A Role for PML3 in Centrosome Duplication and Genome Stability

Molecular Cell, Vol. 17, 721–732, March 4, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.02.014

A Role for PML3 in Centrosome Duplication and Genome Stability Zhi-Xiang Xu,1 Wen-Xin Zou,1 Pei Lin,2 and Kun-Sang Chang1,* 1 Department of Molecular Pathology 2 Department of Hematopathology Division of Pathology and Laboratory Medicine The University of Texas M.D. Anderson Cancer Center 1515 Holcombe Boulevard Houston, Texas 77030

Summary The promyelocytic leukemia gene (PML), which is disrupted by the chromosomal translocation t(15;17) in acute promyelocytic leukemia (APL), encodes a multifunctional protein involved in several important cellular functions. Herein, we demonstrate that PML is localized to centrosomes and that PML deficiency leads to centrosome amplification. By using PML isoformspecific antibodies, we found PML3-specific association with the centrosome and the pole of the mitotic spindle. PML3 deficiency leads to dysregulation of the centrosome duplication checkpoint. Furthermore, PML3 physically interacts with Aurora A and regulates its kinase activity. Specific knockdown of PML3 activates Cdk2/cyclin kinase activity. The results of this study implicate a direct role for PML3 in the control of centrosome duplication through suppression of Aurora A activation to prevent centrosome reduplication. Introduction The promyelocytic leukemia tumor suppressor gene (PML) was originally identified for its involvement in the chromosomal translocation t(15;17) in patients with APL. This fuses the PML gene with the retinoic acid receptor α (RARα) gene to create two fusion genes: PML-RARα and RARα-PML (Melnick and Licht, 1999). PML is a multifunctional protein involved in multiple apoptosis pathways (Wang et al., 1998), tumor growth suppression and cell cycle regulation (Salomoni and Pandolfi, 2002), ras-induced premature cellular senescence (Ferbeyre et al., 2000), and transcriptional regulation (Lin et al., 2001; Zhong et al., 2000). PML is the essential component of the nuclear body (NB) designated PML NB, PML oncogenic domain, or nuclear domain 10 (Melnick and Licht, 1999). SUMO-1 modification of PML is required for the formation of PML NBs and for many of its cellular functions (Seeler and Dejean, 2001). At least seven major PML isoforms generated by alternative splicing have been described (Jensen et al., 2001). Five of them (PML1, PML2, PML3, PML4, and PML5) consist of unique variable C-terminal sequences. The biological significance of these isoforms in mammalian cells has not been well charac*Correspondence: [email protected]

terized. Most of the studies on PML functions reported in the literature have been performed with PML4. A recent study demonstrated an important role for the cytoplasmic PML isoform in regulating transforming growth factor-β signaling events (Lin et al., 2004). Others recently reported PML1-specific functional interaction with the hematopoietic transcription factor AML1 (Nguyen et al., 2004). The functional significance of the other PML isoforms is unknown. Recent findings suggest that PML plays a role in the maintenance of genome stability and DNA damage response (Salomoni and Pandolfi, 2002; Carbone et al., 2002; Bischof et al., 2001; Xu et al., 2003). PML colocalizes in vivo with BLM, a RecQ DNA helicase deficient in patients with Bloom syndrome (Zhong et al., 1999). Deficiency of BLM results in genome instability (Ellis and German, 1996; Watt and Hickson, 1996). PML was found to associate in vivo with several DNA-repair proteins, including Mre11, Rad51, Rad 9, ATM, and γ-H2AX, and localized to the single-stranded DNA repair foci in response to ionizing radiation (Mirzoeva and Petrini, 2001; Carbone et al., 2002; Xu et al., 2003). A role for PML in genome stability was further suggested by the presence of a high percentage of sister chromatid exchange in PML−/− mouse embryo fibroblasts (MEFs) (Zhong et al., 1999). The development and growth of all living organisms involve the faithful reproduction of cells and require that the genome be accurately replicated and equally participate in the two progenies. Genomic instability is characterized by chromosome losses or gains (aneuploidy) as well as chromosome rearrangements. Loss of control of centrosome duplication or amplification is often associated with aneuploidy in most human cancers (Lingle et al., 2002; Ghadimi et al., 2000). In mammalian cells, the centrosome is comprised of a pair of centrioles and amorphous pericentriolar materials. The centrosome is duplicated once during a normal cell cycle starting at the G1 phase; the duplication is completed by the end of the G2 phase (Nigg, 2002). During mitosis, the two centrosomes separate to organize the bipolar mitotic spindle and determine the position and orientation of the cleavage furrow to ensure equal segregation of sister chromatids to each daughter cell. An increasing number of proteins has been found in centrosomes, including cell cycle regulators, protein phosphatases, cell-signaling proteins, and tumor suppressors (Nigg, 2002; D’Assoro et al., 2002; Tarapore and Fukasawa, 2002). Abnormalities in these proteins may result in centrosome amplification, aneuploidy, and genome instability. The molecular mechanism of how centrosome duplication is controlled in mammalian cells is not well understood. Many kinases and phosphatases have been found to be involved in this process, including Cdk2/cyclin E (Hinchcliffe and Sluder, 2002), the Pololike kinases (Glover et al., 1998; Hoffmann 2004), and Aurora A (Zhou et al., 1998; Goepfert and Brinkley, 2000). Cdk2/cyclin E kinase is the major regulator of the early events of centrosome duplication (Hinchcliffe and

