DISCUSSION

Recent studies have revealed a variety of mechanisms that lead to nuclear shape changes.

For example, a defect in lamin and inner and outer nuclear membrane proteins, such as Lem4, Lap2β and LBR (Luke et al., 2008; Prokocimer et al., 2009; Olins et al., 2010; Asencio et al., 2012; Jevtic et al., 2014), and defects in proteins that affect the tension between the cytoskeleton and the nucleus, such as the LINC (linker of nucleoskeleton and cytoskeleton) complex, actin and tubulin (Wang et al., 2009; Lombardi et al., 2011; Jevtic et al., 2014), lead to changes in nuclear shape. In addition, defects in BAF, a core protein that bridges the nuclear envelope and chromatin, induce nuclear deformation (Furukawa et al., 2003; Gorjanacz et al., 2007; Puente et al., 2011), reflecting defects in other core proteins (e.g., emerin, Lap2β, and lamin A (Jevtic et al., 2014). VRK1, the protein kinase of BAF, which phosphorylates Ser-4 on BAF during mitotic entry, a defect in VRK1, in turn, causes BAF to remain on mitotic chromosomes, thus increasing anaphase bridges and multipolar spindles, and ultimately disrupting the morphology of the nuclear envelope (Molitor and Traktman, 2014). Moreover, transcription factors (e.g., GATA6) and chromatin-remodeling factors (e.g., BRG1) that are not directly involved in regulating the structure of the nuclear envelope can also induce changes in nuclear shape by reducing the expression of proteins such as emerin (Capo-chichi et al., 2009; Imbalzano et al., 2013).

Most of the above-mentioned studies have shown that abnormal nuclei are cell cycle-independent events induced by deletion/mutation of specific proteins. Even in the case of ataxia-telangiectasia cells, ROS induce nuclear shape changes by altering the amount of lamin B1 protein (Barascu et al., 2012). Here, my study suggests that pathophysiologically achievable concentrations of H2O2 affect nuclear envelope disassembly and reassembly by decreasing the activity of Cdk1 and PP2A, respectively.

In part Ⅰ, reduction of the activity of PP2A by H2O2 then caused subsequent mislocalization of its substrate, BAF, from its normal core position, leading to changes in nuclear shape (Fig.

22). Mislocalization of BAF appears to affect the proper localization of lamin A/C, another well-known core protein (Table 1), suggesting the possibility that other core proteins involved

in the nuclear envelope reassembly process also fail to be recruited, resulting in an abnormal nuclear envelope reassembly process and a malformed nucleus.

BAF is recruited to the core region via microtubules during early telophase, and provides a platform for LEM-domain proteins (Haraguchi et al., 2008; Barton et al., 2015; LaJoie and Ullman, 2017). In addition to LEM-domain proteins, other proteins, such as SUN2 and A-type lamin, also localize at the core region in a BAF-dependent manner; these proteins are collectively dubbed ‘core’ proteins (Haraguchi et al., 2008; LaJoie and Ullman, 2017).

Consistent with this, a BAF mutant (BAF-G25E) that does not localize to the core region was shown to be unable to recruit emerin, Lap2β, or lamin A to the core region during nuclear envelope reassembly; thus, these proteins remained in the cytosol during the next interphase (Haraguchi et al., 2001). In addition, overexpression of truncated Lap2β has been reported to change lamina assembly and nuclear envelope formation in Xenopus oocyte extracts (Gant et al., 1999; Jevtic et al., 2014). Therefore, BAF-mediated recruitment of core proteins to their correct positions would appear to have a clear effect on nuclear morphology. Here, I showed that the localization of BAF and lamin A/C was altered by H2O2 treatment during mitosis and was rescued by overexpression of PP2A (Fig. 11, Table 1) strongly support the conclusion that H2O2 induces abnormal nuclear shapes through its inhibitory effect on PP2A and subsequent mislocalization of BAF and other core proteins (e.g., lamin A).

