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m-survivin in thymocytes enhances cell proliferation
Molecular Immunology 39 (2002) 289–298
Overexpression of TIAP/m-survivin in thymocytes enhances cell proliferation Satosi Hikita a,b , Masahiko Hatano a , Atsushi Inoue a , Nobuyuki Sekita a , Koichi Kobayashi b , Masayuki Otaki b , Takeshi Ogasawara b , Seiji Okada a , Hiroyuki Hirasawa b , Takeshi Tokuhisa a,∗ b
a Department of Developmental Genetics (H2), Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan Department of Emergency and Critical Care Medicine (J3), Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
Received 8 April 2002; accepted 17 April 2002
Abstract TIAP/m-survivin, a member of the inhibitor of apoptosis (IAP) protein family, is expressed in a cell cycle dependent manner. It is strongly expressed in various subsets of thymocytes. To investigate a role of TIAP/m-survivin in thymocytes, mice carrying the lck-TIAP transgene were established. Two out of six transgenic mice expressed large amounts of TIAP mRNA and protein in thymocytes. Although T cell development and apoptosis of thymocytes were largely unaffected in lck-TIAP mice, transgenic thymocytes displayed hyperproliferation in response to PMA and ionomycin but not to anti-CD3 antibody. Thus, overexpression of TIAP/m-survivin augments cell proliferation of thymocytes to a certain stimulation. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Apoptosis; Cellular proliferation; Thymocytes; TIAP/m-survivin; Transgenic mice
1. Introduction Apoptosis plays a critical role in the development and maintenance of the immune system (Vaux and Korsmeyer, 1999). Dysregulation of lymphocyte apoptosis causes numerous disorders such as cancer and autoimmune diseases (Rinkenberger and Korsmeyer, 1997). Recently many regulators of apoptosis were identified and molecular mechanisms of apoptosis became much clearer. Signals from death receptor such as Fas or TNFR activate caspase cascades that induce apoptosis. Some stresses such as free radicals or growth factor deprivation damage mitochondria, and release of cytochrome c from mitochondria also activate caspase cascades to induce apoptosis. Apoptosis is controlled by some families of intrinsic cellular regulators. Bcl-2 family such as Bcl-2 or -xL is thought to regulate cytochrome c release from mitochondria and functions as an anti-apoptotic manner (Gross et al., 1999). The IAP protein family is recently identified as an intrinsic cellular regulator of apoptosis by binding and inhibiting function of caspases (Deveraux and Reed, 1999). Abbreviations: IAP, inhibitor of apoptosis; BIR, baculovirus IAP repeat; HRP, horseradish peroxidase; PI, propidium iodide; DP, double positive; DN, double negative; SP, single positive ∗ Corresponding author. Tel.: +81-43-226-2181; fax: +81-43-226-2183. E-mail address: [email protected] (T. Tokuhisa).
The IAP genes were originally discovered in baculoviruses (Crook et al., 1993). Their homologues have been identified in other viruses, Drosophila, yeast, Caenorhabditis elegans and mammals, suggesting a common evolutional origin (Duckett et al., 1996, Deveraux and Reed, 1999). A mode of action of the IAP is believed to be by direct binding and inhibition of the key caspases and procaspases, primarily caspases 3, 7 and 9 (Deveraux et al., 1997; Roy et al., 1997; Talanian et al., 1997; Deveraux et al., 1998; Takahashi et al., 1998). The common feature of IAP family proteins is the presence of one to three BIR domains at its N-terminus and a RING finger domain at the C-terminus. The BIR domain is thought to be important for protein–protein interaction and is necessary and sufficient to prevent apoptosis. The RING finger domain is thought to be important for degradation of the protein by ubiqutination (Yang et al., 2000). Although many IAP proteins are expressed in lymphocytes and can inhibit apoptosis induced by a variety of triggers in vitro, a physiological role of IAP proteins in the development and maintenance of the immune system is not yet clear. TIAP, a murine homolog of human survivin, is one of the IAP family proteins (Ambrosini et al., 1997, Kobayashi et al., 1999). TIAP/m-survivin which has one BIR domain interacts with caspases 3, 7 and 9 inhibits their activity in vitro (Tamm et al., 1998; Kobayashi et al., 1999; Banks et al.,
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2000; O’Connor et al., 2000; Verdecia et al., 2000; Wright et al., 2000). It can also inhibit apoptosis caused by anti-Fas, Bax overexpression and anti-cancer drugs when overexpressed in vitro (Tamm et al., 1998). TIAP is expressed in growing tissues such as the thymus, testis, intestinal epithelium, and many tissues of embryos (Adida et al., 1998; Kobayashi et al., 1999). Its expression is regulated in cell cycle dependent manner. Expression of TIAP/m-survivin is induced at S to G2/M phase of the cell cycle (Li et al., 1998, Kobayashi et al., 1999). Furthermore, survivin interacts with microtubules of the mitotic spindle during mitosis (Li et al., 1998). Thus, TIAP/m-survivin may be involved in a regulation of apoptosis during cell proliferation. To study a role of TIAP/m-survivin in T cell development and proliferation, we examined expression of TIAP/m-survivin in subsets of normal thymocytes and generated transgenic mice which overexpress TIAP/m-survivin in thymocytes. Every subset of thymocytes examined expresses the large amount of TIAP/m-survivin. T cell development and susceptibility to various apoptotic stimuli were not altered in thymocytes by overexpression of TIAP/m-survivin. However, proliferation of thymocytes from transgenic mice after stimulation with PMA and ionomycin was augmented. We discuss a role of TIAP/m-survivin in T cell development and proliferation.
