For immunoprecipitation of Cdk1, cells were transfected with HA-Cdk1 plasmid, early mitotic cells obtained by shake off were arrested for prometaphase by treatment with nocodazole. Cells treated with H2O2 depending on concentration for 1 h, and cells were lysed with IP buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 % NP-40, 5 mM EDTA), 400 μg of cell lysates were incubated with 1 μg of HA-antibody at 4 °C for overnight. Each tube was incubated with 30 μl Protein G (GE, 17-0618-01) beads at 4 °C for 1 h. After three times of
washing with IP buffer, beads were boiled with 30 μl non-reducing sample buffer for 5 min.
The samples were separated by SDS-PAGE and then were transferred to PVDF membrane.
After blocking for 1 h at RT with TBST and 5 % (W/V) non-fat milk, the membranes were incubated with indicated antibodies at 4 °C for overnight. After three times washing with TBST, the membranes were incubated with secondary antibody (TrueBlot; ROCKLAND, 18-8817-30) at room temperature for 1 h. Detection was carried out using ECL reagents (Amersham Biosciences, RPN2106) and by exposing them to X-ray film.
O. Phos-tag gel analysis
The phos-tag reagents were purchased from Wako Chemicals, and gels containing phos-tag were prepared according to the manufacture’s instructions. For immunoprecipitation, cells were lysed with mild lysis buffer (50 mM HEPES at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1%
NP-40, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1.5 mM Na3VO4, 1 mM PMSF, 1 mM DTT, protease inhibitor cocktail (Roche)), and centrifuged at 12,000 × g for 15 min at 4 °C. The supernatants were incubated with the appropriate antibodies for 2 h at 4 °C, and protein G or protein A conjugated beads were added in for additional 1 h.
Immunoprecipitates were collected by centrifugation and washed four times with lysis buffer, and proteins were eluted with SDS-PAGE sample buffer.
P. S-glutathionylation assay
Asynchronous or mitotic HeLa cells were preincubated for 1 h with culture medium containing 250 μM biotinylated glutathione ethyl ester (BioGEE) (Invitrogen, G36000) and subsequently exposed to H2O2 for 30 min in a concentration dependent manner. Cells were washed with ice-cold PBS and lysed in a IP buffer containing 10 mM N-ethylmaleimide (NEM) to block further thiol oxidation. 500 μg of cell lysates were incubated for 1 h at 4 °C with 50 μl streptavidin-conjugated agarose beads (Invitrogen, 88816). Then beads were rinsed three times with IP buffer, S-glutathionylated proteins were released from the beads by boiling with 30 μl sample buffer for 5 min. The samples were separated by SDS-PAGE, and Cdk1 levels were evaluated by Western blot analysis.
Q. Statistical analysis
Most data are represented as mean ± standard deviation (SD). Each experiment was performed in triplicates. Statistical differences were analyzed by Student's t-test and the asterisk (*) and sharp (#) indicates a significant difference *,#; P<0.05, **,##;, P<0.01, ***,###;, P<0.001.
III. Result
PART Ⅰ. Inhibition of PP2A activity by H2O2 during mitosis disrupts nuclear envelope reassembly and alters nuclear shape.
