Homotypic Cell–Cell Adhesion Induced by Human T Cell Leukemia Virus Type 1 Tax Protein in T Cell Lines

Virology 302, 132–143 (2002) doi:10.1006/viro.2002.1629

Homotypic Cell–Cell Adhesion Induced by Human T Cell Leukemia Virus Type 1 Tax Protein in T Cell Lines Toshiyuki Takahashi,* ,† Masaya Higuchi,* Masaya Fukushi,* Masayasu Oie,* Masaaki Ito,† and Masahiro Fujii* ,1 *Division of Virology and †Division of Dermatology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-Dori, Niigata 951-8510, Japan Received February 12, 2002; returned to author for revision March 15, 2002; accepted May 28, 2002 Cell–cell adhesion is involved in the processes of cell growth, activation and migration, and inflammation. T cells infected with human T cell leukemia virus type 1 (HTLV-1) exhibit a high degree of homotypic cell–cell adhesion in vitro. In this study, we investigated the involvement of the viral protein Tax in such process. Expression of Tax in an interleukin (IL)-2-dependent mouse T cell line (CTLL-2) increased homotypic cell–cell adhesion; however, less cell adhesion was induced by Tax than that observed in HTLV-1-infected T cell lines. Moreover, Tax induced cell–cell adhesion in a human T cell line, in which the expression of Tax is inducible. Microscopic examination also revealed Tax-induced morphologic changes, including rounding of CTLL-2 cells, increased cell volume, and increased nucleus size. Taken together, our results suggest that Tax induces cell–cell adhesion and morphologic changes in HTLV-1-infected cells. Tax may thus play a role in persistent HTLV-1 infection and the pathogenesis of associated disease. © 2002 Elsevier Science (USA) Key Words: HTLV-1; Tax; adhesion; NF-␬B; CREB; IL-2.

in HTLV-1-infected cells. Various types of stimuli can augment expression of Tax and Rex in infected cells and, in response to Rex accumulation, mRNA expression of the structural genes increases, while that of tax and rex decreases. Tax plays crucial roles in both the regulation of viral genes and the pathogenesis of HTLV-1-induced disease (Yoshida, 2001). For example, Tax can immortalize primary human T cells in vitro and can transform rodent fibroblast cell lines (Tanaka et al., 1990; Grassmann et al., 1992; Akagi and Shimotohno, 1993). In addition, Tax induces various types of autoimmune-like disorders, such as arthritis and exocrinopathies, in transgenic animals (Green et al., 1989; Iwakura et al., 1991; Nakamaru et al., 2001). Consistent with these multivalent functions, the Tax protein exhibits multiple activities; Tax activates a number of cellular genes, such as cytokine receptors [e.g., ␣-chain of interleukin (IL)-2 receptor] (Inoue et al., 1986; Cross et al., 1987; Maruyama et al., 1987), cytokines (e.g., IL-1 and IL-2) (Inoue et al., 1986; Maruyama et al., 1987; Mori and Prager, 1996), nuclear oncogenes (e.g., c-fos, c-jun, fra-1) (Fujii et al., 1991; Tsuchiya et al., 1993), bcl-xl (Nicot et al., 2000; Mori et al., 2001), and cyclin D2 (Akagi et al., 1996; Santiago et al., 1999; Huang et al., 2001). Tax also represses several cellular genes (e.g., DNA polymerase ␤) (Jeang et al., 1990). In addition, Tax forms a protein–protein complex with cyclin D3 (Neuveut et al., 1998). Moreover, Tax inactivates several tumor suppressor gene products (e.g., p16INK4A, p53, and