2. Materials and methods 2.1. Animals C57BL/6Crslc and (C57BL/6 × DBA/2)F1 mice were purchased from Japan SLC Co. (Hamamatsu, Japan). ICR mice were purchased from Japan CLEA Co. (Tokyo Japan). These mice were maintained under specific pathogen free conditions in the animal center of Chiba University School of Medicine. 2.2. Generation of transgenic mice The lck-TIAP transgene was constructed by inserting a BamH1 fragment of TIAP cDNA into the BamH1 site of the lck-hGH plasmid (Allen et al., 1992). Transgenic mice were produced by the method described by Hogan et al. (1986). Briefly, (C57BL/6 × DBA/2)F1 mice were used to obtain fertilized eggs, and the lck-TIAP gene was microinjected into a male pronucleus of fertilized eggs. Injected eggs were returned to oviducts of pseudopregnant females of ICR strain. The transgenes in genomic DNA isolated from the tail were detected by slot blot analysis using the human growth hormone intron DNA as a probe. Transgenic founders were backcrossed to C57BL/6 mice five times. 2.3. Antibodies Anti-CD4, -CD8, -CD3, -CD25, -CD44, and -Fas monoclonal antibodies were purchased from Pharmingen (San
Diego, CA). Rabbit anti-TIAP polyclonal antibodies were described before (Kobayashi et al., 1999). 2.4. FACS analysis Thymocytes and splenocytes were isolated from 4- to 8-week-old transgenic mice or control littermates. Cell suspensions were treated with ACK lysing buffer to lyse erythrocytes before staining. Single cell suspensions were prepared in staining medium and stained with monoclonal antibodies described above. Stained cells were analyzed by a FACSCalibur. 2.5. Northern blot analysis Total RNAs were extracted from thymocytes and splenocytes using the Trizol reagent (Gibco BRL). Northern blot analysis was done using a TIAP specific 351 bp probe as described previously (Kobayashi et al., 1999). 2.6. Western blot analysis Total proteins from thymocytes and splenocytes from transgenic mice or control littermates were obtained as described previously (Kobayashi et al., 1999). The amount of protein in cell lysates was determined using the Bio-Rad protein assay (Bio-Rad Laboratories). Fifteen micrograms of cell lysates were resolved by SDS-PAGE and transferred to a polyvinyl difluoride membrane (Immobilon-P, Millipore Corp., Bedford, MA). Blots were blocked in Blockace (Yukijirushi, Sapporo, Japan) for 1 h, washed four times with Tris-buffered saline with 0.2% Tween 20 (TBST) and incubated with anti-TIAP antibodies in TBST for 1 h at room temperature. The blots were then washed four times with TBST, incubated with affinity-purified donkey anti-rabbit antibodies conjugated with horseradish peroxidase (HRP, Amersham International, Arlington Heights, IL) for 1 h at room temperature, washed four times with TBST, and developed with enhanced chemiluminescence reagents (Amersham International). 2.7. Cell sorting and RT-PCR analysis Thymocytes from 4-week-old C57BL/6 mice were stained with FITC conjugated anti-CD4 and PE conjugated anti-CD8 monoclonal antibodies for 20 min at 4 ◦ C. After washing, the cells were resuspended in staining medium supplemented with PI. Stained cells were analyzed by FACS Vantage, and each subset of thymocytes was sorted. Total RNA was extracted from sorted cells using the Trizol reagent. RNAs were reverse-transcribed using Superscript and oligo dT in a final volume of 20 l, and 1 l of cDNAs was used for PCR. PCR reactions were incubated for 7 min at 94 ◦ C for one cycle and 1 min at 94 ◦ C, 1 min at 55 ◦ C, 1 min at 72 ◦ C for 30 cycles. PCR primers for the cDNA amplification were as
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follows: TIAP primers, 5 -ATGGGAGCTCCGGCGCT-3 and 5 -TTAGGCAGCCAGCTGCT-3 ; G3PDH primers, 5 -TGAAGGTCGGTGTGAACGGATTTGGC-3 and 5 CATGTAGGCCATGAGGTCCACCAC-3 . PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. 2.8. Immunohistochemistry Four-week-old C57BL/6 mice were perfused with a solution of 4% para-formaldehyde in 0.1 M phosphate buffer (pH 7.4). The thymus was dissected from mice and post-fixed with 4% para-formaldehyde for 12 h. The tissue was equilibrated with 20% sucrose and sectioned at 10 m on a cryostat. After quenching the activity of endogenous peroxidase with 3% H2 O2 in methanol, sections were stained with rabbit antibodies against TIAP. Biotinylated goat anti-rabbit antibodies (Nichirei, Tokyo, Japan) and streptABComplex/HRP (Dako, Carpinteria, CA) were used as the second- and third-phase reagents, respectively. Bound HRP activity was visualized using the DAB kit (Nichirei). Hematoxylin staining was done using a standard protocol.
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2.9. Cell culture and apoptosis assay Thymocytes were cultured in RPMI-1640 medium supplemented with 10% FCS. Apoptosis was induced by plate coated anti-CD3 (10 g/ml), anti-Fas (1 g/ml) and cyclohexamide (10 g/ml), dexamethazone (100 nM), or ␥-irradiation (0.5 or 1.0 Gy). Thymocytes (1 × 106 ) were cultured in 24-well plates with various reagents for 24 h and apoptotic cells were determined by FACS after annexin V and PI staining. 2.10. Proliferation assay CD4− CD8− DN thymocytes were enriched by depleting CD4+ CD8+ DP, CD4+ CD8− and CD4− CD8+ SP thymocytes from lck-TIAP transgenic mice and control littermates. Briefly, thymocytes were incubated with mixture of biotinylated monoclonal antibodies to CD4 and CD8. These cells were subsequently reacted with streptavidin coated immunomagnetic beads (Miltenyi Biotec, Gladbach, Germany). Labeled cells were separated by applying them in a magnetic field using a MACS system (Miltenyi Biotec).
Fig. 1. Expression of TIAP in thymocytes. (A) TIAP mRNA expression in subsets of thymocytes. The amount of TIAP mRNA in total RNAs from subsets of thymocytes was measured by the semiquantative RT-PCR; (B) Immunohistochemistry of normal mouse thymus stained with anti-TIAP polyclonal antibodies.
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Splenic T cells were also enriched in a similar manner by depleting non-T lineage cells with monoclonal antibodies to B220, TER119 and Mac-1 from spleen cells of lck-TIAP transgenic mice and control littermates. The resulting T cells contained <0.5% of B220+ cells. Thymocytes or splenic T cells (1 × 105 per well) were cultured with anti-CD3 (5 g/ml), PMA (50 ng/ml) and ionomycin (250 nM), or concanavalin A (1 g/ml) in a 96-well microplates in triplicates for 1–4 days. The cultures were pulsed with 1 Ci of [3 H]thymidine for 6 h, and the cells were harvested onto a glass filter. The [3 H]thymidine uptake was measured in a liquid scintillation counter.
3. Results 3.1. Expression of endogenous TIAP/m-survivin during T cell development Since TIAP is highly expressed in the thymus, we examined the expression during T cell development. Thymocytes and splenocytes were stained with anti-CD4 and -CD8,
and sorted according to their expression profiles. We have used semiquantitative RT-PCR analysis to detect the expression in total RNAs from those sorted cells. As shown in Fig. 1A, TIAP mRNA was detected in early T cell precursors (DN), CD4+ CD8+ (DP) and CD4+ CD8− or CD4− CD8+ (SP) thymocytes. Each fraction expressed almost the same amount of mRNA. The amount of TIAP decreased in splenic SP T cells when compared with that in thymic SP T cells. To determine the amount of TIAP protein, immunohistochemistry was performed using anti-TIAP polyclonal antibodies. TIAP was detected in thymocytes in both cortex and medulla (Fig. 1B). Some epithelial-like cells were also positive for TIAP. Furthermore, positive signal was detected in nucleus of some thymocytes whereas most thymocytes were stained in cytoplasm. 3.2. Generation of lck-TIAP transgenic mice In order to examine a function of TIAP/m-survivin in thymocytes, we generated transgenic mice carrying the TIAP cDNA under the control of the lck proximal promoter (lck-TIAP) (Fig. 2A). We established six transgenic
Fig. 2. Construction and expression of the lck-TIAP transgene. (A) Schematic diagram of the transgenic construct used to generate lck-TIAP mice; (B) Northern blot analysis of RNAs from the thymus and spleen of transgenic mice (#201, #242) and control littermates (WT); (C) Western blot analysis of proteins from the thymus and spleen of transgenic mice (#201, #242) and control littermates (WT).
