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. Author manuscript; available in PMC: 2021 May 1.
Published in final edited form as: J Cell Physiol. 2019 Oct 17;235(5):4508–4519. doi: 10.1002/jcp.29328

Haspin inhibition delays cell cycle progression through interphase in cancer cells

Peiling Wang 1,2, Xiangmei Hua 2, Yuge Han Bryner 2, Sijing Liu 1, Christopher B Gitter 2, Jun Dai 1,2,*
PMCID: PMC7018545  NIHMSID: NIHMS1063347  PMID: 31625162

Abstract

Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) is a serine/threonine kinase pertinent to normal mitosis progression and mitotic phosphorylation of histone H3 at threonine 3 in mammalian cells. Different classes of small molecule inhibitors of haspin have been developed and utilized to investigate its mitotic functions. We report herein that applying haspin inhibitor CHR-6494 or 5-ITu at the G1/S boundary could delay mitotic entry in synchronized HeLa and U2OS cells, respectively, following an extended G2 or S phase. Moreover, late application of haspin inhibitors at S/G2 boundary is sufficient to delay mitotic onset in both cell lines, thereby indicating a direct effect of haspin on G2/M transition. A prolonged interphase duration is also observed with knockdown of haspin expression in synchronized and asynchronous cells. These results suggest that haspin can regulate cell cycle progression at multiple stages at both interphase and mitosis.

Keywords: Haspin, Kinase, Mitosis, Cell cycle

INTRODUCTION

Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) is a Ser/Thr protein kinase required for normal mitosis progression in mammalian cells (Dai et al., 2005). Depletion of haspin by small interfering RNAs (siRNAs) leads to chromosome misalignment, precocious loss of cohesion between sister chromatids, and formation of multipolar spindles (Dai et al., 2009; Dai et al., 2006; Dai et al., 2005). The best-characterized substrate of haspin is the threonine 3 residue of histone H3 (H3T3) (Dai et al., 2005; Nguyen et al., 2014; Panigada et al., 2013; Xie et al., 2015). In mammalian cells, this phosphorylation (H3T3ph) occurs from late G2 throughout anaphase of mitosis and is concentrated at the inner centromere region of mitotic chromosomes (Dai et al., 2006; Dai et al., 2005). In terms of functions, H3T3ph acts as a docking site for the chromosome passenger complex (CPC) at the centromere and ensures the proper kinetochore-microtubule attachment mediated by the Aurora B kinase (Wang et al., 2010). Haspin and H3T3ph are also critical for the maintenance of sister chromatid cohesion via interactions with heterochromatin HP1 and a number of other centromeric proteins (Liang et al., 2018; Yi et al., 2018). These findings substantiate haspin as a member of the select group of mitotic kinases that include cyclin-dependent kinases (Cdks), Aurora kinases, and Polo-like kinases (Plks) (Ma and Poon, 2011).

While possessing essential mitotic functions, the haspin expression is found throughout the entire cell cycle (Dai et al., 2005; Hindriksen et al., 2017). Previously, we have reported that myc- or pEGFP-tagged exogenous haspin is located exclusively in the nucleus at interphase and associated condensed chromosomes throughout mitosis (Dai et al., 2005). With a haspin-YFP knock-in system, Hindriksen et al. have confirmed similar expression patterns of endogenous haspin (Hindriksen et al., 2017). Haspin is an atypical protein kinase and possesses constitutive kinase activity (Eswaran et al., 2009; Villa et al., 2009). The activity of haspin towards H3T3 is enhanced at mitosis following sequential phosphorylations on multiple sites within its N-terminal domain (Ghenoiu et al., 2013). These phosphorylation events are triggered initially by cyclin B/Cdk1 and then by other mitotic kinases including Plk1 and Aurora B (Ghenoiu et al., 2013; Zhou et al., 2014). Although H3T3ph disappears during telophase, haspin maintains its association with mitotic chromosomes (Hindriksen et al., 2017). It remains unclear whether the unphosphorylated form of haspin is also functional and has substrates other than H3T3 at telophase and different stages of interphase. A recent proteomic analysis identified numerous potential haspin interacting proteins and substrates, and components of the spliceosome complex and regulatory factors for gene transcription were among those implicated (Maiolica et al., 2014). This critical analysis further validates the belief that haspin could have other functions as well as targets and play a vital role in regulating the progression of cell cycle outside mitosis.

