Molecular Cancer Research 5, 1304-1311, December 1, 2007. doi: 10.1158/1541-7786.MCR-07-0388
© 2007 American Association for Cancer Research
Cell Cycle, Cell Death, and Senescence
Defective in Mitotic Arrest 1/Ring Finger 8 Is a Checkpoint Protein That Antagonizes the Human Mitotic Exit Network
Robyn L. Tuttle1,
John Bothos1,
Matthew K. Summers1,2,
Francis C. Luca3 and
Thanos D. Halazonetis1,4
1 The Wistar Institute, 2 Biomedical Graduate Studies, 3 Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; and 4 Departments of Molecular Biology and Biochemistry, University of Geneva, Geneva, Switzerland
Requests for reprints: Thanos D. Halazonetis, Department of Molecular Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland. Phone: 41-22-379-61-12; Fax: 41-22-379-68-68. E-mail: thanos.halazonetis{at}molbio.unige.ch
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Abstract
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A molecular pathway homologous to the S. cerevisiae mitotic exit network (MEN) and S. pombe septation initiation network has recently been described in higher eukaryotes and involves the tumor suppressor kinase LATS1 and its subunit MOB1A. The yeast MEN/septation initiation network pathways are regulated by the ubiquitin ligase defective in mitotic arrest 1 (Dma1p), a checkpoint protein that helps maintain prometaphase arrest when cells are exposed to microtubule poisons. We identified here the RING domain protein ring finger 8 (RNF8) as the human orthologue of the yeast protein Dma1p. Like its yeast counterparts, human DMA1/RNF8 localized at the midbody and its depletion by siRNA compromised mitotic arrest of nocodazole-treated cells in a manner dependent on the MEN. Depletion of MAD2, a spindle checkpoint protein, also compromised mitotic arrest, but in a MEN-independent manner. Thus, two distinct checkpoint pathways maintain mitotic arrest in cells exposed to microtubule poisons. (Mol Cancer Res 2007;5(12):1304–11)
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Introduction
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Progress through mitosis is controlled by multiple checkpoints. The best characterized of these is the spindle checkpoint, which delays entry into anaphase until all chromosomes align on the equatorial plane. Central to the function of the spindle checkpoint is the protein MAD2, which inhibits the anaphase-promoting complex (APC), a multi-subunit ubiquitin ligase (1, 2).
A second mitotic checkpoint functions in early prophase and inhibits chromosome condensation in response to microtubule poisons (3). A key effector of this checkpoint is CHFR, a protein with FHA and RING domains. The FHA domain binds phosphopeptides (4) whereas the RING domain confers ubiquitin protein ligase activity (5).
A third mitotic checkpoint that functions late in mitosis has been characterized in yeast. This checkpoint controls the mitotic exit network (MEN; S. cerevisiae terminology), a signaling cascade composed of a GTPase (Tem1p) and two downstream kinases (Cdc15p and Dbf2p; refs. 6, 7). Dbf2p activates the phosphatase Cdc14p, which dephosphorylates cyclin-dependent kinase (CDK) substrates leading to chromosome decondensation and mitotic exit. In S. pombe, the septation initiation network (SIN) is homologous to the S. cerevisiae MEN (8). The activities of the MEN and SIN are inhibited in response to microtubule poisons by checkpoint proteins belonging to the defective in mitotic arrest (DMA) family. S. pombe Dma1p, like human CHFR, is a ubiquitin ligase with FHA and RING domains. Through its FHA domain, Dma1p localizes to the spindle pole bodies and to the yeast "midbody" equivalent and inhibits localization of SIN kinases to these structures (9, 10). In S. cerevisiae, two redundant proteins, Dma1p and Dma2p, are functionally equivalent to S. pombe Dma1p (11).
Unlike yeast, the molecular mechanisms governing mitotic exit in higher eukaryotes have not been well characterized. In human cells, the tumor suppressor LATS1 seems to be the orthologue of the S. cerevisiae Dbf2p MEN kinase. Like Dbf2p, LATS1 associates with a Mob1 subunit, which is referred to as MOB1A in humans; MOB1A can enhance the kinase activity of LATS1; and LATS1 overexpression drives mitotic exit in cells exposed to microtubule poisons in a MOB1A-dependent manner (12-14). Here, we identify a checkpoint pathway that regulates the activity of the human MEN in response to microtubule poisons and show that ring finger 8 (RNF8; refs. 15-18), a protein with structural and functional similarities to yeast DMA proteins, is a component of this checkpoint pathway.
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Results
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RNF8 Has Sequence Similarity to Yeast DMA Proteins
Mining of sequence databases identified CHFR and RNF8 as the human proteins with the highest sequence similarity to S. cerevisiae Dma1p/Dma2p and S. pombe Dma1p (Fig. 1
). Of these two human proteins, CHFR has a COOH-terminal cysteine-rich region, which is absent in S. cerevisiae Dma1p and Dma2p and S. pombe Dma1p, thus suggesting that RNF8 is the human orthologue of the yeast DMA proteins. Indeed, the experiments described below confirm this prediction, and to have consistent terminology across species, we propose that RNF8 be renamed DMA1/RNF8.
