Overexpression of adenovirus-mediated p27^kip1 lacking the Jab1-binding region enhances cytotoxicity and inhibits xenografted human cholangiocarcinoma growth

The cyclin-dependent kinase inhibitor (CDK1) p27^kip1 is a negative regulator of cell cycling and has antitumor effects. In our previous study, the recombinant adenovirus expressing wild-type p27^kip1 (Adp27-wt) induced cell cycle arrest and apoptosis, and proved that p27 is a tumor suppressor gene like p53. Another adenovirus vector expressing mutant p27^kip1 (Adp27-mt), which inhibited degradation by the ubiquitin-proteasome system, showed increased protein stability and caused a stronger induction of apoptosis. Recently, the p27^kip1 protein binding with Jab1 (Jun activating binding protein 1) was found to translocate from the nucleus into the cytosol, and then become degraded by the 26S proteasome system. The inhibition of nuclear-cytoplasmic translocation increases the protein stability of p27^kip1 and p27^kip1 with a deletion of the Jab1-binding region (p27-jab-d) is not translocated and not degraded. Therefore, a new recombinant adenovirus (Adp27-jab-d) expressing p27-jab-d was made which was able to induce greater cytotoxicity. Adp27-jab-d inhibited the growth of human cholangiocarcinoma cell line (TFK-1) cells in vitro at 3.3 times (IC₅₀) lower concentration than Adp27-wt. Moreover, in a xenografted severe combined immuno-deficient (SCID) mouse model injected with TFK-1 cells in the subcutaneous tissue, treatment by intratumor injection of Adp27-jab-d once a day for 3 days after the tumor was established, inhibited tumor growth more strongly than Adp27-wt or Adp27-mt and even induced tumor regression. However, the flow cytometric TUNEL assay showed little enhancement of apoptosis. Adp27-jab-d was thought to induce not only apoptosis but also necrosis, which was due to a specific effect of the Adp27-jab-d. Thus, by enhancing the cytotoxicity through inhibiting the translocaton of p27 ^kip1, p27^kip1 lacking the Jabl-binding region might be useful for cancer therapy. The control protein localization might also be a new target not only for cancer treatment, but also other diseases.