Studies involving other mitotic kinases have been significantly advanced through the discovery of their specific inhibitors (Ditchfield et al., 2003; Lenart et al., 2007). Several selective haspin inhibitors with different scaffolds have recently been identified (Huertas et al., 2012; Kestav et al., 2017; Patnaik et al., 2008). Among them, the nucleotide mimetic inhibitor 5-iodotubercidin (5-ITu) has been found to possess a potent in vitro activity against haspin and based on the co-crystal structure, 5-ITu appears to compete for the ATP-binding site (Cuny et al., 2012). When applied directly to mitotic cells, 5-ITu downregulates nocodazole-stimulated Aurora B activities likely through inhibiting centromeric recruitment of the chromosome passenger complexes and overriding spindle checkpoint activation (De Antoni et al., 2012; Wang et al., 2012). The other potent haspin inhibitor CHR-6494 is an imidazolyl-pyridazine derivative and has shown anti-proliferative activities in multiple cancer cell lines as well as a mouse xenograft model (Han et al., 2017; Huertas et al., 2012). At the cellular level, CHR-6494 also triggers chromosome misalignment and the formation of multipolar spindles (Huertas et al., 2012). Mitotic defects caused by different haspin inhibitors closely resemble phenotypes observed with haspin siRNAs, and thus, these small molecule inhibitors provide an invaluable tool to explore cellular functions of haspin.

In this study, we intend to use haspin inhibitors to investigate further its functions throughout the entire cell cycle. We wish to report that application of haspin inhibitors at G1/S or S/G2 boundary delays mitosis entry in synchronized HeLa and U2OS cell lines. Moreover, similar effects were also observed with siRNA-mediated haspin gene depletion, thereby revealing essential functions of haspin in regulating cell cycle progression at interphase.

MATERIALS AND METHODS

Chemicals and reagents

CHR-6494, thymidine, and Paclitaxel (Taxol) were purchased from MilliporeSigma (St Louis, Missouri). 5-Iodotubercidin (5-Itu) was purchased from Aladdin (Shanghai, China). All compounds were dissolved in dimethyl sulfoxide (DMSO) [Corning-Thermo Fisher Scientific, Waltham, MA) as 1000-fold concentrated stock solutions, and 0.1% DMSO was used as vehicle controls.

Cell culture and synchronization

HeLa and U2OS cells were purchased from ATCC and cultured in the DMEM medium (MilliporeSigma) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL)/streptomycin (100 mg/mL) [Gibco-Thermo Fisher Scientific, Waltham, MA). FBS was from Capricorn (Germany) for Figure 1, 2 and Supplementary Figure S1, from Gibco-Thermo Fisher Scientific for other figures. All cells were maintained at 37°C with 5% CO2. HeLa cells were synchronized at G1/S boundary by two successive thymidine (2 mM) blocks, whereas U2OS cells were synchronized by a single thymidine (2.5 mM) block.

Figure 1.

Figure 1

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Haspin inhibitor CHR-6494 or 5-Itu delays mitotic entry in synchronized HeLa cells. (a) Timeline of cell cycle analysis with flow cytometry. HeLa cells were synchronized at G1/S boundary by double thymidine blocks and released into the medium containing DMSO, CHR-6494 (300 nM), or 5-Itu (450 nM). (b) Cells were fixed at indicated time points and stained with propidium iodide (PI) and the FITC-conjugate anti-phospho-MPM-2 antibody to monitor DNA content and mitotic cells, respectively. Data show flow cytometry profiles of the cell cycle distribution. (c) Quantification of the percentage of cells (from b) at each phase. Cells at M phase were gated as indicated. Data are the representative of 3 independent experiments.

Figure 2.

Figure 2

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Effects of haspin inhibition on the level of cell cycle markers in synchronized HeLa cells. HeLa cells were blocked at G1/S boundary and treated with haspin inhibitors right after thymidine release, as described in Figure 1a. Cells were harvested at indicated time points for protein extraction. (a) Immunoblotting analysis of phosphorylated histone H3, p-H3 (Thr3) and p-H3 (Ser10). (b) Immunoblotting analysis of cyclin A, cyclin B, phosphorylated p-Cdk1 (Tyr15), and unphosphorylated Cdk1. Data are the representative of 3 independent experiments.

SiRNA Transfection

Pre-designed human GSG2 (HASPIN) siRNA (#1093) and negative control siRNA (#4613) were from Ambion-Thermo Fisher. Cells were transfected with 40 nM of siRNA duplexes using Lipofectamine RNAiMax (Invitrogen-Thermo Fisher) according to the manufacturer’s instruction.

Cell cycle analysis

Cells were plated in 60-mm dishes at a density of 6 x105/dish. For cell cycle analysis, cells were fixed with ice-cold 70% ethanol and stained with 10 μg/mL of FITC-conjugated mouse anti-phospho-Ser/Thr-Pro MPM-2 antibody (MilliporeSigma) to label the mitotic cells. After MPM-2 staining, cells were incubated with 50 μg/mL of propidium iodide (PI) and 100 U/mL RNase A (MilliporeSigma). Cell cycle was analyzed by FACSCalibur flow cytometer (Becton Dickinson, San Diego, CA) for Figure 1 or by Attune Nxt Flow Cytometer (Thermo Fisher Scientific) for Supplementary Figure S2, S3 (with PI staining only). Cell populations were analyzed using the FlowJo software.