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Sluder, 2002). Phosphorylation of several of the targets of Cdk2/cyclin E, including nucleophosmin/B32 (Okuda et al., 2000), Mps-1 (Fisk and Winey, 2001), and CP110 (Chen et al., 2002), is essential for initiating centrosome duplication. The p53 tumor suppressor is the major transcription activator of p21, which binds Cdk2/cyclin A or E and inhibits its kinase activity. p53-deficient cells consistently display centrosome amplification through loss of control of the centrosome duplication cycle (Tarapore and Fukasawa, 2002), indicating that the p53 signaling pathway plays a critical role in regulating centrosome duplication, possibly through regulation of Cdk2/cyclin activity. In this study, we found that PML3 is associated with the centrosome in vivo and a high percentage of cells with centrosome amplification in PML-deficient cells. We also observed that PML3-specific knockdown by small interfering RNAs (siRNAs) causes centrosome amplification and that reexpression of PML3 suppresses centrosome amplification in PML−/− MEFs. Furthermore, we found that PML3 physically interacts with Aurora A and inhibits its kinase activity. Our findings indicate a direct role for PML3 in the control of centrosome duplication through suppression of Aurora A activation, which prevents centrosome reduplication. Results PML Is Associated with Centrosomes In Vivo The centrosome is central to the control of cytokinesis. Centrosome dysregulation causes genome instability and tetraploidy. We sought to determine whether PML deficiency affects centrosome function. Figure 1A demonstrates that normal MEFs consisted of two centrosomes on each end of the mitotic poles in cells at metaphase. However, the number of centrosomes increased in PML−/− MEFs. More than 35% of the PML−/− MEFs (passage 5) consisted of amplified centrosomes (Figure 1B). Some of the PML-associated proteins, such as Brca1 and TopBP1, were localized to the centrosomes (Xu et al., 2003; Hsu and White, 1998; Reini et al., 2004). We next examined PML to see whether it is associated with the centrosome in vivo. We performed double-color immunofluorescence (IF) staining of 293T cells with PML polyclonal and γ-tubulin or Aurora A monoclonal antibodies. We analyzed colocalization between PML and γ-tubulin or Aurora A under a confocal microscope. Figure 1C demonstrates that PML was indeed colocalized with these centrosomeassociated proteins in vivo. These results suggest that PML has a role in maintaining genome stability by associating with the centrosomes. PML Function Is Required for Maintaining Centrosome Integrity To further support a role for PML in maintaining centrosome integrity, we treated the diploid cell line 293T and CCD-37 cells (normal human lung fibroblasts; at passage 7) with three consecutive rounds of PMLspecific siRNAs. By using two sets of siRNAs as we described previously (Xu et al., 2003), we successfully knocked down more than 80% of the PML protein expression in these cells. We determined the effects of

PML knockdown on the number of centrosomes by IF staining with a γ-tubulin antibody. The Western blot analysis results presented in Figure 1D show that PML expression decreased substantially in both cell types after treatment. Interestingly, the number of cells with centrosome amplification increased after each round of siRNA treatment (Figures 1E and 1F and Figure S1 available with this article online). More than 30% of the cells exhibited centrosome amplification after three rounds of siRNA treatment. These findings strongly suggest that PML function is required for maintaining the appropriate number of centrosomes in mammalian cells. Isoform-Specific Localization of PML3 to the Centrosomes At least seven major PML isoforms are expressed in human cells (Jensen et al., 2001), five of which consist of unique C-terminal sequences. Taking advantage of this, we successfully produced PML isoform-specific antibodies against the PML1, PML2, PML3, PML4, and PML5 proteins (Figure S2). We performed double-color IF staining with the PML isoform-specific antibodies and a monoclonal antibody against Aurora A in the MO59K cell line. Figure 2A demonstrates that only the PML3-specific antibody recognized a signal that was colocalized with Aurora A. Furthermore, PML3 was the only PML isoform colocalized with γ-tubulin, supporting the conclusion that PML3 is specifically associated with the centrosome in vivo (Figure 2B). Figure 2C shows that PML3 was associated with γ-tubulin in vivo in cells at interphase, prometaphase, metaphase, and anaphase. We performed similar experiments with the Aurora A antibody and observed comparable results (data not shown). Also, we found PML3 colocalization with the centrosomes in several other cell lines (Figure S3). To further confirm the endogenous association between PML3 and centrosomes, we partially purified the centrosomal complex by discontinuous sucrose gradient centrifugation as described previously (Hsu and White, 1998). Figure 2D shows that PML3 was the only PML isoform copurified with the fractions enriched in γ-tubulin. Together, these results conclusively demonstrated PML3-specific localization to the centrosomes. PML3 Function Is Required for Maintaining an Appropriate Number of Centrosomes Figures 1D–1F show that loss of PML function induced centrosome amplification. We next sought to determine whether loss of PML3 function alone is sufficient to induce centrosome amplification. We designed a series of siRNAs to specifically knock down the five major PML isoforms. Figure 3A shows the feasibility of each siRNA in inhibiting the expression of PML1 to PML5, respectively, in both 293T and CCD-37 cells. The siRNAs were able to knock down expression of about 80% of each isoform. Interestingly, specific knockdown of PML3, but not the other isoforms, increased the number of cells with centrosome amplification (Figures 3B and 3C). The results in Figure 1 demonstrate that a significant number of PML−/− MEFs displayed centrosome amplification. We next investigated whether overexpression of PML3 by transient transfection restores the

