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Survivin loss in thymocytes triggers p53-mediated growth arrest and p53-independent cell death.

Okada H, Bakal C, Shahinian A, Elia A, Wakeham A, Suh WK, Duncan GS, Ciofani M, Rottapel R, Zúñiga-Pflücker JC, Mak TW - J. Exp. Med. (2004)

Bottom Line: In response to proliferative stimuli, cycling survivin-deficient DN cells exhibit cell cycle arrest, a spindle formation defect, and increased cell death.Strikingly, loss of survivin activates the tumor suppressor p53.However, the developmental defects caused by survivin deficiency cannot be rescued by p53 inactivation or introduction of Bcl-2.

View Article: PubMed Central - PubMed

Affiliation: Advanced Medical Discovery Institute, University of Toronto, 620 University Avenue, Suite 706, Ontario M5G 2C1, Canada. hokada@uhnres.utoronto.ca

ABSTRACT
Because survivin- embryos die at an early embryonic stage, the role of survivin in thymocyte development is unknown. We have investigated the role by deleting the survivin gene only in the T lineage and show here that loss of survivin blocks the transition from CD4- CD8- double negative (DN) thymocytes to CD4+ CD8+ double positive cells. Although the pre-T cell receptor signaling pathway is intact in survivin-deficient thymocytes, the cells cannot respond to its signals. In response to proliferative stimuli, cycling survivin-deficient DN cells exhibit cell cycle arrest, a spindle formation defect, and increased cell death. Strikingly, loss of survivin activates the tumor suppressor p53. However, the developmental defects caused by survivin deficiency cannot be rescued by p53 inactivation or introduction of Bcl-2. These lines of evidence indicate that developing thymocytes depend on the cytoprotective function of survivin and that this function is tightly coupled to cell proliferation but independent of p53 and Bcl-2. Thus, survivin plays a critical role in early thymocyte development.

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Effect of survivin loss on cell death in vivo and in vitro and on thymocyte proliferation. (A) Increased cell death of proliferating survivin-deficient DN cells. DN thymocytes were prepared ex vivo and stained with anti-CD25, anti-CD44, and anti-Lin Ab followed by annexin. The average number of annexin+ cells in each DN subpopulation was assessed by flow cytometry. Results shown are mean ± SD. Annexin+ cells were significantly increased in Lck-survivinflox/flox DN3L and DN4 populations (ANOVA; n = 3). (B) Normal susceptibility of resting survivin-deficient DN cells to various apoptotic stimuli. Lck-survivinflox/flox and RAG-2−/− DN3E cells were treated with etoposide (Etp), dexamethasone (Dex), γ-irradiation (IR), and staurosporine (STS) at the indicated doses. Cell death was evaluated as described in Materials and Methods. Cell viability was normalized to account for spontaneous cell death. Triplicate samples of each treatment in three independent experiments were assayed. Results shown are mean ± SD. (C) Impaired proliferation in vivo in the absence of survivin. Lck-survivinflox/+ and Lck-survivinflox/flox mice were injected with 1 mg BrdU and DN thymocytes were purified and stained with anti-CD25, anti-CD44, anti-BrdU, and 7AAD. The percentages of cells in the G1, S, and G2/M phases were measured by flow cytometry for the indicated DN subsets. Results shown are representative of three independent experiments.
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fig4: Effect of survivin loss on cell death in vivo and in vitro and on thymocyte proliferation. (A) Increased cell death of proliferating survivin-deficient DN cells. DN thymocytes were prepared ex vivo and stained with anti-CD25, anti-CD44, and anti-Lin Ab followed by annexin. The average number of annexin+ cells in each DN subpopulation was assessed by flow cytometry. Results shown are mean ± SD. Annexin+ cells were significantly increased in Lck-survivinflox/flox DN3L and DN4 populations (ANOVA; n = 3). (B) Normal susceptibility of resting survivin-deficient DN cells to various apoptotic stimuli. Lck-survivinflox/flox and RAG-2−/− DN3E cells were treated with etoposide (Etp), dexamethasone (Dex), γ-irradiation (IR), and staurosporine (STS) at the indicated doses. Cell death was evaluated as described in Materials and Methods. Cell viability was normalized to account for spontaneous cell death. Triplicate samples of each treatment in three independent experiments were assayed. Results shown are mean ± SD. (C) Impaired proliferation in vivo in the absence of survivin. Lck-survivinflox/+ and Lck-survivinflox/flox mice were injected with 1 mg BrdU and DN thymocytes were purified and stained with anti-CD25, anti-CD44, anti-BrdU, and 7AAD. The percentages of cells in the G1, S, and G2/M phases were measured by flow cytometry for the indicated DN subsets. Results shown are representative of three independent experiments.

Mentions: Because the pre-TCR signaling required for DN cell proliferation was normal in Lck-survivinflox/flox thymocytes, we investigated whether the loss of DN4 cells in the mutant mice was due to impaired proliferation or increased apoptosis or both. Annexin V staining for cell viability in vivo showed that 37% of DN3L and 32% of DN4 cells were annexin+ in Lck-survivinflox/flox mice (Fig. 4 A). In contrast, only 5–7% cells were annexin+ in control mice, indicating that loss of survivin induces apoptosis of DN3L and DN4 thymocytes.