Immunoblotting analysis

Whole cell lysates were prepared with or SDS sample buffer or buffer L containing 50 mM Tris/0.5 M NaCl/1% Triton X-100/1% DOC/0.1% SDS/2 mMEDTA at pH 7.4 (Dai et al., 2005). Proteins were separated by SDS-PAGE and transferred onto the 0.45 μm PVDF membrane (Thermo Fisher). Antibodies against phospho-Histone H3 (Thr3) [04-746] and phospho-H2A.X (Ser139) [05-636] were purchased from MilliporeSigma; Antibodies for cyclin A (BM1582) was from Boster (China); Antibodies against phospho-Histone H3 (Ser10), Phospho-Cdk1 (Tyr15) [9111], phospho-Chk1 (Ser345) [2348], and phospho-Chk2 (Thr68) [2661] were from Cell Signaling Technology (Danvers, MA); Antibodies for cyclin B (sc-245), Cdk1 (sc-54), p53 (sc-126), and β-actin (sc-47778) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); antibodies against α-tubulin (T9026) and γ-tubulin (T5192) were from (MilliporeSigma). Chemiluminescence images were acquired with Amersham Imager 600 from GE Healthcare Life Sciences (Pittsburgh, PA) for Figure 2 and Supplementary Figure S1, or with iBright CL1000 (Thermo Fisher) for other figures. The level of target proteins was quantified by densitometry scanning with the ImageJ software and normalized to the amount of α-tubulin or β-actin.

Time-lapse imaging

U2OS cells were seeded in 24-well plates at a density of 7 x104 cells/well. After a 24 h single thymidine block, cells were washed and incubated with normal medium containing 0.1% DMSO or different concentrations of CHR-6494 (Figure 3). Alternatively, 600 nM of CHR-6494 was added to the medium at 7 h or 10 h after thymidine release (Figure 6a). At 12 h after release, 1 μM Taxol was added to the medium to arrest cells at mitosis, and cells were moved onto the stage for time-lapse imaging, using the Lionheart FX automated microscope (Biotek, Winooski, VT). The automated system is contained in a humidified chamber that was maintained at 37°C and supplied with 5% CO2. Bright field images were collected with a 10x/0.3 Plan Fluorite objective (Biotek) at a frequency of 1 frame per hour as 2 x 2 montages. The number of round shaped mitotic cells were counted using the Gen5 3.04 software (Biotek).

Figure 3.

Figure 3

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Haspin inhibitor CHR-6494 delays mitotic entry in synchronized U2OS cells. (a) U2OS cells were synchronized at G1/S boundary by a 24 h single thymidine block and released into the medium containing DMSO or different doses of CHR-6494. Starting from 12 h after thymidine release, time-lapse imaging was performed at a frequency of 1 frame/h in the presence of Taxol (1 μM) to monitor the accumulation of round shaped mitotic cells. Cumulative mitotic index was calculated by the Gen5 3.04 software and presented as mean percentage of cells ± SEM. ***, p < 0.001 (compared with the control of the respective time point), n=3 independent experiments. (b) Immunoblotting analysis of p-H3 (Thr3) and p-H3 (Ser10) of protein lysates from U2OS cells collected at indicated time points. (c) Immunoblotting analysis of indicated cell cycle markers. Relative protein levels (right panel) were quantified by densitometry scanning of target proteins on immunoblots with the ImageJ software and normalized to the amount of α-tubulin. Data are the representative of 3 independent experiments.

Figure 6.

Figure 6

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Haspin inhibitors applied at the late stage of interphase are sufficient to delay mitotic entry in U2OS cells. U2OS cells were synchronized at G1/S boundary by single thymidine block and treated with DMSO or 600 nM of CHR-6494 at 7 h or 10 h after thymidine release. (a) Mitotic cells were monitored by time-lapse imaging, starting from 12 h in the presence of Taxol, as described in Figure 3a. Cumulative mitotic index was calculated by the Gen5 3.04 software and presented as mean percentage of cells ± SEM. ***, p < 0.001 (compared with the control of the respective time point), n=3 independent experiments. (b) U2OS cells were treated with DMSO or 600 nM CHR-6494 at 10 h after thymidine release and harvested at 12 h or after an additional 7 h incubation with Taxol for protein analysis of indicated proteins.