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Figure 1. A Role for PML in Centrosome Function (A) PML deficiency induced centrosome amplification. IF staining was performed with the PML+/+ and PML−/− MEFs using the antibody against Aurora A. Fluorescence images were recorded with a Kodak digital imaging system mounted on top of a Leica fluorescence microscope. (B) Percentage of centrosome amplification in early-passage (passage 5) MEFs from PML+/+ and PML−/− mice. Centrosomes were immunostained with a monoclonal antibody against Aurora A as described above. The results are shown as average ± SEM from three experiments. 200 cells were counted in each experiment. (C) Colocalization of PML and the centrosome proteins Aurora A and γ-tubulin. Double-color IF staining was performed with 293T cells using γ-tubulin and Aurora A monoclonal antibodies and a rabbit PML polyclonal antibody. Chromosomal DNA was counterstained with TOPRO. Images were captured with a Zeiss LSM 5 confocal microscope. (D) 293T and CCD-37 cells were repeatedly treated with two pairs of siRNAs as described in the Experimental Procedures. Western blot analysis was performed with total protein isolated from each round of siRNA treatment to examine the effects on PML expression. (E and F) Percentage of 293T (E) and CCD-37 (F) cells with amplified centrosomes after each round of siRNA treatment. Double-color IF staining was performed with a PML polyclonal antibody and γ-tubulin monoclonal antibody. Results represent an average ± SEM of three independent experiments from 200 cells (PML knockdown) counted per group.

normal number of centrosomes in PML−/− MEFs. We transiently transfected these MEFs with the expression plasmids of different PML isoforms. We performed double-color IF staining with PML isoform-specific antibodies and γ-tubulin monoclonal antibody. We counted 200 positively transfected cells in each group for the presence of excess centrosomes. Figure 3D shows that reexpression of PML3, but not the other isoforms, significantly reduced the number of cells with centrosome amplification. Together, these findings demonstrated an important role for PML3 in maintaining an appropriate number of centrosomes in mammalian cells. PML3 Colocalizes with ␣-Tubulin at the Pole of the Mitotic Spindle We next sought to further understand the role of PML3 in centrosome function. Figure 4A shows that PML3

colocalized with α-tubulin after washing with a cold stripping buffer, indicating that PML3 was located at the spindle pole. We repeated this washing with the human leukemia cell line U937; PML3 remained colocalized with α-tubulin. To further explore whether PML3 colocalization with the spindle pole is microtubules independent, we exposed Mo59K cells to nocodazole for 24 hr to disrupt microtubules. The results show that PML3 remained colocalized with α-tubulin (Figure 4A). These studies thus suggest that PML3 localization to the centrosome is independent of microtubles. Furthermore, in PML-deficient cells, the aberrant centrosomes colocalized with the active sites of microtubule nucleation, indicating that this event leads to aberrant mitosis (Figure 4B). Additionally, a significantly higher number of cells displayed aberrant mitotic spindles in PML−/− MEFs than in PML+/+ MEFs (Figure 4C).

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Figure 2. PML3-Specific Localization to the Centrosome (A and B) Proliferating Mo59K cells were fixed, and double-color IF staining was performed with PML isoform-specific polyclonal antibodies and a monoclonal antibody against the centrosome protein Aurora A or γ-tubulin. Chromosomal DNA was counterstained with DAPI. Images were captured as described for Figure 1. (C) Localization of PML3 to the centrosome during various cell cycle phases. MO59K cells were fixed, and double-color IF staining was performed with the PML3-specific polyclonal antibody and γ-tubulin monoclonal antibody. Chromosomal DNA was counterstained with TOPRO. Images of cells at various cell cycle phases were captured with a Zeiss LSM 5 confocal microscope. (D) Detection of PML3 in the centrosome fraction by discontinuous sucrose gradient centrifugation. Protein isolated from each gradient fraction within the 40%–70% range was subjected to Western blot analysis with PML isoform-specific antibodies and a γ-tubulin antibody.

PML3 Colocalizes with the Centrosomes in APL Cells In APL blasts and APL-derived NB4 cells, the normal functions of PML are disrupted as a result of the chromosomal translocation t(15;17), which fuses the PML gene with the RARα gene. The fusion protein PMLRARα forms a heterodimer with the normal PML protein and sequesters it to a different nuclear location to produce the APL-specific microspeckled staining pattern (Melnick and Licht, 1999). Based on these findings, one would expect loss of PML function in APL to cause genome instability and centrosome amplification. To determine whether centrosome amplification is common in APL cells, we performed double-color IF staining with a PML3-specific polyclonal antibody and γ-tubulin monoclonal antibody in nine APL blasts and NB4 cells. We found normal colocalization between PML3 and

γ-tubulin in the centrosomes in APL cells (Figure 5). Double-color IF staining with PML1-, PML2-, PML4-, and PML5-specific antibodies showed the typical APLspecific nuclear microspeckled staining pattern in both the APL blasts and NB4 cells; staining with a PML3 specific antibody showed a cytoplasmic speckled staining pattern that colocalized with γ-tubulin. These results indicated that although other PML isoforms form heterodimers with PML-RARα and delocalize in the nucleus of APL blasts, PML3 apparently functions normally by associating with the centrosomes in the cytoplasm. Deregulation of Centrosome Reduplication in PML-Deficient Cells In normal cells, centrosome duplication occurs during late G1 to S phase. Significant evidence supports the existence of a centrosome duplication checkpoint, which