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lines with various copy numbers. The amount of TIAP in RNAs and that in proteins from these transgenic mice were examined by Northern and Western blot analysis, respectively (Fig. 2B and C). Transgenic lines which expressed the highest (#201) and the second highest (#242) amounts of TIAP protein in thymocytes were expanded for further experiments. Those two transgenic mice carry approximately 20 and 10 copies of the exogenous gene, respectively. Expression of the transgene was further examined in these two lines. TIAP transgene message was also detectable in the
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spleen but not in the other non-lymphoid organs such as the liver and testis (data not shown). 3.3. T cell development in the thymus of lck-TIAP transgenic mice Subpopulation of T cells was examined in the thymus from 4- to 6-week-old lck-TIAP mice. Total cell number ((2.46±1.36)×108 ) in the thymus from lck-TIAP mice was not significantly different from that ((2.21 ± 0.83) × 108 )
Fig. 3. Flow cytometry analysis of thymocytes and splenocytes from lck-TIAP transgenic mice. (A) Thymocytes and splenocytes were stained with anti-CD4 and -CD8. Numbers in quadrants represent percentage of the total population. (B) Thymocytes were stained with anti-CD4, -CD8, -CD44 and -CD25. CD4− CD8− thymocytes were gated and further analyzed with expression of CD44 and CD25. Numbers in quadrants represent percentage of the total population.
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in control littermates. Thymocytes were stained with developmental surface markers and analyzed by FACS. In lck-TIAP mice, T cell subsets that expressed CD3 (data not shown), CD4 and CD8 (Fig. 3A), or CD44 and CD25 in CD4− 8− thymocytes (Fig. 3B) appeared essentially normal. When the thymus was histologically examined, architecture of cortex and medulla in the thymus from lck-TIAP mice was essentially the same as that from control littermates (data not shown). Size and cell number of the spleen and lymph nodes from lck-TIAP mice were also the same as those of control littermates. Therefore, development of T cells in the thymus is not perturbed by elevating levels of TIAP/m-survivin. 3.4. Apoptosis of thymocytes from lck-TIAP transgenic mice Thymocytes undergo apoptosis in response to many stimuli. Since TIAP/m-survivin inhibits activity of caspase 3, 7 and 9 in vitro (Tamm et al., 1998, Kobayashi et al., 1999, O’Connor et al., 2000), we examined whether overexpression of TIAP could prevent any form of thymocyte apoptosis or not. Thymocytes were cultured with various stimuli and apoptosis was evaluated by annexin V and PI staining. There was no significant difference in thymocyte apoptosis after anti-CD3 or -Fas stimulation, glucocorticoid treatment, or ␥-irradiation between transgenic and control littermates (Fig. 4). Thus, overexpression of TIAP in developing thymocytes did not affect a susceptibility to various apoptotic stimuli examined.
3.5. Proliferative response of thymocytes from lck-TIAP transgenic mice Proliferative responses of thymocytes from lck-TIAP mice to various mitogens were examined. Transgenic thymocytes responded three times more than control thymocytes did after stimulation with PMA and ionomycin (Fig. 5A). Proliferative responses of thymocyte subsets were further examined. DN thymocytes and DP + SP thymocytes from lck-TIAP mice proliferated more than those from control mice (Fig. 5B). Proliferative responses of thymocytes reached maximum between 48 and 72 h after stimulation in both lck-TIAP and control littermates (Fig. 5C). The response in transgenic mice returned almost the same level as control littermates 96 h after stimulation. However, upon stimulation with anti-CD3 antibody (Fig. 5A–C) or with concanavalin A (data not shown), no difference was observed in proliferation of thymocytes between transgenic and control littermates at any time points after stimulation. Proliferative responses of splenic T cells from lck-TIAP mice stimulated with PMA and ionomycin was examined. The responses of splenic T cells from lck-TIAP mice stimulated with PMA and ionomycin or with anti-CD3 were almost the same as those from control littermates (Fig. 6).
4. Discussion In order to elucidate a role of TIAP/m-survivin in T cell development and activation, we have established lck-TIAP
Fig. 4. Apoptosis of thymocytes from lck-TIAP transgenic mice. Thymocytes from lck-TIAP mice (closed bars) and control littermates (open bars) were cultured with various stimuli indicated for 24 h. Viability of stimulated cells was measured by annexin V and PI staining. CHX; cyclohexamide, DEX; dexamethasone.
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Fig. 5. Proliferation of thymocytes from lck-TIAP transgenic mice. (A) Thymocytes were stimulated with anti-CD3 or PMA and ionomycin for 24 h. Closed bars; lck-TIAP mice, open bars; control littermates. The results from three independent experiments were shown. (B) Subsets of thymocytes were stimulated with PMA and ionomycin for 24 h. Closed bars; lck-TIAP mice, open bars; control littermates. (C) Kinetics of thymocyte proliferation induced by anti-CD3 or PMA and ionomycin. Closed circle; lck-TIAP mice, open circle; control littermates. The results are expressed as mean cpm ± S.D. of triplicate cultures.
transgenic mice. Proliferation of transgenic thymocytes in response to PMA and ionomycin was three-fold higher than that of control thymocytes. TIAP/m-survivin displays cell cycle specific expression that is mediated by G1 transcriptional repressor elements in its promoter region (Li and Altieri, 1999, Otaki et al., 2000). Recently, a possible link between cell cycle progression and apoptosis has emerged. Treatment of cells with survivin antisense results in increased apoptosis and inhibition of cell proliferation
(Ambrosini et al., 1998). Furthermore, survivin has been shown to associate with mitotic spindles and may play a critical role in dual control of mitotic spindle checkpoints and apoptosis (Li et al., 1998; Li et al., 1999). Thus, it is possible that overexpression of TIAP/m-survivin accelerates cell cycle progression by affecting mitotic spindle checkpoints. Alternatively, apoptosis during proliferation is rescued and more cells enter cell cycle resulting in increased proliferation. Interestingly, thymocyte proliferation in response to
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Fig. 6. Proliferation of splenic T cells from lck-TIAP transgenic mice. Splenic T cells were stimulated with anti-CD3 or PMA and ionomycin for 24 h. Closed bars; lck-TIAP mice, open bars; control littermates. The results are expressed as mean cpm ± S.D. of triplicate cultures.
