Use of the Ki67 promoter to label cell cycle entry in living cells.

Genetic based reporters have distinct advantages over classical immunocytochemical techniques for probing cellular functions. Most importantly, they enable dynamic real-time visualization and quantification of cellular processes in living cells and tissue. This study was conducted to generate a genetic based reporter to label cells that transitioned from the G(0) to G(1)/S phases of the cell cycle, hypothesizing that the proximal promoter of the Ki67 (Ki67p) gene, a commonly used cytology marker induced during this transition, would contain the suitable regulatory elements to drive marker gene expression. This study reports the cloning and characterization of the 1.5 kb proximal promoter (Ki67p) of the human Ki67 gene. Ki67p driven GFP expression colocalizes in cells with endogenous Ki67 expression and is correlated with cells transitioning through S/G(2)/M phases of the cell cycle. Treatment Ki67p-GFP expressing HT1080 cells with mitomycin C, an antineoplastic agent, induces P21 and P27 expression, G(1)/S/G(2)M block and attenuates Ki67p activity. Attenuation of the Ki67p also occurs during cell-density induced cell cycle arrest. Taken together, these results indicate that the Ki67p can be used to identify proliferating subpopulations of live cells in intact complex three-dimensional cellular aggregates, such as embryoid bodies, thus providing some unique advantages over conventional immunohistochemical approaches.