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Figure 3. PML3-Specific Knockdown by siRNA Induces Centrosome Amplification (A) Specific knockdown expression of different PML isoforms by siRNA. The specific siRNAs against PML isoforms and a mock control were designed and transfected into proliferating 293T and CCD-37 cells as described in the Experimental Procedures. At 72 hr after transfection, total proteins were isolated for Western blot analysis with the PML isoform-specific antipeptide antibodies. (B) Cells treated with PML isoform-specific siRNAs were fixed, and IF staining was performed with a γ-tubulin monoclonal antibody. (C) PML3 knockdown by siRNA induced centrosome amplification in 293T and CCD-37 cells. The fold increase in the number of 293T cells with centrosome amplification was determined by counting an average of 200 cells (PML knockdown) per group. Values are mean ± SEM of two independent experiments. (D) The effects of reexpression of different PML isoforms by transient transfection on centrosome amplification in PML−/− MEFs. MEFs were transiently transfected with the expression plasmids pcDNA3/PML1, pcDNA3/PML2, pcDNA3/PML3, pcDNA3/PML4, and pcDNA3/PML5 by using FuGENE 6. At 72 hr after transfection, cells were fixed, and double-color IF staining was performed with a γ-tubulin monoclonal antibody and PML isoform-specific polyclonal antibodies. The percentage of cells with centrosome amplification was determined by counting an average of 200 positively transfected cells. The results represent an average ± SEM of three independent experiments. Total proteins in each group were isolated, and Western blot analysis was performed with the PML antibody against the GST-PML fusion protein (Xu et al., 2003). The filter was reprobed with an α-tubulin antibody to serve as a loading control.

ensures that centrosome duplication occurs only once per cell cycle to maintain two centrosomes during mitosis and cell division (Nigg, 2002). When DNA replication is arrested by drugs such as hydroxyurea, this checkpoint allows only one round of centrosome duplication. In some cell lines, such as CHO, U2OS, and p53 null cell lines, treatment with hydroxyurea allows for several rounds of centrosome reduplication, generating multiple centrosomes per cell (Nigg, 2002; Tarapore and Fuka-

sawa, 2002). How this checkpoint is regulated is unknown, however. To determine whether PML is involved in regulating centrosome duplication, we treated PML+/+ and PML−/− MEFs with hydroxyurea, harvested them at various time points, and performed IF staining with a γ-tubulin antibody. Figures 6A and S4 show that the number of PML−/− MEFs with amplified centrosomes increased progressively, but that of the PML+/+ MEFs did not. We

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Figure 4. PML3 Is Located at the Pole of the Mitotic Spindle (A) Proliferating Mo59K and U937 cells were fixed and coimmunostained with anti-PML3 (red) and anti-α-tubulin (green) antibodies. Cells were treated with ice-cold stripping buffer or incubated with nocodazole for 24 hr to disrupt the microtubules. Colocalization of PML3 and α-tubulin was determined by double-color IF staining with an α-tubulin monoclonal antibody and PML3 polyclonal antibody. (B) The aberrant centrosomes in PML-deficient cells colocalized with active sites of microtubule nucleation. Double-color IF staining was performed with anti-α-tubulin monoclonal and anti-γ-tubulin polyclonal antibodies in PML+/+ and PML−/− MEFs. (C) Percentage of PML+/+ and PML−/− MEFs containing the aberrant multipolar spindles. The results represent the average percentage ± SEM determined in three independent experiments. In each experiment, at least 200 dividing cells were counted.

repeated this experiment with immortalized PML−/− MEFs established in our previous study (Xu et al., 2003) and T antigen-transformed PML−/− MEFs with similar results. These findings suggest that in the absence of PML, the mechanism that prevents centrosome reduplication was impaired, allowing continued centrosome duplication in the absence of DNA replication. To examine the integrity of the amplified centrosomes as a result of DNA replication arrest, we sought to determine whether they consist of a pair of centrioles as described by Bennett et al. (2004). We performed double-color IF staining with α-tubulin and centrin-1. Because α-tubulin is one of the major components of centrioles, the enlarged views of the α-tubulin staining in Figure 6B (bottom) show that many of the centrosomes indeed consisted of centriole pairs, suggesting that these are intact centrosomes. Furthermore, doublecolor IF staining with centrin-1 and γ-tubulin demonstrated that they colocalize in amplified centrosomes in PML−/− MEFs. To further examine the role of PML3 deficiency in loss of control of the centrosome duplication checkpoint,

we treated 293T and CCD-37 cells with specific siRNAs to knock down PML1 and PML3 in a manner similar to that shown in Figure 3A. Figures 6C and 6D show that PML3, but not PML1, knockdown increased the number of cells with centrosome amplification. Also, treatment with hydroxyurea arrested cells at early S phase, significantly increasing the number of cells with centrosome amplification. These results implicate PML3 in regulation of centrosome duplication. Functional Interaction and Repression of Aurora A Activity by PML3 Aurora A is a serine/threonine protein kinase that is associated with the centrosome and plays a role in regulating mitotic microtubules and centrosome maturation (Hannak et al., 2001; Giet et al., 2002). Overexpression of Aurora A has been shown to induce centrosome amplification (Zhou et al., 1998). To further understand the mechanism of PML3-mediated centrosome function, we sought to determine whether there was a functional interaction between PML3 and Aurora A. We performed coimmunoprecipitation with Aurora A and PML iso-