For time-lapse imaging in Figure 7, HeLa cells were first transfected with H2B-GFP plasmid (Addgene, Cambridge, MA) using Lipofectamine 3000 (Invitrogen-Thermo Fisher) and selected with 2 mg/ml of G418 (MilliporeSigma) for two weeks. Colonies of cells with moderate GFP intensity were collected and transfected with 40 nM control siRNA and haspin siRNA using Lipofectamine RNAiMax. In Figure 7b, time-lapse imaging was performed in asynchronous cells at a frequency of 1 frame per hour between 30 h to 75 h after siRNA transfection, using the Lionheart FX automated microscope (Biotek). Cell cycle progression was examined in more than 200 individual cells from 2 independent experiments for each condition. The interphase duration was defined based on the morphology of histone H2B-GFP, as the gap between chromosome decondensation at the end of previous telophase and chromosome condensation at the beginning of mitosis of the new cell cycle. In Figure 7e, cells were pre-synchronized via the mitotic shake-off. Mitotic cells were collected and replated in 96-well plates. The duration of the first interphase was monitored using time-lapse imaging.

Figure 7.

Figure 7

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Haspin depletion with its siRNA prolongs interphase progression in asynchronous and synchronized HeLa cells. HeLa cells stably expressing histone H2B-GFP were transfected with control or haspin siRNA. (a) At 48 h after transfection, cells were fixed and stained for p-H3 (Thr3) [red]. Bar = 100 μm. (b) Interphase duration in asynchronous cells. Time-lapse imaging was performed at a frequency of 1 frame/h between 30 h to 75 h after siRNA transfection. The duration of interphases was examined in > 200 individual cells/group from two independent experiments and measured as the gap between chromosome decondensation at the end of the previous telophase and chromosome condensation at the beginning of new mitosis. (c) Average interphase duration (h) ± SEM. ***, p < 0.001, n = >200 cells/group from (b). (d) Cell proliferation assay. Fluorescent images of H2B-GFP were collected from the same fields as 2 x 2 montages between 30 h and 75 h after siRNA transfection. Cell numbers at indicated time points were calculated by the Gen5 3.04 software and normalized to that at 30 h. Data are presented as mean-fold over control ± SEM. **, p < 0.01, ***, p < 0.001, n=3 independent experiments. (e) Interphase duration in synchronized cells. The mitotic population of H2B-GFP expressing HeLa cells was collected at 48 h after siRNA transfection via mitotic shake-off and was replated for time-lapse imaging. The duration of the first interphase was examined in >150 cells/group from two independent experiments. The right panel shows the average interphase duration (h) ± SEM. ***, p < 0.001.

Fluorescence microscopy

HeLa cells were seeded onto coverslips. At the end of experiments, cells were fixed with 4% paraformaldehyde (PFA) in Phosphate-Buffered Saline (PBS) followed by 5 min fixation with methanol at −20°C. Cells were stained with the antibodies against Cyclin B (SC-245, Santa Cruz) and γ-tubulin (MilliporeSigma) [Figure 5a] or phospho-histone H3 (Thr3) [MilliporeSigma]. This was followed by staining with Alexa 488- or Alexa 594-conjugated secondary antibodies and Hoechst 33342 (Pierce Biotechnology-Thermo Fisher). Fluorescence images were acquired with the LIONHEART FX automated Microscope (Biotek). The number of cyclin B (green) positive cells at interphase and total cell numbers (based on Hoechst staining) were analyzed by the Gen5 3.04 software (Figure 5a). Cells at prophase, prometaphase/metaphase, or anaphase/telophase were examined with the ImageJ software.

Figure 5.

Figure 5

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Haspin inhibitors applied at the late stage of interphase are sufficient to delay mitotic entry in HeLa cells. HeLa cells were synchronized at the G1/S boundary, as described in Figure 1a. At 7.5 h after release, cells were treated with vehicle (DMSO), CHR-6494, or 5-Itu. (a) After 2 h or 3 h incubation (at 9.5 h or 10.5 h post thymidine release), cells were fixed and co-stained with antibodies against cyclin B (green) and γ-tubulin (red). DNA was counterstained with Hoechst 33342. Bar = 100 μm. The percentage of cyclin B positive cells at interphase was calculated by the Gen5 3.04 software. Different phases of mitotic cells were quantified manually based on the morphology of chromosomes using the ImageJ software. Data are the representative of 3 independent experiments. (b) HeLa cells treated with haspin inhibitors at 7.5 h were harvested at 9 h and 10.5 h for immunoblotting analysis of indicated proteins.

Cell proliferation assay

HeLa H2B-GFP cells were seeded in 24-well plates at a density of 1x104 cells/well and transfected with siRNAs, as described above. Fluorescent images of H2B-GFP were collected from the same fields as 2 x 2 montages between 30 h and 75 h after siRNA transfection. Cell number was calculated by the Gen5 3.04 software and normalized to the cell number at 30 h.

Statistics

All statistical evaluations were carried out using the GraphPad Prism 7.0 Software. Data were analyzed by Student’s t-test for comparison between two groups or two-way ANNOVA for comparison between multiple groups. Combined data were obtained from 3 independent experiments. P-values < 0.05 were considered significant.

