Cell Cycle Arrest and Apoptosis Induced by SART-1 Gene Transduction

The biological function of the SART-1 gene product is demonstrated and its potential as a target for cancer gene therapy is discussed. Materials and Methods: The SART-1 gene was transduced by a recombinant adenovirus vector and its expression was promoted by a CMV promoter. Results: The transduction efficiency by recombinant adenoviruses in A549 and MCF-7 cells was determined using a vector expressing luciferase, which showed high expression in the cells. Cell count analysis using Trypan-Blue dye exclusion showed that SART-1 gene transduction inhibited cell growth. Flow cytometry analysis suggested that SART-1 gene transduction induced cell cycle arrest followed by apoptosis. Western blot analysis confirmed that the apoptosis pathway was activated by SART-1 gene transduction. Conclusion: These results show that SART-1 gene transduction induces cell cycle arrest leading to apoptosis and suggest the possibility of gene therapy against cancer. In addition, SART-1 is known to be a tumor antigen in a range of cancers recognized by T cells, thus a potential strategy would be the combination of suicide gene therapy with immuno-gene therapy. Since the MAGE-1 gene product was identified as a tumor rejection antigen by Boon et al., numerous tumor rejection antigens recognized by cytotoxic T lymphocytes (CTLs) have been identified (1-3). Computer software to predict antigen peptide sequences from cDNA databases has recently become available and researchers seeking tumor antigens have reported that some identified sequences are active in vivo. SART-1 is ubiquitously expressed in various cancers, including breast, esophagus, lung and uterine cancers (4-10). SART-1 encodes both the SART-1259 antigen, expressed in the cytosol of epithelial cancers and the SART-1800 antigen, expressed in the nuclei of most proliferating cells (11-15). Peptides derived from the gene are known to be tumor- derived antigens recognized by HLA A2601- and A2402- restricted CTLs. There have been several reports on SART-1- derived peptides capable of inducing CTLs (16, 17). The HLA-A26 allele is found in 22% of the Japanese population, 17% of Caucasians and 16% of Africans, while the HLA-A24 allele is found in 60% of Japanese, 20% of Caucasians and 12% of Africans (18). Therefore, SART-1 may be an ideal target molecule in specific immunotherapy for cancer patients. However, in contrast to its immunological properties, the function of SART-1 has not been elucidated. Gene transfer into mammalian cells using viral vectors provides a powerful tool for gene therapy. Vectors include retroviruses derived from the Mouse Moloney Leukemia virus, human immunodeficiency virus and herpes virus (19, 20). With regard to safety, efficiency and specificity, adenoviruses are superior to other vectors. Adenovirus vectors are able to transduce genes into non-replicating / poorly-replicating cells, while other viral vectors, including retrovirus vectors, require cell proliferation for sufficient gene transduction. This suggests that adenovirus vectors have many advantages when used as vehicles for gene transfer (21). The RGD-fiber-modified recombinant adenovirus, in which the Arg-Gly-Asp (RGD)-containing peptide is incorporated into the HI-loop of the fiber knob domain, exhibits high transduction and expression efficiency when