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Figure 5. Colocalization of PML3 and the Centrosomes in APL Blasts and in the APL-Derived NB4 Cells APL bone morrow samples and NB4 cells were immobilized on slides by cytocentrifugation. Double-color IF staining was performed with a PML isoform-specific polyclonal antibody and the γ-tubulin monoclonal antibody. The images were captured as described in Figure 1A.

form-specific antibodies. Figure 7A shows that only the PML3-isoform-specific antibody coimmunoprecipitated the Aurora A protein. Similarly, the Aurora A antibody specifically coimmunoprecipitated PML3, but not the other PML isoforms (Figure 7B). This demonstrated a physical interaction between PML3 and the Aurora A protein. Phosphorylation of Aurora A on Thr288, which is located in the activation T loop of the kinase, markedly increases its enzymatic activity (Walter et al., 2000). The phosphorylation site at Thr288 is an indicator of the enzymatic activity of Aurora A (Hirota et al., 2003). We next investigated whether PML3 affects Aurora A kinase activity in vivo. We transiently transfected 293T cells with the expression plasmids of different PML isoforms and examined the effect of Aurora A on Thr288 phosphorylation by using a Thr288 phosphospecific antibody. Figure 7C shows that transient overexpression of PML3, but not the other PML isoforms, substantially reduced Thr288 phosphorylation of Aurora A, but expression of the Aurora A protein was not affected. This suggests that PML3 physically interacted with Aurora A in vivo and repressed Aurora A kinase activity by inhibiting Thr288 phosphorylation. Recent findings demonstrated that Aurora A phosphorylates Ser315 of p53 and induces destabilization of the p53 protein (Katayama et al., 2004). p53 activates transcription of p21, which binds and inhibits Cdk2/ cyclin functions. Cdk2/cyclin E plays an essential role in regulating centrosome duplication (Hinchcliffe and Sluder, 2002). To further elucidate the mechanism of how PML3 is involved in centrosome duplication, we examined whether PML3 affects Cdk2/cyclin E function. We found comparable Cdk2, cyclin E, and p21 expression levels in the cytosol and nuclear fraction be-

tween PML−/− and control PML+/+ MEFs (Figure 7D). However, a coimmunoprecipitation assay with a Cdk2 antibody demonstrated significantly higher levels of cyclin E and much lower levels of p21 protein associated with Cdk2 in the nucleus of PML−/− MEFs (Figure 7E). This indicated that in the absence of PML, a lower level of p21 binds the Cdk2/cyclin E complex, leading to a significantly higher level of Cdk2/cyclin E kinase complex. We next determined whether increased Cdk2 activity could be found in the PML-deficient cells. We used the immunoprecipitated Cdk2-associated complex to assess the levels of kinase activity. In agreement with the results in Figure 7E, this study showed significantly higher levels of Cdk2 kinase activity in the PML−/− MEFs (Figure 7F). To further confirm that PML3 downregulation alone is sufficient for increasing Cdk2 activity, we used a PML3-specific siRNA to knock down PML3 expression as shown in Figure 3A. Figures 7G and 7H show that downregulation of PML3 by this siRNA, but not a control siRNA, increased Cdk2 kinase activity in the kinase assay. Taken together, our results suggest that PML3 regulates Cdk2 kinase activity through inhibition of Aurora A kinase. Discussion PML3 Plays a Role in Maintaining Centrosome Integrity and Genome Stability We found that PML colocalizes with centrosomes in vivo and that PML deficiency induces centrosome amplification. This demonstrates the existence of a mechanism through which PML is directly involved in the maintenance of genome stability by regulating a proper number of centrosomes during cell cycle progression.

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Figure 6. Deregulation of the Centrosome Duplication Cycle in PML-Deficient Cells Leads to Centrosome Amplification (A) PML+/+ and PML−/− MEFs were treated with 2 mM hydroxyurea. Cells were harvested at various time points as indicated and analyzed to determine the number of centrosomes by staining with a γ-tubulin antibody. The results represent an average ± SEM of three independent experiments. (For all the graphs presented in this section, we counted 200 cells per group.) (B) To determine whether the amplified centrosomes consisted of a pair of centrioles, double-color IF staining was performed with polyclonal centrin-1 and monoclonal α-tubulin antibodies at 48 hr after hydroxyurea treatment. Colocalization of centrin-1 and α-tubulin was shown in the amplified centrosome (bottom). (C and D) 293T (C) and CCD-37 (D) cells were treated with PML1- and PML3-specific siRNAs as described for Figure 4. The percentage of cells (PML1 or PML3 knockdown) with more than two centrosomes after treatment with or without 2 mM hydroxyurea (HOU) for 48 hr was determined. The results represent an average ± SEM of three independent experiments.