INTRODUCTION Human T cell leukemia virus type 1 (HTLV-1) is associated with two distinct disease phenotypes, adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Poiesz et al., 1980; Hinuma et al., 1981; Gessain et al., 1985; Osame et al., 1986). ATL is an aggressive form of leukemia arising from monoclonal expansion of CD4-positive T cells (Uchiyama, 1997). HAM/TSP is a chronic inflammatory disease characterized by progressive symmetrical myelopathy associated with autoimmune-like conditions (Gessain et al., 1985; Osame et al., 1986; Izumo et al., 2000). Only 5 and 1–3% of HTLV-1-infected individuals develop ATL and HAM/TSP, respectively, and both conditions have long latent periods of around 40–50 and 10–20 years, respectively (Uchiyama, 1997). In addition to its structural genes (gag, pol, env), HTLV-1 encodes two regulatory genes (tax and rex) (Seiki et al., 1983; Inoue et al., 1987). Tax is a transcriptional activator of viral transcription (Sodroski et al., 1984; Cann et al., 1985; Fujisawa et al., 1985; Felber et al., 1985; Seiki et al., 1986), and Rex is a posttranscriptional regulator (Inoue et al., 1987). These two genes coordinately regulate the latent and lytic phases of HTLV-1 infection. A low level of tax and rex gene expression, but no structural gene expression, is evident during the latent phase 1

To whom correspondence and reprint requests should be addressed. Fax: ⫹81 (25) 227-0763. E-mail: [email protected]. 0042-6822/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

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CELL ADHESION-INDUCING ACTIVITY OF Tax TABLE 1 Characterization of T Cell Lines Used

Cell lines

HTLV-1 provirus

Tax protein

␬B activity

CRE activity

Adhesion

Jurkat HUT78 MOLT-4 H9 HUT-102 MT-4 SLB-1 TL-Su C5/MJ CTLL-2 CTLL/Tax WT CTLL/703 CTLL/M22

⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫺ ⫹⫹ ⫹⫹ ⫹

⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫺ ⫹⫹ ⫹⫹ ⫺

N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. ⫺ ⫹ ⫺ ⫹

⫺ ⫺ ⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫹⫹ ⫹⫹ ⫺

N.D., not determined.

hDlg) (Suzuki et al., 1996, 1999; Akagi et al., 1997; Lee et al., 1997; Pise-Masison et al., 1998). A prominent feature of HTLV-1-infected T cell lines in culture is the appearance of large cell clusters comprising innumerable cells attached to each other by strong homotypic cell–cell adhesion. Since cell-to-cell contact regulates multiple cellular functions, such as cell growth, activation and migration, and inflammation, the adhesion-inducing activity of HTLV-1 in T cells is likely to play a crucial role in persistence of viral infection, and thereby the development of HTLV-1-associated disease. In this study, we demonstrate that Tax alone induces homotypic cell–cell adhesion in an IL-2-dependent mouse T cell line (CTLL-2) and a human T cell line (Jurkat). We discuss these findings in the context of persistent HTLV-1 infection and the pathogenesis of associated diseases. RESULTS Augmented cell–cell adhesion in HTLV-1-infected T cell lines We initially investigated the homotypic cell–cell adhesion activity of HTLV-1-infected T cell lines. Nine human T cell lines (five HTLV-1-infected and four uninfected) were used (Table 1), and expression of the viral protein Tax in these cells was measured by Western blot analysis. An anti-Tax antibody detected a protein of ⬃40 kDa in all HTLV-1-infected T cell lines, but in no uninfected lines, although the expression of Tax in C5/MJ was lower than in the other HTLV-1-infected lines (Fig. 1). To analyze homotypic cell–cell adhesion, cells were dispersed by repetitive pipetting and cultured for 6 or 12 h in growth medium on 24-well plates, after which cell morphology was examined under light microscopy. All five HTLV-1infected T cell lines exhibited strong cell–cell adhesion

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after 6 h of culture and generated large cell clumps (which were absent prior to culturing) (Fig. 2). In contrast, two uninfected T cell lines (Jurkat, HUT78) did not exhibit any cell–cell adhesion within 12 h, and the other two uninfected cell lines (MOLT-4, H9) exhibited only very low levels of cell–cell adhesion. These results indicated that HTLV-1-infected T cell lines have an augmented cell–cell adhesion capacity compared to uninfected leukemic T cell lines. Tax induces homotypic cell–cell adhesion in a mouse T cell line Tax acts at the transcriptional level to induce expression of a number of cellular genes (Yoshida, 2001). We therefore investigated whether Tax could induce homotypic cell–cell adhesion in T cells. For this purpose, we used CTLL-2 cells that expressed Tax protein (CTLL-2/ TaxWT) (Iwanaga et al., 1999). The parental CTLL-2 line is a mouse T cell line that grows only in the presence of IL-2. Western blotting assay using anti-Tax antibody revealed that three CTLL-2 lines transfected with the tax plasmid expressed Tax protein, whereas Tax was undetectable in parental CTLL-2 cells (Fig. 3A). These four cell lines were subjected to repetitive pipetting and then cultured in growth medium containing IL-2. Three Taxexpressing CTLL-2 lines generated large cell clumps, whereas such cell clumps were not observed in parental CTLL-2 cells (Figs. 3B and 3C). These results indicated that Tax alone could induce homotypic cell–cell adhesion in a T cell line. It was notable that the cell clumps induced by Tax were much smaller than those observed in HTLV-1-infected cells (Figs. 2 and 3) and were more easily dispersed by repetitive pipetting (data not shown), indicating that other factor(s) in HTLV-1-infected cells may also induce cell–cell adhesion. Pathways of Tax required for cell–cell adhesion activity in T cell lines Tax can activate the transcription of cellular genes through two distinct enhancers, the cAMP responsive

