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Originally published online as doi:10.1189/jlb.1202607 on May 22, 2003

Published online before print May 22, 2003
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(Journal of Leukocyte Biology. 2003;73:850-861.)
© 2003 by Society for Leukocyte Biology

p53 deficiency and defective mitotic checkpoint in proliferating T lymphocytes increase chromosomal instability through aberrant exit from mitotic arrest

Kwan-Hyuck Baek*, Hyun-Jin Shin*,{dagger}, Jae-Kwang Yoo*, Jae-Ho Cho*, Yo-Han Choi*, Young-Chul Sung*, Frank McKeon{ddagger} and Chang-Woo Lee*,{dagger}

* National Research Laboratory of DNA Medicine, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Korea;
{ddagger} Department of Cell Biology, Harvard Medical School, Boston, Massachusetts; and
{dagger} Research Institute, National Cancer Center, Ilsan-gu, Goyang, Korea

Correspondence: Chang-Woo Lee, Research Institute, National Cancer Center, Ilsan-gu, Goyang, Gyeonggi-do 411-764, Korea. E-mail: cwlee{at}ncc.re.kr


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ABSTRACT
 
During the proliferation of T cells for successful immune responses against pathogens, the fine regulation of cell cycle is important to the maintenance of T cell homeostasis and the prevention of lymphoproliferative disorders. However, it remains to be elucidated how the cell cycle is controlled at the mitotic phase in proliferating T cells. Here, we show that during the proliferation of primary T cells, the disruption of the mitotic spindle leads to cell-cycle arrest at mitosis and that prolonged mitotic arrest results in not only apoptosis but also the form of chromosomal instability observed in human cancers. It is interesting that in response to spindle damage, the phosphorylation of BubR1, a mitotic checkpoint kinase, was significantly induced in proliferating T cells, and the expression of the dominant-negative mutant of BubR1 compromised mitotic arrest and subsequent apoptosis and thus led to the augmentation of polyploidy formation. We also show that in response to prolonged spindle damage, the expression of p53 but not of p73 was significantly induced. In addition, following sustained mitotic arrest, p53-deficient T cells were found to be more susceptible to polyploidy formation than the wild type. These results suggest that during flourishing immune response, mitotic checkpoint and p53 play important roles in the prevention of chromosomal instability and in the maintenance of the genomic integrity of proliferating T cells.

Key Words: cell cycle • proliferation • apoptosis • aneuploid • lymphoma


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INTRODUCTION
 
Under antigen-specific stimulation, T cells, especially memory T cells, are programmed to proliferate at a high division rate to elicit a successful immune response against foreign pathogens [1 2 3 4 5 ]. However, the uncontrolled proliferation of T cells results in lymphoproliferative disorders, such as autoimmune diseases and lymphomas [6 , 7 ]. Thus, during such T cell proliferation, a fine regulation of the cell cycle is required to prevent these disorders and to maintain T cell homeostasis. Moreover, on considering the high division rate and the short cell-cycle period, it is possible that the cell-cycle control of T cells is achieved by using mechanisms that are somewhat distinct from those used by other cells. For this reason, a study of whole cell-cycle regulation during T cell proliferation is important, not only to be able to comprehend the development of proper immune responses and the maintenance of T cell homeostasis but also to allow the elucidation of the pathogenic pathways implicated in many lymphoproliferative disorders.

To date, several extensive studies have been performed on cell-cycle regulation and checkpoint controls in T cells. However, the majority of these studies have focused on the molecular mechanisms implicated in early cell-cycle regulation, such as activation-induced cell death (AICD) or the G1/S and G2 checkpoints. AICD plays a critical role in the maintenance of the homeostasis of the T cell population after the establishment of an immune response against foreign antigens and the development of T cells in the thymus [8 ]. In addition, the G1/S and G2 checkpoints are important in terms of ensuring the genomic integrity of T cells affected by environmental stresses, such as UV irradiation or oxidative damage [9 , 10 ]. Although these cell-cycle and checkpoint regulations during the early cell cycle are important in the prevention of disorders caused by uncontrolled T cell proliferation after antigenic stimulation or the transmission of altered genomic information into daughter cells, they cannot be the whole story in terms of T cell-related diseases. Thus, more studies on cell-cycle regulators other than AICD and checkpoint controls during the early phase of T cell proliferation are required to fully elucidate regulatory mechanisms of T cell cell-cycle control and to understand the pathogenesis of T cell disorders.

During mitosis, the duplicated chromosome pairs are accurately split and subsequently segregated into daughter cells. The mitotic checkpoint ensures this faithful chromosomal segregation by delaying the onset of anaphase until both kinetochores on each duplicated chromosome pair have properly attached to the spindle microtubule and are under tension [11 12 13 14 15 16 ]. For the successful execution of this checkpoint function, several molecular components of mitotic checkpoint machinery are known to be necessary; these include Mps1, Mad1–3, Bub1, Bub3, BubR1 (a homologue of Mad3 and Bub1), and CENP-E (a microtubule-dependent motor protein). These checkpoint proteins are preferentially localized to the kinetochores of unaligned chromosomes and contribute to the production of a diffusible "wait anaphase" signal. This signal delays anaphase by inhibiting the anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase required for the degradation of securin and the subsequent activation of separase for sister chromatid separation [17 , 18 ]. Defects in the mitotic checkpoint lead to the premature separation of sister chromatids, resulting in the loss or gain of chromosomes in daughter cells and subsequent chromosomal instability, which is implicated in the pathogenesis of human cancers [19 20 21 ]. Therefore, an impaired mitotic checkpoint, particularly in rapidly proliferating cells such as activated T cells, seems to exert a more deleterious effect on cellular and normal tissue homeostasis than errors in other checkpoints, such as the DNA damage-dependent G1 checkpoint.