RESULTS

1. Haspin inhibitors delay mitosis entry in synchronized HeLa cells.

The small molecule inhibitor CHR-6494 has shown an inhibitory effect on cell proliferation in several cancer cell lines (Han et al., 2017; Huertas et al., 2012). We commenced our study by first investigating the effect of haspin inhibitors throughout the entire cell cycle. HeLa cells were synchronized at the G1/S boundary by double thymidine block and were released into the medium containing vehicle (DMSO) or two haspin inhibitors, CHR-6494 or 5-ITu (Figure 1a). FACS analysis of DNA contents showed that cells treated with either of haspin inhibitors reached the peak of S phase at 5 h and of G2/M phase at 9 h post thymidine release, which was similar to control cells (Figure 1b, c). However, while the majority of control cells shifted to the next G1 at 13 h, a large percentage of cells treated with haspin inhibitor remained at the G2/M phase for up to 15 h (Figure 1b, c). This extended G2/M duration was associated with a 4 h-delay in the appearance of the mitotic peak as shown by MPM-2 staining (Figure 1b, c). Immunoblotting analysis indicated that both haspin inhibitors efficiently reduced the mitotic specific phosphorylation of histone H3 at Thr3 (H3T3ph), the substrate of haspin kinase activity (Figure 2a). Consistent with results obtained from the FACS analysis, the induction of H3 phosphorylation at Ser10 (H3S10ph), which is the other marker for mitosis, was also delayed by these two compounds (Figure 2a). Despite the delayed mitotic entry, neither haspin inhibitor led to mitotic arrest with a majority of cells moving to the next G1 at 24 h post thymidine release (Figure 1b, c). These results indicate that haspin inhibitors delayed mitosis entry after a temporary G2 arrest in synchronized HeLa cells.

The effects of haspin inhibitors on cell cycle progression were further evaluated by immunoblotting analysis of other cell cycle markers. Cyclin A and cyclin B are expressed from early S phase to prometaphase and from late S phase and metaphase, respectively (Pines and Hunter, 1991). Cdk1 is the master kinase for G2/M transition, and its activation relies on the association with cyclin B as well as the removal of the inhibitory phosphates at Thr14 and Tyr15 (Nurse, 1990). In control cells, protein levels of cyclin A and cyclin B were elevated within 5 h - 11 h and 9 h - 11 h, respectively (Figure 2b). The Tyr15 phosphorylated form of Cdk1 (Cdk1-Y15P) showed a temporal pattern of elevation similar to that of cyclin A (Figure 2b). Despite having no effects on the onset of the induction of cyclin A/B and Cdk1-Y15P, CHR-6494 or 5-ITu kept these markers at high levels up till 15 h (Figure 2b). These results further substantiate that haspin inhibitors could prolong G2 phase progression in HeLa cells.

It has been shown that 5-ITu can cause G2 arrest and cell death by inducing DNA damage and activating DNA damage checkpoint (Zhang et al., 2013). However, in our study, treatment with CHR-6494 or 5-ITu did not increase the level of γ-H2AX, a marker for DNA damage, or levels of other markers of DNA damage responses such as phospho-CHK1 (Ser345) or phospho–CHK2 (Thr68) [Supplementary Figure S1] (Ciccia and Elledge, 2010). Our results suggest that the delayed cell cycle progression by haspin inhibitors was not associated with enhanced DNA damage in HeLa cells.

2. The haspin inhibitor CHR-6494 delays interphase progression in synchronized U2OS cells.

The effect of haspin inhibitors on cell cycle progression was next examined in the U2OS osteosarcoma cell line. In comparison with HeLa cells, U2OS cells are harder to be synchronized. After a single thymidine block for 24 h, cells were released into medium containing DMSO or CHR-6494 at different doses (Figure 3a). The microtubule stabilizer paclitaxel (Taxol) was added to the medium at 12 h after thymidine release to accumulate mitotic cells. Time-lapse imaging showed that the cumulative percentage of mitosis cells (round-shaped) started to increase at 12 h and reached a peak of 64% at 21 h in control U2OS cells (Figure 3a). CHR-6494 delayed the onset of mitotic cell accumulation in a dose-dependent manner. Also, the mitotic index increased slowly and only reached 21% at 28 h post-release in cells treated with 600 nM of CHR-6494 (Figure 3a).