FIG. 1. Expression of Tax in HTLV-1-infected T cell lines. Cell lysates were prepared from the indicated human T cell lines. The amount of Tax protein in each lysate was measured by Western blotting using anti-Tax antibody (TAXY-8).

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FIG. 2. Augmented cell–cell adhesion activity of HTLV-1-infected T cell lines. (A) The indicated cell lines were dispersed by repetitive pipetting, and the cells were then cultured for the indicated time intervals in RPMI/FCS on 24-well plates. Cell images were examined under light microscopy (magnification, ⫻100). (B) The cell–cell adhesion activity (size) was calculated as an average of the top 10 cell clumps in size.

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FIG. 3. Tax induced cell–cell adhesion in a mouse T cell line. (A) Cell lysates were prepared from CTLL-2 cells and CTLL-2 cells transfected with the tax plasmid. The amount of Tax protein in the lysate was measured by Western blotting using anti-Tax antibody (TAXY-8). (B) The adhesion activity of the CTLL-2 and CTLL-2/TaxWT cell lines was examined under light microscopy as described under Materials and Methods (magnification, ⫻100). (C) The cell–cell adhesion activity (size) was calculated as an average of the top 10 cell clumps in size.

element (CRE)-like sequence and the ␬B element (Yoshida, 2001). Two well-known Tax mutants, TaxM22 and Tax703 (also called as M47), exhibit different activities toward these two enhancers (Smith and Greene, 1990). TaxM22 can activate transcription through the CRE-like element but not through ␬B, while Tax703 preferentially activates the ␬B element but not the CRE-like element in various cell lines, including CTLL-2 (Iwanaga et al., 1999). A CTLL-2 clone expressing TaxM22 induced only a few small cell clumps, similar to those seen in parental CTLL-2 cells, although the amount of TaxM22 protein in CTLL-2 was much less than that of CTLL-2-expressing wild-type Tax (Fig. 4). In contrast, all four CTLL-2 clones expressing Tax703 induced multiple large cell clumps. All of the CTLL/703 clones expressed Tax more than CTLL/TaxWT clones (Fig. 4), and CTLL/WT and CTLL/703

clones exhibited equivalent high DNA-binding activity to NF-␬B as measured by mobility shift assay (Iwanaga et al., 1999; Iwai et al., 2001). These results suggested that activation of CREB by Tax is dispensable for induction of adhesion in CTLL-2 cells. TNF-␣ minimally induces cell–cell adhesion in CTLL-2 cells Tax and Tax703 that activate NF-␬B in CTLL-2 cells induced cell–cell adhesion (Fig. 4), suggesting that activation of NF-␬B by Tax is involved in induction of adhesion in CTLL-2 cells. So, we next examined whether tumor necrosis factor (TNF)-␣, another NF-␬B activator, also induces cell–cell adhesion in CTLL-2 cells. The treatment of CTLL-2 cells with TNF-␣, however, minimally

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FIG. 4. Activity associated with cell–cell adhesion induction by Tax. (A) The adhesion activity of the CTLL-2 and CTLL-2/TaxWT cell lines was examined under light microscopy as described under Materials and Methods (magnification, ⫻100). (B) The cell–cell adhesion activity (size) was calculated as an average of the top 10 cell clumps in size. (C) Cell lysates were prepared from CTLL-2 cells and CTLL-2 cells transfected with the tax plasmid. The amount of Tax protein in the lysate was measured by Western blotting using anti-Tax antibody (TAXY-8).

