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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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Normal TCRβ gene rearrangement and pre-TCR signaling in Lck-Cre;survivinflox/flox thymocytes. (A) Normal TCRβ D-J rearrangement. TCRβ Dβ2-Jβ2 recombination in Lck-survivinflox/+ and Lck-survivinflox/flox DN thymocytes was examined by PCR followed by Southern blotting with 32P-labeled oligonucleotide probes as previously described (reference 59). The Cre gene was amplified as an internal control for PCR. (B) Normal TCRβ protein expression. DN3E and DNL3 thymocytes from Lck-survivinflox/+ and Lck-survivinflox/flox mice were surface stained with anti-CD25, anti-CD44, and anti-Lin followed by intracellular staining with TCRβ and flow cytometric analysis. (C) Normal MAPK activation. Lck-survivinflox/+ and Lck-survivinflox/flox DN thymocytes were treated with anti–CD3ε-biotin followed by cross-linking with avidin for the indicated times. Protein lysates were subjected to Western blot analysis using anti–phospho-ERK Abs (top). To control for loading, the blot was stripped and reprobed with anti–ERK Ab (bottom). (D) Impaired in vivo response of survivin-deficient thymocytes to anti-CD3ε. RAG-2−/− (left) and Lck-survivinflox/flox (right) mice were intraperitoneally injected with 100 mg anti-CD3ε. After 72 h, thymocytes were prepared and stained with anti-CD25, anti-CD44, and anti-Lin Abs, and then subjected to flow cytometry. Unlike RAG-2−/− DN3 cells, Lck-survivinflox/flox DN3 cells failed to advance to DN4. Data shown are representative of three independent experiments.
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fig3: Normal TCRβ gene rearrangement and pre-TCR signaling in Lck-Cre;survivinflox/flox thymocytes. (A) Normal TCRβ D-J rearrangement. TCRβ Dβ2-Jβ2 recombination in Lck-survivinflox/+ and Lck-survivinflox/flox DN thymocytes was examined by PCR followed by Southern blotting with 32P-labeled oligonucleotide probes as previously described (reference 59). The Cre gene was amplified as an internal control for PCR. (B) Normal TCRβ protein expression. DN3E and DNL3 thymocytes from Lck-survivinflox/+ and Lck-survivinflox/flox mice were surface stained with anti-CD25, anti-CD44, and anti-Lin followed by intracellular staining with TCRβ and flow cytometric analysis. (C) Normal MAPK activation. Lck-survivinflox/+ and Lck-survivinflox/flox DN thymocytes were treated with anti–CD3ε-biotin followed by cross-linking with avidin for the indicated times. Protein lysates were subjected to Western blot analysis using anti–phospho-ERK Abs (top). To control for loading, the blot was stripped and reprobed with anti–ERK Ab (bottom). (D) Impaired in vivo response of survivin-deficient thymocytes to anti-CD3ε. RAG-2−/− (left) and Lck-survivinflox/flox (right) mice were intraperitoneally injected with 100 mg anti-CD3ε. After 72 h, thymocytes were prepared and stained with anti-CD25, anti-CD44, and anti-Lin Abs, and then subjected to flow cytometry. Unlike RAG-2−/− DN3 cells, Lck-survivinflox/flox DN3 cells failed to advance to DN4. Data shown are representative of three independent experiments.

Mentions: The accumulation of DN3 thymocytes in Lck-survivinflox/flox mice was strikingly similar to that in RAG-1 (43), RAG-2 (44), TCRβ (45), and Lck (46) knockout mice. Therefore, we analyzed the rearrangement of the TCRβ locus, a requirement for the DN3 to DN4 transition. Analysis of DN thymocyte DNA by PCR and Southern blotting showed that somatic recombination of Dβ2-Jβ2 gene segments was not altered in survivin-deficient thymocytes (Fig. 3 A). Furthermore, flow cytometry revealed no differences in intracellular TCRβ protein between DN3E and DN3L cells of control and mutant mice (Fig. 3 B).