A. Treatment of mitotic cells with H2O2 induces abnormal nucleation.
To determine whether the cue that induces abnormal nuclear shape functions in a cell cycle-dependent manner—specifically, whether mitosis is a sensitive period for the formation of abnormal nuclear shape—I compared the effects of H2O2 treatment on asynchronous and mitotic cells, obtained following the scheme shown in Fig. 6A. After 10 h of H2O2 treatment, which provides sufficient time for mitotic cells to enter the next interphase during which altered nuclear morphology is observed, I monitored changes in nuclear shape by immunostaining for lamin B1. Treatment of asynchronous cells with H2O2 had little effect on nuclear shape in most cells except at high concentrations of H2O2 with longer treatment duration. In contrast, treatment with H2O2 during mitosis caused marked, concentration-dependent changes in nuclear shape in the subsequent interphase, inducing significant changes at an H2O2 concentration of 50 μM and reaching a plateau at 100 μM; in both cases, nuclear shape was analyzed at 10 and 24 h after H2O2 treatment. Indeed, mitotic cells showed a significantly higher tendency to form abnormal nuclear shapes than asynchronous cells under every H2O2 treatment condition (Fig. 6B). Notably, neither 50 nor 100 μM H2O2, concentrations that are easily achievable in a pathological setting (e.g., a rat ischemia/reperfusion model (Hyslop et al., 1995)), caused cell death after 24 h, as our lab reported previously (Cho et al., 2017).
Treatment of mitotic cells with H2O2 was followed by a variety of changes in nuclear shape, including folding or fragmentation of the nuclear envelope or adoption of a globular shape.
This was also confirmed in simulated 3-dimensional (i.e., 2.5D) images (Fig. 6C). Furthermore, electron microscopy revealed that the nuclear envelope in cells treated with H2O2 during mitosis formed a curved section with electron-dense sites that may indicate thickening of the nuclear membrane (Fig. 6D).
To measure the abnormal nuclear shape more objectively, the extent of the variability in lamin B1 staining intensity was analyzed. Since the intensity of lamin B1 staining in folded or curved nucleus is more variable than that of normally shaped nucleus, I reasoned that the standard deviation of these values would be an indicator of the degree of nuclear shape alteration. Consistent with the result of counts of abnormal nuclei, lamin B1 staining in mitotic cells exhibited a significantly larger standard deviation than that in asynchronous cells both 10 and 24 h after H2O2 treatment (Fig. 6E). The circularity of the nucleus was quantified as another approach for objectively representing changes in nuclear shape (Fig. 6F). A circularity value of “1” corresponds to a complete circle, whereas smaller values denote greater deviations from circularity. As a reference point, the mean circularity values of control asynchronous and mitotic HeLa cells were both ~0.8. Whereas the mean circularity value at 10 and 24 h after H2O2 treatment of asynchronous cells was maintained at ~0.8 regardless of the concentration of H2O2, it was significantly reduced in mitotic cells, reaching ~0.7.
Fig. 6. H2O2 treatment to mitotic cells forms more abnormal nuclear shape than asynchronous cells. (A) Experimental design to obtain mitotic cells. (B) Left panel:
Asynchronous (upper) or mitotic (lower) HeLa cells were treated with 100 μM H2O2 for 10 h and subjected to immunocytochemistry for lamin B1 (green), α-tubulin (red) and DAPI (blue).
Scale bar: 20 μm. Right panel: Asynchronous or mitotic cells were treated with H2O2 at indicated concentrations, and percentage of cells with abnormal nuclear shape was measured after 10 h or 24 h. Results are shown as the mean ± SD from three independent experiments (n=300), *; Control versus H2O2, #; Asynchronous versus Mitosis, *p<0.05, **, ##p<0.01,
###p<0.001 by Student's t-test. (C) Representative examples of abnormal nuclear shape in H2O2-treated cells. Upper panel; Lamin B1 staining (green). Lower panel; Images from upper panels were converted to 2.5 dimensional images by using ZEISS Microscope software ZEN.
Scale bar: 5 μm. (D) Electron microscopy images of nuclear envelope in cells treated with or without H2O2 for 10 h. Scale bar: 2.5 μm. (E) Standard deviation of lamin B1 intensity inside an imaginary circle in the nucleus was measured by using ZEN software with the same samples in (B). Results are shown as the mean ± SD (n=50), *; Control versus H2O2, #; Asynchronous versus Mitosis, #p<0.05, **, ##p<0.01, ###p<0.001 by Student t-test. (F) Nuclear circularity was calculated by using ImageJ software with the same samples in (B). Results are shown as the mean ± SD (n=30). *; Control versus H2O2, #; Asynchronous versus Mitosis, #p<0.05, **,
##p<0.01, ###p<0.001 by Student's t-test.
