Evidence for Cooperative Transforming Activity of ... - Journal of Virology

JOURNAL OF VIROLOGY, Aug. 2007, p. 7894–7901 0022-538X/07/$08.00⫹0 doi:10.1128/JVI.00555-07 Copyright © 2007, ARTICLE. All Rights Reserved.

Vol. 81, No. 15

Evidence for Cooperative Transforming Activity of the Human Pituitary Tumor Transforming Gene and Human T-Cell Leukemia Virus Type 1 Tax䌤 Sergey V. Sheleg,1† Jean-Marie Peloponese,1† Ya-Hui Chi,1 Yan Li,1 Michael Eckhaus,2 and Kuan-Teh Jeang1* Molecular Virology Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases,1 and Veterinary Resources Program, Office of Research Services,2 National Institutes of Health, Bethesda, Maryland 20892 Received 15 March 2007/Accepted 9 May 2007

Aneuploidy is frequent in cancers. Recently it was found that pituitary tumor transforming gene (PTTG; also called Pds1p or securin) is overexpressed in many different tumors. Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that primarily infects CD4ⴙ T lymphocytes and causes adult T-cell leukemia. Here, we report that overexpression of human PTTG cooperated with the HTLV-I Tax oncoprotein in cellular transformation. Coexpression of Tax and PTTG enhanced chromosomal instability and neoplastic changes to levels greater than overexpression of either factor singularly. Cells that overexpressed both PTTG and Tax induced tumors more robustly in nude mice than cells that expressed either PTTG alone or Tax alone. Recently it was reported that Tax directly binds the Cdc20associated anaphase-promoting complex to induce premature degradation of PPTG/securin and Clb2p/cyclin B1 in yeast and human cells (32, 33). Such an untimely activity by Tax could create chromosomal instability. Because of our interest in understanding Tax, chromosomal instability, and cellular transformation (15, 25, 47), we wished to elucidate the interplay between PTTG and Tax in mammalian cells. Here, we demonstrate that PTTG and Tax cooperate to enhance cellular proliferation and transformation, without evidence that Tax causes premature degradation of PTTG.

Pituitary tumor transforming gene (PTTG; also called Pds1 or securin) was first identified in rat pituitary tumors (46, 60, 68). The human PTTG protein was subsequently found to also be highly expressed in pituitary tumors and many other neoplasms (18, 53, 57). Interestingly, aside from being a cancerassociated factor, PTTG is the securin protein, which normally binds and inactivates separase, a protease that cleaves the cell’s cohesin protein (9). During a normal cell cycle, after chromosomes replicate in S phase, the duplicated chromosomes are “glued” together by a multisubunit cohesin complex (42). As the cell enters mitosis and needs to partition replicated chromosomes into progeny nuclei, cleavage of cohesin by separase occurs. This cleavage liberates tethered sister chromatids, permitting anaphase to ensue. Accordingly, aberration in the securin-separase-cohesin process can produce aneuploid daughter cells with gains and losses of whole chromosomes, a condition frequent in cancers (29, 30, 50, 54). Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that infects CD4⫹ T lymphocytes and is etiologically linked to adult T-cell leukemia (ATL) (62, 64). HTLV-1 encodes a viral oncoprotein, Tax, which is also a transcriptional activator that regulates viral and cellular gene expression. Tax influences host gene expression by interacting with and activating cellular signaling pathways, some of which can lead to dysregulated growth and malignant transformation of cells (2, 12, 14, 36, 37, 44, 65). Tax also modulates the functions of proteins involved in cell cycle control (p53, the mitotic checkpoint regulator MAD1, D cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors [1, 3, 10, 16, 25, 28, 40, 41, 43, 48, 51, 59]).

