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p31comet-Induced Cell Death Is Mediated by Binding and Inactivation of Mad2.

Shin HJ, Park ER, Yun SH, Kim SH, Jung WH, Woo SR, Joo HY, Jang SH, Chung HY, Hong SH, Cho MH, Park JJ, Yun M, Lee KH - PLoS ONE (2015)

Bottom Line: Consistently, wild-type Mad2 interacting with p31comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31comet-induced cell death involves Mad2 inactivation.Our results clearly suggest that the regions of p31comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death.The finding that p31comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Division of Radiation Cancer Research, Korea Institute of Radiological & Medical Sciences, Seoul 139-706, Korea.

ABSTRACT
Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31comet is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31comet-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31comet, the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31comet overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31comet. Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31comet. Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wild-type Mad2 interacting with p31comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31comet-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.

No MeSH data available.


Related in: MedlinePlus

Mad2 binding activity is essential for p31comet inhibition of colony formation.(A) Effects of a p31comet mutant (Q83A/F191A) on apoptosis (lower panel) and repression of colony formation (upper panel) were analyzed in HeLa cells. Expression of p31comet and Q83A/F191A was confirmed via Western blot. (B) The effects of a p31comet mutant (Q83/F191A) on Myc/Ras-mediated colony formation in p53-/- MEFs were compared with those of wild-type (WT) p31comet. (C) The effect of Mad2 on p31comet-induced repression of clonogenic cell survival was analyzed in HeLa cells in the presence of either wild-type or dominant-negative mutant Mad2 (R133E/Q134A) (Mad2mt). (D) Expression of p31comet, Mad2, and R133E/Q134A mutant (Mad2mt) was confirmed using immunoblotting. Values represent mean of triplicate samples. Statistical significance (p-value) was calculated with Student’s t-test. * indicates p>0.05, when each test transfection was compared with that of empty vector.
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pone.0141523.g004: Mad2 binding activity is essential for p31comet inhibition of colony formation.(A) Effects of a p31comet mutant (Q83A/F191A) on apoptosis (lower panel) and repression of colony formation (upper panel) were analyzed in HeLa cells. Expression of p31comet and Q83A/F191A was confirmed via Western blot. (B) The effects of a p31comet mutant (Q83/F191A) on Myc/Ras-mediated colony formation in p53-/- MEFs were compared with those of wild-type (WT) p31comet. (C) The effect of Mad2 on p31comet-induced repression of clonogenic cell survival was analyzed in HeLa cells in the presence of either wild-type or dominant-negative mutant Mad2 (R133E/Q134A) (Mad2mt). (D) Expression of p31comet, Mad2, and R133E/Q134A mutant (Mad2mt) was confirmed using immunoblotting. Values represent mean of triplicate samples. Statistical significance (p-value) was calculated with Student’s t-test. * indicates p>0.05, when each test transfection was compared with that of empty vector.

Mentions: To provide further evidence that p31comet-induced death relies on Mad2 interactions, we examined a p31comet (Q83A/F191A) mutant lacking binding activity to Mad2 [17]. In contrast to wild-type p31comet, the mutant form did not suppress clonal survival when transduced into HeLa cells via retroviral infection (Fig 4A) or MEFs with Myc/Ras co-transfection (Fig 4B). Consistently, HeLa cells infected with the Q83A/F191A mutant were not susceptible to Annexin staining, in contrast to those infected with wild-type p31comet (Fig 4A). These findings imply that p31comet-induced death occurs only when Mad2 binding activity is retained, ultimately leading to inhibition of Mad2 action. To explore this point, we performed an experiment to establish whether p31comet-induced death can be rescued by Mad2. To this end, HeLa cells retrovirally infected with p31comet were reinfected with retroviruses containing wild-type Mad2. As expected, p31comet -induced suppression of clonal survival was reversed by wild-type Mad2 (Fig 4C and 4D). However, the dominant-negative form of Mad2 (R133E/Q134A) lacking the ability to bind to p31comet (and devoid of spindle checkpoint activity) [21] did not affect survival (Fig 4C and 4D). Mad2 or its mutant (R133E/Q134A) alone did not suppress colony formation. These data support the theory that Mad2 inactivation is essential for cell death induction by p31comet. Furthermore, the balance between p31comet and Mad2 levels may determine the cytotoxic effect.


p31comet-Induced Cell Death Is Mediated by Binding and Inactivation of Mad2.