Fig. 22. Schematic diagram showing that inhibition of PP2A activity during mitosis by ROS results in abnormal nuclear shape via mislocalization of BAF, a substrate of PP2A during mitotic exit. Schematic diagram showing how the increase of H2O2 during mitosis induces nuclear shape alteration. BAF localizes at chromosomal core region during telophase through dephosphorylation by PP2A, and subsequently the constituents of other nuclear membranes are recruited around the chromosome to form a normal nuclear shape. However, increasing H2O2 in mitotic cells induces inhibition of PP2A activity. Decrease of PP2A activity seems to prevent BAF from being located in the chromosome core region during telophase, which subsequently mislocalizes other proteins that come into the core region by the BAF, resulting in the formation of abnormal nuclear shape.

In part Ⅱ, I suggest that H2O2 treated at early mitosis induces oxidative modification of the single cysteine residue present in Cdk1, leading to defects in lamin protein disassembly. The resultant lamin aggregates leads to premature lamin reassembly during late mitosis, eventually leading to the formation of an abnormal nucleation (Fig. 23). The possibility of oxidative modification of the cysteine residue of Cdk1, the key kinase of mitosis, and the resulting change in nuclear shape due to decreased activity are not known until now and are very interesting results.

According to previous results of our lab, cysteine 119 of Cdk1 is an essential amino acid residue in binding to cyclin B1 and that other amino acids in the surrounding region have no effect on binding to cyclin B1. In addition, cysteine 118, similar to 119 of Cdk1 in Cdk2, also plays an important role in binding of Cdk2 and cyclin A. It has been reported that the activity of kinases such as ATM, Pyruvate kinase, and AMPK is decreased or increased by oxidative post-translational modification (Ox-PTM) of cysteine residue, such as disulfide bonds, glutathionylation (Guo et al., 2010; Zmijewski et al., 2010; Anastasiou et al., 2011). It is also known that kinases or phosphatases that regulate cell cycle also affect cell proliferation by modification of cysteine residue (Chiu and Dawes, 2012). Thus, I have tried several ways to determine what Ox-PTM of cysteine 119 residue of Cdk1 is, but failed to identify at the endogeneous level (Fig. 18). However, mass spectrometry analysis using recombinant Cdk1 showed that there is a possibility of modification of the cysteine; cysteine was converted to serine or Cys + 64 using recombinant protein (Fig. 19). Recently, development of probes capable of confirming the modification of cysteine has been actively pursued. Therefore, it is necessary to study what kind of cysteine modification occurs in Cdk1 using probes developed in the future.

Recently, it has been shown that senescence-inducing stimulation, such as H2O2, IR, oncogenic RAS, and replicative stress, causes cells to undergo a mitotic skipping before entering a permanent cell cycle arrest state (Johmura et al., 2014). Significantly, mitotic skipping is a necessary and sufficient condition for senescence induction. My data suggested the possibility that reduced activity of Cdk1 by increase in the intracellular ROS level also can lead to mitotic skipping. Furthermore, downregulation of several cell cycle genes in patients with Hutchinson-Gilford Progeria syndrome (HGPS) disease, one of premature aging diseases, was demonstrated by microarray-based analysis (Lee et al., 2016). Cdk1 gene expression was

also decreased along with many cell cycle related genes. The major features of HGPS cells are increased ROS levels and nuclear shape changes. I observed lamin aggregates during mitosis in progerin expressing cells. (Fig. 21). Thus, according to the results of others and my observations, the decreased expression of Cdk1 in HGPS cells may form lamin aggregates during mitosis, which may lead to the formation of an abnormal nucleus, thus providing another evidence that decrease of Cdk1 activity during early mitosis can induce abnormal nuclear shape via formation of lamin aggregates.

Fig. 23. Schematic diagram showing that inhibition of Cdk1 activity during mitosis by H2O2 induces formation of aggregated lamin, leading to abnormal nuclear shape.