Although a previous study reported that all PML isoforms are expressed in the same cells (Fagioli et al., 1992), substantial evidence indicates that different PML isoforms have different cellular functions. For example, our recent report demonstrated PML4-specific repression of survivin expression (Xu et al., 2004). PML4 was also shown to specifically associate with p53 (Fogal et al., 2000), form a complex with MDM2 and regulate p53 stability in response to DNA damage (Louria-Hayon et al., 2003), and be involved in premature cellular senescence (Bischof et al., 2002). An unknown mechanism of alternative splicing of the primary PML transcript is responsible for generating all of the PML isoforms (Jensen et al., 2001). We have successfully produced PML isoform-specific antipeptide antibodies against each of the five major PML isoforms. Herein, we demonstrate that PML3 is localized to the centrosome in vivo during various stages of the cell cycle. We further show that PML3 is localized to the pole of the mitotic spindle. Additionally, we show that an aberrant centrosome co-

localizes with the active sites of microtubule nucleation in PML-deficient cells, strongly suggesting that this event leads to chromosome missegregation and aberrant mitosis. The consequences of such aberrant mitosis will most likely trigger cell death by apoptosis. In cancer, centrosome amplification appears to be a consistent feature of tumor cells with aneuploidy (Nigg, 2002). The exact mechanism of how such cells are capable of completing mitosis to produce two daughter cells with more than two centrosomes is not clear. We designed PML isoform-specific siRNAs capable of knocking down the five major PML isoforms. We showed that loss of PML3 function alone is sufficient to induce centrosome amplification (Figure 3C). Furthermore, reexpression of PML3, but not other PML isoforms, in PML−/− MEFs significantly reduced the number of cells with centrosome amplification (Figure 3D). These findings demonstrate a role for PML3 in maintaining a proper number of centrosomes in mammalian cells. PML functions have been compromised in APL cells as

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Figure 7. PML3 Physically Interacts with Aurora A, Inhibits Its Kinase Activity, and Affects Cdk2/Cyclin Activity (A) Coimmunoprecipitation (CoIP) was performed by using total protein isolated from U2OS cells with PML isoform-specific antibodies. Western blotting (WB) was performed with an Aurora A antibody. (B) CoIP was performed by using an Aurora A antibody, and WB was carried out with different PML isoform-specific antibodies. (C) The effects of various PML isoforms on Aurora A expression and Thr288 phosphorylation. PML expression plasmids were transiently transfected into U2OS cells. Western blot analysis was performed by using the Aurora A or Aurora A Thr288 phosphospecific antibody. The Western blot was reprobed with a PML polyclonal antibody, which recognized all of the PML isoforms. (D) Subcellular localization of Cdk2, cyclin E, and p21 in PML+/+ and PML−/− MEFs. Equal quantities of protein fractions (30 µg) from PML+/+ and PML−/− MEFs isolated from cytoplasm (C), the nucleus (N), and total proteins (T) were analyzed for the expression of Cdk2, cyclin E, and p21 with WB. The same filter was reprobed with an anti-α-tubulin antibody to serve as a cytoplasmic protein loading control and anti-histone H3 antibody as a nuclear protein loading control. (E) Association of cyclin E and Cdk2 in normal and PML−/− MEFs. A coimmunoprecipitation assay was performed by using a protein fraction (100 µg) isolated from C, N, and T with a Cdk2-specific antibody. WB analysis of the precipitated proteins was performed by using cyclin E, Cdk2, and p21 antibodies. (F) Cdk2 kinase activity in PML−/− and normal MEFs. A coimmunoprecipitation assay was performed with a rabbit polyclonal anti-Cdk2 antibody. The immunoprecipitated proteins in protein A-Sepharose beads were used for kinase activity analysis with histone H1 as a substrate. Cdk2/cyclin E kinase was used as a positive control. (G) Inhibition of PML3 expression by a specific siRNA. PML3-specific knockdown by siRNA was performed as described in Figure 3. (H) The effects of PML3 knockdown on Cdk2 activity. The Cdk2 activity in cells treated with a PML3 and control siRNA was determined as described for (F).

a result of fusion of the PML gene with the RARα gene by t(15;17)(q22;q21). Interestingly, our study demonstrates that PML3 was associated with the centrosomes in all nine APL cases tested and the APLderived NB4 cells. This indicates that although the fusion protein PML-RARα forms a heterodimer and sequesters PML to the abnormal microspeckled nuclear domains as shown previously (Melnick and Licht, 1999),

PML3 was not affected by PML-RARα in APL cells and functions normally in the centrosome. Mechanism of PML3-Mediated Centrosome Duplication The centrosome is central to the control of cytokinesis. Centrosome dysregulation causes genome instability and aneuploidy (Nigg, 2002). In normal cells, centro-

