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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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Tissue-specific targeted disruption of the survivin locus. (A) A portion of the murine wild-type survivin locus (top) showing exons 1–3 (open boxes) and a 10-kb XbaI fragment. The targeting vector was designed to generate floxed exon2 (loxP; triangles), flank the PGK-neo cassette with FRT sequences (ovals), and introduce a new XbaI site (X). The targeted allele contains a diagnostic 7.0-kb XbaI fragment. To generate the conditional allele, the neo cassette was removed by transient expression of Flpe recombinase. Cre-mediated recombination resulted in the deleted allele. The positions of the 5′ flanking probe (A) and neo probe (B) used for genotyping are indicated. (B) Southern blot analyses to identify survivinflox/+ ES cells containing neo (left), ES cells with neo removed (middle), and F2 pups (right). Genomic DNA was digested with XbaI and hybridized with probe A or B. (C) Western blot of survivin protein in Lck-survivinflox/+ and Lck-survivinflox/flox DN thymocytes. Wild-type survivin protein (arrowhead) and a nonspecific band (asterisk) are indicated. Actin, loading control. (D) Detection of deleted survivin allele in DN thymocyte subpopulations. The floxed (flox) and deleted (Δflox) survivin alleles were amplified by PCR using primers a and b shown in A. Genomic DNA samples were prepared from the indicated DN subsets.
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fig1: Tissue-specific targeted disruption of the survivin locus. (A) A portion of the murine wild-type survivin locus (top) showing exons 1–3 (open boxes) and a 10-kb XbaI fragment. The targeting vector was designed to generate floxed exon2 (loxP; triangles), flank the PGK-neo cassette with FRT sequences (ovals), and introduce a new XbaI site (X). The targeted allele contains a diagnostic 7.0-kb XbaI fragment. To generate the conditional allele, the neo cassette was removed by transient expression of Flpe recombinase. Cre-mediated recombination resulted in the deleted allele. The positions of the 5′ flanking probe (A) and neo probe (B) used for genotyping are indicated. (B) Southern blot analyses to identify survivinflox/+ ES cells containing neo (left), ES cells with neo removed (middle), and F2 pups (right). Genomic DNA was digested with XbaI and hybridized with probe A or B. (C) Western blot of survivin protein in Lck-survivinflox/+ and Lck-survivinflox/flox DN thymocytes. Wild-type survivin protein (arrowhead) and a nonspecific band (asterisk) are indicated. Actin, loading control. (D) Detection of deleted survivin allele in DN thymocyte subpopulations. The floxed (flox) and deleted (Δflox) survivin alleles were amplified by PCR using primers a and b shown in A. Genomic DNA samples were prepared from the indicated DN subsets.

Mentions: Three independent overlapping genomic survivin clones were isolated from a 129/Sv library and used to construct a targeting vector (see Fig. 1) that was electroporated into E14K embryonic stem (ES) cells (129/Ola). Homologous recombinants were used to generate chimeric mice and survivinflox/flox mice after the removal of the neo cassette by transient expression of Flpe recombinase in vitro (35). Germline transmission was confirmed by Southern blot analysis of tail DNA as previously described (36). The primer sequences for genomic DNA PCR are available upon request.


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)

Tissue-specific targeted disruption of the survivin locus. (A) A portion of the murine wild-type survivin locus (top) showing exons 1–3 (open boxes) and a 10-kb XbaI fragment. The targeting vector was designed to generate floxed exon2 (loxP; triangles), flank the PGK-neo cassette with FRT sequences (ovals), and introduce a new XbaI site (X). The targeted allele contains a diagnostic 7.0-kb XbaI fragment. To generate the conditional allele, the neo cassette was removed by transient expression of Flpe recombinase. Cre-mediated recombination resulted in the deleted allele. The positions of the 5′ flanking probe (A) and neo probe (B) used for genotyping are indicated. (B) Southern blot analyses to identify survivinflox/+ ES cells containing neo (left), ES cells with neo removed (middle), and F2 pups (right). Genomic DNA was digested with XbaI and hybridized with probe A or B. (C) Western blot of survivin protein in Lck-survivinflox/+ and Lck-survivinflox/flox DN thymocytes. Wild-type survivin protein (arrowhead) and a nonspecific band (asterisk) are indicated. Actin, loading control. (D) Detection of deleted survivin allele in DN thymocyte subpopulations. The floxed (flox) and deleted (Δflox) survivin alleles were amplified by PCR using primers a and b shown in A. Genomic DNA samples were prepared from the indicated DN subsets.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211792&req=5

fig1: Tissue-specific targeted disruption of the survivin locus. (A) A portion of the murine wild-type survivin locus (top) showing exons 1–3 (open boxes) and a 10-kb XbaI fragment. The targeting vector was designed to generate floxed exon2 (loxP; triangles), flank the PGK-neo cassette with FRT sequences (ovals), and introduce a new XbaI site (X). The targeted allele contains a diagnostic 7.0-kb XbaI fragment. To generate the conditional allele, the neo cassette was removed by transient expression of Flpe recombinase. Cre-mediated recombination resulted in the deleted allele. The positions of the 5′ flanking probe (A) and neo probe (B) used for genotyping are indicated. (B) Southern blot analyses to identify survivinflox/+ ES cells containing neo (left), ES cells with neo removed (middle), and F2 pups (right). Genomic DNA was digested with XbaI and hybridized with probe A or B. (C) Western blot of survivin protein in Lck-survivinflox/+ and Lck-survivinflox/flox DN thymocytes. Wild-type survivin protein (arrowhead) and a nonspecific band (asterisk) are indicated. Actin, loading control. (D) Detection of deleted survivin allele in DN thymocyte subpopulations. The floxed (flox) and deleted (Δflox) survivin alleles were amplified by PCR using primers a and b shown in A. Genomic DNA samples were prepared from the indicated DN subsets.
Mentions: Three independent overlapping genomic survivin clones were isolated from a 129/Sv library and used to construct a targeting vector (see Fig. 1) that was electroporated into E14K embryonic stem (ES) cells (129/Ola). Homologous recombinants were used to generate chimeric mice and survivinflox/flox mice after the removal of the neo cassette by transient expression of Flpe recombinase in vitro (35). Germline transmission was confirmed by Southern blot analysis of tail DNA as previously described (36). The primer sequences for genomic DNA PCR are available upon request.

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