anti-CD3 stimulation was almost the same between transgenic and control littermates. In contrast, stimulation with PMA and ionomycin bypasses the TCR signaling pathway and activates broad population of thymocytes. In case of anti-CD3 stimulation, responding cells are already competent to enter cell cycle and probably that’s why we could not see the difference in proliferation. PMA and ionomycin force many cells to enter the cell cycle. Overexpression of TIAP/m-survivin may elevate anti-apoptotic threshold during cell division and may rescue incompetent cells to enter cell cycle. T cell development in lck-TIAP mice appeared normal. Numbers of thymocytes and T cell subpopulations in lck-TIAP mice were normal levels, suggesting that excess amounts of TIAP do not affect T cell development. T cell apoptosis was also examined in lck-TIAP mice. Apoptotic responses of transgenic thymocytes to anti-Fas stimulation, anti-CD3 stimulation, dexamethasone treatment, or ␥-irradiation were not inhibited. Previously we and others reported that overexpression of TIAP/m-survivin in cultured cell lines inhibited various forms of apoptosis such as anti-Fas stimulation, Bax overexpression or growth factor withdrawal (Ambrosini et al., 1997; Tamm et al., 1998; Kobayashi et al., 1999). However, the effect of TIAP/m-survivin to prevent apoptosis was not so strong as that of XIAP or Bcl-2 in cultured cell lines. TIAP/m-survivin could bind to caspases 3 and 7 in vitro but its affinity to bind these caspases is not so strong as that of XIAP (Tamm et al., 1998). In lck-TIAP transgenic thymocytes, levels of overexpressed TIAP proteins are sufficient to influence T cell proliferation to PMA and ionomycin
stimulation but may not be enough to inhibit apoptosis. Thus, the regulation of apoptosis may not be the primary role of TIAP/m-survivin in thymocyte development. Recently, BIR domain containing proteins with diverse structure were identified from yeast to mammals. In yeast and C. elegans, structurally related proteins to TIAP/m-survivin named BIR1, or bir-1 were identified (Fraser et al., 1999; Uren et al., 1999). Originally a function of IAP protein family was implicated to prevent apoptosis. The identification of BIR containing proteins in both Saccharomyces cervisiae and Schizosaccharomyces pombe with no identified apoptotic program and no caspase encoded gene raise a possibility that BIR containing proteins play roles in other cellular processes than apoptosis. Indeed, loss of BIR1 function in yeast strains induces cell death at the early cell division and blocked at the metaphase/anaphase transition because of an inability to elongate their mitotic spindle (Uren et al., 1999). In C. elegans, ablation of bir-1 resulted in embryonic lethality and the embryos were multinucleated and failed to form more than two cells (Fraser et al., 1999). These phenotypes resulted from an inability of bir-1 deficient embryos to complete cytokinesis. Furthermore, overexpression of bir-1 in C. elegans did not affect cell death in vivo whereas overexpression of ced-9, the nematode Bcl-2 homologue, markedly suppressed cell death during development (Fraser et al., 1999). The survivin-deficient mice are also embryonic lethal (Uren et al., 2000). Taken together, primary function of some IAP family proteins including TIAP/m-survivin or BIR1 may be a control of cell division rather than inhibition of apoptosis. During a process of evolution, coordinated regulation
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of cell proliferation, differentiation and apoptosis become essential for higher organisms and IAP family proteins may acquire pleiotropic function. Apoptosis plays a critical role in T cell development and activation. In the thymus, unsuccessfully rearranged immature T cells are eliminated by apoptosis. Negative selection takes place at DP stage and self reactive T cells are eliminated by apoptosis. T cells that have little affinity to MHC molecules also die by apoptosis. However, these events are not directly linked to cell proliferation. In periphery, naive T cells are activated by antigen and become effector cells. Some of these T cells proliferate and become memory cells while others die (activation induced cell death). In this process, cell division and apoptosis are closely linked each other. It is interesting to know whether TIAP/m-survivin regulates this process. Survivin expression is reported to be high in various tumors such as lymphoma, neuroblastoma, and colon adenocarcinoma (LaCasse et al., 1998). No obvious chromosomal translocation is associated with their genomic loci in humans. High level of the expression in cancer may reflect a result of accelerated cell proliferation of tumor cells. At this moment, no spontaneous tumor developed in lck-TIAP mice up to 24-month observation. Thus, TIAP/m-survivin itself may not function as an oncogene to initiate carcinogenesis but may play a role in disease progression. Identification of functionally cooperative protein with TIAP/m-survivin will further clarify a mechanism of cancer initiation and progression.
Acknowledgements We would like to extend our thanks to H. Satake for her technical assistance, and K. Ujiie for her secretarial assistance. This work was supported in part by Grant-in-Aid for Scientific Research on Priority areas “Cancer”, from the Ministry of Education, Science, Sports and Culture of Japan.
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Deveraux, Q.L., Reed, J.C., 1999. IAP family proteins-suppressors of apoptosis. Genes Dev. 13, 239–252. Deveraux, Q.L., Roy, N., Stennicke, H.R., Arsdale, T.V., Zhou, Q., Srinivasula, S.M., Alnemri, E.S., Salvesen, G.S., Reed, J.C., 1998. IAP block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J. 17, 2215–2223. Deveraux, Q.L., Takahashi, R., Salvesen, S.G., Reed, J.C., 1997. X-linked IAP is a direct inhibitor of cell-death proteases. Nature 388, 300–304. Duckett, S.C., Nava, E.V., Gedrich, W.R., Clem, J.R., Van Dongen, J.L., Gilfillan, M.C., Shiels, H., Hardwick, J.M., Thompson, C.B., 1996. A conserved family of cellular genes related to the baculovirus IAP gene and encoding apoptosis inhibitor. EMBO J. 15, 2685–2694. Fraser, A.G., James, C., Evan, G.I., Hengartner, M.O., 1999. Caenorhabditis elegans inhibitor of apoptosis protein (IAP) homologue BIR-1 plays a conserved role in cytokinesis. Curr. Biol. 9, 292–301. Gross, A., McDonnell, J.M., Korsmeyer, S.J., 1999. Bcl-2 family members and the mitochondria in apoptosis. Genes Dev. 13, 1899–1911. Hogan, B., Constantini, F., Lacy, E., 1986. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Kobayashi, K., Hatano, M., Otaki, M., Ogasawara, T., Tokuhisa, T., 1999. Expression of a murine homologue of the inhibitor of apoptosis protein is related to cell proliferation. Proc. Natl. Acad. Sci. U.S.A. 96, 1457– 1462. LaCasse, E.C., Baird, S., Korneluk, R.G., MacKenzie, A.E., 1998. The inhibitor of apoptosis (IAPs) and their emerging role in cancer. Oncogene 17, 3247–3259. Li, F., Ackermann, E.J., Bennett, C.F., Rothermel, A.L., Plescia, J., Tognin, S., Villa, A., Marchisio, P.C., Altieri, D.C., 1999. Pleiotropic cell-division defects and apoptosis induced by interference with survivin function. Nat. Cell. Biol. 1, 461–466. Li, F., Altieri, D.C., 1999. The cancer antiapoptosis mouse survivin gene: characterization of locus and transcriptional requirements of basal and cell cycle-dependent expression. Cancer Res. 59, 3143–3151. Li, F., Ambrosini, G., Chu, E.Y., Plescia, J., Tognin, S., Marchisio, P.C., Altieri, D.C., 1998. