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The transcriptional repressor Kaiso localizes at the mitotic spindle and is a constituent of the pericentriolar material.

Soubry A, Staes K, Parthoens E, Noppen S, Stove C, Bogaert P, van Hengel J, van Roy F - PLoS ONE (2010)

Bottom Line: In the present study we monitored Kaiso's subcellular localization during the cell cycle and found the following: (1) during interphase, Kaiso is located not only in the nucleus, but also on microtubular structures, including the centrosome; (2) at metaphase, it is present at the centrosomes and on the spindle microtubules; (3) during telophase, it accumulates at the midbody.We found that Kaiso is a genuine PCM component that belongs to a pericentrin molecular complex.Knockdown of Kaiso accelerated cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department for Molecular Biomedical Research, VIB, Ghent, Belgium.

ABSTRACT
Kaiso is a BTB/POZ zinc finger protein known as a transcriptional repressor. It was originally identified through its in vitro association with the Armadillo protein p120ctn. Subcellular localization of Kaiso in cell lines and in normal and cancerous human tissues revealed that its expression is not restricted to the nucleus. In the present study we monitored Kaiso's subcellular localization during the cell cycle and found the following: (1) during interphase, Kaiso is located not only in the nucleus, but also on microtubular structures, including the centrosome; (2) at metaphase, it is present at the centrosomes and on the spindle microtubules; (3) during telophase, it accumulates at the midbody. We found that Kaiso is a genuine PCM component that belongs to a pericentrin molecular complex. We analyzed the functions of different domains of Kaiso by visualizing the subcellular distribution of GFP-tagged Kaiso fragments throughout the cell cycle. Our results indicate that two domains are responsible for targeting Kaiso to the centrosomes and microtubules. The first domain, designated SA1 for spindle-associated domain 1, is located in the center of the Kaiso protein and localizes at the spindle microtubules and centrosomes; the second domain, SA2, is an evolutionarily conserved domain situated just before the zinc finger domain and might be responsible for localizing Kaiso towards the centrosomal region. Constructs containing both SA domains and Kaiso's aminoterminal BTB/POZ domain triggered the formation of abnormal centrosomes. We also observed that overexpression of longer or full-length Kaiso constructs led to mitotic cell arrest and frequent cell death. Knockdown of Kaiso accelerated cell proliferation. Our data reveal a new target for Kaiso at the centrosomes and spindle microtubules during mitosis. They also strongly imply that Kaiso's function as a transcriptional regulator might be linked to the control of the cell cycle and to cell proliferation in cancer.

