JNK inhibitor SP600125 promotes the formation of polymerized tubulin, leading to G2/M phase arrest, endoreduplication, and delayed apoptosis

Dong-Oh Moon,Chang-Hee Kang,Gi-Young Kim,Jae-Dong Lee,Yung Hyun Choi,Mun-Ock Kim

doi:10.3858/emm.2009.41.9.073

Abstract

The JNK inhibitor SP600125 strongly inhibits cell proliferation in many human cancer cells by blocking cell-cycle progression and inducing apoptosis. Despite extensive study, the mechanism by which SP600125 inhibits mitosis-related effects in human leukemia cells remains unclear. We investigated the effects of SP600125 on the inhibition of cell proliferation and the cell cycle, and on microtubule dynamics in vivo and in vitro. Treatment of synchronized leukemia cells with varying concentrations of SP600125 results in significant G2/M cell cycle arrest with elevated p21 levels, phosphorylation of histone H3 within 24 h, and endoreduplication with elevated Cdk2 protein levels after 48 h. SP600125 also induces significant abnormal microtubule dynamics in vivo. High concentrations of SP600125 (200 microM) were required to disorganize microtubule polymerization in vitro. Additionally, SP600125- induced delayed apoptosis and cell death was accompanied by significant poly ADP-ribose polymerase (PARP) cleavage and caspase-3 activity in the late phase (at 72 h). Endoreduplication showed a greater increase in ectopic Bcl-2-expressing U937 cells at 72 h than in wild-type U937 cells without delayed apoptosis. These results indicate that Bcl-2 suppresses apoptosis and SP600125-induced G2/M arrest and endoreduplication. Therefore, we suggest that SP600125 induces mitotic arrest by inducing abnormal spindle microtubule dynamics.

Highlights

Cell cycle progression to the G1, S, and G2/M phases is controlled by cell cycle checkpoints that ensure the correct order and transition timing (Elledge, 1996)
SP600125 has been implicated in G2/M arrest and apoptosis, but its precise role remains unknown (Potapova et al, 2000; Hideshima et al, 2003; Du et al, 2004; Jacobs-Helber and Sawyer, 2004; Mingo-Sion et al, 2004)
The present study provides the first mechanism to explain the induction of G2/M arrest, endoreduplication, and delayed apoptosis caused by SP600125 in leukemia cells

Summary

Introduction

Cell cycle progression to the G1, S, and G2/M phases is controlled by cell cycle checkpoints that ensure the correct order and transition timing (Elledge, 1996). The spindle assembly checkpoint monitors the segregation of sister chromatids, inhibiting the onset of anaphase until all of the chromosomes are properly attached to the mitotic spindle apparatus (Shah and Cleveland, 2000). It has been shown that there are three main types of agent that induce endoreduplication (Cortes et al, 2004). The first type includes agents that interfere with cytoskeleton assembly (i.e. microtubule inhibitors), e.g., colchicines and colcemid. The third type comprises physical and chemical agents that damage DNA, such as X-rays. Some of these microtubule-interfering agents, such as nocodazole and paclitaxel, induce significant endoreduplication because sister chromatid segregation is interrupted (Andreassen et al, 1996; Jordan et al, 2003).

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