For interest, it was recently reported that the expression of the oncoprotein Tax [encoded by human T cell leukemia virus type-1 (HTLV-1), a causative agent of adult human T cell lymphoma/leukemia (ATLL)] abrogates the mitotic checkpoint by binding with the human homologue of the yeast mitotic checkpoint Mad1 protein in human cervical cancer cells [21 ]. This observation raises the possibility that failures of mitotic checkpoint regulation are implicated not only in the tumorigenesis of T cells by viral infection but also in the pathogenesis of nonviral lymphoproliferative disorders, such as lymphomas. In agreement with this hypothesis, malignant T cells from cutaneous T cell lymphoma (CTCL) patients frequently show an aneuploid phenotype [23 , 24 ]. In addition, mutations of mitotic checkpoint genes, such as hBUB1 and hBUBR1, have been detected at high frequencies in many ATLL cases [25 ]. However, in spite of the importance of the mitotic checkpoint in the maintenance of genomic integrity during cell division and the possible linkage between impaired mitotic checkpoint and lymphoproliferative diseases, few studies have been conducted on checkpoint regulation in proliferating T cells.

In this study, as an initial step to the elucidation of the molecular mechanisms involved in mitotic checkpoint regulation during T cell proliferation, we monitored the fates of activated primary T cells after spindle damage. Of interest, we found that after prolonged mitotic arrest, a significant portion of the activated T cell population escaped arrest without undergoing apoptosis and subsequently formed polyploid cells with DNA contents exceeding 4N. By expressing a dominant-negative mutant, we found that BubR1, a mitotic checkpoint kinase, is required to elicit the checkpoint response to spindle damage during T cell proliferation. In addition, we demonstrate that after prolonged spindle damage, p53 and not p73 plays an important role in monitoring chromosomal instability in primary T cells.


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MATERIALS AND METHODS
 
Animals
Six- to 12-week-old female mice were used for the isolation of primary T cells. Female BALB/c mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). OT-II mice of the 425-2 line [26 ] were kindly provided by Dr. William R. Heath (The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia). Mice heterozygous for p53 deficiency (p53+/-) were kindly provided by Dr. Han W. Lee (SungKyunKwan University, Korea), and normal age- and sex-matched controls, C57BL/6, were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice homozygous for p53 deficiency (p53-/-) were obtained from a cross between p53+/- mice. All mice were bred and maintained in specific pathogen-free conditions.

Reagents and antibodies
General reagents used were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. Fluorescein isothiocyanate (FITC) anti-Thy-1.2, phycoerythrin (PE) anti-V{alpha}2 T cell receptor (TCR), FITC anti-CD4, FITC anti-CD11b, and FITC antiactive caspase-3 monoclonal antibodies (mAb) were obtained from BD PharMingen (San Diego, CA). Anti-p73, anti-c-Myc, anti-p53, anticyclin B, anticyclin A, anti-p27, and anti-p21 Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell culture
Primary T cells purified from mice were cultured in RPMI 1640 (Jeil Biotech, Korea), supplemented with 10% fetal calf serum (Hyclone Laboratories, Logan, UT), 2 mM L-glutamine, 50 µM ß-mercaptoethanol, and antibiotics. Jurkat-TetOn (Clontech, Palo Alto, CA) and JM36 cell lines were maintained in RPMI supplemented with tetracycline-free 10% fetal bovine serum (Hyclone Laboratories).

Preparation of murine primary T cells
Primary T cells of wild-type and p53-/- mice were purified from the spleen or lymph nodes as described previously [27 , 28 ]. Briefly, cell suspensions were treated with J11d (anti-human serum albumin) and M5/114.15.2 [anti-major histocompatibility complex (MHC)-II] for 30 min on ice, centrifuged, and then depleted by treatment with rabbit complement for 40 min at 37°C. Remaining cells were then treated with anti-B220 and anti-MHC-II microbeads (Miltenyi Biotech, Auburn, CA) and were purified by negative selection using the magnetic cell sorter separator system. Purified cell populations were 90–95% Thy-1.2+. OT-II cells were prepared as described previously [29 , 30 ]. Briefly, lymph nodes were removed from OT-II mice, and single-cell suspensions were treated with 3.168 (anti-CD8) and J11d and then complement as above. Purified OT-II cell populations were 70–75% CD4+V{alpha}2+. Remaining 25–30% of cell populations were CD11b+, indicating that these populations were antigen-presenting cells such as dendritic cells and macrophages. Purity was assessed by flow cytometric analysis on a FACScan flow cytometer (BD Biosciences, Heidelberg, Germany) after staining with FITC anti-Thy-1.2, PE anti-V{alpha}2 TCR, FITC anti-CD4, or FITC anti-CD11b mAb.

Generation of inducible cell lines
To generate tetracycline-responsive Jurkat cell lines expressing a dominant-negative mutant of human BubR1 (hBubR1), the amplified Myc epitope-tagged N-terminal domain encoding amino acids 1–344 was cloned into the pTRE2-hygro (Clontech) to create pTRE2-Myc-N-terminal hBubR1 (N-hBubR1). Thereafter, Jurkat-TetOn cells were transfected with this construct by electroporation. To isolate the individual colonies expressing N-hBubR1, transfected cells were serially diluted and added to each well of 96-well plates, selected in 200 µg/ml hygromycin B (Roche, Mannheim, Germany), expanded, and then screened by immunoblotting for expression of N-hBubR1 in the presence of 0.1 µg/ml doxycycline. Three independent cell lines, JM9, JM36, and JM106, were obtained and characterized for further studies. Data using the JM36 cell line were presented here.

