34 JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2008, The Society for Biotechnology, Japan Vol. 105, No. 1, 34–38. 2008 DOI: 10.1263/jbb.105.34 Transient Cell Proliferation with Polyethylenimine-Cationized N-Terminal Domain of Simian Virus 40 Large T-Antigen Hitoshi Murata, 1 Junichiro Futami, 1,2 Midori Kitazoe, 1 Megumi Kosaka, 1 Hiroko Tada, 1 Masaharu Seno, 1,3 and Hidenori Yamada 1 * Department of Bioscience and Biotechnology, Faculty of Engineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan, 1 Engineering Innovation Center, Okayama University, Okayama 700-853, Japan, 2 and Research Center for Biomedical Engineering, Okayama University, Okayama 700-8530, Japan 3 Received 22 August 2007/Accepted 10 October 2007 Polyethylenimine (PEI) cationization is a powerful strategy for protein transduction into cells. In this study, we attempted the artificial regulation of cell proliferation by protein transduction of the N-terminal domain (1–132 amino acids) of the simian virus 40 large T-antigen (SVLT-N), which inactivates retinoblastoma family proteins but not p53. To deliver SVLT-N into cells, we employed an indirect cationization method by forming a complex of biotynylated SVLT-N through disulfide bonds (biotin-SS-SVLT-N) and PEI-cationized avidin (PEI600-avidin). Using this com- plex, SVLT-N was transduced into the nucleus of confluent and quiescent Balb/c 3T3 cells and was found to be complexed with a cellular target protein, pRb. Furthermore, SVLT-N transduction in- duced cell proliferation in spite of confluent conditions. Because SVLT-N thus transduced into cells gradually degraded and was not detectable after a 4-d incubation, transiently transformed cells were obtained by this method. These results suggest that oncogene protein transduction tech- nology has great potential for in vitro regulation of cell proliferation. [Key words: N-terminal domain of simian virus 40 large T-antigen, polyethylenimine cationization, protein transduction, transient cell proliferation, avidin] Although autologous cell-based therapy is promising for treatment of various disorders, it will remain a theoretical prospect unless the requirement for providing a large num- ber of therapeutic cells can be met. Therefore, technology for controllable expansion of primary cells provides an at- tractive means of producing a large number of cells for ther- apeutic purposes. A prototype approach for the induction of proliferation and extension of the life span of primary cul- tured cells is introduction of a cell cycle inducer or elimi- nation of a negative regulator. One of the most commonly used agents is the simian virus 40 large T-antigen (SVLT), which inactivates the retinoblastoma family (pRb, p107, and p130) and the tumor suppressor p53 (1). Transfer of onco- genes such as SVLT can generate indefinitely proliferative cells, known as cell immortalization. However, in vivo in- jection of immortalized cell lines would expose patients to an unacceptable tumorigenic risk (2). Therefore, a revers- ible immortalization procedure that involves a retrovirus- mediated transfer of an oncogene that can be subsequently excised by site-specific recombination such as Cre recombi- nase has been proposed (3). Recently developed protein transduction technology has preferable safety characteristics for controlling cellular growth and differentiation for cell-based therapy, because transiently transduced proteins will degrade after expression of their function in cells without any alternation of genomic DNA (4–10). A number of efficient protein transduction ve- hicles, including cationic cell-penetrating peptides (CPPs) and cationic lipid-based reagents, have been proposed. All of these vehicles appear to transduce the target protein through an endocytosis-like pathway after electrostatic ad- sorption to the negatively charged cellular surface (11, 12). Chemical protein cationization by a synthetic cationic poly- mer of polyethylenimine (PEI) is also a powerful strategy for protein transduction, because highly cationic proteins rap- idly bind to the cellular surface followed by internalization into cells via adsorptive mediated endocytosis (4, 13–15). In this study, we developed an efficient in vitro method for inducing cell proliferation by transient transduction of an oncogenic protein for future cell-based therapy. We em- ployed SVLT as a prototype oncogenic protein and found that the N-terminal domain of 132 amino acids (SVLT-N), which inactivates pRB but not p53, was efficiently deliv- ered into quiescent cells by PEI-cationized carrier proteins and induced cell proliferation. The protein transduction technology of SVLT-N has great potential for in vitro regu- lation of cell proliferation and may pave a new way for ex vivo cell expansion needed for cell-based therapy. * Corresponding author. e-mail: yamadah@cc.okayama-u.ac.jp phone: +81-(0)86-251-8217 fax: +81-(0)86-251-8265