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some duplication occurs during late G1 to S phase. Evidence supports the existence of a centrosome duplication checkpoint, which ensures that this event occurs only once per cell cycle to maintain two centrosomes during mitosis. The cell cycle-dependent kinase Cdk2 is one of the major regulators of centrosome duplication. Evidence demonstrates that the early events of centrosome duplication depend on Cdk2/cyclin activity (Hinchcliffe and Sluder, 2002). One of the important targets of this kinase is nucleophosmin (NPM). NPM is associated with the unduplicated centrosome in early G1 phase but dissociates from the centrosome once phosphorylated by Cdk2/cyclin kinases. Importantly, centrosome duplication fails to occur if NPM phosphorylation is blocked (Okuda et al., 2000). Other Cdk2 substrates involved in centrosome function include Mps1 (Fisk and Winey, 2001) and CP110 (Chen et al., 2002). Our study showed that PML colocalizes with Aurora A during various phases of the cell cycle. PML3, but not other PML isoforms, physically interacts with Aurora A at endogenous levels and regulates Aurora A activity. A recent study demonstrated that Aurora A plays an important role in destabilization of the p53 tumor-suppressor protein (Katayama et al., 2004). Downregulation of Aurora A activity is expected to stabilize and increase p53 expression. A major function of p53 is to act as a transcription activator of p21, a potent inhibitor of Cdk2/cyclin kinase. We demonstrated significantly more cyclin E but less p21 in the nucleus of PML−/− MEFs as compared with normal MEFs control. In agreement with this finding, we found significantly increased Cdk2 activity in PML-deficient cells. Taken together, these findings support the hypothesis that PML3 physically interacts with Aurora A and inhibits its kinase activity. In the absence of PML3, the stability of the p53 protein was reduced and decreases p21 expression, leading to activation of Cdk2/cyclin. A previous report showed that increased Cdk2/cyclin activity leads to continued centrosome duplication in hematopoietic cells (Mantel et al., 1999) and that inhibition of Cdk2/cyclin by p21 overexpression blocks centrosome reduplication (Matsumoto et al., 1999). The present study strongly supports a role for PML3 in regulating centrosome duplication by preventing reduplication through repression of Aurora A activation. PML Functions as a Tumor Suppressor The tumor and growth suppressor functions of PML have been demonstrated in animal and cell-culture models (Mu et al., 1994; Le et al., 1998; Salomoni and Pandolfi, 2002). A recent study by Gurrieri et al. (2004) demonstrated a surprisingly high incidence of PML deficiency in numerous types of tumors, including lymphomas and prostate, breast, colon, central nervous system, and germ cell tumors. They concluded that PML protein expression is frequently lost in human tumors of multiple histologic origins and that this loss is associated with tumor progression. This observation strengthens the role of PML in tumor suppression. Also, a role for PML in multiple pathways of apoptosis has been well documented (Salomoni and Pandolfi, 2002). The present study demonstrates that PML3 is a candidate tumor suppressor, because it maintains a proper

number of centrosomes during cell cycle progression. Loss of PML3 function leads to centrosome amplification and genome instability, an event that eventually fosters tumor progression. Experimental Procedures Cell Lines and Cell Culture U2OS, MO59K, and 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). PML+/+ and PML−/− MEFs were generated from normal and PML-knockout mouse embryos and cultured in DMEM containing 15% FBS in a humidified incubator containing 5% CO2. Immortalized and PML−/− MEFs were maintained and cultured as described previously (Xu et al., 2004). CCD-37 cells were obtained from the American Type Culture Collection (CRL-1496) at passage 5 and cultured in DMEM supplemented with 10% FBS. Plasmid Construction and Gene Transfection The full-length cDNAs of PML1, PML2, PML3, and PML5 were obtained by RT-PCR and subcloned into the pcDNA3 vector. Naming of the different PML isoforms was based on the classification by Jensen et al. (2001). The NCBI accession numbers for PML1, PML2, PML3, PML4, and PML5 are M79462, AF230403, S50913, X63131, and M79463, respectively. The cDNA sequence of each PML isoform was confirmed by direct DNA sequencing. pcDNA3/PML4 was constructed as described previously (Wu et al., 2003; Xu et al., 2004). U2OS cells were cultured to semiconfluence and transfected with mammalian expression plasmid constructs by using the FuGENE 6 transfection reagent (Roche Diagnostics Corp.). After transfection, cells were cultured in a humidified CO2 incubator for 48 hr. Total proteins were then isolated for Western blot analysis, and cells were fixed for IF staining. Antibodies The mouse anti-α and -γ-tubulin monoclonal antibodies were purchased from Sigma-Aldrich. The anti-γ-tubulin polyclonal antibody was obtained from Santa Cruz Biotechnology. The mouse anti-Aurora A monoclonal antibody was purchased from BD Biosciences-Pharmingen. The anti-Aurora A and anti-phosphor-Aurora A (Thr288) polyclonal antibodies were obtained from Cell Signaling Technology. The centrin-1 polyclonal antibody was obtained from Abcam. Fluorescent and horseradish-peroxidase-conjugated secondary antibodies were purchased from Amersham Biosciences. Affinity-purified anti-PML isoform-specific antibodies were raised in rabbits against the following peptides: PML1, LRVLDENLADP QAEDRPLVF; PML2a, TPDAEPHSEPPDHQERPAVH; PML2b, ISPPH RIRGAVRSRSRSLRG; PML3, QSEVLYWKVRGAHGDRRATV; PML4, NESGFSWGYPHPFLI; and PML5, QPQQVTLRLALRLGNFPVRH. siRNA Inhibition of PML Expression Two pairs of siRNAs (Qiagen) were synthesized for targeting total PML RNA as we described previously (Xu et al., 2003). For PML isoform-specific mRNA knockdown, the siRNAs were synthesized for targeting the cDNA sequences as follows: PML1, 5#- AACGT GAGCTTCATGGAGCTG-3#; PML2, 5#-AACATCCTGCCCAGCTGC AAA-3#; PML3, 5#-AAAGTGCATGGAGCCCATGGA-3#; PML4, 5#-AAT GAAAGTGGGTTCTCCTGG-3#; and PML5, 5#-AAGTTCAGCCCAG GACTCCTG-3#. Cells cultured to semiconfluence were transfected with siRNA (3.2 µg/well in a 6-well plate) by using the Transmessager RNA transfection reagents (Qiagen) or X-tremeGENE siRNA transfection reagent (Roche) according to the manufacturer’s protocols. Briefly, cells were seeded in a 6-well microplate at a density of 2–3 × 105 cells/well the day before transfection. Cells were transfected with 3.2 ␮g of siRNAs or control siRNA for each well. At 4 hr after transfection, cells were washed once with phosphate-buffered saline (PBS) and continued to grow in fresh medium for an additional 24–72 hr. Cells were either treated with 2 mM hydroxyurea or subjected to two more rounds of siRNA transfection. IF Staining MO59K, 293T, and U2OS cells and MEFs were grown in 6-well plates with cover slides and fixed in cold 4% neutral paraformalde-