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Related in: MedlinePlus

Knockdown of Kaiso in SK-LMS-1 cells accelerates cell proliferation.(A) SK-LMS-1 parental cells and various SK-LMS-1_shKAISO derivative cell populations were subjected to an SRB cell proliferation assay. Cells with medium to high GFP expression levels (med+ and hi+; with stringent Kaiso knockdown) did proliferate more rapidly and reached significantly higher saturation densities than cells with very low GFP expression (hi– and med–; with poor Kaiso knockdown). Values are means ± SD; n = 6. Letters a and b indicate significant differences (p<0.05; Wilcoxon rank sum test): a, SK-LMS-1hi+ vs. parental cells; b, SK-LMS-1hi+ vs. SK-LMS-1hi- cells. (B) Cell cycle profile from unsynchronized SK-LMS-1_shKAISO cell populations with either low GFP expression (GFP–; with poor Kaiso knockdown) or high GFP expression (GFP+, with stringent Kaiso knockdown). Cells were stained with Hoechst33342 and analyzed by flow cytometry to detect 2n/4n DNA contents. In the GFP+ cells, fewer cells (39.8%) contained 4n amounts of DNA than in the GFP– cells (44.4%). Data shown represent one example of four independent experiments. All analyses were performed on subconfluent cell populations. (C) Flow cytometric analysis of cell proliferation by unsynchronized SK-LMS-1_shKAISO cell populations. At time 0, cells were stained with the red fluorescent cell-tracking dye, PKH26, which incorporates into cell membrane lipids and is thus diluted twofold every cell division. At 24 h and at 48 h after plating, cells were first distinguished according to GFP levels (left panels), where cells with high GFP levels (GFP+) show strong Kaiso knockdown. Dead cells were excluded from analysis by positive staining for SytoxRed. Mean PKH26 fluorescence intensity (MFI) was then measured in each subpopulation (right panels), showing that GFP+ cells underwent 1.56 [log2(9343/3162)] cell divisions in 24 h, whereas GFP– cells underwent 1.20 [log2(10470/4549)] cell divisions.
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pone-0009203-g012: Knockdown of Kaiso in SK-LMS-1 cells accelerates cell proliferation.(A) SK-LMS-1 parental cells and various SK-LMS-1_shKAISO derivative cell populations were subjected to an SRB cell proliferation assay. Cells with medium to high GFP expression levels (med+ and hi+; with stringent Kaiso knockdown) did proliferate more rapidly and reached significantly higher saturation densities than cells with very low GFP expression (hi– and med–; with poor Kaiso knockdown). Values are means ± SD; n = 6. Letters a and b indicate significant differences (p<0.05; Wilcoxon rank sum test): a, SK-LMS-1hi+ vs. parental cells; b, SK-LMS-1hi+ vs. SK-LMS-1hi- cells. (B) Cell cycle profile from unsynchronized SK-LMS-1_shKAISO cell populations with either low GFP expression (GFP–; with poor Kaiso knockdown) or high GFP expression (GFP+, with stringent Kaiso knockdown). Cells were stained with Hoechst33342 and analyzed by flow cytometry to detect 2n/4n DNA contents. In the GFP+ cells, fewer cells (39.8%) contained 4n amounts of DNA than in the GFP– cells (44.4%). Data shown represent one example of four independent experiments. All analyses were performed on subconfluent cell populations. (C) Flow cytometric analysis of cell proliferation by unsynchronized SK-LMS-1_shKAISO cell populations. At time 0, cells were stained with the red fluorescent cell-tracking dye, PKH26, which incorporates into cell membrane lipids and is thus diluted twofold every cell division. At 24 h and at 48 h after plating, cells were first distinguished according to GFP levels (left panels), where cells with high GFP levels (GFP+) show strong Kaiso knockdown. Dead cells were excluded from analysis by positive staining for SytoxRed. Mean PKH26 fluorescence intensity (MFI) was then measured in each subpopulation (right panels), showing that GFP+ cells underwent 1.56 [log2(9343/3162)] cell divisions in 24 h, whereas GFP– cells underwent 1.20 [log2(10470/4549)] cell divisions.

Mentions: We then addressed the functional implications of Kaiso knockdown. Abnormalities in mitotic spindle formation, centrosomal division or PCM composition appeared not to be increased by the knockdown (data not shown). However, in a cell proliferation assay, cell populations with efficient Kaiso knockdown showed significantly increased growth rate and saturation density (Fig. 12A). It was somewhat surprising that DNA content analysis in unsynchronized, subconfluent SK-LMS-1_shKAISO cell subpopulations showed a lower level of 4n cells in GFP+ cells with strong Kaiso reduction (Fig. 12B). In combination with the increased growth rate and density of such cells, this points at a shorter S/G2/M period rather than a shorter G1 phase. The length of the cell cycle was analyzed on the basis of dilution speed of PHK26, a lipophilic dye that stably integrates into the cell membrane (Fig. 12C). Subconfluent GFP+ subpopulations of SK-LMS-1_shKAISO cells underwent 1.56 cell divisions in 24 h, whereas 1.20 divisions were calculated for GFP–cells. Hence, in SK-LMS-1 cells Kaiso slows down the cell cycle.