Cell cycle and cell death analysis
Approximately 106 primary T cells were plated in 12-well plates and stimulated with the combination of phorbol 12-myristate 13-acetate (PMA; 0.5 µM) plus ionomycin (0.5 µg/ml) for 24 h. In the case of CD4+ T cells from OT-II mice, 106 cells of mixed populations of T cells and antigen-presenting cells were plated in 96-well plates and stimulated with 1 µM chicken ovalbumin (OVA) peptides (OVA323–339) for 24 h. Stimulated T cells were then cultured in the absence or presence of 70 ng/ml nocodazole, 0.5 µM taxol, or 5 nM actinomycin D. At various time points, cells were harvested, fixed in 70% ethanol, washed in phosphate-buffered saline (PBS), and stained with propidium iodide (PI; 40 µg/ml final concentration) in the presence of 50 µg/ml RNase A for 30 min at room temperature, followed by the analysis of a cell-cycle profile on FACScan (Becton Dickinson, San Jose, CA). To examine the effect of N-hBubR1 on the mitotic checkpoint, JM36 cells and parental cells (Jurkat-TetOn) were preincubated with 0.1 µg/ml doxycycline for 48 h and then treated with 0.1 µg/ml nocodazole. Then, cells were harvested, treated, and analyzed as described above. To verify the apoptotic cell death, 106 primary T cells were activated with the combination of PMA (0.5 µM) plus ionomycin (0.5 µg/ml) for 24 h and were then cultured in the presence of nocodazole (70 ng/ml). At various time points, cells were harvested, resuspended in Cytofix/CytopermTM solution (BD PharMingen), washed with Perm/WashTM buffer (BD PharMingen), stained with FITC antiactive caspase-3 mAb plus PI (10 µg/ml final concentration), and then subjected to flow cytometry analysis on FACscan according to the manufacturer’s instructions. Data were presented by using Cell Quest software (Becton Dickinson).

Immunoblotting assay
For immunoblot assay, primary T cells (2x107 cells/sample) were stimulated with the combination of PMA (0.5 µM) plus ionomycin (0.5 µg/ml) for 24 h and were then treated with 70 ng/ml nocodazole. Cells were harvested at various time points and used for the preparation of whole-cell lysates. To address the expression of N-hBubR1 in JM36 cell lines, 107 cells of each JM36 and Jurkat-TetOn cell line as a control were treated with 0.1 µg/ml doxycycline for 48 h and then harvested. Whole-cell lysates were prepared by the addition of 500 µl lysis buffer [50 mM HEPES (pH 7.2), 250 mM NaCl, 2 mM EDTA, 0.1% Nonidet P-40, 1 mM dithiothreitol, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 50 µg/ml phenylmethylsulfonyl fluoride]. For immunoblot analysis, equal amounts of protein (quantitated by a Biorad assay) of each sample were loaded on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and analyzed by anti-BubR1 [31 ], anti-p73, anti-c-Myc, anti-p53, antiactin (Sigma Chemical Co.), anticyclin B, anticyclin A, anti-p27, or anti-p21 antibody.

Immunofluorescence
For immunofluorescence analysis, Jurkat-TetOn and JM36 cells were grown on 13 mm round-glass coverslips coated with 1 µg/ml collagen (Iwaki Glass, Tokyo, Japan). Thereafter, cells were fixed with PBS containing 5% formaldehyde for 10 min at room temperature or methanol for 10 min at -20°C and then were washed four times with 0.5 ml PBS. In some experiments, cells were extracted for 1 min at room temperature. Fixed cells were washed and permeabilized in PBS containing 0.1% Triton X-100 and were then incubated in primary antibody at room temperature for 2 h. Subsequently, cells were washed three times and further incubated in secondary antibody, goat anti-mouse immunoglobulin G-conjugated rhodamine (Santa Cruz Biotechnology) for 1 h. Cells were washed and exposed to Hoechst dye to visualize the DNA and were viewed under the fluorescence microscope (Zeiss photomicroscope, Carl Zeiss, Thornwood, NY).


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RESULTS
 
Chromosomal instability of activated primary T cells increases after prolonged mitotic arrest in response to spindle damage
To investigate the fates of activated T cells after spindle disruption, primary T cells from spleen or lymph nodes of BALB/c mice were purified, stimulated with TCR mimetics (PMA plus ionomycin) for 24 h, and then cultured in the absence or presence of nocodazole or taxol, inhibitors for microtubule assembly or disassembly, respectively. As a control, activated T cells were cultured in the presence of actinomycin D, which has been reported to arrest them at G1 [32 ]. At various time points, cells were harvested, fixed in ethanol (EtOH), stained with PI, and subjected to the cell-cycle analysis by flow cytometry (Fig. 1A ). Splenic and lymph node T cells behave in a similar manner after exposure to these drugs (data not shown). Thus, we present the data from splenic T cells here. In control culture, treated with medium only, T cell populations in S and G2/M phases increased up to 32 h. Thereafter, as reported previously [33 , 34 ], the S and G2/M phase cell populations began to decline, and the majority of cells accumulated in the G1 phase as a result of activation-induced cell arrest. In addition, as previously observed [32 ], most of the activated T cells cultured in the presence of actinomycin D were arrested at G1 after 16 h and then remained in this phase. Of interest, after 16 h of treatment with nocodazole or taxol, most of T cells accumulated in mitosis, indicating that in response to spindle damage, the mitotic checkpoint had been activated, as has been observed for non-T cells such as human cervical carcinoma cells. In addition, the maintenance of spindle damage resulted in a dramatic increase in the cell population with a DNA content of less than 2N (Fig. 1A and 1B) . To verify that these subdiploid cells are the result of apoptotic cell death, we examined caspase-3 activation during prolonged mitotic arrest. As expected, the activation of caspase-3 was dramatically induced during incubation with nocodazole, and the majority of the subdiploid cells was found to harbor active caspase-3 (Fig. 1C) . These results demonstrate that following prolonged spindle damage, the majority of activated T cells undergoes apoptotic cell death, as is the case for non-T cells. However, it was also noted that T cells with DNA contents exceeding 4N also increased significantly after sustained mitotic arrest (Fig. 1A and 1B) . In addition, we observed that these polyploid T cells were large and multinucleated, consistent with an 8N DNA content and with having left mitosis without completing chromosome segregation (data not shown). Surprisingly, the proportion of polyploid T cells accounted for 20% of the cell population. Moreover, this increase in the chromosomal instability of primary T cells appeared to be higher than that observed in other cell lines, where the majority of cells underwent apoptosis with no apparent re-entry from mitosis to the S phase (data not shown). These observations suggest that activated primary T cells are more susceptible to polyploidy after prolonged spindle damage, thus raising the possibility that mitotic checkpoint regulation in T cells may be achieved by a somewhat distinct mechanism from those of non-T cells. This possibility, however, should be further addressed, as such an investigation might explain how T cells are able to proliferate with a rapid turnover rate and short cell-cycle period during a flourishing immune response.