Shin HJ, Park ER, Yun SH, Kim SH, Jung WH, Woo SR, Joo HY, Jang SH, Chung HY, Hong SH, Cho MH, Park JJ, Yun M, Lee KH - PLoS ONE (2015)

Mad2 binding activity is essential for p31comet inhibition of colony formation.(A) Effects of a p31comet mutant (Q83A/F191A) on apoptosis (lower panel) and repression of colony formation (upper panel) were analyzed in HeLa cells. Expression of p31comet and Q83A/F191A was confirmed via Western blot. (B) The effects of a p31comet mutant (Q83/F191A) on Myc/Ras-mediated colony formation in p53-/- MEFs were compared with those of wild-type (WT) p31comet. (C) The effect of Mad2 on p31comet-induced repression of clonogenic cell survival was analyzed in HeLa cells in the presence of either wild-type or dominant-negative mutant Mad2 (R133E/Q134A) (Mad2mt). (D) Expression of p31comet, Mad2, and R133E/Q134A mutant (Mad2mt) was confirmed using immunoblotting. Values represent mean of triplicate samples. Statistical significance (p-value) was calculated with Student’s t-test. * indicates p>0.05, when each test transfection was compared with that of empty vector.
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Related In: Results  -  Collection

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pone.0141523.g004: Mad2 binding activity is essential for p31comet inhibition of colony formation.(A) Effects of a p31comet mutant (Q83A/F191A) on apoptosis (lower panel) and repression of colony formation (upper panel) were analyzed in HeLa cells. Expression of p31comet and Q83A/F191A was confirmed via Western blot. (B) The effects of a p31comet mutant (Q83/F191A) on Myc/Ras-mediated colony formation in p53-/- MEFs were compared with those of wild-type (WT) p31comet. (C) The effect of Mad2 on p31comet-induced repression of clonogenic cell survival was analyzed in HeLa cells in the presence of either wild-type or dominant-negative mutant Mad2 (R133E/Q134A) (Mad2mt). (D) Expression of p31comet, Mad2, and R133E/Q134A mutant (Mad2mt) was confirmed using immunoblotting. Values represent mean of triplicate samples. Statistical significance (p-value) was calculated with Student’s t-test. * indicates p>0.05, when each test transfection was compared with that of empty vector.
Mentions: To provide further evidence that p31comet-induced death relies on Mad2 interactions, we examined a p31comet (Q83A/F191A) mutant lacking binding activity to Mad2 [17]. In contrast to wild-type p31comet, the mutant form did not suppress clonal survival when transduced into HeLa cells via retroviral infection (Fig 4A) or MEFs with Myc/Ras co-transfection (Fig 4B). Consistently, HeLa cells infected with the Q83A/F191A mutant were not susceptible to Annexin staining, in contrast to those infected with wild-type p31comet (Fig 4A). These findings imply that p31comet-induced death occurs only when Mad2 binding activity is retained, ultimately leading to inhibition of Mad2 action. To explore this point, we performed an experiment to establish whether p31comet-induced death can be rescued by Mad2. To this end, HeLa cells retrovirally infected with p31comet were reinfected with retroviruses containing wild-type Mad2. As expected, p31comet -induced suppression of clonal survival was reversed by wild-type Mad2 (Fig 4C and 4D). However, the dominant-negative form of Mad2 (R133E/Q134A) lacking the ability to bind to p31comet (and devoid of spindle checkpoint activity) [21] did not affect survival (Fig 4C and 4D). Mad2 or its mutant (R133E/Q134A) alone did not suppress colony formation. These data support the theory that Mad2 inactivation is essential for cell death induction by p31comet. Furthermore, the balance between p31comet and Mad2 levels may determine the cytotoxic effect.

Bottom Line: Consistently, wild-type Mad2 interacting with p31comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31comet-induced cell death involves Mad2 inactivation.Our results clearly suggest that the regions of p31comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death.The finding that p31comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Division of Radiation Cancer Research, Korea Institute of Radiological & Medical Sciences, Seoul 139-706, Korea.

ABSTRACT
Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31comet is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31comet-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31comet, the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31comet overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31comet. Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31comet. Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wild-type Mad2 interacting with p31comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31comet-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.

No MeSH data available.


Related in: MedlinePlus