MATERIALS AND METHODS Cells, vectors, and transfection. HeLa, HCT116, murine embryonic fibroblast, and NIH 3T3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). Where indicated, cells were synchronized in G0/G1 phase by incubation in Dulbecco’s modified Eagle’s medium supplemented with 0.5% fetal calf serum for 24 h, in S phase with 2 mM hydroxyurea (Sigma), and in M phase with 0.2 ␮g/ml nocodazole (Sigma). Some cells were also treated with the proteasome inhibitor MG132 (5 ␮M; Calbiochem). Cell were transfected using Lipofectamine (Invitrogen) and Plus reagent (Invitrogen) according to the manufacturer’s protocols. A pCI-PTTG/securin expression vector was a gift from Shlomo Melmed (Cedars-Sinai Medical Center, David Geffen School of Medicine, University of California, Los Angeles). PCRamplified PTTG was subcloned into the pcDNA3-2HA-neo expression plasmid. Tax plasmid was as described previously (21). Western blot analysis. HTLV-1 Tax oncoprotein was detected with antiHTLV-I Tax rabbit antibody at a final dilution of 1:1,000 (23). Rabbit antiPTTG-1 was purchased from Zymed (San Francisco, CA). Purified mouse anti-cyclin B was purchased from BD Biosciences Pharmingen. Monoclonal anti-␤-actin (clone AC-15) and monoclonal anti-␣-tubulin were purchased from Sigma-Aldrich. Where indicated, densitometry was performed on the autoradiograms. The signals on the scanned film were quantified using the Image Gauge densitometry program (FUJIFILM). Cell proliferation assay. Cell proliferation was determined by using a modified 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium, bromide (MTT) assay employing the Cell Counting kit 8 (CCK-8; Dojindo) according to the manufacturer’s protocol. Briefly, after transfection, 100,000 cells/well were plated in a 96-well plate. After 2, 4, 6, or 8 days, 10 ␮l CCK-8 solution [2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt; Dojindo] was added to each well and incubated for 1 to 4 h. The cell viability in each well was determined by reading the optical density at 450 nm.

* Corresponding author. Mailing address: Molecular Virology Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. Phone: (301) 496-6680. Fax: (301) 480-3686. E-mail: kj7e @nih.gov. † S.V.S. and J.-M.P. made equal contributions. 䌤 Published ahead of print on 16 May 2007. 7894

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FIG. 1. Tax does not increase PTTG degradation. (A) HeLa cells were transfected with a PTTG expression vector (lanes 2 to 4) or a Tax plasmid (lanes 5 to 7). Whole-cell lysates were prepared, resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and probed with anti-PTTG, anti-Tax, or anti-tubulin. The PTTG expression level was quantified by densitometry and normalized against tubulin. (B) HCT116 cells were transfected as indicated with a PTTG expression vector (lanes 2 to 7) or a Tax plasmid (lanes 5 to 7). Whole-cell lysates were prepared, resolved by 12% SDS-PAGE, and probed with anti-PTTG, anti-Tax, or anti-tubulin. The PTTG expression level was quantified by densitometry and normalized against tubulin. (C) NIH 3T3 and HCT116 cells were transfected with either a control pCDNA 3.0 vector (lanes 1 and 3) or 2 ␮g of Tax-expressing vector (lanes 2 and 4). At 48 h later, whole-cell lysates were prepared and resolved by 12% SDS-PAGE. Amounts of PTTG and Tax in cell extracts were assayed using anti-PTTG or anti-Tax. Equal protein loading was verified with anti-actin (lower panel). (D) Expression of cellular endogenous PTTG in synchronized HCT116 cells was monitored in the absence or presence of HTLV-1 Tax. HCT116 cells were transfected with either a control pCDNA 3.0 vector (lanes 1 to 4) or a Tax plasmid (lanes 5 to 8). Three hours after transfection, cells were separately synchronized for 24 h in low FCS (0.5%; for G0/G1 phase), hydroxyurea (2 mM; for S phase), or nocodazole (0.2 ␮g/ml; for G2/M phase). In parallel, cells were also treated with the proteasome inhibitor MG132 for 8 h. Whole-cell lysates were prepared, resolved by 12% SDS-PAGE, and probed with anti-PTTG, anti-Tax, anti-cyclin B1, or anti-tubulin. Equal loading of protein in the cell extract was verified with antiactin antibody (lower panel). The PTTG expression level was quantified by densitometry and normalized against actin.