To determine whether the effects of H2O2 on the cell cycle were limited to continuous-exposure conditions, I also tested the effects of transient continuous-exposure to H2O2. Treatment with H2O2 for 2 h followed by wash-out produced the same susceptibility of mitotic cells to abnormal nucleation compared with asynchronous cells, as shown by measuring the variability of lamin B1 immunostaining intensity and assessing the circularity index (Fig. 6G–I). This enhanced vulnerability of mitotic cells to abnormal nucleation following H2O2 treatment compared with asynchronous cells was observed not only in HeLa cells, but also in U2OS, RPE-1 and HT1080 cells, indicating the generalizability of my observations (Fig. 6J).
Fig. 6. H2O2 treatment to mitotic cells forms more abnormal nuclear shape than asynchronous cells. (G) Asynchronous or mitotic cells were treated with H2O2 at indicated concentrations. Two hours later, cells were washed out, and the percentage of cells with abnormal nuclear shape was determined 10 h or 24 h after H2O2 treatment. Results are shown as the mean ± SD from three independent experiments (n=300), *; Control versus H2O2, #; Asynchronous versus Mitosis, *p<0.05, **, ##p<0.01, ###p<0.001 by Student's t-test. (H) Standard deviation of lamin B1 intensity inside an imaginary circle in the nucleus was measured by using ZEN software with the same samples in (G). Results are shown as the mean
± SD (n=50), *; Control versus H2O2, #; Asynchronous versus Mitosis, #p<0.05, **, ##p<0.01,
###p<0.001 by Student's t-test. (I) Nuclear circularity was analyzed with ImageJ with the same samples in (G). Results are shown as the mean (n=30). *; Control versus H2O2, #; Asynchronous versus Mitosis, *p<0.05, **, ##p<0.01, ###p<0.001 by Student's t-test. (J) Mitotic U2OS, RPE-1 and HT1080 cells were treated with H2O2 at indicated concentrations, and percentage of cells with abnormal nuclear shape was determined after 10 h. Results are shown as the mean ± SD from three independent experiments (n=300), *p<0.05, **p<0.01,
***p<0.001 by Student’s t-test.
B. Formation of abnormal nuclei following H2O2 treatment is prevented by NAC or catalase.
To confirm that the formation of abnormal nuclear shapes in H2O2-treated mitotic cells was actually caused by ROS, I pretreated cells with the antioxidant N-acetyl-L-cysteine (NAC).
Indeed, changes in nuclear shape after H2O2 treatment were almost totally prevented by NAC (Fig. 7A). The ROS-lowering effect of NAC on mitotic cells under these treatment conditions was verified by fluorescence-activated cell sorting (FACS) analysis using the fluorescent ROS indicator, dichlorodihydrofluorescein diacetate (DCF-DA) (Fig. 7B).
To more specifically address whether H2O2 was responsible for the abnormal nucleation, prior to H2O2 treatment, catalase, an enzyme that converts H2O2 to water and oxygen, was transfected into the cells. Whereas cells that did not express catalase exhibited a change in nuclear shape, as expected, nuclear shape change was remarkably reduced in cells overexpressing catalase (Fig. 7C). Interestingly, the formation of abnormal nuclei was also suppressed in catalase-expressing cells in the absence of H2O2 treatment, suggesting that basal levels of H2O2 induce formation of a basal level of abnormal nuclei. The H2O2-lowering effect of ectopically expressed catalase was confirmed using pHyper-Cyto (Fig. 7D), a specific fluorescent protein probe for H2O2 (Belousov et al., 2006). Collectively, these findings indicate that mitotic cells are more prone to abnormal nucleus formation following H2O2 treatment than asynchronous cells, and that this phenomenon is directly attributable to ROS, based on the preventive effect of NAC treatment and catalase overexpression.
