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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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Induction of p53 and p21 in Lck-survivinflox/flox DN thymocytes. (A) p53 and p21 induction in DN thymocytes. Top: Protein samples prepared from Lck-survivinflox/+ and Lck-survivinflox/flox DN cells were subjected to Western blot analysis using anti-p53 Ab followed by reprobing with anti-actin as a loading control. Bottom: Levels of p21 and HPRT (loading control) mRNA in control and mutant DN cells were determined by RT-PCR. (B) p53 induction in DN subpopulations. Lck-survivinflox/+ and Lck-survivinflox/flox thymocytes were surface stained with anti-CD25, anti-CD44, and anti-Lin followed by intracellular staining with anti-p53 or control IgG. p53 protein in each DN subpopulation was assessed by flow cytometry.
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fig6: Induction of p53 and p21 in Lck-survivinflox/flox DN thymocytes. (A) p53 and p21 induction in DN thymocytes. Top: Protein samples prepared from Lck-survivinflox/+ and Lck-survivinflox/flox DN cells were subjected to Western blot analysis using anti-p53 Ab followed by reprobing with anti-actin as a loading control. Bottom: Levels of p21 and HPRT (loading control) mRNA in control and mutant DN cells were determined by RT-PCR. (B) p53 induction in DN subpopulations. Lck-survivinflox/+ and Lck-survivinflox/flox thymocytes were surface stained with anti-CD25, anti-CD44, and anti-Lin followed by intracellular staining with anti-p53 or control IgG. p53 protein in each DN subpopulation was assessed by flow cytometry.

Mentions: Increased cell death and cell cycle arrest are hallmarks of p53 function. To test whether loss of survivin induced p53, we investigated the status of p53 protein in Lck-survivinflox/flox DN cells. We found that p53 protein expression was indeed induced in Lck-survivinflox/flox DN cells but not in Lck-survivinflox/+ DN cells (Fig. 6 A, top). Flow cytometric analysis of intracellular p53 protein confirmed that p53 was expressed in survivin-deficient DN2 to DN4 cells (Fig. 6 B). In addition, RT-PCR analysis showed that expression of the p53 target gene p21 was induced in Lck-survivinlox/lox DN cells (Fig. 6 A, bottom). These results suggested that p53 might be responsible for the increased apoptosis and arrest observed in Lck-survivinflox/flox mice.


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)

Induction of p53 and p21 in Lck-survivinflox/flox DN thymocytes. (A) p53 and p21 induction in DN thymocytes. Top: Protein samples prepared from Lck-survivinflox/+ and Lck-survivinflox/flox DN cells were subjected to Western blot analysis using anti-p53 Ab followed by reprobing with anti-actin as a loading control. Bottom: Levels of p21 and HPRT (loading control) mRNA in control and mutant DN cells were determined by RT-PCR. (B) p53 induction in DN subpopulations. Lck-survivinflox/+ and Lck-survivinflox/flox thymocytes were surface stained with anti-CD25, anti-CD44, and anti-Lin followed by intracellular staining with anti-p53 or control IgG. p53 protein in each DN subpopulation was assessed by flow cytometry.
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Related In: Results  -  Collection

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fig6: Induction of p53 and p21 in Lck-survivinflox/flox DN thymocytes. (A) p53 and p21 induction in DN thymocytes. Top: Protein samples prepared from Lck-survivinflox/+ and Lck-survivinflox/flox DN cells were subjected to Western blot analysis using anti-p53 Ab followed by reprobing with anti-actin as a loading control. Bottom: Levels of p21 and HPRT (loading control) mRNA in control and mutant DN cells were determined by RT-PCR. (B) p53 induction in DN subpopulations. Lck-survivinflox/+ and Lck-survivinflox/flox thymocytes were surface stained with anti-CD25, anti-CD44, and anti-Lin followed by intracellular staining with anti-p53 or control IgG. p53 protein in each DN subpopulation was assessed by flow cytometry.
Mentions: Increased cell death and cell cycle arrest are hallmarks of p53 function. To test whether loss of survivin induced p53, we investigated the status of p53 protein in Lck-survivinflox/flox DN cells. We found that p53 protein expression was indeed induced in Lck-survivinflox/flox DN cells but not in Lck-survivinflox/+ DN cells (Fig. 6 A, top). Flow cytometric analysis of intracellular p53 protein confirmed that p53 was expressed in survivin-deficient DN2 to DN4 cells (Fig. 6 B). In addition, RT-PCR analysis showed that expression of the p53 target gene p21 was induced in Lck-survivinlox/lox DN cells (Fig. 6 A, bottom). These results suggested that p53 might be responsible for the increased apoptosis and arrest observed in Lck-survivinflox/flox mice.

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