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Polyoma small T antigen triggers cell death via mitotic catastrophe.

Pores Fernando AT, Andrabi S, Cizmecioglu O, Zhu C, Livingston DM, Higgins JM, Schaffhausen BS, Roberts TM - Oncogene (2014)

Bottom Line: In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis.These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, thereby resulting in the activation of the spindle assembly checkpoint.These data suggested, and our results confirmed, that PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA [2] Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.

ABSTRACT
Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, thereby resulting in the activation of the spindle assembly checkpoint. Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed, that PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors.

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PyST expressing cells exhibit aberrant spindle pole formation and loss of cohesion(A) Control cells and PyST expressing cells analyzed with epifluorescence microscopy revealed that there was an increase in the incidence of multipolar spindle formation in PyST expressing cells. Cells were fixed and stained with anti- alpha- tubulin and anti-gamma tubulin followed by corresponding FITC/TRITC secondary antibodies (Ai) or Alexa 488 and 568 secondary antibodies (Aii). Cells were counterstained with DAPI. Quantification from three such experiments is plotted in Aii. Control cells were mostly bipolar whereas PyST expressing cells showed an increased prevalence of multipolar spindles and decreased levels of bipolar spindle formation. (B) Z stacks of anti-gamma tubulin staining in control cells and PyST expressing cells imaged at 0.3 micron intervals. Representative images at 0.9 micron intervals are shown. (C) Categorization of mitotic chromosome spreads from control U2OS and PyST-expressing cells reveals defects in chromosome cohesion upon PyST expression. Means +/- SD are shown (n=4). At least 50 cells were examined in each replicate. See also See also Supplementary Figure 4.
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Figure 4: PyST expressing cells exhibit aberrant spindle pole formation and loss of cohesion(A) Control cells and PyST expressing cells analyzed with epifluorescence microscopy revealed that there was an increase in the incidence of multipolar spindle formation in PyST expressing cells. Cells were fixed and stained with anti- alpha- tubulin and anti-gamma tubulin followed by corresponding FITC/TRITC secondary antibodies (Ai) or Alexa 488 and 568 secondary antibodies (Aii). Cells were counterstained with DAPI. Quantification from three such experiments is plotted in Aii. Control cells were mostly bipolar whereas PyST expressing cells showed an increased prevalence of multipolar spindles and decreased levels of bipolar spindle formation. (B) Z stacks of anti-gamma tubulin staining in control cells and PyST expressing cells imaged at 0.3 micron intervals. Representative images at 0.9 micron intervals are shown. (C) Categorization of mitotic chromosome spreads from control U2OS and PyST-expressing cells reveals defects in chromosome cohesion upon PyST expression. Means +/- SD are shown (n=4). At least 50 cells were examined in each replicate. See also See also Supplementary Figure 4.

Mentions: We next used α- and γ- tubulin immunostaining to assess the status of the mitotic spindles. There was an increase in the incidence of multipolar spindle formation in PyST expressing cells (30% in PyST expressing cells compared to 3% in control cells), which correlated with the failure of chromosome alignment, while control cells had normal bipolar spindles (Figure 4A). In general, the spindle axis is thought to lie parallel to the substratum (reviewed in 19). However, we observed that PyST expressing cells frequently contained misaligned spindle axes. Images of γ-tubulin staining for different z-stacks (0.3 microns intervals) were obtained and the orientation of the mitotic spindle in relation to the coverslip was determined. While we were able to observe both the spindle poles of the control cells on the same z plane (or very close to each other), they were frequently represented in different z planes for PyST expressing cells that had bipolar spindles. Since PyST-expressing cells contained misaligned chromosomes, we also asked whether they had lost their centromeric cohesion. We prepared mitotic chromosome spreads for control and PyST expressing U2OS cells and co-stained with anti-centromere antibodies and Hoechst 33342 for DNA visualization. In control cells only ∼5% of the cells had lost cohesion while in PyST expressing cells almost 60% exhibited separated or completely scattered sister chromatids (Supplementary Figure 4, Figure 4C).


Polyoma small T antigen triggers cell death via mitotic catastrophe.