Survivin loss in thymocytes triggers p53-mediated growth arrest and p53-independent cell death.

Okada H, Bakal C, Shahinian A, Elia A, Wakeham A, Suh WK, Duncan GS, Ciofani M, Rottapel R, Zúñiga-Pflücker JC, Mak TW - J. Exp. Med. (2004)

Effect of survivin loss on cell death in vivo and in vitro and on thymocyte proliferation. (A) Increased cell death of proliferating survivin-deficient DN cells. DN thymocytes were prepared ex vivo and stained with anti-CD25, anti-CD44, and anti-Lin Ab followed by annexin. The average number of annexin+ cells in each DN subpopulation was assessed by flow cytometry. Results shown are mean ± SD. Annexin+ cells were significantly increased in Lck-survivinflox/flox DN3L and DN4 populations (ANOVA; n = 3). (B) Normal susceptibility of resting survivin-deficient DN cells to various apoptotic stimuli. Lck-survivinflox/flox and RAG-2−/− DN3E cells were treated with etoposide (Etp), dexamethasone (Dex), γ-irradiation (IR), and staurosporine (STS) at the indicated doses. Cell death was evaluated as described in Materials and Methods. Cell viability was normalized to account for spontaneous cell death. Triplicate samples of each treatment in three independent experiments were assayed. Results shown are mean ± SD. (C) Impaired proliferation in vivo in the absence of survivin. Lck-survivinflox/+ and Lck-survivinflox/flox mice were injected with 1 mg BrdU and DN thymocytes were purified and stained with anti-CD25, anti-CD44, anti-BrdU, and 7AAD. The percentages of cells in the G1, S, and G2/M phases were measured by flow cytometry for the indicated DN subsets. Results shown are representative of three independent experiments.
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Related In: Results  -  Collection

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fig4: Effect of survivin loss on cell death in vivo and in vitro and on thymocyte proliferation. (A) Increased cell death of proliferating survivin-deficient DN cells. DN thymocytes were prepared ex vivo and stained with anti-CD25, anti-CD44, and anti-Lin Ab followed by annexin. The average number of annexin+ cells in each DN subpopulation was assessed by flow cytometry. Results shown are mean ± SD. Annexin+ cells were significantly increased in Lck-survivinflox/flox DN3L and DN4 populations (ANOVA; n = 3). (B) Normal susceptibility of resting survivin-deficient DN cells to various apoptotic stimuli. Lck-survivinflox/flox and RAG-2−/− DN3E cells were treated with etoposide (Etp), dexamethasone (Dex), γ-irradiation (IR), and staurosporine (STS) at the indicated doses. Cell death was evaluated as described in Materials and Methods. Cell viability was normalized to account for spontaneous cell death. Triplicate samples of each treatment in three independent experiments were assayed. Results shown are mean ± SD. (C) Impaired proliferation in vivo in the absence of survivin. Lck-survivinflox/+ and Lck-survivinflox/flox mice were injected with 1 mg BrdU and DN thymocytes were purified and stained with anti-CD25, anti-CD44, anti-BrdU, and 7AAD. The percentages of cells in the G1, S, and G2/M phases were measured by flow cytometry for the indicated DN subsets. Results shown are representative of three independent experiments.
Mentions: Because the pre-TCR signaling required for DN cell proliferation was normal in Lck-survivinflox/flox thymocytes, we investigated whether the loss of DN4 cells in the mutant mice was due to impaired proliferation or increased apoptosis or both. Annexin V staining for cell viability in vivo showed that 37% of DN3L and 32% of DN4 cells were annexin+ in Lck-survivinflox/flox mice (Fig. 4 A). In contrast, only 5–7% cells were annexin+ in control mice, indicating that loss of survivin induces apoptosis of DN3L and DN4 thymocytes.

Bottom Line: In response to proliferative stimuli, cycling survivin-deficient DN cells exhibit cell cycle arrest, a spindle formation defect, and increased cell death.Strikingly, loss of survivin activates the tumor suppressor p53.However, the developmental defects caused by survivin deficiency cannot be rescued by p53 inactivation or introduction of Bcl-2.

View Article: PubMed Central - PubMed

Affiliation: Advanced Medical Discovery Institute, University of Toronto, 620 University Avenue, Suite 706, Ontario M5G 2C1, Canada. hokada@uhnres.utoronto.ca

ABSTRACT
Because survivin- embryos die at an early embryonic stage, the role of survivin in thymocyte development is unknown. We have investigated the role by deleting the survivin gene only in the T lineage and show here that loss of survivin blocks the transition from CD4- CD8- double negative (DN) thymocytes to CD4+ CD8+ double positive cells. Although the pre-T cell receptor signaling pathway is intact in survivin-deficient thymocytes, the cells cannot respond to its signals. In response to proliferative stimuli, cycling survivin-deficient DN cells exhibit cell cycle arrest, a spindle formation defect, and increased cell death. Strikingly, loss of survivin activates the tumor suppressor p53. However, the developmental defects caused by survivin deficiency cannot be rescued by p53 inactivation or introduction of Bcl-2. These lines of evidence indicate that developing thymocytes depend on the cytoprotective function of survivin and that this function is tightly coupled to cell proliferation but independent of p53 and Bcl-2. Thus, survivin plays a critical role in early thymocyte development.

Show MeSH
Related in: MedlinePlus