of certain disease, but excessive production of the gene prod- uct may be toxic in some cases [14,15]. For better treat- ment, it may be necessary to control the product of therapeutic genes within a window of appropriate time and dose. The Tet-On regulatable system can tightly regu- late gene expression in response to induction and be switched off when necessary, which is of value in gene therapy [16]. The system is based on the interaction between the tetracycline-responsive element (TRE), con- sisting of seven copies of the prokaryotic tetracycline oper- ator site (tetO) fused to a cytomegalovirus minimal promoter region, and the reverse tetracycline-transactiva- tor (rtTA), consisting of the prokaryotic tetracycline repressor protein (TetR) fused to the activator domain of the herpes simplex virus VP16 protein. In the presence of tetracycline (tet) or its derivative Dox, rtTA binds TRE and activates gene expression. Compared with several drug- dependent regulatable systems, the Tet-On system shows several superior characteristics for applications in patients, including high induction, rapid reaction and high specificity [17,18]. This system had been studied in the context of numerous viral and non-viral vectors to reg- ulate expression of various genes, and diseases needed intermittent treatment [19]. For suicide gene therapy, the transfer of therapeutic genes into the tumor cells and the maintenance of their expres- sion in tumors require efficient vector systems. The adeno- associated viral (AAV) is a promising gene transfer vector for human gene therapy because of its unique combina- tion of attractive properties. These include: (1) the effi- ciency of in vivo gene transfer to a number of different cell types, including skeletal muscle cells, hepatocytes, neu- rons, and photoreceptors; (2) the ability to transduce dividing and growth arrested cells; (3) the availability of procedures for high-titre, helper virus-free preparation; and (4) a lack of significant toxicity or immune response. Furthermore, AAV-mediated gene delivery produces a long-term gene expression following prenatal administra- tion and after administration in adult tissues [20,21]. In our previous study, the pseudoviruses, pRevTRE/HSVtk and pRevTet-On, in which the expression of suicide tk gene is tightly controlled by Dox, were used to infect MCF- 7 cells or were directly delivered into the model of BALB/ c nude mice bearing human breast cancer. The findings were that HSVtk gene expression was controlled by the Dox-induced Tet-On system, and it is feasible for treating human breast cancer [22,23]. However, TRE and rtTA were respectively cloned into two vectors, which were transferred into host cells at their appropriate ratios. There is a latent danger in insertion mutation and activation of oncogenes in human being using retroviral vector system [24]. We explored the treatment of human breast cancer by AAV-2 expressing HSVtk gene under the regulation of Tet-On, the rAAV (rAAV-2/TRE/HSVtk/Tet-On) was con- structed and transduced into MCF-7 cell line or BALB/c nude mice. In order to test the antitumor effect with this system, nude mice bearing breast cancer were treated with GCV followed by rAAV intratumoral injection and consec- utive induction by Dox. This antitumor treatment may also offer a new approach and ideal model in human breast cancer or other solid tumor gene therapy. Methods Cell line, cell culture and vectors Packaging cell line 293 (ATCC # CRL-11268™), human breast cancer cell line MCF-7 (ATCC # HTB-22™) and mouse fibroblast cell line NIH 3T3 (ATCC # CRL-1658™) originated from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % of fetal bovine serum (FBS) plus 100 units of peni- cillin per ml, and 0.1 mg of streptomycin per ml. The cells were grown at 37°C in a humidified incubator with an atmosphere of 5 % CO2. The AAV-2 Helper-Free system, including the plasmids of pAAV-MCS, pAAV-RC, pAAV- Page 2 of 8 (page number not for citation purposes)