doi:10.1016/j.mrfmmm.2008.06.001 
Copyright © 2008 Elsevier B.V. All rights reserved.
Two structurally distinct inhibitors of glycogen synthase kinase 3 induced centromere positive micronuclei in human lymphoblastoid TK6 cells
Masayuki Mishima
, a, 
, Kenji Tanakaa, Akira Takeiria, Asako Haradaa, Chiyomi Kuboa, Sachiko Sonea, Yoshikazu Nishimuraa, Yukako Tachibanaa and Makoto Okazakia
aFuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
Received 20 February 2008;
revised 26 May 2008;
accepted 3 June 2008.
Available online 12 June 2008.
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Abstract
Glycogen synthase kinase 3 (GSK3) is an attractive novel pharmacological target. Inhibition of GSK3 is recently regarded as one of the viable approaches to therapy for Alzheimer's disease, cancer, diabetes mellitus, osteoporosis, and bipolar mood disorder. Here, we have investigated the aneugenic potential of two potent and highly specific inhibitors of GSK3 by using an in vitro micronucleus test with human lymphoblastoid TK6 cells. One inhibitor was a newly synthesized maleimide derivative and the other was a previously known aminopyrimidine derivative. Both compounds elicited statistically significant and concentration-dependent increases in micronucleated cells. One hundred micronuclei (MN) of each were analyzed using centromeric DNA staining with fluorescence in situ hybridization. Both the two structurally distinct compounds induced centromere-positive micronuclei (CMN). Calculated from the frequency of MN cells and the percentage of CMN, CMN cell incidence after treatment with the maleimide compound at 1.2 μM, 2.4 μM, and 4.8 μM was 11.6, 27.7, and 56.3 per 1000 cells, respectively; the negative control was 4.5. CMN cell incidence after the treatment with the aminopyrimidine compound at 1.8 μM, 3.6 μM, and 5.4 μM was 6.7, 9.8 and 17.2 per 1000 cells, respectively. Both compounds exhibited concentration-dependent increase in the number of mitotic cells. The frequency of CMN cells correlated well with mitotic cell incidence after treatment with either compound. Furthermore, both inhibitors induced abnormal mitotic cells with asymmetric mitotic spindles and lagging anaphase chromosomes. These results lend further support to the hypothesis that the inhibition of GSK3 activity affects microtubule function and exhibits an aneugenic mode of action.
Keywords: Glycogen synthase kinase 3; Inhibitor; FISH; Micronucleus test; Aneugen
Fig. 1. Chemical structures of CH3310395 and CHIR98014 [11].
Fig. 2. Inhibition of GSK3 and CDK1 activity by CH3310395 in cell free system. Closed and open circles respectively represent inhibition of GSK3 and CDK1 at various concentrations. CH3310395 reached to maximum inhibition to GSK3 at 0.1 μM where no inhibition was seen to CDK1.
Fig. 3. Western blot analysis for actin (A) and β-catenin (B) after treatment with CH3310395 or CHIR98014. Each lane contained cell lysate corresponding to 6 × 104 TK6 cells. Protein bands were recognized by anti-β-catenin or anti-actin antibodies and visualized with alkaline phosphatase-conjugated secondary antibody and staining substrate.
Fig. 4. FISH analysis using a FITC-conjugated pancentromeric DNA probe on TK6 cells. Cells were counterstained with PI. (A) FITC foci on centromere of chromosomes in metaphase cell. (B) Centromere-positive micronucleus with FITC signal. (C) Centromere-negative micronucleus without FITC signal. Cells were observed under fluorescence microscopy at ×1000 magnitude.
Fig. 5. Concentration response of β-catenin accumulation and MN induction after treatment with CH3310395 and CHIR98014. Bars represent mean fluorescent intensities of β-catenin from FCM analysis on 20,000 cells and diamonds represent frequencies of MN cells from microscopic observation of 1000 cells. Significance of increase in MN frequency was indicated; *p < 0.05, **p < 0.01 (Fischer's exact test).
Fig. 6. Metaphase and anaphase of TK6 cells after treatment withCH3310395. Cells were stained with FITC-conjugated anti-α-tubulin antibody and Hoechst-33258 and observed under confocal microscopy. An asymmetric spindle structure was seen in metaphase treated with CH3310395. Arrowheads indicate chromosomes that dropped out from two major assemblies in late anaphase.
Fig. 7. Alignment of amino acid repeats at the tubulin-binding region between human tau and MAP4. Sequences of tau correspond to the first, second and third repeats of the four repeating domains of tau protein. Boldface S represents the phosphorylation site of tau by GSK3.
Table 1.
Inhibition of kinases with CH3310395
Table 2.
Induction of kinetochore positive micronuclei with GSK3 inhibitors
Abbreviations: Relative cell count, RCC; population doubling, PD; micro nuclei, MN; centromere-positive MN, CMN; non-centromeric MN, NMN; dimethyl sulfoxide, DMSO; methylmethanesulfonate, MMS; not examined, NE.
1) Calculated from a) and b) at each treatment; CMN = a × 10 × b/100, NMN = a × 10-CMN.
*p < 0.05, **p < 0.01, Fischer's exact test (MN cells and mitotic cells), Chi square test (FISH analysis) and Cochran–Armitage trend test (when significant difference was seen at any concentration).
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