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  • br Materials and methods br Results br

    2019-11-15


    Materials and methods
    Results
    Discussion Human Chk, a member of the Csk-family, was initially cloned from two different megakaryocytic cell lines [2], [3]. Structural comparison of human Chk with human Csk shows that human Chk has the additional N-terminal unique domain [2], [3], [25]. Chk is distributed to the nucleus and the cytoplasm (Fig. 1B; [17]) whereas Csk is mainly present in the cytoplasm and focal adhesions ([26], [27]; our unpublished data), implicating that Chk has a distinct function in the nucleus. In our previous study, overexpression of Chk brought about retardation of proliferation and aberration of cell division, including multi-lobulation of the nucleus, multinucleation, and polyploidization, in IL-3/GM-CSF-dependent human immature myeloid KMT-2 imidazoline receptors [17]. Ectopic expression of Chk inhibited proliferation of SV40-transformed simian kidney COS-1 cells as well as KMT-2 cells [17]. Moreover, it should be noted from this study that ectopic expression of Chk in COS-1 and HeLa cells induces nuclear multi-lobulation and concomitant inhibition of proliferation (Fig. 1; data not shown). These results suggest that there is common machinery reactive to Chk among such cell types. Multi-lobulated nuclei can be observed in epithelial cells under certain cancerous conditions, e.g., X-ray-irradiated p53−/−p21−/− HCT116 colorectal cancer cells, HeLa epitheloid carcinoma cells expressing a noncleavable SCC1 cohesin-subunit mutant, and MCF10A mammary epithelial cells co-expressing oncogenic Ras and active RhoA [28], [29], [30]. Those data suggest that the abortive mitosis caused by aberration of the cell-cycle control system leads to multi-lobulation of the nucleus and concomitant multinucleation/polyploidization without disturbing re-entry into S phase. On the other hand, we showed by time-lapse fluorescence microscopy and flow cytometry that nuclear expression of Chk induces multi-lobulation of the nucleus without multinucleation and polyploidization (Fig. 1, Fig. 4). Cell sorting experiments showed that multi-lobulated nuclei are observed predominantly in late S phase (Fig. 5). Thus, these results suggest that multi-lobulation of the nucleus induced by Chk can take place in late S phase independently of multinucleation and polyploidization. It is intriguing to hypothesize that Chk might play a role in multi-lobulation of megakaryocytes and polymorphonuclear leukocytes. There is increasing evidence in support of the interaction between the nucleus and microtubules. Nuclear positioning, which is crucial for cell division in budding yeasts and Drosophila syncytial embryos, is a microtubule-dependent process [31], [32]. The forces produced by polymerization and depolymerization of microtubules together with dynein and kinesin are able to control migration of the nucleus. At G2/M transition, microtubules pull the prophase nuclear envelope for the onset of nuclear envelope breakdown, giving rise to deformation and tearing of the nuclear envelope. Prophase nuclear envelope is invaginated by the action of nuclear envelope-associated dynein and centrosome-associated microtubules [33], [34], [35]. However, given that Chk-induced multi-lobulation of the nucleus takes place in S phase in a microtubule-dependent manner (Fig. 5, Fig. 6), it should be noted that the nucleus can interact with microtubules in S phase as well as M phase. Chk-induced multi-lobulation is incompletely abolished by treatment with nocodazole for 3 h, although microtubules are disrupted thoroughly in this condition (Fig. 6). These results indicate that complete abolishment of the multi-lobulation entails many hours after disruption of microtubules. This delayed recovery from the multi-lobulation leads to the idea that the action of microtubule-dependent forces is influenced by a degree of the rigidity of nuclear structure. Nuclear lamins, the major components comprising the nuclear lamina [36], play an important role in maintaining nuclear shape. Nuclear lamins are phosphorylated by Cdc2 and are rapidly disassembled during the prophase/metaphase transition. Expression of an unphosphorylated lamin mutant inhibits nuclear envelope breakdown, resulting in formation of lobulated nuclei [37]. In fact, the nuclear lamina is suggested to play a role in the nuclear envelope organization and nuclear shape because expression of a truncated lamin mutant perturbs the substructure of the nuclear lamina accompanying nuclear lobulation [38]. Perturbation of the nuclear lamina in cells expressing Chk (Fig. 6A) may be explained by the facts that nuclear-localized Chk influences the distribution of lamin B1 and that an increase in nuclear localization of Chk promotes multi-lobulation of the nucleus (Fig. 1, Fig. 2). Collectively, we hypothesize that multi-lobulation of the nucleus involves Chk-induced perturbation of the nuclear lamina in concert with the forces produced by microtubule dynamics.