The transcriptional repressor Kaiso localizes at the mitotic spindle and is a constituent of the pericentriolar material.

Soubry A, Staes K, Parthoens E, Noppen S, Stove C, Bogaert P, van Hengel J, van Roy F - PLoS ONE (2010)

Knockdown of Kaiso in SK-LMS-1 cells accelerates cell proliferation.(A) SK-LMS-1 parental cells and various SK-LMS-1_shKAISO derivative cell populations were subjected to an SRB cell proliferation assay. Cells with medium to high GFP expression levels (med+ and hi+; with stringent Kaiso knockdown) did proliferate more rapidly and reached significantly higher saturation densities than cells with very low GFP expression (hi– and med–; with poor Kaiso knockdown). Values are means ± SD; n = 6. Letters a and b indicate significant differences (p<0.05; Wilcoxon rank sum test): a, SK-LMS-1hi+ vs. parental cells; b, SK-LMS-1hi+ vs. SK-LMS-1hi- cells. (B) Cell cycle profile from unsynchronized SK-LMS-1_shKAISO cell populations with either low GFP expression (GFP–; with poor Kaiso knockdown) or high GFP expression (GFP+, with stringent Kaiso knockdown). Cells were stained with Hoechst33342 and analyzed by flow cytometry to detect 2n/4n DNA contents. In the GFP+ cells, fewer cells (39.8%) contained 4n amounts of DNA than in the GFP– cells (44.4%). Data shown represent one example of four independent experiments. All analyses were performed on subconfluent cell populations. (C) Flow cytometric analysis of cell proliferation by unsynchronized SK-LMS-1_shKAISO cell populations. At time 0, cells were stained with the red fluorescent cell-tracking dye, PKH26, which incorporates into cell membrane lipids and is thus diluted twofold every cell division. At 24 h and at 48 h after plating, cells were first distinguished according to GFP levels (left panels), where cells with high GFP levels (GFP+) show strong Kaiso knockdown. Dead cells were excluded from analysis by positive staining for SytoxRed. Mean PKH26 fluorescence intensity (MFI) was then measured in each subpopulation (right panels), showing that GFP+ cells underwent 1.56 [log2(9343/3162)] cell divisions in 24 h, whereas GFP– cells underwent 1.20 [log2(10470/4549)] cell divisions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2821401&req=5

pone-0009203-g012: Knockdown of Kaiso in SK-LMS-1 cells accelerates cell proliferation.(A) SK-LMS-1 parental cells and various SK-LMS-1_shKAISO derivative cell populations were subjected to an SRB cell proliferation assay. Cells with medium to high GFP expression levels (med+ and hi+; with stringent Kaiso knockdown) did proliferate more rapidly and reached significantly higher saturation densities than cells with very low GFP expression (hi– and med–; with poor Kaiso knockdown). Values are means ± SD; n = 6. Letters a and b indicate significant differences (p<0.05; Wilcoxon rank sum test): a, SK-LMS-1hi+ vs. parental cells; b, SK-LMS-1hi+ vs. SK-LMS-1hi- cells. (B) Cell cycle profile from unsynchronized SK-LMS-1_shKAISO cell populations with either low GFP expression (GFP–; with poor Kaiso knockdown) or high GFP expression (GFP+, with stringent Kaiso knockdown). Cells were stained with Hoechst33342 and analyzed by flow cytometry to detect 2n/4n DNA contents. In the GFP+ cells, fewer cells (39.8%) contained 4n amounts of DNA than in the GFP– cells (44.4%). Data shown represent one example of four independent experiments. All analyses were performed on subconfluent cell populations. (C) Flow cytometric analysis of cell proliferation by unsynchronized SK-LMS-1_shKAISO cell populations. At time 0, cells were stained with the red fluorescent cell-tracking dye, PKH26, which incorporates into cell membrane lipids and is thus diluted twofold every cell division. At 24 h and at 48 h after plating, cells were first distinguished according to GFP levels (left panels), where cells with high GFP levels (GFP+) show strong Kaiso knockdown. Dead cells were excluded from analysis by positive staining for SytoxRed. Mean PKH26 fluorescence intensity (MFI) was then measured in each subpopulation (right panels), showing that GFP+ cells underwent 1.56 [log2(9343/3162)] cell divisions in 24 h, whereas GFP– cells underwent 1.20 [log2(10470/4549)] cell divisions.
Mentions: We then addressed the functional implications of Kaiso knockdown. Abnormalities in mitotic spindle formation, centrosomal division or PCM composition appeared not to be increased by the knockdown (data not shown). However, in a cell proliferation assay, cell populations with efficient Kaiso knockdown showed significantly increased growth rate and saturation density (Fig. 12A). It was somewhat surprising that DNA content analysis in unsynchronized, subconfluent SK-LMS-1_shKAISO cell subpopulations showed a lower level of 4n cells in GFP+ cells with strong Kaiso reduction (Fig. 12B). In combination with the increased growth rate and density of such cells, this points at a shorter S/G2/M period rather than a shorter G1 phase. The length of the cell cycle was analyzed on the basis of dilution speed of PHK26, a lipophilic dye that stably integrates into the cell membrane (Fig. 12C). Subconfluent GFP+ subpopulations of SK-LMS-1_shKAISO cells underwent 1.56 cell divisions in 24 h, whereas 1.20 divisions were calculated for GFP–cells. Hence, in SK-LMS-1 cells Kaiso slows down the cell cycle.