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  		Figure 1. Increased chromosomal instability of proliferating primary T cells following prolonged spindle damage. (A) Primary T cells were obtained from the spleen of BALB/c mice and stimulated with a combination of PMA (0.5 µM) plus ionomycin (0.5 µg/ml) for 24 h. The cells were then cultured in the absence or presence of nocodazole (70 ng/ml), taxol (0.5 µM), or actinomycin D (Act. D; 5 nM). At the time indicated on the right, the cells were harvested, fixed with EtOH, stained with PI, and analyzed by flow cytometry to determine their DNA contents. (B) Relative percentages of the cell populations undergoing apoptosis (Upper) and with a polyploid phenotype (>4N; Lower). (C) Primary T cells were treated as described in Panel A and then harvested at the time indicated. They were then fixed, permeabilized, stained with a combination of FITC-conjugated mAb against active caspase-3 and PI (10 µg/ml), as described in Materials and Methods, and analyzed by flow cytometry.

Although we observed the activation of the mitotic checkpoint in T cells stimulated with TCR mimetics, it was not clear as to whether those stimulated by specific antigens presented on antigen-presenting cells would also undergo the same checkpoint regulation during their proliferation. Therefore, we investigated whether the mitotic checkpoint also could be activated in T cells stimulated with specific antigens in the same manner as those treated with PMA plus ionomycin. As mentioned above, no apparent difference exists between splenic and lymph node T cells in terms of their response to spindle damage, and therefore, we used primary T cells from the lymph nodes for further experimentation. OVA-specific CD4+ T cells were purified from the lymph nodes of OT-II (CD4) TCR transgenic mice by depleting B cells and CD8+ T cells and were then stimulated with specific OVA peptides recognized by OT-II CD4+ T cells (OVA323–339) in the presence of autologous antigen-presenting cells for 24 h. Thereafter, the cells were cultured in the absence or presence of nocodazole or actinomycin D as a control (Fig. 2 ). In cells cultured in medium only, cell populations in the S and G2/M phases slightly increased after 16 h and then began to decrease. In addition, the sub-G1 cell population, indicative of AICD, increased after 64 h on medium (Fig. 2A and 2B) . In the case of cells cultured in the presence of actinomycin D, most were arrested at G1 phase at 16 h and then underwent apoptosis over the following 48 h. Of note, in activated T cells cultured in the presence of nocodazole, cells gradually accumulated in mitosis with time. In addition, the apoptotic subdiploid cell population (<2N) began to increase after 32 h, and this continued for a further 32 h. Over the same period, the polyploid cell population (>4N) appeared after 32 h, further increased until 48 h, and then slightly decreased after 64 h, indicating that proliferating T cells stimulated with specific antigens are also under mitotic checkpoint surveillance, as is the case of those stimulated with TCR mimetics. However, although T cells stimulated with specific antigens were found to behave in a manner similar to those stimulated with TCR mimetics, the mitotic arrest of T cells under antigenic stimulation seems to occur more slowly than those stimulated by TCR mimetics (Figs. 1A and 2A) . This discrepancy is presumably attributable to the different stimulants, i.e., in terms of their abilities to activate T cells, because in control cultures treated with medium only, T cells stimulated with OVA peptides showed retarded activation compared with those stimulated with TCR mimetics. Alternatively, differences in the interphase microtubule damage checkpoint may also contribute to this discrepancy under our experimental condition, using specific antigen as a stimulant [35 , 36 ]. Moreover, the antigen-presenting cells, added for the presentation of antigens to T cells, may also have contributed to the apparent retardation of T cell accumulation at mitosis, as the percentage of antigen-presenting cells in purified lymph node cells was ~25% of the whole cell population (data not shown).



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  		Figure 2. Increased chromosomal instability of OVA-specific T cells stimulated with specific antigen after prolonged spindle damage. (A) CD4+ T cells and autologous antigen-presenting cells were purified from the lymph nodes of OT-II mice by removing B cells and CD8+ T cells, as described in Materials and Methods. The resultant purified cells were stimulated with OVA peptide (OVA323–339) for 24 h and then cultured in the absence or presence of nocodazole (70 ng/ml) or actinomycin D (Act. D; 5 nM). At the time indicated on the right, cells were harvested, fixed, stained with PI, and analyzed by flow cytometry to determine their DNA contents. (B) Relative percentages of the cell population undergoing apoptosis (Upper) and with the polyploid phenotype (>4N; Lower).

