Construction and characterization of regulated cycle inhibiting factors induced upon Tet-On system in human colon cancer cell lines

A previous study has proven that cycle inhibiting factors (Cifs) inhibit Cullin E3 ubiquitin ligases, resulting in cell cycle arrest. More importantly, Cifs are also involved in cancer progression by deamidating Nedd8. Here we aimed to explore a novel insight into the treatment implications of Cifs in colon cancers by Tet-on system. The anticancer activity of Cif by doxycycline induction was investigated in the colon cell lines based upon Tet-On system. The expression of Cif in the colon cancer cells was determined by western blot. Furthermore, the cell viability and flow cytometry analysis were respectively performed to evaluate the cell proliferation and survival of colon cells. More importantly, the p21 and p27 levels were also evaluated after the induction of Cif with Tet-On system. Multiple clones of colon cancer cells for doxycycline-regulated Cif expression were constructed for maintenance purposes including HCT116 and SW480 cell lines. The result of western blot displayed good inducibility of expressing Cif in the cell lines. The clones with CifWT preserved their transformed phenotype compared with the control group (clones with GFP or with CifC/A), in terms of the inhibition of cancer cell proliferation and survival. Furthermore, western blot analysis showed that p27 and p21 were accumulated in the clones with CifWT, compared with the colon cancer cell lines with GFP or with CifC/A. Using the Tet-On system, we developed an efficient approach toward generation of colon cancer cells induced with Cif. These engineered colons tightly controlled Cif expression in vitro, which is a good inducible model system for cancer treatment.

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