Bottom Line: In the present study we monitored Kaiso's subcellular localization during the cell cycle and found the following: (1) during interphase, Kaiso is located not only in the nucleus, but also on microtubular structures, including the centrosome; (2) at metaphase, it is present at the centrosomes and on the spindle microtubules; (3) during telophase, it accumulates at the midbody.We found that Kaiso is a genuine PCM component that belongs to a pericentrin molecular complex.Knockdown of Kaiso accelerated cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department for Molecular Biomedical Research, VIB, Ghent, Belgium.

ABSTRACT
Kaiso is a BTB/POZ zinc finger protein known as a transcriptional repressor. It was originally identified through its in vitro association with the Armadillo protein p120ctn. Subcellular localization of Kaiso in cell lines and in normal and cancerous human tissues revealed that its expression is not restricted to the nucleus. In the present study we monitored Kaiso's subcellular localization during the cell cycle and found the following: (1) during interphase, Kaiso is located not only in the nucleus, but also on microtubular structures, including the centrosome; (2) at metaphase, it is present at the centrosomes and on the spindle microtubules; (3) during telophase, it accumulates at the midbody. We found that Kaiso is a genuine PCM component that belongs to a pericentrin molecular complex. We analyzed the functions of different domains of Kaiso by visualizing the subcellular distribution of GFP-tagged Kaiso fragments throughout the cell cycle. Our results indicate that two domains are responsible for targeting Kaiso to the centrosomes and microtubules. The first domain, designated SA1 for spindle-associated domain 1, is located in the center of the Kaiso protein and localizes at the spindle microtubules and centrosomes; the second domain, SA2, is an evolutionarily conserved domain situated just before the zinc finger domain and might be responsible for localizing Kaiso towards the centrosomal region. Constructs containing both SA domains and Kaiso's aminoterminal BTB/POZ domain triggered the formation of abnormal centrosomes. We also observed that overexpression of longer or full-length Kaiso constructs led to mitotic cell arrest and frequent cell death. Knockdown of Kaiso accelerated cell proliferation. Our data reveal a new target for Kaiso at the centrosomes and spindle microtubules during mitosis. They also strongly imply that Kaiso's function as a transcriptional regulator might be linked to the control of the cell cycle and to cell proliferation in cancer.

Show MeSH
Related in: MedlinePlus