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396, 580–584. O’Connor, D.S., Grossman, D., Plescia, J., Li, F., Zhang, H., Villa, A., Tognin, S., Marchisio, P.C., Altieri, D.C., 2000. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc. Natl. Acad. Sci. 97, 13103–13107. Otaki, M., Hatano, M., Kobayashi, K., Ogasawara, T., Tokuhisa, T., 2000. Cell cycle-dependent regulation of TIAP/m-survivin expression. Biochem. Biophys. Acta 1493, 188–194. Rinkenberger, J.L., Korsmeyer, S.J., 1997. Errors of homeostasis and deregulated apoptosis. Curr. Opin. Genet. Dev. 7, 589–596. Roy, N., Deveraux, Q.L., Takahashi, R., Salvesen, G.S., Reed, J.C., 1997. The c-IAP-1 and -2 proteins are direct inhibitors of specific caspases. EMBO J. 16, 6914–6925. Takahashi, R., Deveraux, Q., Tamm, I., Welsh, K., Assa-Munt, N., Salvesen, G.S., Reed, J.C., 1998. A single BIR domain of XIAP sufficient for inhibiting caspases. J. Biol. Chem. 273, 7787–7790. Talanian, R.V., Quinlan, C., Trautz, S., Hackett, M.C., Mankovich, J.A., Banach, D., Ghayur, T., Brady, K.D., Wong, W.W., 1997. Substrate specificities of caspase family proteases. J. Biol. Chem. 272, 9677–9682. Tamm, I., Wang, Y., Sausville, E., Scudiero, D.A., Vigna, N., Oltersdorf, T., Reed, J.C., 1998. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res. 58, 5315–5320. Uren, A.G., Beilharz, T., O’Connell, M.J., Bugg, S.J., van Driel, R., Vaux, D.L., Lithgow, T., 1999. Role for yeast inhibitor of apoptosis (IAP)-like proteins in cell division. Proc. Natl. Acad. Sci. 96, 10170– 10175. Uren, A.G., Wong, L., Pakusch, M., Fowler, K.J., Burrows, F.J., Vaux, D.L., Choo, K.H., 2000. Related articles, nucleotide, protein survivin and the inner centromere protein INCENP show similar
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Overexpression of TIAP/m-survivin in thymocytes enhances cell proliferation Satosi Hikita a,b , Masahiko Hatano a , Atsushi Inoue a , Nobuyuki Sekita a , Koichi Kobayashi b , Masayuki Otaki b , Takeshi Ogasawara b , Seiji Okada a , Hiroyuki Hirasawa b , Takeshi Tokuhisa a,∗ b
a Department of Developmental Genetics (H2), Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan Department of Emergency and Critical Care Medicine (J3), Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
Received 8 April 2002; accepted 17 April 2002
Abstract TIAP/m-survivin, a member of the inhibitor of apoptosis (IAP) protein family, is expressed in a cell cycle dependent manner. It is strongly expressed in various subsets of thymocytes. To investigate a role of TIAP/m-survivin in thymocytes, mice carrying the lck-TIAP transgene were established. Two out of six transgenic mice expressed large amounts of TIAP mRNA and protein in thymocytes. Although T cell development and apoptosis of thymocytes were largely unaffected in lck-TIAP mice, transgenic thymocytes displayed hyperproliferation in response to PMA and ionomycin but not to anti-CD3 antibody. Thus, overexpression of TIAP/m-survivin augments cell proliferation of thymocytes to a certain stimulation. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Apoptosis; Cellular proliferation; Thymocytes; TIAP/m-survivin; Transgenic mice
1. Introduction Apoptosis plays a critical role in the development and maintenance of the immune system (Vaux and Korsmeyer, 1999). Dysregulation of lymphocyte apoptosis causes numerous disorders such as cancer and autoimmune diseases (Rinkenberger and Korsmeyer, 1997). Recently many regulators of apoptosis were identified and molecular mechanisms of apoptosis became much clearer. Signals from death receptor such as Fas or TNFR activate caspase cascades that induce apoptosis. Some stresses such as free radicals or growth factor deprivation damage mitochondria, and release of cytochrome c from mitochondria also activate caspase cascades to induce apoptosis. Apoptosis is controlled by some families of intrinsic cellular regulators. Bcl-2 family such as Bcl-2 or -xL is thought to regulate cytochrome c release from mitochondria and functions as an anti-apoptotic manner (Gross et al., 1999). The IAP protein family is recently identified as an intrinsic cellular regulator of apoptosis by binding and inhibiting function of caspases (Deveraux and Reed, 1999). Abbreviations: IAP, inhibitor of apoptosis; BIR, baculovirus IAP repeat; HRP, horseradish peroxidase; PI, propidium iodide; DP, double positive; DN, double negative; SP, single positive ∗ Corresponding author. Tel.: +81-43-226-2181; fax: +81-43-226-2183. E-mail address: [email protected] (T. Tokuhisa).
The IAP genes were originally discovered in baculoviruses (Crook et al., 1993). Their homologues have been identified in other viruses, Drosophila, yeast, Caenorhabditis elegans and mammals, suggesting a common evolutional origin (Duckett et al., 1996, Deveraux and Reed, 1999). A mode of action of the IAP is believed to be by direct binding and inhibition of the key caspases and procaspases, primarily caspases 3, 7 and 9 (Deveraux et al., 1997; Roy et al., 1997; Talanian et al., 1997; Deveraux et al., 1998; Takahashi et al., 1998). The common feature of IAP family proteins is the presence of one to three BIR domains at its N-terminus and a RING finger domain at the C-terminus. The BIR domain is thought to be important for protein–protein interaction and is necessary and sufficient to prevent apoptosis. The RING finger domain is thought to be important for degradation of the protein by ubiqutination (Yang et al., 2000). Although many IAP proteins are expressed in lymphocytes and can inhibit apoptosis induced by a variety of triggers in vitro, a physiological role of IAP proteins in the development and maintenance of the immune system is not yet clear. TIAP, a murine homolog of human survivin, is one of the IAP family proteins (Ambrosini et al., 1997, Kobayashi et al., 1999). TIAP/m-survivin which has one BIR domain interacts with caspases 3, 7 and 9 inhibits their activity in vitro (Tamm et al., 1998; Kobayashi et al., 1999; Banks et al.,
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2000; O’Connor et al., 2000; Verdecia et al., 2000; Wright et al., 2000). It can also inhibit apoptosis caused by anti-Fas, Bax overexpression and anti-cancer drugs when overexpressed in vitro (Tamm et al., 1998). TIAP is expressed in growing tissues such as the thymus, testis, intestinal epithelium, and many tissues of embryos (Adida et al., 1998; Kobayashi et al., 1999). Its expression is regulated in cell cycle dependent manner. Expression of TIAP/m-survivin is induced at S to G2/M phase of the cell cycle (Li et al., 1998, Kobayashi et al., 1999). Furthermore, survivin interacts with microtubules of the mitotic spindle during mitosis (Li et al., 1998). Thus, TIAP/m-survivin may be involved in a regulation of apoptosis during cell proliferation. To study a role of TIAP/m-survivin in T cell development and proliferation, we examined expression of TIAP/m-survivin in subsets of normal thymocytes and generated transgenic mice which overexpress TIAP/m-survivin in thymocytes. Every subset of thymocytes examined expresses the large amount of TIAP/m-survivin. T cell development and susceptibility to various apoptotic stimuli were not altered in thymocytes by overexpression of TIAP/m-survivin. However, proliferation of thymocytes from transgenic mice after stimulation with PMA and ionomycin was augmented. We discuss a role of TIAP/m-survivin in T cell development and proliferation.