Schematic diagram showing how the increase of ROS during mitosis induces nuclear shape alteration through decrease of Cdk1 activity. H2O2 treatment during mitosis causes oxidative modification of cysteine 119 in Cdk1, interfering with its binding to cyclin B1. Reduced activity of Cdk1 by H2O2 diminishes the phosphorylation of lamin during early mitosis and leads to formation of lamin aggregates (green fragments). In addition, decrease of Cdk1 activity by H2O2 contributes to the premature reassembly of lamin, during mitotic exit. Thus, the inhibition of Cdk1 by H2O2 during mitosis leads to the formation of abnormal nuclear shape by preventing the disassembly and reassembly of lamin.

Combining the results of Part Ⅰ and Ⅱ in terms of changes in nuclear shape, the treatment of H2O2 during mitosis causes the inhibition of Cdk1 during early mitosis and the inhibition of PP2A during late mitosis, resulting in defects in lamin disassembly and/or reassembly, respectively, which contribute to the formation of abnormal nuclear shape. On the other hand, Cdk1 and PP2A are known as kinases and phosphatases that regulate phosphorylation levels of substrates during mitosis progression (Wurzenberger and Gerlich, 2011; Mochida and Hunt, 2012). Therefore, to determine whether PP2A counteracts with Cdk1 to regulate the phosphorylation level of lamin during mitosis, lamin aggregates were observed after treatment of H2O2 and okadaic acid, a PP2A inhibitor, simultaneously. As a result, H2O2-induced lamin aggregates decreased by PP2A inhibition depending on the concentration of okadaic acid (Fig.

24A). Conversely, lamin aggregates, which were induced by H2O2 or RO3306, increased by overexpression of PP2A, contrary to PP2A inhibition (Fig. 24B). Furthermore, the degree of phosphorylation of the entire substrates of Cdk1 was reduced by RO3306 or H2O2 treatment, whereas it was increased by okadaic acid treatment and slightly increased by H2O2 and okadaic acid co-treatment compared with H2O2 treatment alone (Fig. 24C). Formation of lamin aggregates in each condition also showed the same tendency as shown in Fig. 24C, but the phosphorylation of lamin showed a more significant effect than the effect seen in the whole substrate phosphorylation (Fig. 24D). This is probably because each substrate has different phosphorylation and dephosphorylation thresholds. Overall, these data support the notion that Cdk1 and PP2A play a key role in regulating mitotic progresstion by balancing kinase and phosphatase, respectively. Therefore, phosphorylation of lamin is also regulated by both Cdk1 and PP2A, contributing to the formation of lamin aggregates.

Fig. 24. Cdk1 and PP2A contribute to the formation of the lamin aggregates by counterbalancing during mitosis. (A) Early mitotic cells obtained from shake off were treated with 100 μM H2O2 or okadaic acid (O.A) at indicated concentration, respectively or both. After 30 min, the percentage of cyclin B1 positive cells with aggregated lamin was determined. Results are shown as the mean ± SD from three independent experiments (n=100),

**p<0.01 by Student's t-test. (B) HeLa cells were infected with a pCDH or CFP-PP2A lenti virus. Left panel; mitotic cells were treated with 100 μM H2O2 (H) and 3 μM RO3306 (R3) for 30 min. The percentage of cyclin B1 positive cells with aggregated lamin was determined.

Results are shown as the mean ± SD from three independent experiments (n=100), *p<0.05 by Student's t-test. Right panel; western blotting showed CFP-PP2A expression. (C and D) Early mitotic cells obtained from shake off were arrested for prometaphase by treatment with nocodazole. Cells were treated with indicated drugs for 1 h. C; control, R3, 5; RO3306 3, 5 μM, O; okadaic acid 40 nM, H; H2O2 100 μM. (C) Upper panel; Cell lysates were analyzed by western blot with the indicated antibodies. Lower panel; the intensity of pSer-Cdk normalized to tubulin was shown. (D) The percentage of cyclin B1 positive cells with aggregated lamin was determined after 1 h. Results are shown as the mean ± SD from three independent experiments (n=100), **p<0.01 by Student's t-test.

