Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway

Abstract

The mitogen-activated protein kinase (MAPK) cascade plays a crucial role in the transduction of extracellular signals into responses governing growth and differentiation. The effects of a specific inhibitor of the MAPK kinase (MEK)/MAPK pathway (PD98059) on nerve growth factor (NGF)-induced growth arrest and inhibition of cell cycle-dependent kinases (CDKs) have been examined. Treatment of NIH 3T3 cells expressing TRKA with PD98059 dramatically reversed the complete inhibition of growth of these cells caused by NGF. PD98059 also blocked the ability of NGF to inhibit the activities of CDK4 and CDK2, while partially preventing NGF induction of p21Cip1/WAF1. To independently evaluate the involvement of the MEK/MAPK pathway in growth arrest, an inducible activated form of the Raf-1 protooncogene (delta RAF-1:ER) was expressed in these cells. Activation of delta RAF-1:ER resulted in a prolonged increase in MAPK activity and growth arrest of these cells, with concomitant induction of p21Cip1/WAF1 and inhibition of CDK2 activity. These effects of delta RAF-1:ER activation were all reversed by treatment of cells with PD98059. These data indicate that in addition to functioning as a positive effector of growth, stimulation of the MEK/MAPK pathway can result in an inhibition of CDK activity and cell cycle arrest.

Figure 1

Reversal of NGF-mediated growth arrest by the MEK-specific inhibitor PD98059. (A) Treatment with PD98059 produces a dose-dependent reversal of the growth arrest induced by NGF treatment. TRK1 cells were grown for 4 days in normal growth medium (▪) or growth medium supplemented with 20 nM NGF added (□). Vehicle [dimethyl sulfoxide (DMSO), 0.2%] or increasing concentrations of PD98059 were included throughout the 4-day incubation as indicated. Cell numbers represent the average of duplicate determinations. Data are representative of two independent experiments. (B) PD98059 blocks NGF stimulation of MAPK activity. Cells were grown in complete growth medium in the presence or absence of 20 nM NGF (as indicated) for 16 hr following addition of vehicle (DMSO, 0.2%) or 40 μM PD98059.

Figure 2

MEK inhibitor PD09059 prevents inhibition of CDK by NGF. TRK1 cells were grown for 16 hr in normal growth medium in the presence or absence of 20 nM NGF. Simultaneous to the addition of NGF, cells were treated with vehicle (DMSO, 0.2%) or 40 μM PD98059. The activity of the CDKs was assayed by immunoprecipitation kinase assay (12), using histone H1 (A) or glutathione S-transferase-Rb (aa 379–928) (B) as substrates. Similar results were obtained in at least four independent experiments.

Figure 3

Effects of the MEK inhibitor, PD98059, on NGF-mediated p21^Cip1/WAF1 induction and changes in cyclin expression. Levels of p21^Cip1/WAF1 (A) or cyclin proteins involved in the activation of CDK2 and CDK4 (B) were analyzed by Western blot analysis of whole cell lysates (20) following treatment with NGF (20 nM) or with a combination of NGF and the MEK inhibitor, PD98059 (40 μM) for 16 hr. Similar results were found in at least three independent experiments.

Figure 4

Activation of the MEK/MAPK pathway by ΔRaf-1:ER results in p21^Cip1/WAF1 induction, inhibition of CDK2 activity, and growth arrest. (A) TRK:ER cells were analyzed for the expression of ΔRaf-1:ER protein by blotting with anti-estrogen receptor antibody. (B–D) TRK:ER cells were pretreated for 5 min with either DMSO (0.1%) or PD98059 (30 μM) prior to addition of vehicle (0.1% ethanol) or 1 μM 17-β-estradiol for 16 hr. Cells were then analyzed for MAPK activity (B), whole cell levels of p21^Cip1/WAF1 (C), and CDK2 activity (D) as described. (E) TRK:ER cells were seeded at 1 × 10⁴ cells per dish and pretreated for 5 min with either vehicle (0.1% DMSO) or 30 μM PD98059, prior to the addition of 0.1% ethanol or 1 μM 17-β-estradiol for 4 days. Cells were counted in duplicate using a hemocytometer. For all data shown, similar results were obtained in at least three independent experiments.