The data were collected from three independents experiments. Results are presented as the mean ⫾ standard error. Soft agar assays. Ten independent clones selected from each transfection were pooled. A soft agar assay was performed with slight modification from that described previously (46). Briefly, 103 stably transfected cells were placed into 1 ml of DMEM-FCS with 0.5% low-melting-point agarose (Invitrogen). The cells were overlaid on a 6-cm tissue culture dish containing 1 ml of bottom agar (DMEM–10% FCS with 0.1% Bacto agar; Difco) and incubated at 37°C with 5% CO2 for 14 days, when visible colonies were counted. Tumor growth in athymic nude mice. A nude mouse tumorigenesis assay was performed as described elsewhere (24). Briefly, 6-week-old athymic nude mice (Harlan, Indianapolis, IN) were injected with 5 ⫻ 106 cells in 0.1 ml of phosphate-buffered saline subcutaneously in the neck region. Following injection, the animals were observed every 2 days for the development of tumors. Animals were sacrificed when a tumor became approximately 3 cm in its largest diameter or when the tumor interfered with the animal’s daily movement. The tumors were resected for histopathological examination. Metaphase spread and chromosome counting. Metaphase spreads were prepared as described previously (13). Cells were incubated in the presence of 0.25 ␮g/ml nocodazole (Sigma) for 8 h to enrich for cells in mitosis. Following centrifugation, the cells were gently resuspended into hypotonic solution (0.075 M KCl) and incubated for 20 min at 37°C. After removal of hypotonic solution, the cells were fixed in a methanol-acetic acid solution (3:1) for 5 min. Fresh fixative was slowly added after discarding the old fixative. The procedure was repeated a total of three times. After the last change of fixative, a few drops were put on positively charged glass slides (Fisher Scientific) and dried completely on

a 68°C hot plate. The dried samples were stained with Hoechst 33342 (0.1 ␮g/ml), mounted in Prolong Antifade (Molecular Probes) with glass coverslips, and visualized using a Leica TCS-NP/SP confocal microscope. For each cell sample, more than 300 cells were counted from three independent experiments.

RESULTS Effect of Tax on PTTG expression. We began our studies by first characterizing the specificity of protein detection by antiPTTG antiserum. By transfecting increasing amounts of a PTTG expression plasmid, pCDNA3-2HA-hPTTG, into HeLa cells, we confirmed the specificity of anti-PTTG antibody. As expected, the antiserum detected with increasing intensity a ⬃28-kDa protein band in PTTG-transfected cells (Fig. 1A, lanes 2 to 4) which comigrated with its cell endogenous counterpart in control cells (Fig. 1A, lane 1). In the same experiment we also transfected increasing amounts of Tax into HeLa cells. However, while increasing Tax expression was documented in the transfected cells (Fig. 1A, middle panel, lanes 5 to 7), we observed that the level of cell endogenous PTTG remained unchanged (Fig. 1A, upper panel, lanes 5 to 7). This finding was surprising, since it differed from previous descrip-

