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. 2016 Feb 4:9:7.
doi: 10.1186/s13045-016-0238-5.

Haplo-insufficiency of both BubR1 and SGO1 accelerates cellular senescence

Sung-Hyun Park  1 , Steve Xie  2 , Chinthalapally V Rao  3 , Wei Dai  4
Affiliations

Haplo-insufficiency of both BubR1 and SGO1 accelerates cellular senescence

Sung-Hyun Park et al. J Hematol Oncol. .

Abstract

Background: Spindle assembly checkpoint components BubR1 and Sgo1 play a key role in the maintenance of chromosomal instability during cell division. These proteins function to block the anaphase entry until all condensed chromosomes have been attached by the microtubules emanating from both spindle poles. Haplo-insufficiency of either BubR1 or SGO1 results in enhanced chromosomal instability and tumor development in the intestine. Recent studies show that spindle checkpoint proteins also have a role in slowing down the ageing process. Therefore, we want to study whether haplo-insufficiency of both BubR1 and SGO1 accelerates cellular senescence in mice.

Methods: We took advantage of the availability of BubR1 and SGO1 knockout mice and generated primary murine embryonic fibroblasts (MEFs) with mutations in either BubR1, SGO1, or both and analyzed cellular senescence of the MEFs of various genetic backgrounds.

Results: We observed that BubR1(+/-) SGO(+/-) MEFs had an accelerated cellular senescence characterized by morphological changes and expressed senescence-associated β-galactosidase. In addition, compared with wild-type MEFs or MEFs with a single gene deficiency, BubR1(+/-) SGO1(+/-) MEFs expressed enhanced levels of p21 but not p16.

Conclusions: Taken together, our observations suggest that combined deficiency of BubR1 and Sgo1 accelerates cellular senescence.

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Figures

Fig. 1
Fig. 1
BubR1 +/− SGO1 +/− MEFs exhibit senescent-like morphologies at an accelerated rate. Primary MEFs of various genetic backgrounds (wild-type, BubR1 +/−, SGO1 +/−, and BubR1 +/− SGO1 +/−) were cultured in DMEM supplemented with FBS and antibiotics as described in the Methods section. Serial cultures were carried out at a density of 106 cells/100-mm dish every 7 days. At each passage, images of MEFs were recorded with a light microscope. Images of MEFs of wild-type (WT), BubR1 +/−, SGO1 +/−, and BubR1 +/− SGO1 +/− at passages 4, 6, and 8 are shown
Fig. 2
Fig. 2
BubR1 +/− or SGO1 +/− deficiency leads to enhanced expression of β-galactosidase. Primary MEFs of various genetic backgrounds (wild-type, BubR1 +/−, SGO1 +/−, and BubR1 +/− SGO1 +/−) at passage 6 were stained for senescence-associated β-Gal activity (blue according to the manufacturer’s protocol. β-Gal-positive cells were stained with blue
Fig. 3
Fig. 3
BubR1 +/− SGO1 +/− MEFs express SA-β-Gal much earlier than wild-type BubR1 +/− or SGO1 +/− MEFs. a Primary MEFs of various genetic backgrounds (wild-type and BubR1 +/− SGO1 +/−) at passage 3 were stained for senescence-associated β-Gal activity. b Primary MEFs of various genetic backgrounds (wild-type BubR1 +/−, SGO1 +/−, and BubR1 +/− SGO1 +/−) at passage 3 were stained for senescence-associated β-Gal activity. β-Gal-positive cells were recorded. Summarized data from three independent experiments are shown
Fig. 4
Fig. 4
BubR1 +/− SGO1 +/− compound mutations accelerate senescence. Primary MEFs of various genetic backgrounds (wild-type, BubR1 +/−, SGO1 +/−, and BubR1 +/− SGO1 +/−) were cultured in DMEM as described in the Methods section. MEFs of different passages were stained for SA-β-Gal. Percents of SA-β-Gal cells were recorded. Data summarized from three independent experiments are presented
Fig. 5
Fig. 5
a BubR1 +/− SGO1 +/− MEFs express enhanced levels of p21. MEFs of various genetic backgrounds (wildtype, BubR1 +/−, SGO1 +/−, and BubR1 +/− SGO1 +/−) were cultured and passed in vitro as described in the Methods section. MEFs of different passages were collected and lysed. Equal amounts of cell lysates were blotted for p21 and β-actin. b The same set of lysates were blotted with antibody to p16
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