Limits...
Sgo1 is a potential therapeutic target for hepatocellular carcinoma.

Wang LH, Yen CJ, Li TN, Elowe S, Wang WC, Wang LH - Oncotarget (2015)

Bottom Line: Shugoshin-like protein 1 (Sgo1) is an essential protein in mitosis; it protects sister chromatid cohesion and thereby ensures the fidelity of chromosome separation.In contrast, cell viability and mitotic progression of immortalized cells were not significantly affected.Notably, mitotic cell death induced upon Sgo1 depletion was suppressed upon inhibitions of cyclin-dependent kinase-1 and Aurora kinase-B, or the depletion of mitotic arrest deficient-2.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan.

ABSTRACT
Shugoshin-like protein 1 (Sgo1) is an essential protein in mitosis; it protects sister chromatid cohesion and thereby ensures the fidelity of chromosome separation. We found that the expression of Sgo1 mRNA was relatively low in normal tissues, but was upregulated in 82% of hepatocellular carcinoma (HCC), and correlated with elevated alpha-fetoprotein and early disease onset of HCC. The depletion of Sgo1 reduced cell viability of hepatoma cell lines including HuH7, HepG2, Hep3B, and HepaRG. Using time-lapse microscopy, we showed that hepatoma cells were delayed and ultimately die in mitosis in the absence of Sgo1. In contrast, cell viability and mitotic progression of immortalized cells were not significantly affected. Notably, mitotic cell death induced upon Sgo1 depletion was suppressed upon inhibitions of cyclin-dependent kinase-1 and Aurora kinase-B, or the depletion of mitotic arrest deficient-2. Thus, mitotic cell death induced upon Sgo1 depletion in hepatoma cells is mediated by persistent activation of the spindle assembly checkpoint. Together, these results highlight the essential role of Sgo1 in the maintenance of a proper mitotic progression in hepatoma cells and suggest that Sgo1 is a promising oncotarget for HCC.

Show MeSH

Related in: MedlinePlus

Sgo1 depletion reduced cell viabilities in HeLa and various hepatoma cells(A) Endogenous levels of Sgo1 in two immortalized and five different transformed cell lines were reduced upon the depletion Sgo1 by RNA interference. Blot for β-actin was included as an internal control. (B) Cell viabilities of indicated cell lines treated with control or Sgo1 siRNA were examined by WST-1 assay. (C) Mitotic progressions of HuH-7, NeHepLxHT, HeLa, and RPE-1 cells depleted with Sgo1 were monitored by time-lapse live cell microscopy. Cells were synchronized with thymidine and released after 24 hours when acquisition of differential interference contrast images began as illustrated by the experimental design. Representative time-point images were selected to show mitotic entry (time stamps displayed as 00:00 for hr:min) and progression of indicated cell lines. White arrows followed the same cell and its progenies over time within same panels. Overall number of cell death (D) and cells death in mitosis (E) during time-lapse imaging. (F) HuH-7 cells were treated with control and Sgo1 siRNA for 48 hrs followed by fixation and staining with 4′,6-diamidino-2-phenylindole (DAPI). Representative images of cells without or with micronucleation (indicated by white arrows, enlarged images shown in right panels). The depletion of Sgo1 increased micronucleation by approximately 3-fold in interphase cells. Asterisks represent a significance difference by Student's t-test (*, p < 0.05; **, p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4385833&req=5

Figure 4: Sgo1 depletion reduced cell viabilities in HeLa and various hepatoma cells(A) Endogenous levels of Sgo1 in two immortalized and five different transformed cell lines were reduced upon the depletion Sgo1 by RNA interference. Blot for β-actin was included as an internal control. (B) Cell viabilities of indicated cell lines treated with control or Sgo1 siRNA were examined by WST-1 assay. (C) Mitotic progressions of HuH-7, NeHepLxHT, HeLa, and RPE-1 cells depleted with Sgo1 were monitored by time-lapse live cell microscopy. Cells were synchronized with thymidine and released after 24 hours when acquisition of differential interference contrast images began as illustrated by the experimental design. Representative time-point images were selected to show mitotic entry (time stamps displayed as 00:00 for hr:min) and progression of indicated cell lines. White arrows followed the same cell and its progenies over time within same panels. Overall number of cell death (D) and cells death in mitosis (E) during time-lapse imaging. (F) HuH-7 cells were treated with control and Sgo1 siRNA for 48 hrs followed by fixation and staining with 4′,6-diamidino-2-phenylindole (DAPI). Representative images of cells without or with micronucleation (indicated by white arrows, enlarged images shown in right panels). The depletion of Sgo1 increased micronucleation by approximately 3-fold in interphase cells. Asterisks represent a significance difference by Student's t-test (*, p < 0.05; **, p < 0.01).