Immunoblotting analysis revealed a dose-dependent suppression of CHR-6494 on Taxol-induced H3T3ph and H3S10ph (Figure 3b), which is consistent with the reduced mitotic accumulation shown in live cell imaging (Figure 3a). With cell cycle progression after thymidine release, the level of cyclin A and cyclin B was increased at 12 h in control U2OS cells, and CHR-6494 did not prevent this elevation (Figure 3c). However, after 7 h incubation with Taxol, while cyclin A and the inhibitory Cdk1-Y15P were dramatically downregulated in control cells, these markers remained unchanged in CHR-6494 treated cells (Figure 3c). The sustained high level of cyclin A/B and Cdk1-Y15P confirmed that the reduced mitotic index through haspin inhibition was due to a prolonged G2 phase and not the mitotic slippage in the presence of Taxol. Moreover, CHR-6494 treatment did not affect the γ-H2AX level (Figure 3c). Instead, it led to a moderate induction of p53 protein at the later time points (Figure 3c), thereby indicating a G2 delay without DNA damage. FACS analysis showed that CHR-6494 caused a delay in G1 exit and S phase progression (Supplementary Figure S2), indicating an action at the early stages of interphase progression in U2OS cells.

3. Knockdown of haspin expression delays interphase progression in synchronized U2OS cells.

We next investigated whether knockdown of haspin expression could affect the interphase progression similar to that observed for haspin inhibitors. U2OS cells transfected with control or haspin siRNAs were synchronized at G1/S boundary by a single thymidine block, and the mitotic cells were monitored by time-lapse live imaging in the presence of Taxol (Figure 4a). The mitotic population of control siRNA transfected cells started to increase from 12.5 h and reached a peak of 67% at 19.5 h post-release. With haspin deficiency, only 19% of cells were able to enter mitosis till 23.5 h (Figure 4a). Western blot analysis showed that haspin depletion actively blocked Taxol-induced mitotic induction of H3T3ph and H3S10ph in U2OS cells (Figure 4b), which is consistent with the reduced accumulation of mitotic cells (Figure 4a).

Figure 4.

Figure 4

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Haspin depletion with its siRNA delays mitotic entry in synchronized U2OS cells. (a) U2OS cells were transfected with a control (CONT.) siRNA or a siRNA against haspin (HASP.). After 36 h, cells were synchronized at G1/S boundary by a 24 h single thymidine block. Starting from 11 h after thymidine release, time-lapse imaging was performed at a frequency of 1 frame/h in the presence of Taxol (1 μM). Cumulative mitotic index was calculated by the Gen5 3.04 software and presented as mean percentage of cells ± SEM. ***, p < 0.001 (compared with the control of the respective time point), n=3 independent experiments. (b-c) Immunoblotting analysis of indicated cell cycle markers. Relative protein levels (right panel) were quantified as described in Figure 3c. Data are the representative of 3 independent experiments.

Similar to CHR-6494, haspin siRNA kept cyclin A and Cdk1-Y15P at high levels after 7 h incubation with Taxol (Figure 4c). However, in contrast to CHR-6494 treated cells, haspin deficient cells expressed a low level of cyclin B before and after thymidine release (Figure 4c). We speculated that the cell cycle could have been arrested at the earlier stage. In support of this speculation, FACS analysis showed that cells with haspin depletion were less progressive between 0 h and 18 h post thymidine release by maintaining the same high G1/low S population (Supplementary Figure S3). The cell cycle arrest was accompanied by marked induction of p53 protein as early as 0 h (Figure 4c). At later points after thymidine release, while the level of p53 and γ-H2AX was decreased in control cells, haspin deficiency prevented such downregulation (Figure 4c). Thus, we anticipate that in response to the thymidine block, knockdown of haspin could arrest U2OS cells at the early stage of the cell cycle via activation of G1/S checkpoint mediated by p53 (Vogelstein et al., 2000).

4. Application of haspin inhibitors at the late stage of interphase is sufficient to delay mitosis entry.

Since haspin inhibitors could delay mitotic entry without causing a noticeable delay in S phase progression in HeLa cells, we postulate that haspin may directly regulate G2 to M transition. To examine this possibility, we applied vehicle or haspin inhibitors at 7.5 h after release from the second thymidine block. At this time point, 96% of cells were at interphase with 76% being positive for cyclin B (Figure 5a). Between 9.5 h and 10.5 h after thymidine release, the percentage of cyclin B positive interphase cells dropped sharply in control cells, along with the arrival of the mitotic peak at 9.5 h (Figure 5a). These results suggested that majority of control cells had passed the first mitosis and entered the next G1 phase to become negative for cyclin B. It is noteworthy that HeLa cells showed a faster cell cycle progression in this set of experiments than those shown in Figure 1, 2. The difference was likely due to different sources of serum in the culture medium. In contrast with control cells, short treatment of HeLa cells with either CHR-6494 or 5-ITu was sufficient to delay mitotic entry, as reflected by the prolonged existence of cyclin B positive cells at interphase (Figure 5a), the postponed appearance of H3S10ph (Figure 5b), and a slight increase in Cdk1-Y15P at 10.5 h post thymidine release (Figure 5b). Immunoblotting analysis also confirmed the efficacy of these compounds in reducing H3T3ph after the short treatment (Figure 5b).

