Generation of Thymocyte-specific Survivin-deficient Mice.
We constructed a survivin
-targeting vector in which the neo
cassette was flanked by FRT sequences and survivin
exon 2 was flanked by loxP sequences ( A). Therefore, Cre-mediated removal of exon 2 resulted in an early frameshift and translation termination. The conditional targeting vector was used to generate two independent ES cell lines carrying a floxed survivin
exon 2 and a flrted neo
gene ( B). Transient transfection of these clones with Flpe recombinase (35
) resulted in successful recombination that removed neo
( B). Recombined ES clones were used for blastocyst injection followed by standard breeding steps to produce animals homozygous for the floxed survivin allele (survivinflox/flox
mice were born at the expected Mendelian ratio and displayed no abnormalities (not depicted), indicating that genetic manipulation of the survivin
gene did not interfere with survivin function. Survivinflox/flox
mice were crossed with Lck-Cre transgenic mice (41
) to generate thymocyte-specific survivin-deficient mice (Lck-survivinflox/flox
). The lack of survivin protein in DN thymocytes was demonstrated by Western blot analysis of survivin protein levels ( C). In addition, genomic DNA prepared from sorted DN cells was analyzed by PCR for the detection of the floxed or deleted (Δfloxed) survivin allele. The floxed allele was deleted in the majority of DN2 and DN3E cells and almost all DN3L and DN4 cells in the mutants ( D).
Defects in Thymocyte Development in Lck-survivinflox/flox Mice.
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 ( 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 ( B). This time frame is consistent with Lck proximal promoter activity (42
Figure 2. 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 (more ...)
Next, we determined numbers of peripheral T cells and thymocyte subpopulation proportions in Lck-survivinflox/+ and Lck-survivinflox/flox mice at 6–8 wk of age. Flow cytometric analysis revealed that the number of peripheral TCRαβ+ cells was dramatically reduced in Lck-survivinflox/flox mice ( C, top). The average number of thymocytes recovered from Lck-survivinflox/flox mice was 2.41 ± 0.5 × 106 (n = 6), ~2.0% of the number of thymocytes in Lck-survivin+/+ mice (1.22 ± 0.6 × 108; n = 6). Moreover, ~95% of Lck-survivinflox/flox thymocytes were DN cells ( C, middle), suggesting that the loss of survivin affected either the DN to DP transition or the production of DN cells. To distinguish between these possibilities, we stained DN cells from control and mutant mice with the early developmental markers CD25 and CD44 ( C, bottom). DN3 (CD25+ CD44−) cells represented ~80% of Lck-survivinflox/flox DN cells compared with <50% of DN cells in Lck-survivin+/+ or Lck-survivinflox/+ mice. DN4 (CD25− CD44−) cells were reduced to 4% in Lck-survivinflox/flox mice compared with 30 and 34% in Lck-survivin+/+ and Lck-survivinflox/flox mice, respectively.
Histological findings confirmed the block in the DN3 to DN4 transition in the absence of survivin. Thymi in Lck-survivinflox/+ mice had a typical structure and contained a cortex ( D, left, dark purple area) with immature T cells and a medulla ( D, left, light purple area) with smaller DP cells. In contrast, thymi of Lck-survivinflox/flox mice were much smaller and lacked cortex medulla compartmentalization ( D, right). The vast majority of thymic cells in the mutants were large and stained light purple, which are characteristics of immature thymocytes. These results indicate that in the absence of survivin, DN thymocytes fail to expand and progress to the DP stage.
Normal TCRβ Gene Rearrangement and In Vitro Pre-TCR Signaling in Survivinflox/flox DN Thymocytes.
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 ( A). Furthermore, flow cytometry revealed no differences in intracellular TCRβ protein between DN3E and DN3L cells of control and mutant mice ( B).
Figure 3. 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 (more ...)