Taken together, these results imply that the mitotic checkpoint plays a critical role in the proper segregation of chromosomes during the proliferation of primary T cells, whether triggered by antigenic stimulation or TCR mimetics, and that this leads to the maintenance of the genomic integrity of the rapidly proliferating T cells during immune response.

The phosphorylation of BubR1 and the expression of p53 are significantly induced in primary T cells in response to prolonged spindle damage
Next, to identify the cellular components involved in the mitotic checkpoint in proliferating T cells, we monitored regulatory proteins, known to participate in cell-cycle control and the mitotic checkpoint, in T cells after nocodazole treatment. Primary T cells from BALB/c mice were treated as described in Figure 1 and then immunoblotted (Fig. 3 ). It has been previously reported that the phosphorylation of some kinetochore proteins, such as BubR1, may be used as a marker of mitotic checkpoint activation and be of importance in terms of their checkpoint functionalities [37 38 39 ]. Therefore, we investigated the involvement of BubR1 protein, a mitotic checkpoint kinase, in the regulation of the mitotic checkpoint in proliferating T cells. In control cultures, the expression of mBubR1 protein was not detected in resting T cells, but it increased significantly in proliferating T cells some 16 h after activation and then decreased as the cells were arrested in the G1 phase (Figs. 1A and 3) . These results suggest that mBubR1 is required during the proliferation of activated T cells. In parallel with this finding, cells cultured in the presence of actinomycin D showed a slightly lower level of mBubR1 expression, presumably because the majority of cells were in G1 arrest. Of note, although mBubR1 was detected in control cells as a single band of ~120 kDa, a new form of mBubR1 with a retarded mobility indicative of phosphorylation was detected in cells treated with nocodazole. These data further support the notion that in response to spindle damage, BubR1 is required for mitotic checkpoint activation in proliferating primary T cells.



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  		Figure 3. Induction of p53 and the phosphorylation of BubR1/Mad3 in response to prolonged spindle damage. Primary T cells were stimulated and treated as described in Figure 1A . At the times indicated, cells were harvested and then subjected to immunoblot analysis with antimurine BubR1 (mBubR1), anti-p53, anti-p21, anti-p73, anti-p27, anti-Bcl2, and antiactin antibodies. The polypeptide corresponding to each antibody is indicated on the right.

We then monitored the expression patterns of regulatory proteins reported to be important for T cell cell-cycle regulation during the activation and proliferation triggered by antigenic stimulation. In particular, it was reported that p53 protein, a tumor suppressor, plays a pivotal role not only in the maintenance of genomic stability in response to genotoxic stresses but also in the prevention of chromosomal instability by halting the cell cycle in a pseudo-G1 phase after prolonged mitotic arrest [40 41 42 ]. However, it was recently reported that p73 but not p53 executes an important function in activation-induced G1 arrest and the subsequent apoptosis of T cells stimulated by the conjugation of TCR with specific antigens presented on antigen-presenting cells [43 ]. Therefore, we also investigated which of the p53 family participates in the regulation of the mitotic checkpoint in primary T cells. Consistent with the findings of a previous report [44 ], p27Kip1, an inhibitor of cyclin-dependent kinase, was detected in resting T cells but not in proliferating T cells cultured in medium only. In addition, we did not detect p27Kip1 induction as a result of spindle or DNA damage, indicating that this cell-cycle inhibitor does not participate in the mitotic and G1 arrests induced by nocodazole or actinomycin D, respectively. However, p53 and p21Waf1/Cip1 were strongly induced in T cells treated with actinomycin D, and these cells were predominantly arrested in the G1 phase of the cell cycle (Figs. 1A and 3) . These results are consistent with the findings of previous studies, i.e., that p53 halts the cell cycle to provide time to repair DNA damage in a p21Waf1/Cip1-dependent manner [42 ]. Of importance, in the nocodazole-treated cells, the expression of p53 began to increase at 16 h and peaked at approximately 32 h. This pattern is consistent with the gradual, observed increases of polyploid (<4N) and sub-G1 (<2N) cell populations (Figs. 1A and 3) . However, although the expression of p73 was strongly induced initially and then remained relatively constant in control cells, as previously reported [43 ], p73 levels were significantly attenuated in cells treated with nocodazole. These results suggest that in response to prolonged spindle damage, p53 and not p73 plays an important role in arresting activated T cells in mitosis.