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tions that Tax induced premature PTTG/securin degradation (32, 33). We next investigated Tax-PTTG interaction. Previously, Tax was reported to induce PTTG degradation in HeLa cells (19, 32, 33) which were aneuploid and/or defective in p53 function (1, 3, 26, 40, 48, 70). Because we did not see Tax-induced degradation of PTTG in our HeLa cells (Fig. 1A), we wished to check this phenomenon using cells which are euploid and intact for p53 function. HCT116 cells are euploid human cells with intact DNA damage checkpoint and p53 activities (6, 31); we wondered how Tax would affect PTTG stability in this cell line. As an additional comparison, we also examined the TaxPTTG interaction in mouse NIH 3T3 cells. HCT116 (Fig. 1B and C) and NIH 3T3 (Fig. 1C) cells were transfected with PTTG and Tax expression vectors, and protein levels were assessed by Western blotting (Fig. 1B and C). Consistent with our HeLa results (above), robust expression of Tax was achieved, but this failed to influence the stability of PTTG in either HCT116 or NIH 3T3 cells (Fig. 1B and C). PTTG expression is cell cycle regulated (5). Because the above assays largely checked the effect of Tax on PTTG expressed from exogenously transfected plasmids, we next examined the effect of Tax on cellular endogenous PTTG during various phases of the cell cycle. Mock- or Tax-transfected HCT116 cells were synchronized in G0/G1 by serum deprivation (lanes 1 and 5), in S phase by hydroxyurea (lanes 2 and 6), and in M phase using nocodazole (lanes 3 and 7) (Fig. 1D; see also Materials and Methods). We then quantified PTTG levels by Western blotting (Fig. 1D). Under synchronized conditions, again no difference in the levels of cell endogenous PTTG was seen in Tax-expressing cells versus control cells (Fig. 1D, compare lanes 1 to 4 with lanes 5 to 8). Although it was previously suggested that Tax induces proteasome-mediated degradation of PTTG (33), we noted that treatment of Tax-expressing cells with the proteasome inhibitor MG132 did not increase the amount of PTTG (Fig. 1D, lane 8). We next investigated the stability of cellular endogenous PTTG in ATL cells that physiologically express Tax. To address this question, we compared the amounts of PTTG in HTLV-1-transformed T-cell lines C8166-45 (C81) and MT4 to that in control non-ATL T-cell lines, Jurkat and CEM-SS (Fig. 2). C81 and MT4 express easily detected amounts of Tax (Fig. 2, middle panel) and, when normalized to actin (Fig. 2, bottom panel), both cell types actually had increased PTTG levels compared to Jurkat and CEM-SS cells (Fig. 2, top panel, compare lanes 1 and 2 to lanes 3 and 4). These results suggest that ATL cells, rather than having reduced PTTG levels, are actually enhanced for PPTG expression, a result consistent with findings from other cancers (18, 53, 57). PTTG and Tax cooperate to induce chromosomal instability. Many cancers that overexpress PTTG are aneuploid (53, 66). One possibility is that increased PTTG, rather than its premature degradation, causes chromosomal instability in tumors. We and others have shown that Tax induces chromosomal instability in cells (15, 25, 47, 49). To compare effects on genetic stability exerted by PTTG, or Tax, or PTTG together with Tax, we generated HCT116 cell lines that stably express Tax alone, PTTG alone, or PTTG plus Tax (Fig. 3A). Expression of PTTG and Tax proteins was confirmed by Western blotting (Fig. 3A), and we asked if overexpressed PTTG and/or

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FIG. 2. Stability of cellular endogenous PTTG is not affected by Tax in ATL cells. Whole-cell lysates from the non-ATL cell lines Jurkat and CEM-SS (lanes 1 and 2) and HTLV-1-transformed C816645 and MT4 cells (lanes 3 and 4) were prepared, resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and probed with anti-PTTG, anti-Tax, or antiactin (for a loading control). PTTG protein expression was measured by densitometry. A representative experiment is shown in panel A, and the means and standard deviations of three experiments are shown in panel B.