The pre-TCR signaling pathway necessary for the DN3 to DN4 transition can be activated in vitro by ligating CD3ε on the surface of DN cells (47
). This activation can be assessed by determining the extent of phosphorylation of the ERKs, downstream targets of pre-TCR signaling (38
). DN thymocytes from Lck-survivinflox/flox
mice were activated using anti-CD3ε Ab followed by detection of ERK activation via anti–phospho-ERK Abs. As shown in C, ERK1 and ERK2 were phosphorylated equally and with comparable kinetics in control and mutant DN cells. Intraperitoneal injection of anti-CD3ε Ab can mimic pre-TCR signaling in vivo and induce DN cells to proliferate and differentiate into DP cells in RAG-2−/−
mice in the absence of pre-TCR signaling (48
). Therefore, we treated Lck-survivinflox/flox
mice with anti-CD3ε Ab and isolated DN thymocytes 72 h after injection. Although anti-CD3ε Ab induced the differentiation and proliferation of DN3 cells in RAG-2−/−
mice, it failed to do so in Lck-survivinflox/flox
mice ( D). These results indicate that the loss of survivin does not affect TCRβ rearrangement, intracellular TCRβ expression, or the pre-TCR signaling pathway itself. However, the DN3 to DN4 transition cannot be rescued by a surrogate pre-TCR signal in the absence of survivin, suggesting that survivin plays a critical role in highly proliferative cells.
Impaired Proliferation and Increased Cell Death of Lck-survivinflox/flox DN Thymocytes.
Because the pre-TCR signaling required for DN cell proliferation was normal in Lck-survivinflox/flox thymocytes, we investigated whether the loss of DN4 cells in the mutant mice was due to impaired proliferation or increased apoptosis or both. Annexin V staining for cell viability in vivo showed that 37% of DN3L and 32% of DN4 cells were annexin+ in Lck-survivinflox/flox mice ( A). In contrast, only 5–7% cells were annexin+ in control mice, indicating that loss of survivin induces apoptosis of DN3L and DN4 thymocytes.
Figure 4. Effect of survivin loss on cell death in vivo and in vitro and on thymocyte proliferation. (A) Increased cell death of proliferating survivin-deficient DN cells. DN thymocytes were prepared ex vivo and stained with anti-CD25, anti-CD44, and anti-Lin Ab (more ...)
To test whether loss of survivin impaired cellular responses to external apoptotic stimuli, we subjected purified DN cells from Lck-survivinflox/flox and RAG- 2−/− mice to treatment with etoposide ( B, Etp), dexamethasone (Dex), γ-irradiation (IR), or staurosporine (STS). About 90% of thymocytes in both mutant strains are DN3E cells. When apoptosis was evaluated at 16 h after treatment, no significant differences in the numbers of apoptotic cells were observed under any conditions tested ( B). These results indicate that survivin is not essential in DN3E cells for the execution of apoptosis in response to various external stimuli in vitro. However, a failure in survivin function triggers the death of proliferating cells in vivo.
To examine the effect of survivin loss on the cell cycle, DN thymocytes were pulse labeled in vivo with BrdU and stained with anti-BrdU Ab and 7AAD in vitro ( C). The cell cycle profile of DN3E cells was comparable in Lck-survivin+/+ and Lck-survivinflox/flox mice (Lck-survivin+/+: G1, 77.1%; S, 21.0%; G2/M, 0.3% vs. Lck-survivinflox/flox: G1, 77.6%; S, 20.2%; G2/M, 0.6%). However, in Lck-survivinflox/flox DN3L cells, the G1 population was increased to 33.3% (Lck-survivin+/+: 14.8%) and the S population was decreased to 63% (Lck-survivin+/+: 80%). In Lck-survivinflox/flox DN4 cells, the G1 population was increased still further to 67.4% (Lck-survivin+/+: 43.7%), whereas the S population dropped to 19.6% (Lck-survivin+/+: 53.0%). In addition, sub-G1 cells represented 3.0% of DN4 thymocytes in Lck-survivinflox/flox mice but only 0.1% of control DN4 cells. Thus, survivin is required for the proliferation and survival of DN3L and DN4 thymocytes, and a lack of survivin leads to cell cycle arrest and cell death at these stages.
Defects in Cytokinesis in Lck-survivin flox/flox DN Thymocytes.
Embryos of mice with a null 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
) and anti-survivin Ab injection results in spindle defects (49
). We examined spindle formation in DN3L and DN4 cells from Lck-survivinflox/flox
mice by staining thymocytes with α-tubulin. Lck-survivin+/+
) cells had well-organized and symmetrical spindles ( A, a), whereas Lck-survivinflox/flox
cells showed shorter and thicker spindles ( 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 ( B). Thus, the organization and assembly of mitotic spindles is severely impaired in the absence of survivin.
Figure 5. 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 (more ...)