The short-term effect of haspin inhibitors on mitosis entry was also examined in U2OS cells. We chose to apply DMSO or CHR-6494 at 7 h and 10 h after thymidine release (Figure 6a) and when cells carried low S and high G2/M populations (Supplementary Figure S2). Mitosis entry was monitored by live cell imaging, commencing from 12 h post thymidine release in the presence of Taxol. At 16 h, the percentage of mitotic cells rapidly rose to 40% in control cells, in comparison with 23% and 12% in cells treated with 600 nM of CHR-6494 at 10 h and 7 h, respectively (Figure 6a). The reduced mitotic cells at this early time point after Taxol incubation may reflect the direct effect of CHR-6494 on the mitosis entry of pre-existing G2 population. As the mitotic index continued to grow sharply to 69% at 20 h in control cells, it only reached the maximum level of 43% throughout 28 h in CHR-6494 treated groups (Figure 6a). Immunoblotting analysis showed that late application of CHR-6494 efficiently blocked the Taxol induction of H3T3ph in U2OS cells (Figure 6b). Moreover, after 7 h of incubation with Taxol, cells with late treatment of CHR-6494 expressed a lower level of H3S10ph than control cells but with higher levels of cyclin A/B and Cdk1-Y15P, thereby implicating a prolonged G2 phase duration (Figure 6b). These results suggest that haspin activities may be directly involved in G2 to M transition in both HeLa and U2OS cells.

5. Depletion of haspin by its siRNA prolongs interphase duration in undisturbed HeLa cells.

The effect of haspin depletion on the interphase progression was further examined in asynchronous HeLa cells expressing histone H2B-GFP through live cell imaging. In these cells, haspin siRNA was able to decrease the level of H3T3ph (Figure 7a) but did not cause an apparent mitotic arrest, which may due to incomplete depletion of endogenous haspin. The duration of interphase was monitored in single cells and measured by the gap between chromosome decondensation at the first telophase and chromosome condensation at the beginning of the next mitosis. Between 30 h and 75 h post siRNA transfection, two complete cell cycles were captured in 97% of control cells, comparing to 70% in cells with haspin depletion (Figure 7b). Moreover, haspin deficient cells showed an average 3 h delay in the interphase progression (Figure 7c), as well as a decreased rate of cell proliferation when compared with control cells (Figure 7d). Given the broad range of variations among individual cells with haspin depletion, we also examined the interphase duration in cells that were pre-synchronized by mitotic shake-off (Figure 7e). Under this setting, haspin deficient cells experienced an average of 6.25 h longer duration at interphase than control cells (Figure 7e). The persistent variation is likely due to the differential knockdown efficiency of haspin siRNA among individual cells. Overall, these results further confirm the involvement of haspin functions outside mitosis in undisturbed cells.

DISCUSSION

In this study, small molecule inhibitors of haspin have been used to investigate its functions outside mitosis. More specifically, we have found that applying haspin inhibitors CHR-6494 and 5-ITu at the G1/S boundary or later S/G2 stage could delay cells entering mitosis in synchronized HeLa and U2OS cells. The delayed cell cycle progression occurred at earlier stages in synchronized U2OS cells following G1/S treatment with CHR-6494 or haspin gene depletion with its siRNA. A prolonged interphase duration was also observed with haspin deficiency in asynchronous HeLa cells. These results suggest that haspin activities are involved in the cell cycle progression at both interphase and mitosis.

In theory, a prolonged interphase progression between G1/S and mitosis could occur during G1/S transition, S phase, or G2/M transition. In HeLa cells, the mitotic delay induced by haspin inhibitors was preceded by the timely onset of G2 entry and extended G2 duration. Consistent with the cell cycle analysis, early treatment of haspin inhibitors did not impact on the starting time for cyclin A and cyclin B elevations but significantly postponed their down regulations (Figure 2). It has been shown that 5-ITu can cause G2 arrest by introducing DNA damage and activating p53-mediated cell cycle checkpoint (Zhang et al., 2013). However, we did not observe any increase in the level of γ-H2AX or other markers of DNA damage response in cells treated with CHR-6494 or 5-ITu (Supplementary Figure S1). This finding suggests that the G2 delay induced by haspin inhibitors is not a consequence of enhanced DNA damage in HeLa cells. Furthermore, mitotic entry was also delayed when cells were exposed to haspin inhibitors at a later stage when most of them already at the late S or G2 phase (Figure 5). These results further imply a direct involvement of haspin activity in G2-M transition in HeLa cells.

By adopting the late application of inhibitors, the effect of CHR-6494 on G2-M transition was also tested in U2OS cells. Based on the cell cycle analysis, about 45% of cells were at the G2 phase at 11 h post thymidine release (Supplemental Figure 2). It took control cells an additional 5 h with Taxol incubation to accumulate 43% of mitotic cells. This portion of mitotic cells was likely originated from the pre-existing G2 cells. We found that the late application of CHR-6494 at 10 h was sufficient to prevent mitotic accumulation during the initial 5 h period, supporting the direct regulation of haspin on G2-M transition of the pre-existing G2 population in U2OS cells (Figure 6a).