These data suggests that if PP2A is activated during early mitosis, it can form lamin aggregates. However, since the activity of PP2A is inhibited by the Greatwall-ENSA pathway during early mitosis in which Cdk1 is normally active, dephosphorylation of lamin by PP2A is unlikely to occur. Meanwhile, PP2A can be activated when Cdk1 is inhibited by H2O2

treatment. But, since PP2A activity is also reduced by H2O2, the formation of lamin aggregates by H2O2 treatment is thought to be caused by the inhibition of Cdk1. And, the reduction of lamin aggregates when treated with both H2O2 and okadaic acid may be thought to be because of an increase in the relative phosphorylation level of lamin due to a decrease in the activity of the remaining PP2A. On the contrary, in late mitosis, the activity of PP2A is increased because the activity of Cdk1 is decreased. Therefore, dephosphorylation of BAF occurs normally by PP2A. However, treatment of H2O2 or okadaic acid causes the dephosphorylation of BAF to fail, resulting in the formation of an abnormal nucleus (Fig. 25).

Fig. 25. Schematic diagram showing how H2O2 induces abnormal nuclear shape via both inhibition of Cdk1 and PP2A during mitosis.

My model of abnormal nucleation suggests the interesting possibility that environmental cues like ROS can efficiently induce changes in nuclear shape by altering the function and/or intracellular localization of certain proteins, and that these environmental cues affect the cells in a specific stage of the cell cycle because they target the nuclear envelope disassembly and reassembly process, which occur only during mitosis. Since it is well known that ROS are involved in many pathological conditions, including cancer, their capacity to induce nuclear shape changes might provide novel insights into the role of ROS in these pathological conditions.

What are the consequences of changes in nuclear shape? It has been shown that structural defects in the nuclear envelope in ovarian cancer cells directly lead to chromosomal numerical instability and aneuploidy (Capo-chichi et al., 2011). In addition, several studies have shown that the NPC, a component of the nuclear envelope, is closely related to genome integrity (Bukata et al., 2013; Rodriguez-Bravo et al., 2014). Given that the numerical instability of chromosomes as well as derangements caused by genomic instability are widely accepted as causes of tumorigenesis and tumor progression (Hanahan and Weinberg, 2011; Gordon et al., 2012), abnormalities in nuclear shape or the NPC might contribute to tumor formation and/or tumor progression. My observation that normal cells (RPE1) as well as various cancer cells (HeLa, U2OS, HT1080) showed nuclear shape changes in response to H2O2 exposure during mitosis (Fig. 6J) strengthens these inferences. The nuclear envelope has also been reported to regulate gene expression through interactions with transcription factors as well as effects on chromatin organization (Peric-Hupkes et al., 2010; Wilson and Foisner, 2010). I found H2O2

treatment caused no structural changes in the cytoskeleton or ER (Fig. 8C, D), but did appear to cause aggregation of NPC subunits (Fig. 8G). In addition, electron microscopy images revealed an electron-dense region in nuclei with abnormal shapes (Fig. 6D). In this region, the NPC might also change, and both genomic stability and gene expression are expected to be different in these cells compared with normal cells, a possibility that warrants further investigation.

Many existing anticancer therapeutics, as well as those under development, are antimitotic agents. But cancer cells often adapt to these drugs, resulting in mitotic ‘slippage’ and subsequent survival of cancer cells; these drugs are also cytotoxic to normal dividing cells (Andreassen et al., 1996; Rieder and Maiato, 2004). As these side effects have emerged, new

studies have been conducted to identify new cancer cell-specific drugs (Gorjanacz, 2014). To specifically kill cancer cells, it is necessary to identify and target cellular characteristics that are unique to cancer cells, such as nuclear deformation. Therefore, investigating the phenomenon of nuclear shape change—one of the defining characteristics of cancer cells—

might foster the development of future anticancer therapies.

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