2. Materials and methods 2.1. Animals C57BL/6Crslc and (C57BL/6 × DBA/2)F1 mice were purchased from Japan SLC Co. (Hamamatsu, Japan). ICR mice were purchased from Japan CLEA Co. (Tokyo Japan). These mice were maintained under specific pathogen free conditions in the animal center of Chiba University School of Medicine. 2.2. Generation of transgenic mice The lck-TIAP transgene was constructed by inserting a BamH1 fragment of TIAP cDNA into the BamH1 site of the lck-hGH plasmid (Allen et al., 1992). Transgenic mice were produced by the method described by Hogan et al. (1986). Briefly, (C57BL/6 × DBA/2)F1 mice were used to obtain fertilized eggs, and the lck-TIAP gene was microinjected into a male pronucleus of fertilized eggs. Injected eggs were returned to oviducts of pseudopregnant females of ICR strain. The transgenes in genomic DNA isolated from the tail were detected by slot blot analysis using the human growth hormone intron DNA as a probe. Transgenic founders were backcrossed to C57BL/6 mice five times. 2.3. Antibodies Anti-CD4, -CD8, -CD3, -CD25, -CD44, and -Fas monoclonal antibodies were purchased from Pharmingen (San
Diego, CA). Rabbit anti-TIAP polyclonal antibodies were described before (Kobayashi et al., 1999). 2.4. FACS analysis Thymocytes and splenocytes were isolated from 4- to 8-week-old transgenic mice or control littermates. Cell suspensions were treated with ACK lysing buffer to lyse erythrocytes before staining. Single cell suspensions were prepared in staining medium and stained with monoclonal antibodies described above. Stained cells were analyzed by a FACSCalibur. 2.5. Northern blot analysis Total RNAs were extracted from thymocytes and splenocytes using the Trizol reagent (Gibco BRL). Northern blot analysis was done using a TIAP specific 351 bp probe as described previously (Kobayashi et al., 1999). 2.6. Western blot analysis Total proteins from thymocytes and splenocytes from transgenic mice or control littermates were obtained as described previously (Kobayashi et al., 1999). The amount of protein in cell lysates was determined using the Bio-Rad protein assay (Bio-Rad Laboratories). Fifteen micrograms of cell lysates were resolved by SDS-PAGE and transferred to a polyvinyl difluoride membrane (Immobilon-P, Millipore Corp., Bedford, MA). Blots were blocked in Blockace (Yukijirushi, Sapporo, Japan) for 1 h, washed four times with Tris-buffered saline with 0.2% Tween 20 (TBST) and incubated with anti-TIAP antibodies in TBST for 1 h at room temperature. The blots were then washed four times with TBST, incubated with affinity-purified donkey anti-rabbit antibodies conjugated with horseradish peroxidase (HRP, Amersham International, Arlington Heights, IL) for 1 h at room temperature, washed four times with TBST, and developed with enhanced chemiluminescence reagents (Amersham International). 2.7. Cell sorting and RT-PCR analysis Thymocytes from 4-week-old C57BL/6 mice were stained with FITC conjugated anti-CD4 and PE conjugated anti-CD8 monoclonal antibodies for 20 min at 4 ◦ C. After washing, the cells were resuspended in staining medium supplemented with PI. Stained cells were analyzed by FACS Vantage, and each subset of thymocytes was sorted. Total RNA was extracted from sorted cells using the Trizol reagent. RNAs were reverse-transcribed using Superscript and oligo dT in a final volume of 20 l, and 1 l of cDNAs was used for PCR. PCR reactions were incubated for 7 min at 94 ◦ C for one cycle and 1 min at 94 ◦ C, 1 min at 55 ◦ C, 1 min at 72 ◦ C for 30 cycles. PCR primers for the cDNA amplification were as
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follows: TIAP primers, 5 -ATGGGAGCTCCGGCGCT-3 and 5 -TTAGGCAGCCAGCTGCT-3 ; G3PDH primers, 5 -TGAAGGTCGGTGTGAACGGATTTGGC-3 and 5 CATGTAGGCCATGAGGTCCACCAC-3 . PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. 2.8. Immunohistochemistry Four-week-old C57BL/6 mice were perfused with a solution of 4% para-formaldehyde in 0.1 M phosphate buffer (pH 7.4). The thymus was dissected from mice and post-fixed with 4% para-formaldehyde for 12 h. The tissue was equilibrated with 20% sucrose and sectioned at 10 m on a cryostat. After quenching the activity of endogenous peroxidase with 3% H2 O2 in methanol, sections were stained with rabbit antibodies against TIAP. Biotinylated goat anti-rabbit antibodies (Nichirei, Tokyo, Japan) and streptABComplex/HRP (Dako, Carpinteria, CA) were used as the second- and third-phase reagents, respectively. Bound HRP activity was visualized using the DAB kit (Nichirei). Hematoxylin staining was done using a standard protocol.
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2.9. Cell culture and apoptosis assay Thymocytes were cultured in RPMI-1640 medium supplemented with 10% FCS. Apoptosis was induced by plate coated anti-CD3 (10 g/ml), anti-Fas (1 g/ml) and cyclohexamide (10 g/ml), dexamethazone (100 nM), or ␥-irradiation (0.5 or 1.0 Gy). Thymocytes (1 × 106 ) were cultured in 24-well plates with various reagents for 24 h and apoptotic cells were determined by FACS after annexin V and PI staining. 2.10. Proliferation assay CD4− CD8− DN thymocytes were enriched by depleting CD4+ CD8+ DP, CD4+ CD8− and CD4− CD8+ SP thymocytes from lck-TIAP transgenic mice and control littermates. Briefly, thymocytes were incubated with mixture of biotinylated monoclonal antibodies to CD4 and CD8. These cells were subsequently reacted with streptavidin coated immunomagnetic beads (Miltenyi Biotec, Gladbach, Germany). Labeled cells were separated by applying them in a magnetic field using a MACS system (Miltenyi Biotec).