Mentions: Sgo1 is essential for sister chromatid cohesion during mitosis and its depletion has been shown to induce cell death in cervical carcinoma and lung cancer cells [8, 21]. With regard to the relatively high expression level of Sgo1 observed in HCC and hepatoma cell lines, we suggest that hepatoma cells may demand a high level of Sgo1 for the maintenance of sister chromatid cohesion. To test this notion, we investigated cell proliferation and mitotic cell progression in the absence of Sgo1. Using a defined small interfering RNA oligonucleotide targeting Sgo1 [7], we successfully reduced endogenous levels of Sgo1 by 60~85% in two immortalized cell lines (RPE-1 and NeHepLxHT) and five different transformed cell lines (HeLa, HuH-7, HepG2, Hep3B, HepaRG) (Fig. 4A). Cell viability of these five transformed cells was significantly reduced upon Sgo1 depletion. In contrast, Sgo1 depletion had no significant impact on RPE-1 and NeHepLxHT cells (Fig. 4B). To understand the nature of reduced cell viability in these transformed cells, we followed cell cycle progression by time-lapse microscopy. Upon Sgo1 depletion, both HuH-7 and HeLa cells displayed a delay in mitosis for several hours and ultimately, these cells died without exiting mitosis, a phenomenon known as mitotic catastrophe (Fig. 4C, 4D). Specifically, about 80% of HeLa cells and 50% of HuH-7 cells were delayed in mitosis for hours before ultimately dying (Fig. 4E). We noted that micronucleation, another morphological trait of mitotic catastrophe, was significantly increased in HuH-7 cells upon Sgo1 depletion (Fig. 4F). In striking contrast, immortalized NeHepLxHT and RPE-1 cells progressed through mitosis without a significant defect upon Sgo1 depletion (Fig. 4C, 4D). Similar results were obtained in other three hepatoma lines (data not shown). Together, these results highlight the crucial role of Sgo1 in the maintenance of cell viability and a proper mitotic progression in transformed hepatoma cells.


Sgo1 is a potential therapeutic target for hepatocellular carcinoma.

Wang LH, Yen CJ, Li TN, Elowe S, Wang WC, Wang LH - Oncotarget (2015)

Sgo1 depletion reduced cell viabilities in HeLa and various hepatoma cells(A) Endogenous levels of Sgo1 in two immortalized and five different transformed cell lines were reduced upon the depletion Sgo1 by RNA interference. Blot for β-actin was included as an internal control. (B) Cell viabilities of indicated cell lines treated with control or Sgo1 siRNA were examined by WST-1 assay. (C) Mitotic progressions of HuH-7, NeHepLxHT, HeLa, and RPE-1 cells depleted with Sgo1 were monitored by time-lapse live cell microscopy. Cells were synchronized with thymidine and released after 24 hours when acquisition of differential interference contrast images began as illustrated by the experimental design. Representative time-point images were selected to show mitotic entry (time stamps displayed as 00:00 for hr:min) and progression of indicated cell lines. White arrows followed the same cell and its progenies over time within same panels. Overall number of cell death (D) and cells death in mitosis (E) during time-lapse imaging. (F) HuH-7 cells were treated with control and Sgo1 siRNA for 48 hrs followed by fixation and staining with 4′,6-diamidino-2-phenylindole (DAPI). Representative images of cells without or with micronucleation (indicated by white arrows, enlarged images shown in right panels). The depletion of Sgo1 increased micronucleation by approximately 3-fold in interphase cells. Asterisks represent a significance difference by Student's t-test (*, p < 0.05; **, p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385833&req=5

