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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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Flow cytometric and histological analyses of peripheral T cells and thymi of Lck-Cre;survivinflox/flox mice. (A) Survivin mRNA expression in DN cells. Levels of survivin mRNA were analyzed in the indicated DN subsets by RT-PCR. RNA was prepared from DN cells sorted from wild-type thymocytes. (B) Survivin protein expression in DN cells. Surface staining of Lck-survivin+/+ (thick line) and Lck-survivinflox/flox (dotted line) thymocytes with anti-CD25, anti-CD44, and anti-Lin Abs was followed by intracellular staining with anti-survivin Ab. Lin+ cells in the indicated thymocyte subpopulations were electrically gated out. (C) Impaired development of survivin-deficient peripheral T cells and DP thymocytes. Peripheral blood cells from Lck-survivin+/+, Lck-survivinflox/+, and Lck-survivinflox/flox mice were stained with anti-TCRαβ and anti-B220 (top). Thymocytes were stained with anti-CD4 and anti-CD8 (middle), or anti-CD25 and anti-CD44 (bottom), and the Lin− population was examined. (D) Reduced size and cellularity of the thymus in Lck-survivinflox/flox mice. Hematoxylin and eosin staining of transverse sections of thymus from Lck-survivinflox/+ (left) and Lck-survivinflox/flox (right) mice. The mutant thymus is much smaller and lacks the typical cortex medulla structure.
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fig2: Flow cytometric and histological analyses of peripheral T cells and thymi of Lck-Cre;survivinflox/flox mice. (A) Survivin mRNA expression in DN cells. Levels of survivin mRNA were analyzed in the indicated DN subsets by RT-PCR. RNA was prepared from DN cells sorted from wild-type thymocytes. (B) Survivin protein expression in DN cells. Surface staining of Lck-survivin+/+ (thick line) and Lck-survivinflox/flox (dotted line) thymocytes with anti-CD25, anti-CD44, and anti-Lin Abs was followed by intracellular staining with anti-survivin Ab. Lin+ cells in the indicated thymocyte subpopulations were electrically gated out. (C) Impaired development of survivin-deficient peripheral T cells and DP thymocytes. Peripheral blood cells from Lck-survivin+/+, Lck-survivinflox/+, and Lck-survivinflox/flox mice were stained with anti-TCRαβ and anti-B220 (top). Thymocytes were stained with anti-CD4 and anti-CD8 (middle), or anti-CD25 and anti-CD44 (bottom), and the Lin− population was examined. (D) Reduced size and cellularity of the thymus in Lck-survivinflox/flox mice. Hematoxylin and eosin staining of transverse sections of thymus from Lck-survivinflox/+ (left) and Lck-survivinflox/flox (right) mice. The mutant thymus is much smaller and lacks the typical cortex medulla structure.

Mentions: We first compared the level of survivin expression in DN subpopulations in mutant and control mice. RT-PCR analysis showed that survivin mRNA was maximally expressed at the DN3L stage in control cells (Fig. 2 A). Flow cytometric analysis demonstrated that compared with Lck-survivin+/+ cells, intracellular survivin expression was decreased in Lck-survivinflox/flox cells starting at the DN2 stage (Fig. 2 B). This time frame is consistent with Lck proximal promoter activity (42).


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)

Flow cytometric and histological analyses of peripheral T cells and thymi of Lck-Cre;survivinflox/flox mice. (A) Survivin mRNA expression in DN cells. Levels of survivin mRNA were analyzed in the indicated DN subsets by RT-PCR. RNA was prepared from DN cells sorted from wild-type thymocytes. (B) Survivin protein expression in DN cells. Surface staining of Lck-survivin+/+ (thick line) and Lck-survivinflox/flox (dotted line) thymocytes with anti-CD25, anti-CD44, and anti-Lin Abs was followed by intracellular staining with anti-survivin Ab. Lin+ cells in the indicated thymocyte subpopulations were electrically gated out. (C) Impaired development of survivin-deficient peripheral T cells and DP thymocytes. Peripheral blood cells from Lck-survivin+/+, Lck-survivinflox/+, and Lck-survivinflox/flox mice were stained with anti-TCRαβ and anti-B220 (top). Thymocytes were stained with anti-CD4 and anti-CD8 (middle), or anti-CD25 and anti-CD44 (bottom), and the Lin− population was examined. (D) Reduced size and cellularity of the thymus in Lck-survivinflox/flox mice. Hematoxylin and eosin staining of transverse sections of thymus from Lck-survivinflox/+ (left) and Lck-survivinflox/flox (right) mice. The mutant thymus is much smaller and lacks the typical cortex medulla structure.
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Related In: Results  -  Collection

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fig2: Flow cytometric and histological analyses of peripheral T cells and thymi of Lck-Cre;survivinflox/flox mice. (A) Survivin mRNA expression in DN cells. Levels of survivin mRNA were analyzed in the indicated DN subsets by RT-PCR. RNA was prepared from DN cells sorted from wild-type thymocytes. (B) Survivin protein expression in DN cells. Surface staining of Lck-survivin+/+ (thick line) and Lck-survivinflox/flox (dotted line) thymocytes with anti-CD25, anti-CD44, and anti-Lin Abs was followed by intracellular staining with anti-survivin Ab. Lin+ cells in the indicated thymocyte subpopulations were electrically gated out. (C) Impaired development of survivin-deficient peripheral T cells and DP thymocytes. Peripheral blood cells from Lck-survivin+/+, Lck-survivinflox/+, and Lck-survivinflox/flox mice were stained with anti-TCRαβ and anti-B220 (top). Thymocytes were stained with anti-CD4 and anti-CD8 (middle), or anti-CD25 and anti-CD44 (bottom), and the Lin− population was examined. (D) Reduced size and cellularity of the thymus in Lck-survivinflox/flox mice. Hematoxylin and eosin staining of transverse sections of thymus from Lck-survivinflox/+ (left) and Lck-survivinflox/flox (right) mice. The mutant thymus is much smaller and lacks the typical cortex medulla structure.
Mentions: We first compared the level of survivin expression in DN subpopulations in mutant and control mice. RT-PCR analysis showed that survivin mRNA was maximally expressed at the DN3L stage in control cells (Fig. 2 A). Flow cytometric analysis demonstrated that compared with Lck-survivin+/+ cells, intracellular survivin expression was decreased in Lck-survivinflox/flox cells starting at the DN2 stage (Fig. 2 B). This time frame is consistent with Lck proximal promoter activity (42).

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