Fig. 7. Nuclear shape alteration induced by H2O2 treatment during mitosis is rescued by antioxidants. (A) N-acetyl-L-cysteine (NAC) was pretreated (NAC+H2O2) or not (H2O2) for 30 min. Then, mitotic cells were obtained through shake-off, and treated with 100 μM H2O2
for 10 h. Left panel; After 10 h, the nuclear shape was observed by lamin B1 staining (red).
Scale bar: 20 μm. Right panel; percentage of the cells with abnormal nuclear shape. Results are shown as the mean ± SD from three independent experiments (n=300), *p<0.05, **p<0.01 by Student's t-test. (B) Mitotic cells were pretreated with NAC or not for 30 min, and then incubated with 100 μM H2O2 for1h. Intracellular ROS level was measured by FACS analysis using DCF-DA. (C) HeLa cells were transfected with an HA-catalase expressing vector, and then mitotic cells were treated with or without 100 μM H2O2 for 10 h. Left panel; nuclear shape of HA-catalase (red)- transfected cells. Scale bar: 20 μm. Right panel; percentage of the cells with abnormal nuclear shape according to HA-catalase expression. Results are shown as the mean ± SD from three independent experiments (n=100), *p<0.05, **p<0.01, ***p<0.001 by Student's t-test. (D) HeLa cells were transfected with pHyper-Cyto, and then with pcDNA3 or HA-Catalase. Changes in intracellular H2O2 level is expressed as the ratio upon excitation by 488 and 405 nm laser. Representative time-lapse fluorescence images (30 s interval for 25 min) of pHyper-cyto sensors in 100 μM H2O2-treated mitotic HeLa cells.
C. Quantitative changes in lamin B1, structural changes in the cytoskeleton and ER, and DNA damage are not major contributors to H2O2-induced nuclear shape changes.
Previous studies have reported that p38 MAPK (mitogen-activated protein kinase) is activated by ROS in ataxia-telangiectasia cells, and that the level of endogenous lamin B1 is increased in these cells, resulting in nuclear deformation and senescence (Barascu et al., 2012).
Therefore, I investigated whether the formation of abnormal nuclei in response to H2O2
exposure under my experimental conditions was accompanied by changes in the level of lamin B1. Lamin B1 levels were not noticeably changed at 10 or 24 h after treatment with H2O2 (Fig.
8A). And, similar results were obtained when the cells were transiently treated with H2O2 (Fig.
8B), excluding the possibility that quantitative changes in lamin B1 levels are involved in H2O2-induced abnormal nucleation.
The nucleus is connected directly or indirectly with cytoskeletal elements, including actin filaments, microtubules and intermediate filaments, and these physical connections are known to determine nuclear shape by creating tension between the nucleus and the cytoskeleton (Wang et al., 2009; Lombardi et al., 2011; Jevtic et al., 2014). To address whether alterations in nuclear shape were induced by changes in the cytoskeleton, mitotic cells were treated with H2O2 and assessed the appearance of the cytoskeleton under a microscope. These experiments showed no significant changes in the appearances of F-actin or microtubules (Fig. 8C). I also investigated possible structural changes in the ER, which is connected to the outer nuclear membrane and thus it could affect nuclear shape (Guttinger et al., 2009; Alvarez-Fernandez and Malumbres, 2014). Changes in ER shape were observed by monitoring ectopically expressed GFP-Sec61β, a fluorescently tagged ER membrane protein, following treatment of mitotic cells with H2O2. These experiments revealed no significant changes in ER structure (Fig. 8D). Taken together, these results show that the changes in nuclear shape induced by H2O2 were not accompanied by quantitative changes in lamin B1, or structural changes in the cytoskeleton or ER. In contrast, I did detect changes in the immunocytochemical images of other elements that constitute the nuclear membrane, such as lamin A/C, emerin, and the NPC in response to increases in ROS during mitosis that were associated with changes in nuclear shape (Fig. 8E–G).