Figure 4: Sgo1 depletion reduced cell viabilities in HeLa and various hepatoma cells(A) Endogenous levels of Sgo1 in two immortalized and five different transformed cell lines were reduced upon the depletion Sgo1 by RNA interference. Blot for β-actin was included as an internal control. (B) Cell viabilities of indicated cell lines treated with control or Sgo1 siRNA were examined by WST-1 assay. (C) Mitotic progressions of HuH-7, NeHepLxHT, HeLa, and RPE-1 cells depleted with Sgo1 were monitored by time-lapse live cell microscopy. Cells were synchronized with thymidine and released after 24 hours when acquisition of differential interference contrast images began as illustrated by the experimental design. Representative time-point images were selected to show mitotic entry (time stamps displayed as 00:00 for hr:min) and progression of indicated cell lines. White arrows followed the same cell and its progenies over time within same panels. Overall number of cell death (D) and cells death in mitosis (E) during time-lapse imaging. (F) HuH-7 cells were treated with control and Sgo1 siRNA for 48 hrs followed by fixation and staining with 4′,6-diamidino-2-phenylindole (DAPI). Representative images of cells without or with micronucleation (indicated by white arrows, enlarged images shown in right panels). The depletion of Sgo1 increased micronucleation by approximately 3-fold in interphase cells. Asterisks represent a significance difference by Student's t-test (*, p < 0.05; **, p < 0.01).
Mentions: Sgo1 is essential for sister chromatid cohesion during mitosis and its depletion has been shown to induce cell death in cervical carcinoma and lung cancer cells [8, 21]. With regard to the relatively high expression level of Sgo1 observed in HCC and hepatoma cell lines, we suggest that hepatoma cells may demand a high level of Sgo1 for the maintenance of sister chromatid cohesion. To test this notion, we investigated cell proliferation and mitotic cell progression in the absence of Sgo1. Using a defined small interfering RNA oligonucleotide targeting Sgo1 [7], we successfully reduced endogenous levels of Sgo1 by 60~85% in two immortalized cell lines (RPE-1 and NeHepLxHT) and five different transformed cell lines (HeLa, HuH-7, HepG2, Hep3B, HepaRG) (Fig. 4A). Cell viability of these five transformed cells was significantly reduced upon Sgo1 depletion. In contrast, Sgo1 depletion had no significant impact on RPE-1 and NeHepLxHT cells (Fig. 4B). To understand the nature of reduced cell viability in these transformed cells, we followed cell cycle progression by time-lapse microscopy. Upon Sgo1 depletion, both HuH-7 and HeLa cells displayed a delay in mitosis for several hours and ultimately, these cells died without exiting mitosis, a phenomenon known as mitotic catastrophe (Fig. 4C, 4D). Specifically, about 80% of HeLa cells and 50% of HuH-7 cells were delayed in mitosis for hours before ultimately dying (Fig. 4E). We noted that micronucleation, another morphological trait of mitotic catastrophe, was significantly increased in HuH-7 cells upon Sgo1 depletion (Fig. 4F). In striking contrast, immortalized NeHepLxHT and RPE-1 cells progressed through mitosis without a significant defect upon Sgo1 depletion (Fig. 4C, 4D). Similar results were obtained in other three hepatoma lines (data not shown). Together, these results highlight the crucial role of Sgo1 in the maintenance of cell viability and a proper mitotic progression in transformed hepatoma cells.

Bottom Line: Shugoshin-like protein 1 (Sgo1) is an essential protein in mitosis; it protects sister chromatid cohesion and thereby ensures the fidelity of chromosome separation.In contrast, cell viability and mitotic progression of immortalized cells were not significantly affected.Notably, mitotic cell death induced upon Sgo1 depletion was suppressed upon inhibitions of cyclin-dependent kinase-1 and Aurora kinase-B, or the depletion of mitotic arrest deficient-2.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan.

ABSTRACT
Shugoshin-like protein 1 (Sgo1) is an essential protein in mitosis; it protects sister chromatid cohesion and thereby ensures the fidelity of chromosome separation. We found that the expression of Sgo1 mRNA was relatively low in normal tissues, but was upregulated in 82% of hepatocellular carcinoma (HCC), and correlated with elevated alpha-fetoprotein and early disease onset of HCC. The depletion of Sgo1 reduced cell viability of hepatoma cell lines including HuH7, HepG2, Hep3B, and HepaRG. Using time-lapse microscopy, we showed that hepatoma cells were delayed and ultimately die in mitosis in the absence of Sgo1. In contrast, cell viability and mitotic progression of immortalized cells were not significantly affected. Notably, mitotic cell death induced upon Sgo1 depletion was suppressed upon inhibitions of cyclin-dependent kinase-1 and Aurora kinase-B, or the depletion of mitotic arrest deficient-2. Thus, mitotic cell death induced upon Sgo1 depletion in hepatoma cells is mediated by persistent activation of the spindle assembly checkpoint. Together, these results highlight the essential role of Sgo1 in the maintenance of a proper mitotic progression in hepatoma cells and suggest that Sgo1 is a promising oncotarget for HCC.

Show MeSH
Related in: MedlinePlus