Blockade of the Extracellular Signal-regulated Kinase Pathway Induces Marked G1 Cell Cycle Arrest and Apoptosis in Tumor Cells in Which the Pathway Is Constitutively Activated

Abstract

The 41-/43-kDa mitogen-activated protein (MAP) 1 kinase pathway, also called the extracellular signal-regulated kinase (ERK) pathway, is activated in a variety of cell types by diverse extracellular stimuli and is among the most thoroughly studied of signaling pathways that connect different membrane receptors to the nucleus (1, 2). Activation of the ERK pathway involves the activation of Ras at the plasma membrane, and the sequential activation of a series of protein kinases. Initially, Ras interacts with and activates Raf-1, which in turn activates MAP kinase/ERK kinase (MEK)-1 and -2 by serine phosphorylation. MEK-1/2 then catalyze the phosphorylation of 41-and 43-kDa MAP kinases (ERK2 and ERK1, respectively) on tyrosine and threonine residues, and these activated MAP kinases can phosphorylate cytoplasmic and nuclear targets. The ERK pathway participates in a wide range of cellular programs including proliferation, differentiation, and movement (1, 2).Aberrant activation of signal transducing proteins has been linked with cancer. For example, constitutively active mutants of Ras (3) and Raf-1 (4) have been observed in several human tumors, and constitutively active mutants of MEK-1 have been shown to transform mammalian cells (5, 6). We recently examined whether constitutive activation of the ERK pathway is associated with the neoplastic phenotype of human tumor cells. Constitutive activation of ERKs and MEK was observed in a relatively large number of tumors; tumor cells derived from pancreas, colon, lung, ovary, and kidney tissues showed especially high frequencies (30 -50%) and a high degree of kinase activation (7,8). Activation of the ERKs is also associated with prostate cancer progression (9). The precise cause of constitutive activation of the ERK pathway in many of these tumor cells remains unclear. However, such high frequencies of ERK/ MEK activation in human tumors indicate that specific inhibitors might be developed against these protein kinases for cancer therapy, especially for treatment of tumors showing constitutive activation of the ERK pathway.In the present study, we have examined the effect of blockade of the ERK pathway on the proliferation of human tumor cells. We utilized small molecule inhibitors of this pathway, PD98059 (10) and U0126 (11), which specifically inhibit MEK activity. Our results demonstrate that these MEK inhibitors induce a remarkable G 1 cell cycle arrest, followed by a modest apoptotic response, in tumor cells in which the ERK pathway is constitutively activated. Up-regulation of the CDK inhibitor p27Kip1 was observed in these G 1 -arrested tumor cells.

Role of the pro-survival molecule Bfl-1 in melanoma

“…In all our cell-lines I observed a marked arrest in the G1 phase of the cell cycle, sometimes leading to cell death. This agrees with previous findings that the blockade of the MAPK pathway results in G1 arrest and apoptosis in tumours which had an activating mutation in the pathway 468 .…”

Blockade of constitutively activated ERK signaling enhances cytotoxicity of microtubule-destabilizing agents in tumor cells

“…Eagle's medium supplemented with 10% fetal bovine serum [4]. Cells exposed to various reagents were harvested by treatment with trypsin, fixed with 70% ethanol, treated with DNase-free RNase A (100 µg/ml, Sigma), stained with propidium iodide (20 µg/ml), and analyzed for DNA content with the use of a FACSCalibur flow cytometer and Cell Quest Pro software (Becton Dickinson) [10].…”

“…Cell lysates were prepared as described previously [10,11] and fractionated by SDS-polyacrylamide gel electrophoresis, and the separated proteins were transferred to a polyvinylidene difluoride membrane and probed with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Promega).…”

“…We have previously shown that specific blockade of the ERK pathway by MEK inhibitors results in marked suppression not only of the proliferation [10] but also of the invasiveness [11] of tumor cells in which the pathway is constitutively activated.…”

“…However, blockade of the ERK pathway by itself was found to be largely cytostatic, rather than cytotoxic, resulting in only a moderate induction of apoptosis in these tumor cells [10]. Efficient induction of apoptotic cell death is essential for the development of effective cancer chemotherapy [9].…”