Oxidative stress is well known to cause DNA damage (Barzilai and Yamamoto, 2004; Acilan
et al., 2007), and our group has previously reported that H2O2 induces DNA damage and subsequent chromatin bridge formation in mitotic cells, changes that appear to be related to binucleation (Cho et al., 2017). To determine whether DNA damage is involved in abnormal nucleus formation, I compared the effects of H2O2 and etoposide, an inhibitor of topoisomerase II that induces DNA double-strand breaks. Notably, treatment with a high concentration of etoposide induced an increase in the number of cells with abnormal nuclei, suggesting that DNA damage does contribute to the formation of an abnormal nucleus. However, although expression of the DNA damage marker, γ-H2A.X, was increased to a greater extent by 10 μM etoposide than by 50 μM H2O2, the percentage of cells that formed abnormal nuclei in response to 10 μM etoposide was significantly less than that in response to 50 μM H2O2. A comparison of 40 μM etoposide and 100 μM H2O2 also showed the same tendency (Fig. 8H), indicating that DNA damage plays at most a modest role in the formation of abnormal nuclei. Therefore, mechanisms other than DNA damage appear to be of primary importance in the nuclear shape changes induced by H2O2.
Fig. 8. H2O2 - induced abnormal nuclear shape is mainly neither due to changes in lamin B1 level, nor in cytoskeleton network, nor in DNA damage. (A) Asynchronous or mitotic HeLa cells were treated with H2O2 at indicated concentrations for 10 h or 24 h. Cell lysates were harvested and subjected to western blot analysis by using indicated antibodies. (B) Asynchronous or mitotic HeLa cells were treated with H2O2 at indicated concentrations. Two hours later, cells were washed out. Cell lysates were harvested 10 h or 24 h after H2O2 treatment, and subjected to western blot analysis by using indicated antibodies. (C) Mitotic HeLa cells were treated with 100 μM H2O2 for 10 h, and cytoskeleton fibers were detected by immunocytochemistry. Actin filaments and microtubules were visualized by using Phalloidin-TRITC (red) and α-tubulin (red) antibody, respectively. Lamin B1 (green), DAPI (blue). Scale bar: 30 μm. Dotted boxes were magnified as shown. (D and E) HeLa cells were transfected with expression vectors of GFP-Sec61β (D), an ER membrane protein, or GFP-Emerin (E), an inner nuclear membrane protein, and then mitotic cells were treated with 100 μM H2O2 for 10 h. Structure of ER and nuclear envelope were visualized by using GFP (green) antibody. Lamin B1 (red), DAPI (blue). Scale bar: 20 μm. (F and G) Mitotic HeLa cells were treated with 100 μM H2O2 for 10 h, and structure of the nuclear envelope was assessed. Lamin A/C (F) and nuclear pore complexs (G) were visualized by using lamin A/C (green) and mAb414 (red) antibody, respectively. α-tubulin (red), lamin B1 (green), DAPI (blue). Scale bar: 10 μm. (H) Mitotic HeLa cells were treated with H2O2 or etoposide at indicated concentrations. Upper panel; Mitotic cells were co-treated with H2O2 or etoposide and 100ng/ml nocodazole, respectively, for 1 h, and subjected to western blot analysis by using indicated antibodies.
Lower panel; Mitotic HeLa cells were treated with H2O2 or etoposide for 10 h, and then percentage of the cells with abnormal nuclear shape was determined. Results are shown as the mean ± SD from three independent experiments (n=300). ***p<0.001 by Student's t-test. S.E;
short exposure, L.E; long exposure.
