Rb Independent Inhibition of Cell Growth by p15INK4B

Abstract

The INK4 cyclin dependent kinase inhibitors (CDKI), such as p15INK4B and p16INK4A, block cell cycle progression from G to S phase. This is mediated by inhibition of phosphorylation of proteins, including the retinoblastoma susceptibility protein (Rb), by cyclin dependent kinases. Ectopic over-expression of the p16INK4A CDKI can inhibit growth of cell lines depending on Rb status. Cell lines lacking Rb, with few exceptions, are resistant to growth inhibition by p16INK4A. The effects of ectopic over-expression of p15INK4B in cell lines with and without wild type Rb were examined by measuring cell recovery. Proliferation was inhibited in cells lacking Rb as well as in cells with wild type Rb expression. Experiments analyzing the effectiveness of chimeric p15INK4B/p16INK4A proteins indicated that the Rb independent growth inhibition required N-terminal residues of p15INK4B. Linker insertion mutation of p15INK4B showed that the inhibition was dependent on intact ankyrin structures. Double staining flow cytometry found that the growth inhibition correlated with a decrease in cells in G2/M phases of the cell cycle. These findings are consistent with Rb independent inhibition of the progression from G1 to S caused by overexpression of p15INK4B.