Dominant-negative mutant of BubR1 compromises mitotic arrest and increases the chromosome instability of T cells
We observed that the phosphorylation of BubR1 is significantly induced in response to spindle damage (Fig. 3) . Thus, to further address the function of BubR1 in the T cell mitotic checkpoint, we generated a mutant hBubR1 with a dominant-negative phenotype. hBubR1, a Bub1-related protein kinase, has three functional domains: an N-terminal homology domain, a Bub3-binding domain, and a C-terminal domain corresponding to the catalytic domain of protein kinase [37 ]. As the N-terminal homology domain is highly conserved in all Bub1- and Mad3-related proteins and is functionally essential, the N-terminal domain seems to mediate interactions with other components of the mitotic checkpoint [45 , 46 ]. Thus, we hypothesized that if BubR1 is required for mitotic checkpoint function in proliferating T cells, a mutant BubR1 containing only an N-terminal homology domain might behave as a dominant-negative mutant in proliferating T cells. To confirm this hypothesis, we established a Jurkat T cell line, JM36, expressing a Myc epitope-tagged N-hBubR1 (amino acids 1–344) under the control of a tetracycline-responsive promoter (Fig. 4A and 4B ). However, it has also been reported that a mutant hBubR1, lacking the Bub3-binding domain, fails to localize at kinetochores in rodent cells, although the ectopic expression of hBub3 is required for the kinetochore localization of wild-type hBubR1 in these cells [37 ]. In addition, the kinetochore localization of a mutant mitotic checkpoint protein seemed to be important for the expression of a dominant-negative phenotype [45 ]. Thus, we next investigated the kinetochore localization of our deletion mutant by comparing the subcellular distribution of N-hBubR1 with the wild type in asynchronous parental Jurkat T and JM36 cells (Fig. 4C and 4D) . In Jurkat T cells, 18 h after nocodazole treatment, wild-type hBubR1 was exclusively localized in the cytoplasm of interphase cells and concentrated at the kinetochores of mitotic cells (Fig. 4C) . Of interest, in JM36 cells grown in the presence of doxycycline, N-hBubR1 was also detected at kinetochores, but no expression of this mutant was observed in the absence of doxycycline (Fig. 4D) . These observations suggest that in human cells, a deletion mutant of hBubR1 containing only the N-terminal homology domain is also able to localize to the kinetochore by binding to kinetochore proteins other than hBub3 and that it might have a dominant-negative phenotype. Thus, to further confirm this notion and to investigate the effect of N-hBubR1 expression on mitotic checkpoint regulation in T cells, JM36 and the parental cell line were treated with doxycycline for 48 h and then further cultured in the absence or presence of nocodazole. Thereafter, resultants were harvested and analyzed by flow cytometry to determine their DNA contents (Fig. 4E) . In both cell lines cultured in medium only, no apparent differences were observed in their DNA contents. Of note, in cells treated with nocodazole, most cells of both cell lines were arrested in the mitotic phase (4N) at 16 h. However, after 32 h, the apoptotic cell population (<2N) of JM36 increased more slowly than that of the parental cell line (Fig. 4F , Left). Moreover, the polyploid cell population (>4N) of JM36 dramatically increased after 48 h, whereas those of the parental cell line remained constant (Fig. 4F , Right), implying that the expression of N-hBubR1 compromises mitotic arrest and subsequent apoptotic cell death and increases the chromosomal instability of T cells after prolonged spindle damage. These results demonstrate that N-hBubR1 acts as a dominant-negative mutant and provide a first description of a dominant-negative form of hBubR1. In addition, these results suggest that BubR1 is required for proper mitotic checkpoint function during T cell proliferation and that a defect in its function leads to chromosomal instability.



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  		Figure 4. The dominant-negative mutant of hBubR1 compromises mitotic arrest and subsequent apoptotic cell death. (A) A schematic diagram of wild-type hBubR1 and Myc epitope-tagged N-hBubR1, which had a dominant-negative phenotype. (B) Parental Jurkat T cells and T cells expressing a dominant-negative BubR1 (JM36) were incubated with doxycycline for 24 h. Cell lysates were then prepared and analyzed by immunoblot using anti-Myc antibody. (C). Subcellular distribution of wild-type hBubR1 in interphase and mitotic cells. Jurkat T cells were cultured in the presence of 0.1 µg/ml nocodazole for 18 h. Thereafter, cells were fixed and stained with a mAb against hBubR1 (Left) and Hoechst dye to visualize the DNA (Right). * and –, Interphase and mitotic cells, respectively. (D) Subcellular localization of Myc-tagged N-BubR1 in mitotic cells. JM36 cells were cultured for 48 h in the absence (Tet-) or presence (Tet+) of doxycycline, and this followed by treatment with 0.1 µg/ml nocodazole for 18 h. The cells were then fixed and stained with anti-Myc antibody (Left) and Hoechst dye for DNA visualization (Right). N-BubR1 expression is only detectable in the presence of doxycycline (Tet+). All pictures were taken at the same magnification. (E) JM36 and parental Jurkat T cells were incubated with doxycycline for 48 h before treatment with 70 ng/ml nocodazole. At the time indicated, the cells were harvested, fixed, stained with PI, and analyzed by flow cytometry to determine their DNA contents. (F) Relative percentages of apoptotic (Left) or polyploid (Right) cells.

To further understand how the expression of dominant-negative BubR1/Mad3 increases the chromosomal instability of T cells, we compared the expression patterns of cyclin A, cyclin B, and hBubR1 in JM36 cells with those in parental cells after treatment with nocodazole in the presence of doxycycline (Fig. 5 ). Of interest, whereas the phosphorylation of hBubR1 decreased rapidly in parental cells at 40 h, its phosphorylation was slightly reduced but nevertheless sustained at significant levels in JM36 cells for up to 56 h. Moreover, while the level of cyclin B in parental cells gradually diminished to an almost undetectable level over the duration of the experiment, that of cyclin B in JM36 cells initially reduced at 32 h but increased again after 40 h. Similarly, cyclin A levels also gradually decreased in parental and in JM36 cells but began to accumulate again after 48 h in JM36 cells, indicating that JM36 cells had escaped mitotic arrest and re-entered a new round of the cell cycle. Thus, these results imply that the expression of dominant-negative BubR1 mutant abrogates mitotic arrest at the mitotic checkpoint by hindering endogenous BubR1 and thereby allowed cells to prematurely enter anaphase and undergo aberrant DNA endoreplication without cytokinesis.



