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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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Effects of Bcl-2 gain or p53 loss on phenotypes of survivin-deficient thymocytes. (A) Gain of Bcl-2 does not restore DP cells in the absence of survivin. Thymocytes prepared from Lck-survivinflox/+;Bcl-2, Lck-survivinflox/flox, and Lck-survivinflox/flox;Bcl-2 mice were stained with anti-CD4 and anti-CD8 followed by flow cytometric analysis. One experiment representative of four independent trials is shown. (B) Loss of p53 does not rescue DN thymocyte development in the absence of survivin. CD4/CD8 expression was determined as in A for thymocytes from Lck-survivinflox/+;p53−/−, Lck-survivinflox/flox;p53+/−, and Lck-survivinflox/flox;p53−/− mice. (C) Decreased G1 arrest of survivin-deficient DN3L cells in the p53−/− and p21−/− genetic backgrounds. Cell cycle profiles of DN cells prepared from Lck-survivinflox/+, Lck-survivinflox/flox, Lck-survivinflox/flox;p53−/−, and Lck-survivinflox/flox;p21−/− mice were determined as in Fig. 4 B. (D) Increased cell death of survivin-deficient DN cells in the p53−/− and p21 genetic backgrounds. The percentages of annexin+ cells in the indicated DN subpopulations were determined as in Fig. 4 A. Results shown are mean ± SD. Annexin+ cells were significantly increased in p53−/− and p21−/− genetic backgrounds (ANOVA; n = 3).
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fig7: Effects of Bcl-2 gain or p53 loss on phenotypes of survivin-deficient thymocytes. (A) Gain of Bcl-2 does not restore DP cells in the absence of survivin. Thymocytes prepared from Lck-survivinflox/+;Bcl-2, Lck-survivinflox/flox, and Lck-survivinflox/flox;Bcl-2 mice were stained with anti-CD4 and anti-CD8 followed by flow cytometric analysis. One experiment representative of four independent trials is shown. (B) Loss of p53 does not rescue DN thymocyte development in the absence of survivin. CD4/CD8 expression was determined as in A for thymocytes from Lck-survivinflox/+;p53−/−, Lck-survivinflox/flox;p53+/−, and Lck-survivinflox/flox;p53−/− mice. (C) Decreased G1 arrest of survivin-deficient DN3L cells in the p53−/− and p21−/− genetic backgrounds. Cell cycle profiles of DN cells prepared from Lck-survivinflox/+, Lck-survivinflox/flox, Lck-survivinflox/flox;p53−/−, and Lck-survivinflox/flox;p21−/− mice were determined as in Fig. 4 B. (D) Increased cell death of survivin-deficient DN cells in the p53−/− and p21 genetic backgrounds. The percentages of annexin+ cells in the indicated DN subpopulations were determined as in Fig. 4 A. Results shown are mean ± SD. Annexin+ cells were significantly increased in p53−/− and p21−/− genetic backgrounds (ANOVA; n = 3).

Mentions: We determined whether Bcl-2 could rescue survivin deficiency by crossing Lck-survivinflox/flox mice to Eμ-Bcl-2 transgenic mice to generate Lck-survivinflox/flox;Bcl-2 animals. The total number of thymocytes in Lck-survivinflox/flox;Bcl-2 mice (2.2 ± 0.7 × 106; n = 4) was comparable to that in Lck-survivinflox/flox mice (2.4 ± 0.5 × 106; n = 4; Fig. 7 A). The number of DN cells was also unaltered in the presence of the Bcl-2 transgene (Lck-survivinflox/flox vs. Lck-survivinflox/flox;Bcl-2; 2.3 ± 0.5 × 106 vs. 2.1 ± 0.6 × 106; n = 4), suggesting that Bcl-2 overexpression cannot overcome cell death induced by an absence of survivin.


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)

Effects of Bcl-2 gain or p53 loss on phenotypes of survivin-deficient thymocytes. (A) Gain of Bcl-2 does not restore DP cells in the absence of survivin. Thymocytes prepared from Lck-survivinflox/+;Bcl-2, Lck-survivinflox/flox, and Lck-survivinflox/flox;Bcl-2 mice were stained with anti-CD4 and anti-CD8 followed by flow cytometric analysis. One experiment representative of four independent trials is shown. (B) Loss of p53 does not rescue DN thymocyte development in the absence of survivin. CD4/CD8 expression was determined as in A for thymocytes from Lck-survivinflox/+;p53−/−, Lck-survivinflox/flox;p53+/−, and Lck-survivinflox/flox;p53−/− mice. (C) Decreased G1 arrest of survivin-deficient DN3L cells in the p53−/− and p21−/− genetic backgrounds. Cell cycle profiles of DN cells prepared from Lck-survivinflox/+, Lck-survivinflox/flox, Lck-survivinflox/flox;p53−/−, and Lck-survivinflox/flox;p21−/− mice were determined as in Fig. 4 B. (D) Increased cell death of survivin-deficient DN cells in the p53−/− and p21 genetic backgrounds. The percentages of annexin+ cells in the indicated DN subpopulations were determined as in Fig. 4 A. Results shown are mean ± SD. Annexin+ cells were significantly increased in p53−/− and p21−/− genetic backgrounds (ANOVA; n = 3).
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fig7: Effects of Bcl-2 gain or p53 loss on phenotypes of survivin-deficient thymocytes. (A) Gain of Bcl-2 does not restore DP cells in the absence of survivin. Thymocytes prepared from Lck-survivinflox/+;Bcl-2, Lck-survivinflox/flox, and Lck-survivinflox/flox;Bcl-2 mice were stained with anti-CD4 and anti-CD8 followed by flow cytometric analysis. One experiment representative of four independent trials is shown. (B) Loss of p53 does not rescue DN thymocyte development in the absence of survivin. CD4/CD8 expression was determined as in A for thymocytes from Lck-survivinflox/+;p53−/−, Lck-survivinflox/flox;p53+/−, and Lck-survivinflox/flox;p53−/− mice. (C) Decreased G1 arrest of survivin-deficient DN3L cells in the p53−/− and p21−/− genetic backgrounds. Cell cycle profiles of DN cells prepared from Lck-survivinflox/+, Lck-survivinflox/flox, Lck-survivinflox/flox;p53−/−, and Lck-survivinflox/flox;p21−/− mice were determined as in Fig. 4 B. (D) Increased cell death of survivin-deficient DN cells in the p53−/− and p21 genetic backgrounds. The percentages of annexin+ cells in the indicated DN subpopulations were determined as in Fig. 4 A. Results shown are mean ± SD. Annexin+ cells were significantly increased in p53−/− and p21−/− genetic backgrounds (ANOVA; n = 3).
Mentions: We determined whether Bcl-2 could rescue survivin deficiency by crossing Lck-survivinflox/flox mice to Eμ-Bcl-2 transgenic mice to generate Lck-survivinflox/flox;Bcl-2 animals. The total number of thymocytes in Lck-survivinflox/flox;Bcl-2 mice (2.2 ± 0.7 × 106; n = 4) was comparable to that in Lck-survivinflox/flox mice (2.4 ± 0.5 × 106; n = 4; Fig. 7 A). The number of DN cells was also unaltered in the presence of the Bcl-2 transgene (Lck-survivinflox/flox vs. Lck-survivinflox/flox;Bcl-2; 2.3 ± 0.5 × 106 vs. 2.1 ± 0.6 × 106; n = 4), suggesting that Bcl-2 overexpression cannot overcome cell death induced by an absence of survivin.

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