TAT-apoptin/GFP into the culture medium from HUVEC cells infected by lentivirus LV-SP-TAT-apoptin/GFP. To obtain sustained apoptin-induced tumor cell death in vivo, we injected the LV-SP-TAT-apoptin viruses via the tail vein for systemic delivery of the viruses; viruses expressing LV-SP-

Apoptin, a chicken anemia virus-derived protein, has been shown to induce apoptosis in various human cancer cell lines, but not in normal cells, thus making it a candidate for the development of novel therapeutic strategies. To enable the efficient transduction of tumor cells with apoptin, we have developed a novel mammalian expression system for the secretion of apoptin in vitro. We have previously shown the efficient and tumor-specific killing of cells by adding a secretory signal peptide (SP) to the N terminus of transacting activator of transcription (TAT)-apoptin (SP-TAT-apoptin). In addition, our report showed the successful secretion of high levels of TAT-apoptin/GFP into the culture medium from HUVEC cells infected by lentivirus LV-SP-TAT-apoptin/GFP. To obtain sustained apoptin-induced tumor cell death in vivo, we injected the LV-SP-TAT-apoptin viruses via the tail vein for systemic delivery of the viruses; viruses expressing LV-SPTAT-GFP were used as a negative control. Markedly, almost all the hepatocellular carcinoma xenograft tumors disappeared following the treatment while the xenografts that received the control LV-SP-TAT-GFP viruses continued to grow. Moreover, the animal studies presented in this paper demonstrate a low toxicity of SP-TAT-apoptin in vivo, confirming and extending the results of the in vitro studies. Taken together, our data strongly suggest that systemic delivery of lentivirus-mediated secretable TAT-apoptin is feasible to eradicate liver cancer in vivo. Introduction Apoptin, a chicken anemia virus-encoded protein, consists of 121 amino acids (1). An attractive feature of apoptin is that it induces apoptosis in a large number of human tumor or transformed cells but not in their normal, healthy counterparts (2,3). This is correlated with the subcellular localization of the protein. In normal cells, apoptin is found in the cytoplasm whereas in tumor cells it is found in the nucleus (4-6). This suggests that apoptin may be used as an agent that selectively eliminates malignant cells, if it can be delivered into target cells in vivo in sufficient amounts. One straightforward strategy to reach this goal would be the application of the apoptin gene, rather than the protein itself. The transacting activator of transcription (TAT) protein transduction domain (PTD) from human immunodeficiency virus type 1 (aa 47-57; YGRKKRRQRRR) is one of the most commonly used protein transduction systems (7,8). In some studies, this TAT-PTD transduction domain has been successfully used to deliver apoptin into cancer cells in vitro. The data from these studies show that TAT-apoptin efficiently kills human tumor, but not normal cells (9-11). However, one disadvantage of using the TAT sequence in in vivo studies is that physical delivery of TAT fusion gene such as direct injection into the tumor bed is inefficient, as the produced recombinant protein may not reach all the cells within the tumor mass. To enable the efficient transduction of tumor cells with apoptin, we have developed a novel mammalian expression system for the in vitro secretion of proteins (12,13). We have previously shown the efficient and tumor-specific killing of cells by TAT-apoptin fused to a signal peptide (SP) whose amino acid sequence and corresponding cDNA sequences were generated by an SP hidden Markov model (SP-HMM) (14). This SP directs strong protein secretion and expression and the TAT-apoptin fusion protein secreted from transfected cells can re-enter the adjacent untransfected cells. It should be noted that in normal cells resistant to apoptin-mediated cell death, fusion protein is localized in the cytoplasm, whereas in sensitive cells it is in the nucleus. Nuclear localization was shown to be crucial for the cell-killing ability of apoptin (12). We have demonstrated that SP-TAT-apoptin induces apoptosis only in malignant cells, and its secretory property might greatly increase its potency, once it is delivered in vivo for cancer therapy. We recently generated a lentivirus-based Systemic delivery of lentivirus-mediated secretable TAT-apoptin eradicates hepatocellular carcinoma xenografts in nude mice JIN-LU MA1, SU-XIA HAN1, JING ZHAO1, DAN ZHANG1, LI WANG1, YAO-DONG LI2 and QING ZHU1 1Department of Oncology, the First Affiliated Hospital of Xi'an Jiaotong University Medical College, Xi'an; 2Department of General Surgery, the Affiliated Hospital of Xi'an Medical College, Xi'an, P.R. China Received March 26, 2012; Accepted June 1, 2012 DOI: 10.3892/ijo.2012.1547 Correspondence to: Dr Qing Zhu, Department of Oncology, the First Affiliated Hospital, Xi'an Jiaotong University Medical College,