RESULTS

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