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  		Figure 5. Effects of N-hBubR1 expression on the phosphorylation of endogenous hBubR1 and the expression of mitotic cyclins after prolonged spindle damage. JM36 and parental Jurkat T cells were synchronized and then treated as described in Figure 4E . The cells were then harvested and subjected to immunoblot analysis with anti-hBubR1, anticyclin B, anticyclin A, and antiactin antibodies.

p53 plays an important role in spindle damage-induced mitotic arrest in proliferating T cells
As p53 but not p73 is significantly induced in proliferating T cells after nocodazole treatment (Fig. 3) , we also further investigated whether p53 is required for the mitotic arrest of proliferating T cells. To do so, we purified primary T cells from the lymph nodes of p53-deficient mice and syngenic wild-type mice. The cells were then stimulated, treated, and analyzed by flow cytometry and immunoblotting (Fig. 6 ). As a control experiment, we confirmed the absence of p53 expression in p53 KO mice. As expected, treatment of the wild-type T cells with a microtubule inhibitor or a DNA-damaging agent induced p53, whereas p53 was undetectable in p53-deficient T cells (Fig. 6A) . Under these conditions, we investigated the effects of p53 on the DNA content of proliferating T cells after prolonged spindle damage (Fig. 6B) . When cells were cultured in medium only, no significant differences were observed in the DNA contents of p53-deficient and wild-type cells. However, cells treated with actinomycin D showed different cell-cycle profiles, which depended on the p53 status. After exposure to actinomycin D, while the wild-type cells were arrested at the G1 phase and undergoing apoptosis, p53-deficient T cells showed no significant arrest at the G1 phase or apoptotic cell death. In addition, in p53-deficient T cells, the population of cells arrested in the G2/M phase gradually decreased, possibly as a result of activation-induced cell arrest by p73, as has been previously reported [43 ]. In cells cultured in the presence of nocodazole, irrespective of p53 status, both types of T cells arrested in mitosis at 16 h. However, after 32 h, the formation of polyploid cells was dramatically enhanced in p53-deficient T cells compared with the wild type (Fig. 6B and 6C) . In addition, cells with a 16N DNA content were observed only in p53-deficient T cells. These results suggest that in proliferating primary T cells, p53 is required for cell-cycle arrest at a pseudo-G1 phase to prevent endoreplication without proper mitosis completion after aberrant exit from mitotic arrest and is thus dispensable during the initiation of mitotic arrest by spindle damage, as previously shown in cervical carcinoma cells [40 41 42 ]. Moreover, in line with previous reports on fibroblasts [41 42 43 44 45 46 47 ], following prolonged spindle damage, apoptotic cell death was also observed in p53-deficient T cells, although the percentage of cells undergoing apoptosis was slightly higher in wild-type cells (Fig. 6C) . These results indicate that following prolonged mitotic arrest, apoptotic cell death in proliferating T cells is induced by a p53-independent pathway.



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  		Figure 6. p53 prevents chromosomal instability in primary T cells by blocking DNA endoreplication after aberrant exit from mitosis. Primary p53-deficient and wild-type T cells were prepared from p53-/- and syngenic wild-type mice, respectively, as described in Materials and Methods. Thereafter, the resultants were treated as described in Figure 1A . (A) At the times indicated, the cells were harvested and analyzed by immunoblot using anti-p53 antibodies. Noco, Nocodazole; Act D, actinomycin D; WT, wild type; KO, knockout. (B) At the same times, cells were fixed with EtOH, stained with PI, and analyzed by flow cytometry to determine their DNA contents. (C) Relative percentages of apoptotic (Upper) or polyploid (Lower) cells. (D) To compare the mitotic progress of p53-deficient and wild-type T cells, the cells were treated as described above and then harvested for immunoblot analysis using anti-mBubR1, anticyclin B1, anticyclin A1, and antiactin Ab as a control. (E) The gel density of each protein was quantitated by densitometry, and the values obtained were normalized against the gel density of actin; results are presented as relative gel densities.

Next, to further address how p53 deficiency increases the chromosomal instability of proliferating T cells, we monitored the well-known mitotic marker proteins, cyclin A, cyclin B, and BubR1 (Fig. 6D and 6E) . It was noted that the phosphorylation of mBubR1 was induced in p53-deficient and wild-type T cells within 16 h. Thereafter, the phosphorylation of mBubR1 decreased rapidly in wild-type cells; however, that in p53-deficient T cells was maintained at significant levels. Similarly, cyclin B had accumulated in both T cells at 16 h; however, the level of cyclin B was rapidly reduced in wild-type cells but was sustained at significant levels in p53-deficient T cells for up to 48 h. It has been previously shown that cyclin B is degraded by activated APC/C during mitosis and that the degradation of cyclin B is required for the transition from the metaphase into the anaphase [48 , 49 ]. Thus, these results suggest that p53-deficient T cells prematurely exit from mitotic arrest, re-enter the cell cycle, while undergoing a novel round of DNA endoreplication, leading to chromosomal instability.

As was observed for cyclin B, cyclin A expression in p53-deficient T cells was sustained for up to 32 h and then decreased after 48 h, and cyclin A expression in wild-type cells peaked at 16 h and then decreased rapidly. However, the degradation of cyclin A occurred more slowly than that of cyclin B in the wild-type T cells. According to previously published results, in cells other than T cells, the degradation of cyclin A occurs before that of cyclin B, and the disruption of the mitotic spindle does not alter the relative stability of this cyclin [50 , 51 ]. Thus, our results raise the possibility that the regulation of the cyclin A degradation pathway in T cells might be achieved through pathways that are somewhat different from those in other cells.


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DISCUSSION
 
Extensive studies have established that improper chromosome segregation during mitosis results in the form of chromosomal instability observed in many human cancers [19 20 21 ]. Thus, in many cases of lymphoma, the failures of mitotic checkpoint control during the proliferation of T cells might act as a critical step in tumorigenesis. In a study that supported this hypothesis, Jin et al. [22 ] found that the Tax protein of HTLV-1, a causative agent of ATLL, interacts with TXBP181, the human homologue of the yeast mitotic checkpoint Mad1 protein. Moreover, the expression of Tax or a transdominant-negative TXBP181 in HeLa cells results in multinucleation, indicative of chromosomal instability [22 ]. However, to date, the majority of studies on the mitotic checkpoint and its regulation has been performed in human carcinoma cell lines. Therefore, studies of mitotic checkpoint regulation in primary T cells, which reflect in vivo conditions, are required to clarify the pathogenic mechanism of T cell lymphoma and to further investigate cycle control during T cell proliferation.