Because the intracellular localization of lpl1/Aurora kinase in Caenorhabditis elegans
is affected by the loss of the survivin
), we examined the localization of murine Aurora-B kinase (Aurora-B) at each mitotic stage in DN cells of control and survivin-deficient mice. In wild-type cells, Aurora-B localized at the centromeres until metaphase ( C, b), transferred to the central spindle during anaphase ( C, c), and finally accumulated at the spindle midbody during telophase and cytokinesis ( C, d). However, in survivin-deficient cells, Aurora-B could not localize at either the midzone or midbody at either anaphase or telophase ( C, e–h). Indeed, no typical telophase cells could be found among survivin-deficient DN thymocytes.
p53 Induction in Lck-survivin flox/flox DN Thymocytes.
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 ( A, top). Flow cytometric analysis of intracellular p53 protein confirmed that p53 was expressed in survivin-deficient DN2 to DN4 cells ( B). In addition, RT-PCR analysis showed that expression of the p53 target gene p21 was induced in Lck-survivinlox/lox DN cells ( A, bottom). These results suggested that p53 might be responsible for the increased apoptosis and arrest observed in Lck-survivinflox/flox mice.
Figure 6. 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 (more ...)
Gain of Bcl-2 or Loss of p53 Expression Does Not Rescue DN Thymocyte Developmental Defects Induced by Survivin Loss.
We determined whether Bcl-2 could rescue survivin deficiency by crossing Lck-survivinflox/flox mice to Eμ-Bcl-2 transgenic mice to generate Lck-survivinflox/flox;Bcl-2 animals. The total number of thymocytes in Lck-survivinflox/flox;Bcl-2 mice (2.2 ± 0.7 × 106; n = 4) was comparable to that in Lck-survivinflox/flox mice (2.4 ± 0.5 × 106; n = 4; A). The number of DN cells was also unaltered in the presence of the Bcl-2 transgene (Lck-survivinflox/flox vs. Lck-survivinflox/flox;Bcl-2; 2.3 ± 0.5 × 106 vs. 2.1 ± 0.6 × 106; n = 4), suggesting that Bcl-2 overexpression cannot overcome cell death induced by an absence of survivin.
Figure 7. Effects of Bcl-2 gain or p53 loss on phenotypes of survivin-deficient thymocytes. (A) Gain of Bcl-2 does not restore DP cells in the absence of survivin. Thymocytes prepared from Lck-survivinflox/+;Bcl-2, Lck-survivinflox/flox, and Lck-survivin (more ...)
To test whether survivin deficiency had a direct impact on p53-mediated apoptosis, we generated compound conditional survivin mutants by crossing Lck-survivinflox/flox mice to p53−/− mice to generate Lck-survivinflox/flox;p53−/− animals. In mice lacking both survivin and p53, the total number of thymocytes was decreased (0.33 ± 0.106; n = 3) compared with Lck-survivinflox/flox;p53+/− controls (1.23 ± 0.12 × 106; n = 3). The percentage of DN thymocytes in the double mutants was decreased by 10% at most (n = 3), whereas the percentages of DP and SP thymocytes were slightly (not significantly) increased ( B), indicating that loss of p53 cannot restore DN thymocyte development in the absence of survivin.
Loss of p53 or p21 Expression Releases Cell Cycle Arrest but Accelerates Apoptosis Induced by Survivin Loss.
Next, we examined cell cycle status and apoptosis in survivin-deficient thymocytes also lacking either p53 or p21. In both cases, the G1 subpopulation of DN3L thymocytes was restored to the control level (Lck-survivinflox/+ vs. Lck-survivinflox/flox vs. Lck-survivinflox/flox;p53−/− vs. Lck-survivinflox/flox;p21−/−; 35.3 vs. 51.1 vs. 35.6 vs. 37.0%; C, middle). Thus, cell cycle arrest induced by loss of survivin is mediated by the p53 pathway and involves p21 induction. The G1 subpopulation of DN4 thymocytes was reduced by >80% regardless of genetic background, suggesting profound cell damage resulted in these cells. These results suggest that loss of survivin, as well as triggering p53-mediated growth arrest, precipitates aberrant mitosis that inexorably causes the death of affected cells regardless of their p53 or p21 status. Indeed, annexin staining showed that the viability of DN3E and DN3L cells was significantly decreased when both survivin and either p53 or p21 were missing ( D). Thus, the loss of the cytoprotective function of survivin appears to trigger p53 induction and cell cycle arrest, but the death of survivin-deficient cells involves a mechanism that is independent of p53.