Fig. 1. Expression of TIAP in thymocytes. (A) TIAP mRNA expression in subsets of thymocytes. The amount of TIAP mRNA in total RNAs from subsets of thymocytes was measured by the semiquantative RT-PCR; (B) Immunohistochemistry of normal mouse thymus stained with anti-TIAP polyclonal antibodies.
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Splenic T cells were also enriched in a similar manner by depleting non-T lineage cells with monoclonal antibodies to B220, TER119 and Mac-1 from spleen cells of lck-TIAP transgenic mice and control littermates. The resulting T cells contained <0.5% of B220+ cells. Thymocytes or splenic T cells (1 × 105 per well) were cultured with anti-CD3 (5 g/ml), PMA (50 ng/ml) and ionomycin (250 nM), or concanavalin A (1 g/ml) in a 96-well microplates in triplicates for 1–4 days. The cultures were pulsed with 1 Ci of [3 H]thymidine for 6 h, and the cells were harvested onto a glass filter. The [3 H]thymidine uptake was measured in a liquid scintillation counter.
3. Results 3.1. Expression of endogenous TIAP/m-survivin during T cell development Since TIAP is highly expressed in the thymus, we examined the expression during T cell development. Thymocytes and splenocytes were stained with anti-CD4 and -CD8,
and sorted according to their expression profiles. We have used semiquantitative RT-PCR analysis to detect the expression in total RNAs from those sorted cells. As shown in Fig. 1A, TIAP mRNA was detected in early T cell precursors (DN), CD4+ CD8+ (DP) and CD4+ CD8− or CD4− CD8+ (SP) thymocytes. Each fraction expressed almost the same amount of mRNA. The amount of TIAP decreased in splenic SP T cells when compared with that in thymic SP T cells. To determine the amount of TIAP protein, immunohistochemistry was performed using anti-TIAP polyclonal antibodies. TIAP was detected in thymocytes in both cortex and medulla (Fig. 1B). Some epithelial-like cells were also positive for TIAP. Furthermore, positive signal was detected in nucleus of some thymocytes whereas most thymocytes were stained in cytoplasm. 3.2. Generation of lck-TIAP transgenic mice In order to examine a function of TIAP/m-survivin in thymocytes, we generated transgenic mice carrying the TIAP cDNA under the control of the lck proximal promoter (lck-TIAP) (Fig. 2A). We established six transgenic
Fig. 2. Construction and expression of the lck-TIAP transgene. (A) Schematic diagram of the transgenic construct used to generate lck-TIAP mice; (B) Northern blot analysis of RNAs from the thymus and spleen of transgenic mice (#201, #242) and control littermates (WT); (C) Western blot analysis of proteins from the thymus and spleen of transgenic mice (#201, #242) and control littermates (WT).
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lines with various copy numbers. The amount of TIAP in RNAs and that in proteins from these transgenic mice were examined by Northern and Western blot analysis, respectively (Fig. 2B and C). Transgenic lines which expressed the highest (#201) and the second highest (#242) amounts of TIAP protein in thymocytes were expanded for further experiments. Those two transgenic mice carry approximately 20 and 10 copies of the exogenous gene, respectively. Expression of the transgene was further examined in these two lines. TIAP transgene message was also detectable in the
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spleen but not in the other non-lymphoid organs such as the liver and testis (data not shown). 3.3. T cell development in the thymus of lck-TIAP transgenic mice Subpopulation of T cells was examined in the thymus from 4- to 6-week-old lck-TIAP mice. Total cell number ((2.46±1.36)×108 ) in the thymus from lck-TIAP mice was not significantly different from that ((2.21 ± 0.83) × 108 )
Fig. 3. Flow cytometry analysis of thymocytes and splenocytes from lck-TIAP transgenic mice. (A) Thymocytes and splenocytes were stained with anti-CD4 and -CD8. Numbers in quadrants represent percentage of the total population. (B) Thymocytes were stained with anti-CD4, -CD8, -CD44 and -CD25. CD4− CD8− thymocytes were gated and further analyzed with expression of CD44 and CD25. Numbers in quadrants represent percentage of the total population.
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in control littermates. Thymocytes were stained with developmental surface markers and analyzed by FACS. In lck-TIAP mice, T cell subsets that expressed CD3 (data not shown), CD4 and CD8 (Fig. 3A), or CD44 and CD25 in CD4− 8− thymocytes (Fig. 3B) appeared essentially normal. When the thymus was histologically examined, architecture of cortex and medulla in the thymus from lck-TIAP mice was essentially the same as that from control littermates (data not shown). Size and cell number of the spleen and lymph nodes from lck-TIAP mice were also the same as those of control littermates. Therefore, development of T cells in the thymus is not perturbed by elevating levels of TIAP/m-survivin. 3.4. Apoptosis of thymocytes from lck-TIAP transgenic mice Thymocytes undergo apoptosis in response to many stimuli. Since TIAP/m-survivin inhibits activity of caspase 3, 7 and 9 in vitro (Tamm et al., 1998, Kobayashi et al., 1999, O’Connor et al., 2000), we examined whether overexpression of TIAP could prevent any form of thymocyte apoptosis or not. Thymocytes were cultured with various stimuli and apoptosis was evaluated by annexin V and PI staining. There was no significant difference in thymocyte apoptosis after anti-CD3 or -Fas stimulation, glucocorticoid treatment, or ␥-irradiation between transgenic and control littermates (Fig. 4). Thus, overexpression of TIAP in developing thymocytes did not affect a susceptibility to various apoptotic stimuli examined.
3.5. Proliferative response of thymocytes from lck-TIAP transgenic mice Proliferative responses of thymocytes from lck-TIAP mice to various mitogens were examined. Transgenic thymocytes responded three times more than control thymocytes did after stimulation with PMA and ionomycin (Fig. 5A). Proliferative responses of thymocyte subsets were further examined. DN thymocytes and DP + SP thymocytes from lck-TIAP mice proliferated more than those from control mice (Fig. 5B). Proliferative responses of thymocytes reached maximum between 48 and 72 h after stimulation in both lck-TIAP and control littermates (Fig. 5C). The response in transgenic mice returned almost the same level as control littermates 96 h after stimulation. However, upon stimulation with anti-CD3 antibody (Fig. 5A–C) or with concanavalin A (data not shown), no difference was observed in proliferation of thymocytes between transgenic and control littermates at any time points after stimulation. Proliferative responses of splenic T cells from lck-TIAP mice stimulated with PMA and ionomycin was examined. The responses of splenic T cells from lck-TIAP mice stimulated with PMA and ionomycin or with anti-CD3 were almost the same as those from control littermates (Fig. 6).
4. Discussion In order to elucidate a role of TIAP/m-survivin in T cell development and activation, we have established lck-TIAP
Fig. 4. Apoptosis of thymocytes from lck-TIAP transgenic mice. Thymocytes from lck-TIAP mice (closed bars) and control littermates (open bars) were cultured with various stimuli indicated for 24 h. Viability of stimulated cells was measured by annexin V and PI staining. CHX; cyclohexamide, DEX; dexamethasone.