induced cell–cell adhesion activity (Fig. 5). These results suggested that activation of NF-␬B by Tax is not sufficient for the induction of cell–cell adhesion in CTLL-2 cells.

adhesion potencies of HTLV-1-infected T cell lines and CTLL-2/Tax cells appear to be unrelated to IL-2. Tax induces cell–cell adhesion in a human T cell line

IL-2 does not affect cell–cell adhesion activity in CTLL-2 cells The HTLV-1-infected T cell lines used here were all cultured in the absence of IL-2, while all of the CTLL-2 cell lines were cultured in the presence of IL-2. Since IL-2 modulates various activities in T cells, we next investigated whether IL-2 affected the cell–cell adhesion activity in CTLL-2 cells. For this purpose, we used CTLL2/WT-14 and CTLL-2/703 cell lines (Iwanaga et al., 1999). When highly expressed, Tax can often convert CTLL-2 cells from being IL-2-dependent for growth to being IL2-independent. CTLL-2/WT-14 and CTLL-2/Tax703-2 are such clones that can grow without IL-2, and we therefore cultured these two cell lines in the absence or presence of IL-2 and assayed their homotypic cell–cell adhesion activity. IL-2 did not alter the cell adhesion activity of either cell line (Fig. 6A). In addition, IL-2 had minimal effect on Tax expression (Fig. 6B). Thus, the differing

The above data showed that the long-term expression of Tax in a mouse T cell line induces homotypic cell–cell adhesion. We next examined whether Tax affects cell– cell adhesion activity in human T cells. JPX-9 and JPX/M are derivatives of a human T cell line Jurkat, and they have the wild-type tax and the inactive mutant gene under the control of a metallothionein promoter, respectively (Ohtani et al., 1989). Addition of CdCl 2 to the culture medium induced Tax and the mutant protein in JPX-9 and JPX/M but not Jurkat cells (Fig. 7A). Such cells were subjected to repetitive pipetting and further cultured for 6 h in the presence or absence of CdCl 2. JPX-9 cells treated with CdCl 2 exhibited cell–cell adhesion activity, whereas such adhesion was little observed in JPX-9 cells without CdCl 2 treatment (Fig. 7B). The induction of cell– cell adhesion in JPX-9 cells was due to Tax, since the cell–cell adhesion was not changed by CdCl 2 treatment in Jurkat and in JPX/M cells which express the mutant

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FIG. 5. Effect of TNF-␣ on cell–cell adhesion activity in CTLL-2. CTLL-2 cells were cultured in the absence or presence of 10 or 20 ng/ml of TNF-␣ for 3 days. Then, the cells were dispersed by repetitive pipetting and cultured for 6 or 12 h in the same medium. The morphology of the cultured cells was examined under light microscopy (magnification, ⫻100).

Tax protein (Fig. 7B). These results indicated that Tax induces cell–cell adhesion in a human T cell line. Morphological changes induced by Tax in CTLL-2 cells In addition to the differences in adhesion activity, microscopic examination revealed morphological differences between CTLL-2 and CTLL-2/TaxWT cells. CTLL2/TaxWT cells were bigger and more rounded than the droplet-shaped parental CTLL-2 cells (Fig. 8). Propidium iodine staining revealed that CTLL-2/TaxWT cells possessed much bigger nucleus than parental CTLL-2 cells (Fig. 9). These morphological differences were also observed in CTLL-2 cells expressing Tax703. The effect of Tax703 on cell morphology was equivalent to that of wild-type Tax. In contrast, the morphological features of CTLL-2/TaxM22 cells were very similar to those of parental CTLL-2 cells (Figs. 8 and 9). DISCUSSION In the present study, HTLV-1-infected T cell lines exhibited strong homotypic cell–cell adhesion activity in vitro and formed large cell clusters consisting of innu-