Here, we document that the mitotic checkpoint plays an important role in the fidelity of chromosome segregation during the proliferation of primary T cells. Moreover, our unpublished observations show that following prolonged mitotic arrest, primary T cells stimulated with TCR mimetics or specific antigens are more prone to polyploidy than other cell types. One possible explanation for this higher tendency toward polyploidy is their rapid turnover rate on antigenic stimulation [1 2 3 4 5 ]. A rapid clonal expansion of T cells, especially memory T cells, in response to specific antigens is necessary for the immediate establishment of protective immunity against invading foreign pathogens. Thus, it is possible that to proliferate rapidly upon antigenic stimulation, T cells have evolved to decrease the cell-cycle period by adopting cell-cycle regulatory pathways different from those in other cell types. This discrepancy might enable T cells to overcome apoptotic cell death after prolonged mitotic arrest and allow them to enter the next round of the cell cycle without cell division, termed endoreplication. Alternatively, as previously suggested [45 ], the strength of the mitotic checkpoint signal at the starting point of mitosis may act as a determinant of cell fate after aberrant exit from mitosis with a damaged spindle. Thus, it is also possible that the strength of the mitotic checkpoint signals in T cells might be weaker than those of non-T cells such as fibroblasts and epithelial cells and that this results in the increased susceptibility of T cells to chromosomal instability and their decreased apoptotic cell death after prolonged spindle damage. Furthermore, the different sensitivities of T cells and nonlymphoid cells to spindle inhibitors might be another possible explanation for our observations. The different cell fates of primary T cells and other types of cells in response to spindle damage should be further examined to elucidate a proper explanation.

p53, a tumor suppressor, is known to be required for G1 cell-cycle arrest. However, G1 arrest after TCR-mediated T cell activation requires p73 rather than p53 [43 ]. In the present study, we also showed that in activated T cells cultured in medium only, the expression of p73 and not p53 is dramatically induced by treatment with TCR mimetics and that this occurs in parallel with a shift in the T cell population toward the G1 phase. However, it was of interest to find that the expression of p53 and not of p73 is significantly induced in T cells treated with nocodazole after the phosphorylation of BubR1, a mitotic checkpoint kinase. Furthermore, chromosomal instability was dramatically higher in p53-deficient T cells than in wild type. Therefore, these results suggest that in response to spindle damage, p53 and not p73 is implicated in the cell-cycle regulation of primary T cells at the mitotic phase. In partial agreement with our suggestion, mice that lack both copies of p53 (p53-/- mice) spontaneously develop lymphoid malignancies in the thymus and spleen at high frequency. Mice with only one copy of the wild-type allele (p53+/- mice) also spontaneously develop lymphoid malignancies but at a much lower frequency than p53-/- mice [52 ]. Thus, the function of p53 in T cells following mitotic checkpoint activation may be connected to the pathogenesis of lymphoma, and further examination of the role of p53 during mitosis would help our understanding of the pathogenic mechanisms of T cell tumorigenesis.

In eukaryotic cells, cyclin A and cyclin B are required for entry into mitosis, and their degradation through APC/C-mediated ubiquitination is also critical for exit from mitosis [49 50 51 ]. Recently, it was reported that the mitotic checkpoint protein complex inhibits the activity of APC/C, leading to the delay of the onset of anaphase until all chromosomes have aligned properly at the metaphase plate [17 , 18 ]. Here, we observed that cyclin B accumulates transiently in wild-type T cells after spindle damage but that its expression is sustained in p53-deficient T cells. This discrepancy might be a result of the resynthesis of cyclin B in p53-deficient T cells during progression into a new round of the cell cycle after aberrant exit from mitosis, rather than being the result of APC/C inactivation as a result of a delayed mitotic checkpoint signal or of the up-regulation of this protein as a result of the lack of p53 [53 , 54 ]. As observed for cyclin B, cyclin A was also observed to accumulate transiently in wild-type T cells and to be sustained in p53-deficient T cells throughout the process of spindle disruption. However, it was of interest to find that the degradation of cyclin A seems to occur more slowly than that of cyclin B in wild-type T cells, indicating that the control of cyclin A levels in proliferating T cells may be regulated in a different manner than that in other cell types [50 , 51 , 55 , 56 ]. This discrepancy in the regulation of cyclin A expression may be one of the possible explanations for the rapid turnover rate of T cells upon antigenic stimulation and the increased susceptibility of T cells to chromosomal instability after prolonged spindle damage. This possibility needs further investigation to allow us to understand T cell cell-cycle regulation.

In conclusion, our results imply that following prolonged spindle damage, a p53 deficiency and a defective mitotic checkpoint in proliferating T cells compromise mitotic arrest and allow cells to progress into new rounds of DNA endoreplication without undergoing apoptotic cell death, thus leading to the form of chromosomal instability implicated in the tumorigenesis of T cell lymphomas. However, the sensitivity of T cells to mitotic spindle inhibitors also has significant implications for anticancer treatment strategies, as these inhibitors are potential anticancer drugs.


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ACKNOWLEDGEMENTS
 
We thank Drs. William R. Heath and Han W. Lee for materials and express our gratitude to Sang C. Lee and So Y. Choi for managing the animals and technical assistance. This work was supported by grant (RII-2001-098-02003-0) from Korea Science & Engineering Foundation (KOSEF) through RRC.

Received December 16, 2002; revised February 12, 2003; accepted February 13, 2003.


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