Limits...
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.

Show MeSH

Related in: MedlinePlus

Defects in cytokinesis of Lck-survivinflox/flox DN thymocytes. (A) Impaired spindle formation. Purified DN3L and DN4 cells from Lck-survivin+/+ (a) and Lck-survivinflox/flox (b) mice were stained with anti–α-tubulin (green). DNA was visualized with Hoechst 33258 (blue) staining. Data shown are representative of at least three independent preparations per genotype. (B) Severe impairment of spindle assembly in mitotic cells. Spindle assembly in cells from Lck-survivinflox/flox mice was compared at interphase (bottom left) and mitosis (top right). Cells were stained with anti–α-tubulin (green), anti–phospho-histone H3 (p-H3) Ab (red), and Hoechst (blue). a, α-tubulin; b, anti–p-H3 and Hoechst; c, merge. (C) Impaired cytokinesis and localization of Aurora-B kinase. Mitotic DN3L and DN4 cells from Lck-survivin+/+ (a–d) and Lck-survivinflox/flox (e–h) mice were stained with anti–p-H3 Ab (red), anti–Aurora-B kinase Ab (green), and Hoechst (blue). a and e, prometaphase; b and f, metaphase; c and g, anaphase; d and h, telophase/cytokinesis.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211792&req=5

fig5: Defects in cytokinesis of Lck-survivinflox/flox DN thymocytes. (A) Impaired spindle formation. Purified DN3L and DN4 cells from Lck-survivin+/+ (a) and Lck-survivinflox/flox (b) mice were stained with anti–α-tubulin (green). DNA was visualized with Hoechst 33258 (blue) staining. Data shown are representative of at least three independent preparations per genotype. (B) Severe impairment of spindle assembly in mitotic cells. Spindle assembly in cells from Lck-survivinflox/flox mice was compared at interphase (bottom left) and mitosis (top right). Cells were stained with anti–α-tubulin (green), anti–phospho-histone H3 (p-H3) Ab (red), and Hoechst (blue). a, α-tubulin; b, anti–p-H3 and Hoechst; c, merge. (C) Impaired cytokinesis and localization of Aurora-B kinase. Mitotic DN3L and DN4 cells from Lck-survivin+/+ (a–d) and Lck-survivinflox/flox (e–h) mice were stained with anti–p-H3 Ab (red), anti–Aurora-B kinase Ab (green), and Hoechst (blue). a and e, prometaphase; b and f, metaphase; c and g, anaphase; d and h, telophase/cytokinesis.

Mentions: Embryos of mice with a mutation of the survivin gene show a cytokinesis defect and die early during embryogenesis (28, 34, and unpublished data). Overexpression of dominant negative survivin or antisense inactivation of survivin also lead to cytokinetic defects (25, 27) and anti-survivin Ab injection results in spindle defects (49). We examined spindle formation in DN3L and DN4 cells from Lck-survivinflox/flox and Lck-survivin+/+ mice by staining thymocytes with α-tubulin. Lck-survivin+/+ (and Lck-survivinflox/+) cells had well-organized and symmetrical spindles (Fig. 5 A, a), whereas Lck-survivinflox/flox cells showed shorter and thicker spindles (Fig. 5 A, b). The number of spindle fibers was reduced in many cases, suggesting a defect in the assembly of spindle microtubules. Significantly, we found that microtubule assembly defects were enhanced in mitotic mutant cells compared with interphase mutant cells (Fig. 5 B). Thus, the organization and assembly of mitotic spindles is severely impaired in the 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)

Defects in cytokinesis of Lck-survivinflox/flox DN thymocytes. (A) Impaired spindle formation. Purified DN3L and DN4 cells from Lck-survivin+/+ (a) and Lck-survivinflox/flox (b) mice were stained with anti–α-tubulin (green). DNA was visualized with Hoechst 33258 (blue) staining. Data shown are representative of at least three independent preparations per genotype. (B) Severe impairment of spindle assembly in mitotic cells. Spindle assembly in cells from Lck-survivinflox/flox mice was compared at interphase (bottom left) and mitosis (top right). Cells were stained with anti–α-tubulin (green), anti–phospho-histone H3 (p-H3) Ab (red), and Hoechst (blue). a, α-tubulin; b, anti–p-H3 and Hoechst; c, merge. (C) Impaired cytokinesis and localization of Aurora-B kinase. Mitotic DN3L and DN4 cells from Lck-survivin+/+ (a–d) and Lck-survivinflox/flox (e–h) mice were stained with anti–p-H3 Ab (red), anti–Aurora-B kinase Ab (green), and Hoechst (blue). a and e, prometaphase; b and f, metaphase; c and g, anaphase; d and h, telophase/cytokinesis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211792&req=5

fig5: Defects in cytokinesis of Lck-survivinflox/flox DN thymocytes. (A) Impaired spindle formation. Purified DN3L and DN4 cells from Lck-survivin+/+ (a) and Lck-survivinflox/flox (b) mice were stained with anti–α-tubulin (green). DNA was visualized with Hoechst 33258 (blue) staining. Data shown are representative of at least three independent preparations per genotype. (B) Severe impairment of spindle assembly in mitotic cells. Spindle assembly in cells from Lck-survivinflox/flox mice was compared at interphase (bottom left) and mitosis (top right). Cells were stained with anti–α-tubulin (green), anti–phospho-histone H3 (p-H3) Ab (red), and Hoechst (blue). a, α-tubulin; b, anti–p-H3 and Hoechst; c, merge. (C) Impaired cytokinesis and localization of Aurora-B kinase. Mitotic DN3L and DN4 cells from Lck-survivin+/+ (a–d) and Lck-survivinflox/flox (e–h) mice were stained with anti–p-H3 Ab (red), anti–Aurora-B kinase Ab (green), and Hoechst (blue). a and e, prometaphase; b and f, metaphase; c and g, anaphase; d and h, telophase/cytokinesis.
Mentions: Embryos of mice with a mutation of the survivin gene show a cytokinesis defect and die early during embryogenesis (28, 34, and unpublished data). Overexpression of dominant negative survivin or antisense inactivation of survivin also lead to cytokinetic defects (25, 27) and anti-survivin Ab injection results in spindle defects (49). We examined spindle formation in DN3L and DN4 cells from Lck-survivinflox/flox and Lck-survivin+/+ mice by staining thymocytes with α-tubulin. Lck-survivin+/+ (and Lck-survivinflox/+) cells had well-organized and symmetrical spindles (Fig. 5 A, a), whereas Lck-survivinflox/flox cells showed shorter and thicker spindles (Fig. 5 A, b). The number of spindle fibers was reduced in many cases, suggesting a defect in the assembly of spindle microtubules. Significantly, we found that microtubule assembly defects were enhanced in mitotic mutant cells compared with interphase mutant cells (Fig. 5 B). Thus, the organization and assembly of mitotic spindles is severely impaired in the 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