merable cells (Fig. 2). Tax has been shown to induce adhesion of HTLV-1-infected cells to other cells, such as endothelial and HTLV-1 uninfected T cells (Uchiyama, 1997), but whether Tax alone can induce adhesion of HTLV-1-infected cells to other HTLV-1-infected cells has not yet been elucidated. In this study we demonstrated that Tax alone can induce such homotypic cell–cell adhesion in a mouse T cell line (CTLL-2) and a human T cell line (JPX-9). Since cell–cell adhesion regulates various activities such as cell growth, activation status, cell migration, and inflammation, the present results suggest that Tax, through inducing cell–cell adhesion of infected cells, plays a crucial role in persistent HTLV-1 infection and pathogenesis of HTLV-1-associated diseases. Tax-induced cell–cell adhesion in CTLL-2 and JPX-9 cells was weaker than that induced by HTLV-1 (Figs. 2, 3, and 7). Thus, other viral protein(s) may also contribute to cell–cell adhesion-inducing activity in HTLV-1-infected cells. The viral envelope protein is such a candidate, since it can interact with the putative viral receptor on the cell surface. If this is the case, the cell–cell adhesion induced by each of these two viral genes would be likely to have distinct roles, since Tax is expressed during

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FIG. 6. Effect of IL-2 on cell–cell adhesion activity in CTLL-2. CTLL-2/WT-14 and CTLL-2/703-2 cell lines were dispersed by repetitive pipetting and then cultured in the presence or absence of IL-2 for 6 or 12 h. The morphology of the cultured cells was examined under light microscopy (magnification, ⫻100).

latent and lytic phases of infection, while the envelope protein is expressed only in the lytic phase. Kitajima et al. (1996) showed that Tax increased homotypic cell–cell adhesion in a rat pheochromocytoma cell line (PC12). Thus, Tax-induced cell–cell adhesion is not specific to T cells, which are the main natural host for latent HTLV-1 infection. They also showed that Tax-induced E-cadherin-mediated homotypic cell–cell adhesion in PC12 cells (Kitajima et al., 1996). Matsuyoshi et al. (1998), however, showed that HUT-102 cells with augmented Tax expression (Fig. 2) did not express E-cadherin (Fig. 2). Thus, it is unlikely that E-cadherin mediated the homotypic cell–cell adhesion of HTLV-1-infected T cell lines observed in the present study. Our present results indicated that activation of NF-␬B may be involved in Tax-dependent induction of cell–cell adhesion in CTLL-2 cells but the activation alone is not

sufficient for the induction (Figs. 4 and 5, Table 1). Several adhesion molecules and their ligands, including LFA-1, ICAM-1 (a ligand for LFA1), and OX40 and OX40 ligand, are expressed in HTLV-1-infected T cell lines. Tax has been shown to induce ICAM-1, VCAM-1, OX40, and OX40 ligand in T cells (Higashimura et al., 1996; Tanaka et al., 1996; Ohtani et al., 1998; Valentin et al., 2001), and at least VCAM1, OX40, and OX40 ligand were activated by Tax through NF-␬B (Valentin et al., 1997; Ohtani et al., 1998; Pankow et al., 2000). Thus, these genes are candidates for mediating homotypic cell–cell adhesion in HTLV-1-infected T cell lines. Our results here also demonstrated that Tax induced morphological changes in a mouse T cell line, as seen by an increased cell volume, rounded cell shape, and an increased nucleus size (Figs. 8 and 9). Jin et al. (1998) showed that Tax increases DNA content per cell by

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FIG. 7. Tax-induced cell–cell adhesion in a human T cell line. (A) Cell lysates were prepared from Jurkat, JPX-9, and JPX/M cells cultured with CdCl 2 or without it. The amount of Tax protein in the lysate was measured by Western blotting using anti-Tax antibody (TAXY-8). (B) The adhesion activity of the Jurkat, JPX-9, and JPX/M cell lines was examined under light microscopy as described under Materials and Methods (magnification, ⫻100).

abrogating checkpoint function at G2-M cell cycle. Since increase of the cellular DNA content is generally associated with increase of the cell volume, these two phenotypes may be related to each other. On the other hand, Trihn et al. (1997) showed that Tax interacted with an intermediate filament (keratin 8), and that such interaction was associ-

ated with alteration of the keratin network. Further analysis is required to elucidate the mechanism by which Tax alters the structure of infected T cells. The tax gene is always expressed in HTLV-1-infected cells, such as peripheral blood lymphocytes in vivo. Thus, our results here suggest that HTLV-1-infected T