PML3 Regulates Centrosome Duplication 731

hyde in PBS for 30 min on ice, washed in PBS, permeabilized in a 1% Triton X-100/0.5% NP40/PBS solution, and blocked in 1% bovine serum albumin (BSA) in PBS. Alternatively, cells were treated with cold cytoskeleton stripping buffer. Briefly, cells were placed on ice for 15 min and then treated with cytoskeleton buffer (10 mM piperazine-N,N#-bis[2-ethanesulfonic acid] [PIPES], [pH 6.8], 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, and 0.5% Triton X-100) for 5 min. After three washes with PBS, cells were treated with cytoskeleton stripping buffer (10 mM Tris-HCl, [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 1% Tween-20, and 0.25% sodium deoxycholate) for another 3 min. Cells were then fixed and permeabilized as described above. Incubation with a primary antibody was carried out for 2 hr at room temperature. Incubation with a secondary antibody was carried out for 1 hr at room temperature followed by staining of DNA with 4,6-diamidino-2-phenylindole (DAPI) for 5–10 min or TOPRO for 1 hr. Slides were mounted with Vectashield antifade medium (Vector Laboratories) after three washes with washing buffer and examined with a Kodak digital imaging system (Eastman Kodak) mounted on top of a Leica DM LB fluorescence microscope or LSM 5 confocal microscope (Carl Zeiss, Inc.). To determine whether the amplified centrosomes in PML−/− MEFs are intact after treatment with hydroxyurea and contain a pair of intact centrioles, double-color IF was performed with centrin-1, α-tubulin polyclonal, and γ-tubulin monoclonal antibodies according to the procedure described by Bennett et al. (2004). Cdk2 Kinase Activity Assay Wild-type and PML−/− MEFs were cultured in DMEM with or without 10% FBS for 24 hr. Cell lysates were prepared by adding lysis buffer containing 50 mM Tris-HCl, (pH 7.4), 250 mM NaCl, 5 mM EGTA, 0.1% NP-40, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ ml aprotinin, 10 µg/ml leupeptin, 60 mM β-glycerophosphate, 50 mM NaF, and 0.5 mM sodium vanadate. An immunoprecipitation assay was performed by incubating 500 µg of total proteins with a rabbit polyclonal anti-Cdk2 antibody for 2 hr at 4°C. The immunocomplexes binding to Protein A-Sepharose beads were resuspended in 30 µl of kinase buffer (50 mM Tris-HCl, [pH 7.4], 10 mM MgCl2, 1 mM dithiothreitol, and 0.1 mg/ml BSA) and used directly in a kinase activity assay with histone-1 (H1) as a substrate. The kinase reaction mixture contained 1 µg of H1, 30 µM ATP, and 5 µCi of [γ-32P] ATP. The kinase assay was carried out at 37°C for 30 min and terminated by adding sample buffer. Subcellular Fractionation of Nuclear and Cytoplasmic Proteins and Immunoprecipitation Nuclear and cytoplasmic protein fractions were prepared from PML+/+ and PML−/− MEFs; coimmunoprecipitation and Western blotting were performed essentially as we described previously (Wu et al., 2003). Isolation of Centrosomes Centrosomes from proliferating MO59K cells were partially purified by discontinuous sucrose gradient centrifugation essentially as described by Hsu and White (1998). Centrosomes recovered in each fraction were collected by centrifugation at 15,000 rpm for 10 min in a refrigerated microcentrifuge and resuspended in gel loading buffer for Western blot analysis.

Supplemental Data Supplemental Data include four figures and can be found with this article online at http://www.molecule.org/cgi/content/full/17/5/721/ DC1/.

Acknowledgments −/−

We are grateful to Pier Paolo Pandolfi for the PML MEFs, Gerd G. Maul for the T antigen-transformed PML−/− MEFs, and Subrata Sen for the Aurora A antibody and reagents. We are also grateful to Thanh T. Tran for technical assistance, Don Norwood for editing and critically reading the manuscript, and Dong-Er Zhang for helpful discussion and reagents. The DNA sequencing and confocal

microscopy facilities were supported by research grant CA 16672 from the National Institutes of Health. This work was supported by grants CA 55577 and CA 099963 from the National Institutes of Health, and a grant from the Charlotte Geyer Foundation (to K.-S.C.).

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