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Fig. 5. Proliferation of thymocytes from lck-TIAP transgenic mice. (A) Thymocytes were stimulated with anti-CD3 or PMA and ionomycin for 24 h. Closed bars; lck-TIAP mice, open bars; control littermates. The results from three independent experiments were shown. (B) Subsets of thymocytes were stimulated with PMA and ionomycin for 24 h. Closed bars; lck-TIAP mice, open bars; control littermates. (C) Kinetics of thymocyte proliferation induced by anti-CD3 or PMA and ionomycin. Closed circle; lck-TIAP mice, open circle; control littermates. The results are expressed as mean cpm ± S.D. of triplicate cultures.
transgenic mice. Proliferation of transgenic thymocytes in response to PMA and ionomycin was three-fold higher than that of control thymocytes. TIAP/m-survivin displays cell cycle specific expression that is mediated by G1 transcriptional repressor elements in its promoter region (Li and Altieri, 1999, Otaki et al., 2000). Recently, a possible link between cell cycle progression and apoptosis has emerged. Treatment of cells with survivin antisense results in increased apoptosis and inhibition of cell proliferation
(Ambrosini et al., 1998). Furthermore, survivin has been shown to associate with mitotic spindles and may play a critical role in dual control of mitotic spindle checkpoints and apoptosis (Li et al., 1998; Li et al., 1999). Thus, it is possible that overexpression of TIAP/m-survivin accelerates cell cycle progression by affecting mitotic spindle checkpoints. Alternatively, apoptosis during proliferation is rescued and more cells enter cell cycle resulting in increased proliferation. Interestingly, thymocyte proliferation in response to
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Fig. 6. Proliferation of splenic T cells from lck-TIAP transgenic mice. Splenic T cells were stimulated with anti-CD3 or PMA and ionomycin for 24 h. Closed bars; lck-TIAP mice, open bars; control littermates. The results are expressed as mean cpm ± S.D. of triplicate cultures.
anti-CD3 stimulation was almost the same between transgenic and control littermates. In contrast, stimulation with PMA and ionomycin bypasses the TCR signaling pathway and activates broad population of thymocytes. In case of anti-CD3 stimulation, responding cells are already competent to enter cell cycle and probably that’s why we could not see the difference in proliferation. PMA and ionomycin force many cells to enter the cell cycle. Overexpression of TIAP/m-survivin may elevate anti-apoptotic threshold during cell division and may rescue incompetent cells to enter cell cycle. T cell development in lck-TIAP mice appeared normal. Numbers of thymocytes and T cell subpopulations in lck-TIAP mice were normal levels, suggesting that excess amounts of TIAP do not affect T cell development. T cell apoptosis was also examined in lck-TIAP mice. Apoptotic responses of transgenic thymocytes to anti-Fas stimulation, anti-CD3 stimulation, dexamethasone treatment, or ␥-irradiation were not inhibited. Previously we and others reported that overexpression of TIAP/m-survivin in cultured cell lines inhibited various forms of apoptosis such as anti-Fas stimulation, Bax overexpression or growth factor withdrawal (Ambrosini et al., 1997; Tamm et al., 1998; Kobayashi et al., 1999). However, the effect of TIAP/m-survivin to prevent apoptosis was not so strong as that of XIAP or Bcl-2 in cultured cell lines. TIAP/m-survivin could bind to caspases 3 and 7 in vitro but its affinity to bind these caspases is not so strong as that of XIAP (Tamm et al., 1998). In lck-TIAP transgenic thymocytes, levels of overexpressed TIAP proteins are sufficient to influence T cell proliferation to PMA and ionomycin
stimulation but may not be enough to inhibit apoptosis. Thus, the regulation of apoptosis may not be the primary role of TIAP/m-survivin in thymocyte development. Recently, BIR domain containing proteins with diverse structure were identified from yeast to mammals. In yeast and C. elegans, structurally related proteins to TIAP/m-survivin named BIR1, or bir-1 were identified (Fraser et al., 1999; Uren et al., 1999). Originally a function of IAP protein family was implicated to prevent apoptosis. The identification of BIR containing proteins in both Saccharomyces cervisiae and Schizosaccharomyces pombe with no identified apoptotic program and no caspase encoded gene raise a possibility that BIR containing proteins play roles in other cellular processes than apoptosis. Indeed, loss of BIR1 function in yeast strains induces cell death at the early cell division and blocked at the metaphase/anaphase transition because of an inability to elongate their mitotic spindle (Uren et al., 1999). In C. elegans, ablation of bir-1 resulted in embryonic lethality and the embryos were multinucleated and failed to form more than two cells (Fraser et al., 1999). These phenotypes resulted from an inability of bir-1 deficient embryos to complete cytokinesis. Furthermore, overexpression of bir-1 in C. elegans did not affect cell death in vivo whereas overexpression of ced-9, the nematode Bcl-2 homologue, markedly suppressed cell death during development (Fraser et al., 1999). The survivin-deficient mice are also embryonic lethal (Uren et al., 2000). Taken together, primary function of some IAP family proteins including TIAP/m-survivin or BIR1 may be a control of cell division rather than inhibition of apoptosis. During a process of evolution, coordinated regulation
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of cell proliferation, differentiation and apoptosis become essential for higher organisms and IAP family proteins may acquire pleiotropic function. Apoptosis plays a critical role in T cell development and activation. In the thymus, unsuccessfully rearranged immature T cells are eliminated by apoptosis. Negative selection takes place at DP stage and self reactive T cells are eliminated by apoptosis. T cells that have little affinity to MHC molecules also die by apoptosis. However, these events are not directly linked to cell proliferation. In periphery, naive T cells are activated by antigen and become effector cells. Some of these T cells proliferate and become memory cells while others die (activation induced cell death). In this process, cell division and apoptosis are closely linked each other. It is interesting to know whether TIAP/m-survivin regulates this process. Survivin expression is reported to be high in various tumors such as lymphoma, neuroblastoma, and colon adenocarcinoma (LaCasse et al., 1998). No obvious chromosomal translocation is associated with their genomic loci in humans. High level of the expression in cancer may reflect a result of accelerated cell proliferation of tumor cells. At this moment, no spontaneous tumor developed in lck-TIAP mice up to 24-month observation. Thus, TIAP/m-survivin itself may not function as an oncogene to initiate carcinogenesis but may play a role in disease progression. Identification of functionally cooperative protein with TIAP/m-survivin will further clarify a mechanism of cancer initiation and progression.
Acknowledgements We would like to extend our thanks to H. Satake for her technical assistance, and K. Ujiie for her secretarial assistance. This work was supported in part by Grant-in-Aid for Scientific Research on Priority areas “Cancer”, from the Ministry of Education, Science, Sports and Culture of Japan.
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