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)

Normal TCRβ gene rearrangement and pre-TCR signaling in Lck-Cre;survivinflox/flox thymocytes. (A) Normal TCRβ D-J rearrangement. TCRβ Dβ2-Jβ2 recombination in Lck-survivinflox/+ and Lck-survivinflox/flox DN thymocytes was examined by PCR followed by Southern blotting with 32P-labeled oligonucleotide probes as previously described (reference 59). The Cre gene was amplified as an internal control for PCR. (B) Normal TCRβ protein expression. DN3E and DNL3 thymocytes from Lck-survivinflox/+ and Lck-survivinflox/flox mice were surface stained with anti-CD25, anti-CD44, and anti-Lin followed by intracellular staining with TCRβ and flow cytometric analysis. (C) Normal MAPK activation. Lck-survivinflox/+ and Lck-survivinflox/flox DN thymocytes were treated with anti–CD3ε-biotin followed by cross-linking with avidin for the indicated times. Protein lysates were subjected to Western blot analysis using anti–phospho-ERK Abs (top). To control for loading, the blot was stripped and reprobed with anti–ERK Ab (bottom). (D) Impaired in vivo response of survivin-deficient thymocytes to anti-CD3ε. RAG-2−/− (left) and Lck-survivinflox/flox (right) mice were intraperitoneally injected with 100 mg anti-CD3ε. After 72 h, thymocytes were prepared and stained with anti-CD25, anti-CD44, and anti-Lin Abs, and then subjected to flow cytometry. Unlike RAG-2−/− DN3 cells, Lck-survivinflox/flox DN3 cells failed to advance to DN4. Data shown are representative of three independent experiments.
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fig3: Normal TCRβ gene rearrangement and pre-TCR signaling in Lck-Cre;survivinflox/flox thymocytes. (A) Normal TCRβ D-J rearrangement. TCRβ Dβ2-Jβ2 recombination in Lck-survivinflox/+ and Lck-survivinflox/flox DN thymocytes was examined by PCR followed by Southern blotting with 32P-labeled oligonucleotide probes as previously described (reference 59). The Cre gene was amplified as an internal control for PCR. (B) Normal TCRβ protein expression. DN3E and DNL3 thymocytes from Lck-survivinflox/+ and Lck-survivinflox/flox mice were surface stained with anti-CD25, anti-CD44, and anti-Lin followed by intracellular staining with TCRβ and flow cytometric analysis. (C) Normal MAPK activation. Lck-survivinflox/+ and Lck-survivinflox/flox DN thymocytes were treated with anti–CD3ε-biotin followed by cross-linking with avidin for the indicated times. Protein lysates were subjected to Western blot analysis using anti–phospho-ERK Abs (top). To control for loading, the blot was stripped and reprobed with anti–ERK Ab (bottom). (D) Impaired in vivo response of survivin-deficient thymocytes to anti-CD3ε. RAG-2−/− (left) and Lck-survivinflox/flox (right) mice were intraperitoneally injected with 100 mg anti-CD3ε. After 72 h, thymocytes were prepared and stained with anti-CD25, anti-CD44, and anti-Lin Abs, and then subjected to flow cytometry. Unlike RAG-2−/− DN3 cells, Lck-survivinflox/flox DN3 cells failed to advance to DN4. Data shown are representative of three independent experiments.
Mentions: The accumulation of DN3 thymocytes in Lck-survivinflox/flox mice was strikingly similar to that in RAG-1 (43), RAG-2 (44), TCRβ (45), and Lck (46) knockout mice. Therefore, we analyzed the rearrangement of the TCRβ locus, a requirement for the DN3 to DN4 transition. Analysis of DN thymocyte DNA by PCR and Southern blotting showed that somatic recombination of Dβ2-Jβ2 gene segments was not altered in survivin-deficient thymocytes (Fig. 3 A). Furthermore, flow cytometry revealed no differences in intracellular TCRβ protein between DN3E and DN3L cells of control and mutant mice (Fig. 3 B).

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