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Inhibition of pRb phosphorylation and cell-cycle progression by a 20-residue peptide derived from p16CDKN2/INK4A
R Fåhraeus1, J M Paramio, K L Ball
1Cell Transformation Group, University of Dundee, UK. RFAHRAES/bad.dundee.ac.uk
Background:
The CDKN2/INK4A tumour suppressor gene is deleted or mutated in a large number of human cancers. Overexpression of its product, p16, has been shown to block the transition through the G1/S phase of the cell cycle in a pRb-dependent fashion by inhibiting the cyclin D-dependent kinases cdk4 and cdk6. Reconstitution of p16 function in transformed cells is therefore an attractive target for anti-cancer drug design.
Results:
We have identified a 20-residue synthetic peptide--corresponding to amino acids 84-103 of p16--that interacts with cdk4 and cdk6, and inhibits the in vitro phosphorylation of pRb mediated by cdk4-cyclin D1. The amino-acid residues of p16 important for its interaction with cdk4 and cdk6 and for the inhibition of pRb phosphorylation were defined by an alanine substitution series of peptides. In normal proliferating human HaCaT cells and in cells released from serum starvation, entry into S phase was blocked by the p16-derived peptide when it was coupled to a small peptide carrier molecule and applied directly to the tissue culture medium. This cell-cycle block was associated with an inhibition of pRb phosphorylation in vivo.
Conclusions:
These results demonstrate that a p16-derived peptide can mediate three of the known functions of p16: firstly, it interacts with cdk4 and cdk6; secondly, it inhibits pRb phosphorylation in vitro and in vivo; and thirdly, it blocks entry into S phase. The fact that one small synthetic peptide can enter the cells directly from the tissue culture medium to inhibit pRb phosphorylation and block cell-cycle progression makes this an attractive approach for future peptidometic drug design. Our results suggest a novel and exciting means by which the function of the p16 suppressor gene can be restored in human tumours.
Insights
A synthetic p16 peptide inhibits cell cycle progression by blocking cdk4/cdk6 activity, offering a potential anti-cancer therapy. This peptide restores tumor suppressor gene function by inhibiting pRb phosphorylation.
Area of Science:
- Molecular Biology
- Cancer Research
- Drug Discovery
Background:
- The CDKN2/INK4A tumor suppressor gene (p16) is frequently altered in human cancers.
- p16 inhibits cell cycle progression by targeting cyclin-dependent kinases cdk4 and cdk6.
- Restoring p16 function is a promising anti-cancer strategy.
Purpose of the Study:
- To identify a peptide-based therapeutic targeting p16's tumor suppressor function.
- To investigate the ability of a p16-derived peptide to inhibit cell cycle progression.
Main Methods:
- A 20-residue synthetic peptide from p16 (amino acids 84-103) was designed.
- Alanine scanning mutagenesis defined key residues for cdk4/cdk6 interaction and pRb inhibition.
- Peptide efficacy was tested in human HaCaT cells and serum-starved cells.
Main Results:
- The p16 peptide specifically bound and inhibited cdk4 and cdk6 in vitro.
- Inhibition of pRb phosphorylation was observed both in vitro and in vivo.
- The peptide blocked cell-cycle entry into S phase in normal human cells.
Conclusions:
- A synthetic p16 peptide mimics key functions of the native tumor suppressor.
- The peptide inhibits cdk4/cdk6, pRb phosphorylation, and cell cycle progression.
- This p16 peptide represents a novel peptidometic approach for cancer therapy.