Tax affected chromosome stability. Using prometaphase chromosome spreads, chromosome numbers were counted in PTTG, Tax, or PTTG plus Tax cells. Results showed that while less than 1% of control HCT116-neo cells (Fig. 2B, lane 1) were aneuploid, approximately 15% of PTTG-alone or Taxalone cells (Fig. 3B, lanes 2 and 3) and 60% of PTTG plus Tax HCT116 cells were aneuploid (Fig. 2B, lane 4). To investigate the molecular reason for aneuploidy, we visualized microscopically the chromosome spreads from PTTG plus Tax cells (Fig. 3C). When this was performed, we observed a high frequency of tethered sister chromatids. Because chromosome untethering requires cohesin degradation by separase, the failure of attached chromatids to separate is consistent with increased PTTG/securin expression in PTTG plus Tax cells, which blunts the ability of separase to cleave cohesin. To directly verify this reasoning, we next harvested HCT116-neo and HCT116-PTTG plus Tax cells at 0, 20, 40, 60, and 90 min after release from nocodazole-induced prometaphase arrest. Cohesin stability in cells was then compared by Western blotting (Fig. 3D, top panel). Supporting the interpretation that PTTG/securin overexpression slows separase activity, cohesin degradation in HCT116 PTTG plus Tax cells (normalized to cyclin B1 expression) (Fig. 3D, lanes 8 to 12) significantly lagged that seen in control HCT116-neo cells (Fig. 3D, lanes 2 to 6). Thus, it is conceivable that a failure to degrade cohesin in a timely manner explains the increased frequency of aneuploidy in HCT116 PTTG plus Tax cells (Fig. 3B). PTTG cooperates with Tax to enhance cellular proliferation and tumorigenesis. Tax expression correlates with chromosomal instability (39). The above findings suggest that PTTG expression would further increase Tax-induced instability in cells. Because ectopic expression of PTTG alone has been shown to transform mouse fibroblasts (61), we wondered next how expression of PTTG together with Tax would affect cellular proliferation, cell growth in soft agar, and tumorigenesis in mice. To answer these points, we introduced PTTG alone, Tax alone, or PTTG plus Tax into NIH 3T3 fibroblasts and selected for stable cell lines that expressed these proteins.

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FIG. 3. HCT116 cells expressing PTTG plus Tax have increased chromosomal instability. (A) HCT116 cells stably expressing a neo vector, HTLV-1 Tax-neo, HA-PTTG-neo, or Tax plus HA-PTTG-neo were selected using G418. Whole-cell lysates from HCT116-neo (lane 1), HCT116-Tax (lane 2), HCT116-PTTG (lane 3), and HCT116-PTTG plus Tax (lane 4) were prepared, resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and probed with anti-Tax, anti-HA, or antiactin. The PTTG expression level was quantified by densitometry and normalized against actin. (B) Quantification of the frequency of aneuploidy observed from metaphase spreads of HCT116 cells stably expressing a control neo vector, Tax alone, PTTG alone, or Tax plus PTTG. (C) An example of chromosome spreads of HCT116 stably expressing Tax plus PTTG. Before harvesting, cells were treated with nocodazole for 18 h to enrich for mitotic cells, which were collected by mitotic shake-off. Note the frequent finding of tethered sister chromatids (left), with one example shown in an enlarged view (right). (D) Immunoblot analysis of the kinetics of cohesin degradation. Before harvesting, cells were treated with nocodazole to enrich for mitotic cells. Shown are the times in minutes after removal of nocodazole (top). Whole-cell lysates from HCT116-neo (lanes 1 to 6) and HCT116-PTTG plus Tax (lanes 7 to 12) were prepared, resolved by 10% SDS-PAGE, and probed with anticohesin, anti-cyclin A1, anti-cyclin B1, or antiactin (for loading control). Note the delayed cohesin degradation in PTTG plus Tax compared to Neo cells in the top panel, while the degradation kinetics of control cyclin A1 and B1 were unchanged.

Integration of PTTG and Tax DNA in stable transfectants was confirmed by PCR (data not shown), and protein expression was verified by Western blotting (Fig. 4A). The growth of stable cell transfectants in tissue culture was next analyzed. PTTG alone and Tax alone stimulated the proliferation of NIH 3T3 cells (Fig. 4B); however, PTTG plus Tax together showed the strongest growth stimulation (Fig. 4B), as measured by a modified MTT cell proliferation assay (see Materials and Methods) and by cell counting using trypan blue (data not shown). When anchorage-independent growth characteristics in soft agar were monitored, NIH 3T3 cells which overexpressed both PTTG and Tax showed a two- to threefold increase in numbers of colonies formed in soft agar over cells expressing PTTG alone or Tax alone (Fig. 4C and D). Moreover, whereas the numbers of small colonies formed in soft agar were similar between Tax alone or PTTG alone, the ability to form large colonies, a characteristic of malignant cells (11), was primarily seen with cells expressing both PTTG and Tax (Fig. 4C).

