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. 2006 Jul 11;103(28):10660-5.
doi: 10.1073/pnas.0600447103. Epub 2006 Jul 3.

Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1

Lyubomir T Vassilev  1 , Christian Tovar, Shaoqing Chen, Dejan Knezevic, Xiaolan Zhao, Hongmao Sun, David C Heimbrook, Li Chen
Affiliations

Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1

Lyubomir T Vassilev et al. Proc Natl Acad Sci U S A. .

Abstract

CDK1 is a nonredundant cyclin-dependent kinase (CDK) with an essential role in mitosis, but its multiple functions still are poorly understood at a molecular level. Here we identify a selective small-molecule inhibitor of CDK1 that reversibly arrests human cells at the G(2)/M border of the cell cycle and allows for effective cell synchronization in early mitosis. Inhibition of CDK1 during cell division revealed that its activity is necessary and sufficient for maintaining the mitotic state of the cells, preventing replication origin licensing and premature cytokinesis. Although CDK1 inhibition for up to 24 h is well tolerated, longer exposure to the inhibitor induces apoptosis in tumor cells, suggesting that selective CDK1 inhibitors may have utility in cancer therapy.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
RO-3306 is a selective CDK1 inhibitor. (A) Chemical structure of RO-3306. (B) Activity of RO-3306 against CDK1/cyclin B1, CDK2/cyclin E, and CDK4/cyclin D. (C) Activity of RO-3306 against CDK1 complexes with cyclin A or cyclin B1. (D) A presumed model of RO-3306 binding at the ATP pocket of CDK2 using computation data and structure coordinates. (E) Activity of RO-3306 against eight diverse kinases measured by the IMAP assay.
Fig. 2.
Fig. 2.
RO-3306 reversibly arrests cells at the G2/M phase border. (A) Cell cycle profile of proliferating human cells (HCT116, SW480, and HeLa) treated with solvent (Upper) or RO-3306 (9 μM) (Lower) for 20 h. (B) Mitotic spreads of RO-3306-arrested HCT116 cells treated as above. (C) Cell transition through the G1 and S phase in the presence of RO-3306. HeLa cells were synchronized in mitosis by a shake-off after 14 h of incubation in 100 nM nocodazole, washed, and allowed to transition through the G1 and S phase in the presence or absence of RO-3306 (9 μM). Cells were collected at the indicated times after release from nocodazole block, and total pRB, phospho-pRB, and cyclins E, A, and B1 were analyzed by Western blotting. (D) Kinetics of S phase entry in the presence of RO-3306. HeLa cells synchronized in mitosis by nocodazole as above were allowed to transition through the G1 phase in the presence or absence of RO-3306 (9 μM), and the number of S phase cells was determined by flow cytometry of BrdU-labeled cells. (E) Cell cycle distribution of HeLa cells treated as above. Cells were analyzed 3 h after release from nocodazole block (c), 21 h after readdition of 100 nM nocodazole at 3 h after release from nocodazole block (noc), or 24 h after release from nocodazole block in the presence of 9 μM RO-3306 (3306). (F) Cells synchronously enter mitosis after release from RO-3306 block. HCT116 cells were arrested by RO-3306 (9 μM for 20 h), washed, and incubated in fresh prewarmed medium. Mitotic spreads were prepared at the indicated times and used to calculate mitotic index. Inset shows mitotic spreads from a 30-min sample. (G) RO-3306-synchronized cells transition normally through mitosis. Cell cycle profile of HCT116 cells 1 and 6 h after release from a 20-h RO-3306 block compared with untreated (C) and RO-3306-arrested cells. (H) Western blot analysis of cyclin B1 and phospho-histone H3 in HCT116 cells after release from RO-3306 block.
Fig. 3.
Fig. 3.
CDK1 activity is critical for maintaining the mitotic state. (A) Nocodazole-arrested cells rapidly exit mitosis in the presence of RO-3306. HeLa cells arrested in prometaphase by nocodazole treatment (16 h) were followed by time-lapse photography after the addition of 9 μM RO-3306. (B) Inhibition of CDK1 activity in nocodazole-arrested mitotic cells triggers chromosome decondensation and nuclei reformation. Purified population of nocodazole-arrested metaphase cells was collected by shake-off and treated with RO-3306 (9 μM). Mitotic spreads were prepared at different times and stained with Giemsa. (C) HeLa cells were treated as above but stained for DNA with Hoechst 33258. (D) Western blot analysis of phospho-H3 and phospho-pRB during RO-3306-induced exit from mitosis. (E) Inhibition of cyclin B1 degradation does not prevent mitotic exit in RO-3306-treated HeLa cells. Purified nocodazole-arrested metaphase cells were treated with solvent (a and b) or 10 μM MG132 (c and d) for 30 min followed by the addition of 9 μM RO-3306 for 2 h (b and d). (F) Western blot analysis of cyclin B1 and phospho-H3 in the cells treated as above. (Scale bars: 20 μm.)
Fig. 4.
Fig. 4.
CDK1 activity is essential for prevention of replication origin licensing and cytokinesis before completion of mitosis. (A) CDK1 inhibition in nocodazole-arrested mitotic cells resets them for a new replication cycle without cell division. Prometaphase HeLa cells purified from nocodazole-arrested population by a shake-off were divided into two fractions and either washed and incubated in drug-free medium to allow transition through the G1 phase or incubated in the presence of nocodazole (100 nM) and RO-3306 (9 μM). Cells were harvested at different times, lysed, fractionated into chromatin and soluble fractions, and analyzed for Cdc6, Orc2, Mcm2, cyclin B1, and phospho-histone H3 by Western blotting. (B) HeLa cells were treated as above in the presence of nocodazole and RO-3306, and the S phase cells were labeled by BrdU-immunostaining. Inset shows a representative image (green BrdU-labeled cells) taken at 18 h under fluorescence (FITC filter) and phase contrast. (C) Cell cycle analysis of HeLa cells treated as in B at different times after the addition of 9 μM RO-3306 in the presence of 100 nM nocodazole.
Fig. 5.
Fig. 5.
Inhibition of CDK1 during mitosis causes premature cytokinesis. HeLa cells were enriched in mitotic cells by release from RO-3306 block (9 μM for 18 h) and followed for morphological changes in the absence (Top) or presence of 9 μM RO-3306 (Middle and Bottom). Cells were fixed and immunostained for β-tubulin (red) and DNA by Hoechst 33258 (blue). Images were captured by using a Nikon Eclipse TE2000-U inverted microscope with a CF1 DAPI/FITC/tetramethylrhodamine B isothiocyanate fluorescence filter cube and phase contrast under low light. (Scale bar: 20 μm.)
Fig. 6.
Fig. 6.
RO-3306 induces apoptosis in cancer cells. (A) Exponentially growing SW480 cells were treated with 4 μM RO-3306 for 48 h and stained with propidium iodide (red, dead cells) and acridine orange (green, live cells). (Scale bar: 50 μm.) (B) Proliferating HCT116, SW480, MCF10A, and MCF12A cells were treated with 9 μM RO-3306, and the percentage of Annexin V-positive (apoptotic) cells minus background was determined by flow cytometry.
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