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Involvement of p27KIP1 in G1 arrest mediated by an anti-epidermal growth factor receptor monoclonal antibody
1Laboratory of Receptor Biology, Memorial Sloan-Kettering Cancer Center, NY, NY 10021, USA.
Abstract:
Activation of the cyclin dependent kinases (CDK4/CDK6 and CDK2) is required for G1 phase progression and entry into S-phase. The activation of these kinases is regulated by checkpoints that monitor environmental and intracellular conditions. Progression into S-phase is controlled, in part, by the availability of growth factors, and we have investigated the relationship between growth factor availability and the activation of the CDK kinases. Blocking activation of epidermal growth factor (EGF) receptor tyrosine kinase with anti-EGF receptor monoclonal antibody (mAb) 225 induces G1 phase cell cycle arrest in DiFi human colon adenocarcinoma cells. When DiFi cells are treated with mAb 225 for 24 h, we observe marked decreases in the activities of CDK2 kinase and cyclin E-associated CDK kinase which are not accompanied by reduced levels of cyclin E and CDK2 proteins. However, the amount of cyclin/CDK kinase inhibitor p27KIP1 increases in the mAb-treated cells and p27KIP1 is bound to CDK2 in increasing amounts. Immunodepletion of p27KIP1 removes an inhibitory activity from lysates of mAb-treated cells: the immunodepleted and heated lysates lose the capacity to inhibit cyclin E/CDK2 activity in an in vitro assay. The results suggest that G1 arrest in the cell cycle induced by EGF receptor blockade involves p27KIP1.
Insights
Blocking epidermal growth factor (EGF) receptor signaling halts cell cycle progression at G1 phase. This arrest is mediated by increased levels of the p27KIP1 protein, which inhibits cyclin-dependent kinases (CDKs).
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Cyclin-dependent kinases (CDKs) regulate cell cycle progression from G1 to S phase.
- Growth factor availability influences CDK activation and cell cycle entry.
- Epidermal growth factor (EGF) receptor signaling is crucial for cell proliferation.
Purpose of the Study:
- To investigate the relationship between growth factor availability and CDK kinase activation.
- To determine the mechanism by which EGF receptor blockade induces G1 cell cycle arrest.
Main Methods:
- Treatment of DiFi human colon adenocarcinoma cells with anti-EGF receptor monoclonal antibody (mAb) 225.
- Assay of CDK2 and cyclin E-associated CDK kinase activities.
- Analysis of cyclin E, CDK2, and p27KIP1 protein levels.
- Immunodepletion of p27KIP1 to assess its inhibitory role.
Main Results:
- mAb 225 treatment induced G1 phase cell cycle arrest.
- CDK2 and cyclin E-associated CDK kinase activities decreased without changes in protein levels.
- p27KIP1 protein levels increased and bound to CDK2.
- Immunodepletion of p27KIP1 restored kinase activity.
Conclusions:
- EGF receptor blockade leads to G1 cell cycle arrest in colon cancer cells.
- The G1 arrest is mediated by the cyclin-dependent kinase inhibitor p27KIP1.
- p27KIP1 plays a critical role in regulating cell cycle progression downstream of EGF receptor signaling.