We next compared tumorigenicity in athymic mice. Five million (5 ⫻ 106) NIH 3T3 cells, NIH 3T3 Tax-alone cells, NIH 3T3 PTTG-alone cells, or NIH 3T3 PTTG plus Tax cells were injected subcutaneously into the necks of four groups of athymic nude mice (three mice per group) (Fig. 5A). We monitored tumor development and measured the size of tumors which developed (Fig. 5B). Six weeks after injection, the mice were euthanized and tumors were excised and stained with hematoxylin and eosin (Fig. 5C). No major difference in tumor incidence or size between Tax-alone or PTTG-alone cells was seen (Fig. 5A and B). On the other hand, PTTG plus Tax cells produced tumors that were nearly twice as large in volume at the time of sacrifice (Fig. 5A and B). Histological analyses of the tumors revealed poorly differentiated sarcomas composed of many pleomorphic mitotic cells (Fig. 5C). These findings verify that PTTG and Tax are individually sufficient to initiate a tumorigenic phenotype in NIH 3T3 cells and that coexpression of PTTG plus Tax increases the robustness of both anchorage-independent cell growth and tumorigenicity.

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FIG. 4. Coexpression of PTTG plus Tax enhances cellular transformation. (A) Stable expression of neo, PTTG, Tax, and PTTG plus Tax in G418-selected NIH 3T3 clones. Whole-cell lysates from NIH 3T3 neo (lane 1), NIH 3T3 Tax-neo (lane 2), NIH 3T3 PTTG-neo (lane 3), and NIH 3T3 PTTG plus Tax-neo (lane 4) were prepared, resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and probed with anti-Tax, anti-HA (for PTTG detection), or antiactin (for loading control). (B) Tax, PTTG, and PTTG plus Tax expression enhance cell proliferation. NIH 3T3 cells expressing NIH 3T3 neo, NIH 3T3 Tax-neo, NIH 3T3 PTTG-neo, and NIH 3T3 PTTG plus Tax-neo were cultured in DMEM plus low serum (0.1% FCS). Cells were monitored for proliferation using WST-8 [2-(2-methoxy-4-nitrophenyl)3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] coloration (Dojindo) according to the manufacturer’s protocol. Relative absorbances at 450 nm shown in arbitrary values are means ⫾ standard deviations of three independent experiments. (C) NIH 3T3 cells were stably transfected with control vector (panels 1 and 2), Tax (panels 3 and 4), PTTG (panels 5 and 6), or Tax plus PTTG (panels 7 and 8). Cells were plated at a concentration of 1,000 cells/ml and tested for their ability to form colonies in soft agar (see Materials and Methods). (D) Colonies, as indicated, were counted after 14 days. The mean values from three different experiments are shown.

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FIG. 5. In vivo tumorigenicity of PTTG plus Tax NIH 3T3 cells in nude mice. (A) Six-week-old female athymic nude mice (Harlan, Indianapolis, IN) were injected subcutaneously in the neck region with 5 ⫻ 106 G418 NIH 3T3 neo (panel 1), NIH 3T3 PTTG-neo (panel 2), NIH 3T3 Tax-neo (panel 3), or NIH 3T3 PTTG plus Tax-neo (panel 4) cells. (B) Following injection, mice were monitored with calipers, and mice were sacrificed as described in the text. (C) The tumors were resected and stained with hematoxylin and eosin for histopathological examination. Tax or PTTG alone induced poorly differentiated fibrosarcomas (panels 1 and 2) with mitotic figures (arrows); tumors induced by coexpression of Tax plus PTTG displayed fibrosarcomas and carcinomas with foci of necrosis (panel 3).