D. H2O2 inhibits PP2A activity during mitosis.
Since nuclear envelope disassembly and reassembly occur during mitotic entry and exit, respectively, I hypothesized that the observed propensity for mitotic cells to undergo changes in nuclear shape in response to H2O2 was attributable to effects of H2O2 on nuclear envelope disassembly and/or reassembly processes. It has previously been shown that PP2A plays an important role in the nuclear envelope reassembly process during mitotic exit (Asencio et al., 2012). To determine whether PP2A is involved in the formation of abnormal nuclei in my experimental system, I investigated changes in nuclear shape following treatment of mitotic cells with different concentrations of the PP2A inhibitor, okadaic acid. Although both PP1 and PP2A are inhibited by okadaic acid, it has been reported that PP2A is more sensitive to okadaic acid (in vitro IC50 ≈ 0.5 nM) than PP1 (IC50 ≈ 42 nM) (Foley et al., 2007; Zhuang et al., 2014).
Treatment of mitotic cells with okadaic acid for 2 h caused robust, concentration-dependent changes in nuclear shape, affecting ~94% of cells at the highest concentration tested (150 nM);
by contrast, okadaic acid had little effect on nuclear shape in asynchronous cells at any concentration (Fig. 9A). Thus, inhibition of PP2A results in abnormal nucleus formation, but only when applied during mitosis, a phenomenon comparable to the observed greater vulnerability of mitotic cells to H2O2-induced nuclear shape changes. Since H2O2 is known to decrease PP2A activity in asynchronous cells (Rao and Clayton, 2002; Kim et al., 2003; Foley et al., 2007), I investigated whether PP2A activity was also reduced by H2O2 in mitotic cells (Fig. 9B). In vitro PP2A activity was assayed after treatment of mitotic cells with H2O2 or okadaic acid for different durations. Indeed, both H2O2 and okadaic acid decreased the activity of PP2A in mitotic cells. In addition, the H2O2-induced decrease in PP2A activity was found to be dependent on H2O2 concentration, and showed a tendency towards recovery in cells treated with NAC (Fig. 9C). Therefore, H2O2 inhibits the activity of PP2A in mitotic cells, potentially affecting the nuclear envelope reassembly process and causing changes in nuclear shape.
Fig. 9. Inhibition of protein phosphatase 2A activity is involved in H2O2 - induced abnormal nuclear formation. (A) Mitotic HeLa cells were treated with okadaic acid in a dose-dependent manner for 2 h. Cells were washed, and the nuclear shape alteration was observed after 8 h. Left panel; representative examples of the nuclear shape change in cells treated with okadaic acid. Scale bar: 5 μm. Right panel; the percentage of cells with abnormal nuclear shape by okadaic acid (O.A) at indicated concentrations (n=100). (B) Mitotic HeLa cells were treated with 100 μM H2O2 or 100 nM okadaic acid (O.A) for indicated periods.
Cells were harvested and PP2A activity was determined using the PP2A activity assay kit from R&D Systems. Results are shown as the mean ± SD from three independent experiments,
*p<0.05, **p<0.01 by Student's t-test. (C) NAC was pretreated (NAC+H2O2) or not (H2O2) for 30 min before H2O2 treatment. Mitotic cells were obtained through shake-off, and treated with or without H2O2 for 30 min. Cells were harvested and PP2A activity was determined using the PP2A activity assay kit. Results are shown as the mean ± SD from three independent experiments, *p<0.05 by Student's t-test.
E. Ectopic expression of PP2A rescues H2O2-induced aberrant nuclear shape changes.
To verify the relationship between the decrease in PP2A activity and abnormal nucleation, I investigated whether ectopic expression of PP2A rescued H2O2-induced nuclear shape changes. After overexpression of a Flag-tagged PP2A catalytic subunit, mitotic cells were collected and treated with 50 μM H2O2 to induce changes in nuclear shape. In the absence of H2O2 treatment, exogenously expressed PP2A had no effect on nuclear shape (~17% and 14%
cells with aberrant nuclei with low- and high-level PP2A expression, respectively). In contrast, overexpression of PP2A partially abrogated H2O2-induced abnormal nucleation; treatment of
cells with aberrant nuclei with low- and high-level PP2A expression, respectively). In contrast, overexpression of PP2A partially abrogated H2O2-induced abnormal nucleation; treatment of