Pores Fernando AT, Andrabi S, Cizmecioglu O, Zhu C, Livingston DM, Higgins JM, Schaffhausen BS, Roberts TM - Oncogene (2014)

PyST expressing cells exhibit aberrant spindle pole formation and loss of cohesion(A) Control cells and PyST expressing cells analyzed with epifluorescence microscopy revealed that there was an increase in the incidence of multipolar spindle formation in PyST expressing cells. Cells were fixed and stained with anti- alpha- tubulin and anti-gamma tubulin followed by corresponding FITC/TRITC secondary antibodies (Ai) or Alexa 488 and 568 secondary antibodies (Aii). Cells were counterstained with DAPI. Quantification from three such experiments is plotted in Aii. Control cells were mostly bipolar whereas PyST expressing cells showed an increased prevalence of multipolar spindles and decreased levels of bipolar spindle formation. (B) Z stacks of anti-gamma tubulin staining in control cells and PyST expressing cells imaged at 0.3 micron intervals. Representative images at 0.9 micron intervals are shown. (C) Categorization of mitotic chromosome spreads from control U2OS and PyST-expressing cells reveals defects in chromosome cohesion upon PyST expression. Means +/- SD are shown (n=4). At least 50 cells were examined in each replicate. See also See also Supplementary Figure 4.
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Related In: Results  -  Collection

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Figure 4: PyST expressing cells exhibit aberrant spindle pole formation and loss of cohesion(A) Control cells and PyST expressing cells analyzed with epifluorescence microscopy revealed that there was an increase in the incidence of multipolar spindle formation in PyST expressing cells. Cells were fixed and stained with anti- alpha- tubulin and anti-gamma tubulin followed by corresponding FITC/TRITC secondary antibodies (Ai) or Alexa 488 and 568 secondary antibodies (Aii). Cells were counterstained with DAPI. Quantification from three such experiments is plotted in Aii. Control cells were mostly bipolar whereas PyST expressing cells showed an increased prevalence of multipolar spindles and decreased levels of bipolar spindle formation. (B) Z stacks of anti-gamma tubulin staining in control cells and PyST expressing cells imaged at 0.3 micron intervals. Representative images at 0.9 micron intervals are shown. (C) Categorization of mitotic chromosome spreads from control U2OS and PyST-expressing cells reveals defects in chromosome cohesion upon PyST expression. Means +/- SD are shown (n=4). At least 50 cells were examined in each replicate. See also See also Supplementary Figure 4.
Mentions: We next used α- and γ- tubulin immunostaining to assess the status of the mitotic spindles. There was an increase in the incidence of multipolar spindle formation in PyST expressing cells (30% in PyST expressing cells compared to 3% in control cells), which correlated with the failure of chromosome alignment, while control cells had normal bipolar spindles (Figure 4A). In general, the spindle axis is thought to lie parallel to the substratum (reviewed in 19). However, we observed that PyST expressing cells frequently contained misaligned spindle axes. Images of γ-tubulin staining for different z-stacks (0.3 microns intervals) were obtained and the orientation of the mitotic spindle in relation to the coverslip was determined. While we were able to observe both the spindle poles of the control cells on the same z plane (or very close to each other), they were frequently represented in different z planes for PyST expressing cells that had bipolar spindles. Since PyST-expressing cells contained misaligned chromosomes, we also asked whether they had lost their centromeric cohesion. We prepared mitotic chromosome spreads for control and PyST expressing U2OS cells and co-stained with anti-centromere antibodies and Hoechst 33342 for DNA visualization. In control cells only ∼5% of the cells had lost cohesion while in PyST expressing cells almost 60% exhibited separated or completely scattered sister chromatids (Supplementary Figure 4, Figure 4C).

Bottom Line: In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis.These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, thereby resulting in the activation of the spindle assembly checkpoint.These data suggested, and our results confirmed, that PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA [2] Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.

ABSTRACT
Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, thereby resulting in the activation of the spindle assembly checkpoint. Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed, that PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors.

Show MeSH
Related in: MedlinePlus