DISCUSSION Genomic instability develops in a wide variety of tumors, such as colon, breast, prostate, or lung cancers, as well as in adult T-cell leukemia (4, 29, 37, 38). For genetic damage to emerge and become sustained, multiple discrete events in a normal cell need to be disrupted. Dysregulated cellular functions involving centrosome amplification, silencing of checkpoints, and progression of the cell cycle have all been described to contribute to cellular transformation (29, 50). In published reports, HTLV-1 Tax has been shown to promote genomic instability through impairment of these various processes (22, 25, 27, 35, 47). A tightly regulated separase activity is critical for proper chromosome segregation. Separase triggers partitioning of sister chromatids and influences mitotic spindle stability and mitotic exit (55, 56). PTTG/securin is a physiological inhibitor of separase, and aberrant PTTG function can alter the mechanics of chromosome segregation and create aneuploidy (52). Previously, it was suggested that Tax promotes premature degradation of PTTG by activating the APC proteasome ahead of schedule (32, 33). However, the notion that the HTLV-1 Tax oncoprotein destabilizes and reduces the amount of PTTG is incongruent with the many reports of PTTG overexpression in numerous other human malignancies, including pituitary, thyroid, and colon cancers (18, 53, 57, 63). Moreover, PTTG overexpression has been linked directly to transformation of NIH 3T3 fibroblasts (61). In our current study, we found that Tax did not increase PTTG degradation (Fig. 1). This result is different from that

previously reported. The reason for the discrepancy between our results and the previous work (32, 33) is unknown but could be due to the different experimental systems used. Earlier, Liu et al. employed both Saccharomyces cerevisiae and HeLa cells for their studies. It is worth noting several differences between mitosis in yeast and mammalian cells. In yeast, cohesin persists at centrosomes and along chromosome arms until the onset of anaphase, whereupon it is removed by the APC/Cdc20 pathway (58). In mammalian cells, however, the bulk of cohesin dissociates from chromosome arms but not from centrosomes during prophase and prometaphase. Further, it is possible, because HeLa cells are heterogeneous cells, that Liu et al. used a HeLa clone with characteristics different from ours. Nevertheless, the findings we saw in our HeLa cells (Fig. 1A) are consistent with our results in HCT116 cells (Fig. 1B), NIH 3T3 cells (Fig. 1C), and C81 and MT4 cells (Fig. 2). An important mechanistic question raised by our study is how do Tax and PTTG/securin cooperate to increase cellular transformation? Several groups have shown that increased amounts of PTTG extend the duration of metaphase, promote improper separation of sister chromatids, and create abnormal cytokinesis (8, 17, 67, 69). At the same time, overexpression of Tax alone has also been shown to induce aneuploidy and binucleated cells (7, 20, 25, 34, 45, 47). Taken together, a reasonable interpretation that reconciles extant observations is that aneuploidy in PTTG plus Tax cells arises not from premature degradation but from increased stability of securin, which delays cleavage of cohesin (Fig. 3) and creates failed separation of sister chromatids (Fig. 3). Unsegregated sister

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chromatids that migrate into progeny nuclei would account for aneuploidy. Additionally, an increase in PTTG/securin (Fig. 2), rather than a decrease in PPTG/securin (33), makes our HTLV-1/ ATL findings consistent with other cancers which generally show increased PTTG. Our results add to the body of evidence which suggests complexity in mechanisms regulating euploidy (29, 30, 50, 54). Multiple mechanisms, such as improper amplification of the centrosome and loss of the spindle assembly checkpoint, contribute to the emergence of aneuploidy (29, 47, 50, 54). Here it was shown that reduced separase activity due to overexpression of PTTG/securin can be another independent route used by HTLV-1 to create aneuploidy. Understanding the discrete mechanisms for creating aneuploidy is important, given the increasing recognition that aneuploidy may be causal for carcinogenesis (29, 30, 50, 54). ACKNOWLEDGMENTS

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We thank Ron Plishka and Alicia Buckler-White (NIAID, NIH) for DNA sequencing and oligonucleotide synthesis, Shlomo Melmed (David Geffen School of Medicine, University of California, Los Angeles) for the pCI-PTTG plasmid, Jorge Chavez Robles and Erik Michael Lasker (Charles River Laboratories) for their help in photography of the mice, and members of the Jeang laboratory for critical readings of the manuscript. Work in K.-T.J.’s laboratory is funded by intramural funds from the NIAID, NIH.

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