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FIG. 8. Changes in cell morphology induced by Tax in CTLL-2. The indicated cell lines were dispersed by repetitive pipetting, and cell morphology was examined under light microscopy (magnification, ⫻400).

cells exist in vivo as cell clusters. Since HTLV-1-infected cells produce several cytokines promoting T cell growth, including IL-2 (Inoue et al., 1986; Maruyama et al., 1987), these secreted cytokines may be utilized both in an autocrine and in a paracrine manner. Thus, adhesion between HTLV-1-infected cells may create advantageous conditions for growth and survival of HTLV-1-infected cells in vivo. MATERIALS AND METHODS Cell culture The human T cell lines used in the present experiments have been characterized previously (Sugamura et al., 1984; Mori et al., 2001). MT-4, TL-Su, SLB-1, C5/MJ, and HUT-102 are human T cell lines transformed by HTLV-1. Jurkat, MOLT-4, H9, and HUT78 are HTLV-1negative human T cell lines. JPX-9 and JPX/M cells are derivatives of Jurkat and have a stably integrated tax and tax mutant gene under the control of a methallothionein promoter, respectively (Ohtani et al., 1989). To express the tax gene, the cells were cultured in the presence of CdCl 2 (10 or 20 ␮M) at 37°C for 3 days. These human cell lines were cultured in RPMI1640 supplemented with 10% fetal calf serum (RPMI/FCS). CTLL-2 is a mouse T cell line, whose growth is dependent on IL-2. CTLL-2 clones expressing Tax, Tax703, and TaxM22 were established by transfection with the respective expression plasmids (Iwanaga et al., 1999). CTLL-2 cell lines expressing Tax and parental CTLL-2 were cultured in RPMI/FCS with 1 nM human recombinant IL-2 and 50 ␮M 2-mercaptoethanol.

Western blotting Cell lysates (20 ␮g) prepared from T cell lines were resolved by electrophoresis on 10% polyacrylamide gels and then transferred to PVDF membranes (Bio-Rad Technologies, Richmond, CA). The membrane was incubated with 5% skim milk in TBS-T [10 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20] for 1 h at room temperature to inhibit nonspecific binding and further incubated with anti-Tax mouse monoclonal antibody (TAXY-8). After washing with TBS-T, the membrane was further incubated with anti-mouse immunoglobulin conjugated with horseradish peroxidase (Bio-Rad Technologies). Proteins recognized by the antibodies were visualized using the ECL Western blotting detection system (Amersham Pharmacia Biotech, Piscataway, NJ). Dr. Yuetsu Tanaka (Ryukyu University, Okinawa) kindly provided the TAXY-8 antibody. Cell–cell adhesion assay Cells were dispersed by repetitive pipetting, after which cells (2 ⫻ 10 5/ml) were cultured in 1.5 ml of RPMI/FCS on a 12- or 24-well plate for 6 or 12 h. Cell morphology was examined by inverted light microscopy (Axiovert 200; Zeiss, Oberkochen, Germany) and photographed with a digital camera (Axiocam Digital Camera, Zeiss). The cell–cell adhesion activity (size) was calculated as an average of the top 10 cell clumps in size. Propidium iodine staining Cells were washed once with PBS and plated on slides. The cells on the slides were air-dried and fixed by

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FIG. 9. Changes in nucleus morphology induced by Tax in CTLL-2. The indicated cell lines were stained with propidium iodine (red), and cell and nucleus morphology were examined under fluorescence light microscopy (magnification, ⫻400).

methanol for 10 min at ⫺20°C. After air-drying again, the fixed cells were treated with 2 ␮g/ml of propidium iodine for 1 h at room temperature. After washing with PBS, morphology of the stained nucleus was examined by inverted fluorescence light microscopy (Axiovert 200) and photographed with a digital camera (Axiocam Digital Camera).

ACKNOWLEDGMENTS We thank Yuetsu Tanaka and Masataka Nakamura for kindly providing anti-Tax1 antibody (TAXY-8) and JPX-9 cell lines, respectively. We also thank Ryoko Fujita for the excellent technical assistance. This work was supported in part by Grant-in-Aid for Scientific Research on Priority Areas (C) and for Scientific Research (C) of Japan.

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