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Mitogen-activated protein kinases control p27/Kip1 expression and growth of human melanoma cells
M Kortylewski1, P C Heinrich, M E Kauffmann
1Department of Biochemistry, RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany.
Abstract:
The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated protein kinase (ERK)1 and ERK2, involved in regulating cell growth and differentiation, are constitutively active in A375 and WM239 human melanoma cells. Using PD098059, an inhibitor of MAPK kinase (MEK), we investigated the role of persistently activated ERK1/2 in cell growth. The inhibition of MAPK activity induced a dose-dependent growth arrest in G(0)/G(1) phase. Correspondingly, we observed the up-regulation of the cyclin-dependent kinase (Cdk) inhibitor p27/Kip1 and hypophosphorylation of the retinoblastoma protein. Further studies showed that PD098059 treatment significantly decreased Cdk2 kinase activity, most probably owing to an augmented level of p27/Kip1 associated with cyclin E-Cdk2 complexes. The accumulation of p27/Kip1 protein in A375 cells was attributed to its increased stability. Our findings suggest that constitutively active ERK1/2 kinases contribute to the growth of melanoma cells by negative regulation of the p27/Kip1 inhibitor.
Insights
Constitutively active mitogen-activated protein kinases (MAPKs) drive melanoma cell growth. Inhibiting these kinases with PD098059 causes cell cycle arrest by increasing the p27/Kip1 inhibitor levels.
Area of Science:
- Cell Biology
- Molecular Oncology
- Cancer Research
Background:
- Mitogen-activated protein kinases (MAPKs), specifically extracellular signal-regulated protein kinases (ERK)1 and ERK2, are crucial for cell growth and differentiation.
- Constitutive activation of ERK1/2 is observed in human melanoma cell lines (A375 and WM239).
Purpose of the Study:
- To investigate the role of persistently activated ERK1/2 in melanoma cell proliferation.
- To understand the molecular mechanisms by which ERK1/2 influences cell cycle progression.
Main Methods:
- Utilized PD098059, a specific inhibitor of MAPK kinase (MEK), to block ERK1/2 activity.
- Assessed cell cycle progression using flow cytometry (G0/G1 arrest).
- Quantified protein levels and activity of cell cycle regulators, including p27/Kip1, retinoblastoma protein, and Cdk2/cyclin E complexes.
Main Results:
- PD098059 treatment induced a dose-dependent cell growth arrest in the G0/G1 phase.
- Inhibition of MAPK activity led to the upregulation of the cyclin-dependent kinase (Cdk) inhibitor p27/Kip1.
- PD098059 decreased Cdk2 kinase activity, associated with increased p27/Kip1 levels and hypophosphorylation of the retinoblastoma protein.
- The accumulation of p27/Kip1 was attributed to enhanced protein stability.
Conclusions:
- Constitutively active ERK1/2 signaling promotes melanoma cell growth.
- The persistent activation of ERK1/2 negatively regulates the p27/Kip1 inhibitor, contributing to uncontrolled proliferation.
- Targeting MEK/ERK pathway represents a potential therapeutic strategy for melanoma.