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Novel form of p21(WAF1/CIP1/SDI1) protein in phorbol ester-induced G2/M arrest
W W Tchou1, W N Rom, K M Tchou-Wong
1Division of Hematology, New York University Medical Center, New York, New York 10016, USA.
Abstract:
Cell cycle progression requires activation of different cyclin-dependent kinases (CDKs) which are positively regulated by cyclins and negatively regulated by CDK inhibitors. Growth inhibition of the Calu-1 lung carcinoma cells induced with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, is associated with G2/M arrest and induction of expression of a novel, faster-migrating form of p21(WAF1/CIP1/SDI1) (p21) protein, an inhibitor of cyclin-dependent kinases. This faster-migrating p21 protein was also expressed in TPA-treated A549 lung carcinoma cells which also exhibited G2/M arrest but not in TPA-treated U937 leukemia cells, which only expressed a slower-migrating form of p21 protein. However, reverse transcriptase-polymerase chain reaction and Southern analysis demonstrated no evidence of novel splice in TPA-treated Calu-1 cells. On the other hand, immunoblotting analysis demonstrated that the faster-migrating p21 protein could be detected only by peptide antibody directed against the N terminus but not the C terminus, suggestive of truncation of the latter or protein modification that results in the loss of the C-terminal epitope. Correlation of G2/M arrest with expression of the faster-migrating p21 protein suggests that this novel form of p21 protein may be a mediator of G2/M arrest and growth inhibition.
Insights
A novel, faster-migrating p21 protein induces G2/M arrest and growth inhibition in lung carcinoma cells. This modified p21 protein, identified in TPA-treated Calu-1 and A549 cells, may mediate cell cycle arrest.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Cell cycle progression relies on cyclin-dependent kinases (CDKs), regulated by cyclins and CDK inhibitors.
- Phorbol ester (TPA) induces growth inhibition and G2/M arrest in Calu-1 lung carcinoma cells.
- A novel, faster-migrating form of p21(WAF1/CIP1/SDI1) (p21) protein is induced during TPA-mediated growth inhibition.
Purpose of the Study:
- To investigate the role of a novel p21 protein variant in TPA-induced cell cycle arrest and growth inhibition.
- To characterize the molecular nature of the faster-migrating p21 protein.
- To determine if this novel p21 form is responsible for G2/M arrest in lung carcinoma cells.
Main Methods:
- Treatment of Calu-1, A549, and U937 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA).
- Analysis of cell cycle progression using flow cytometry (implied by G2/M arrest).
- Detection and characterization of p21 protein expression using immunoblotting with specific antibodies and RT-PCR/Southern analysis for splicing.
Main Results:
- TPA treatment induced G2/M arrest and a faster-migrating p21 protein in Calu-1 and A549 cells, but not U937 cells.
- The faster-migrating p21 protein was detected by N-terminal antibodies but not C-terminal ones, suggesting C-terminal modification or truncation.
- No evidence of novel splicing was found in TPA-treated Calu-1 cells, indicating post-translational modification.
Conclusions:
- A novel, potentially truncated or modified, faster-migrating p21 protein is associated with TPA-induced G2/M arrest and growth inhibition in lung carcinoma cells.
- This modified p21 protein is a likely mediator of the observed cell cycle arrest and growth inhibition.
- The findings suggest post-translational modification of p21 plays a role in regulating cell cycle progression in cancer cells.