Unlike most of the other protein kinases, haspin is intrinsically active, and its activation does not require the phosphorylation at the activation loop (Eswaran et al., 2009). Instead, the mitotic specific kinase activity of haspin towards H3T3 is activated by Cdk1 and Plk1, which phosphorylates multiple sites at its N-terminal domain (Ghenoiu et al., 2013; Moutinho-Santos and Maiato, 2014; Zhou et al., 2014). Both Cdk1 and Plk1 kinases are the master regulators for G2-M transition and mitosis progression (Archambault and Glover, 2009; Barr et al., 2004; Pines and Hunter, 1991). Future studies are needed to investigate whether activated haspin can act as a downstream effector of Cdk1 and Plk1 and mediate their ability in controlling mitotic entry. As H3T3ph occurs at late G2 (Dai et al., 2005), it is also possible that this special histone modification and the resulting alteration in chromatin structure are critical for the timely onset of mitosis.

In U2OS cells, G1/S application of CHR-6494 interrupted the cell cycle progression at an earlier stage by slowing down G1 exit and S phase progression (Supplementary Figure S2). In contrast, siRNA-mediated knockdown of haspin expression showed a stronger effect in blocking G1 to S transition (Supplementary Figure S3) and inducing p53 expression (Figure 4c). Such differential effects could imply the existence of other haspin functions independent or unrelated to its intrinsic kinase activity. On the other hand, as thymidine blocks the cell cycle after the G1/S checkpoint (Bostock et al., 1971), application of haspin inhibitors post thymidine release could only affect its functions after that checkpoint. Nevertheless, these results substantiate the critical functions of haspin at multiple stages during interphase progression in U2OS cells.

We have previously found that interphase haspin is partially located in the nucleolus (Dai et al., 2005), the organelle for ribosome assembly and protein biosynthesis (Hernandez-Verdun et al., 2010). A recent proteomic analysis has also identified numerous candidates as interacting proteins or substrates of haspin, including several nucleolar proteins and factors related to gene transcription (Maiolica et al., 2014). Since protein synthesis and gene transcription occur mainly outside mitosis, this critical analysis supports the notion that haspin possesses crucial functions during interphase either through its kinase activity or via its interaction with other proteins.

As a tool for cell cycle synchronization, an excessive amount of thymidine is used to arrest cells at the entrance of S phase by inhibiting DNA replication. This treatment can introduce replication stress and activate the DNA damage response (Halicka et al., 2017). Therefore, the delayed cell cycle progression in synchronized cells may reflect a functional defect in the recovery of DNA damage following haspin inhibition. However, the extended interphase duration was also observed in asynchronous HeLa cells with haspin depletion (Figure 7). This outcome further substantiates the role of haspin in regulating the interphase progression in undisturbed cells.

Although 5-ITu has been characterized as an effective inhibitor of haspin, it also has a few significant off-targets that would include adenosine kinase, CDC like kinase (CLK), and the Dual Specificity Tyrosine Phosphorylation Regulated Kinases (Dyrks) (Balzano et al., 2011; De Antoni et al., 2012; Massillon et al., 1994). We found that late application of 5-ITu at 1 μM was sufficient to delay the mitotic entry in HeLa cells (Figure 5). Both in vivo and in vitro assays in previous reports indicate that 1 μM of 5-ITu has no activity against other mitotic kinases, including Cdk1/Cyclin B (Antoni 2012, Balzano 2011). Therefore, the short term effect of 5-ITu is likely related to the real function of haspin in regulating the G2-M transition. Further investigations are underway to understand whether the effect of haspin inhibitors on other stages of interphase is truely due to its inhibition on the haspin activity.

In summary, this study uncovers the essential functions of haspin in controlling cell cycle progression at multiple stages of interphase in HeLa and U2OS cells. Further studies are required to illustrate underlying molecular mechanisms of its functions outside mitosis. Thorough understanding of haspin functions and identification of its cell cycle specific targets should help to gain insight into its potential as an anti-tumor target either through single or combination inhibition.

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ACKNOWLEDGMENTS

Authors thank Professor Richard P. Hsung of the School of Pharmacy at the University of Wisconsin–Madison for invaluable discussions.

Grant information:

  • Grant sponsor: The National Institutes of Health

  • Grant number: K01AR062132

FUNDINGS

Authors thank the School of Pharmacy at the University of Wisconsin–Madison and the School of Pharmaceutical Science and Technologies at Tianjin University for generous support. JD also acknowledges partial support of this work by The National Institutes of Health (K01AR062132).

Footnotes

COMPETING INTERESTS

Authors declare that they have no competing interests.

DATA SHARING

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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