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FIGURE 1. Domain organization and sequence similarity of human DMA1/RNF8 (DMA1_hs) to DMA proteins in S. pombe (Dma1_sp) and S. cerevisiae (Dma1_sc and Dma2_sc) and to human CHFR (CHFR_hs). A. Domain organization. FHA, forkhead-associated domain; RD, RING domain; CR, cysteine-rich region. B and C. Alignment of the FHA (B) and RING (C) domain sequences of the members of the DMA1-CHFR family.
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DMA1/RNF8 Localizes to the Midbody
In interphase, green fluorescent protein (GFP)–tagged DMA1/RNF8 localizes in the nucleus (16). To study the intracellular localization of DMA1/RNF8 in mitotic cells, we expressed GFP-tagged DMA1/RNF8 protein in U2OS osteosarcoma cells and monitored its intracellular localization at various time points after release from a thymidine-induced cell cycle block. In live cells that were in late anaphase and telophase, DMA1/RNF8 localized to the midbody (Fig. 2A
). The same intracellular localization was also observed in fixed cells (Fig. 2B), in which the midbody can be unambiguously identified by immunostaining for Aurora-B (19). The endogenous DMA1/RNF8 protein also localized to the midbody (data not shown). Thus, in late anaphase and telophase, DMA1/RNF8 and S. pombe Dma1p exhibit similar intracellular localizations (10).
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FIGURE 2. Localization of DMA1/RNF8 at the midbody. A. Localization of a GFP-DMA1/RNF8 fusion protein in live cells. DNA was stained with Hoechst dye. B. Colocalization of GFP-DMA1/RNF8 with Aurora-B at the midbody in fixed cells. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI).
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DMA1/RNF8 Is a Mitotic Checkpoint Protein That Regulates the Human MEN
S. cerevisiae Dma1p/Dma2p and S. pombe Dma1p help maintain mitotic arrest in response to microtubule poisons (9-11). To determine whether human DMA1/RNF8 has a similar function, we studied U2OS osteosarcoma cells in which DMA1/RNF8 expression was silenced by siRNA (Fig. 3A
). After the siRNA treatment, the cells were synchronized at the G1-S boundary, released into the cell cycle, and 8 h later challenged with the microtubule poison nocodazole. In the presence of nocodazole, cells cannot undergo cytokinesis and so retain a 4N DNA content. Of these 4N cells, those that stay arrested in mitosis will be round and refractile and will have condensed chromosomes and histone H3 phosphorylated on Ser10, whereas those that exit mitosis will flatten, decondense their chromosomes, and stain negative for histone H3 phosphorylation on Ser10. Analysis of the cell morphology indicated that many more control cells were arrested in mitosis than DMA1/RNF8-depleted cells (Fig. 3B). Similar results were obtained by flow cytometry monitoring histone H3 phosphorylation on Ser10 and using two different siRNAs to deplete DMA1/RNF8 (Fig. 3C), as well as other cell lines such as HeLa (data not shown).
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FIGURE 3. Depletion of DMA1/RNF8 compromises mitotic arrest. A. Suppression of endogenous DMA1/RNF8 protein levels by siRNA specific for DMA1/RNF8 (d) but not by control siRNA (c). U2OS cells were examined 72 h after siRNA transfection, which corresponds to a time between the 24- and 36-h time points of the experiment shown in C. B. Phase-contrast images of U2OS cells treated with control siRNA or siRNA specific for DMA1/RNF8 (DMA1-1) 24 h after release from a G1-S cell cycle block with nocodazole added at the 8-h time point. C. U2OS cells treated with two different siRNAs specific for DMA1/RNF8 (DMA1-1 and DMA1-2) or with control siRNA were synchronized at the G1-S boundary and then released (0 h). Nocodazole was added at 8 h and progression through the cell cycle was monitored by flow cytometry analysis for DNA content (propidium iodide staining) and for phosphorylation of histone H3 on Ser10 (H3 S10p). Note that the analysis of H3 phosphorylation was gated on the cells with 4N DNA content. The peaks of cells staining negative and positive for histone H3 Ser10 phosphorylation are colored black and gray, respectively.
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The low fraction of DMA1/RNF8-depleted cells staining positive for histone H3 phosphorylated on Ser10 in the presence of nocodazole could indicate an inability of these cells to enter mitosis, an inability to maintain mitotic arrest, or both. To distinguish between these possibilities, synchronized nocodazole-treated control and DMA1/RNF8-depleted cells were examined by time-lapse microscopy. Mitotic entry and exit were scored on the basis of cell morphology, which was monitored for up to 36 h after release from the G1-S arrest. By examining all the acquired images, we could calculate the fraction of cells that had entered mitosis at any time during the 36-h time period, as well as the fraction of cells that remained arrested in mitosis at the 36-h time point (Fig. 4A
). Somewhat fewer (1.6-fold) DMA1/RNF8-depleted cells entered mitosis as compared with the control cells. Importantly, of the DMA1/RNF8-depleted cells that had entered mitosis, only 24.6% were still in mitosis at the 36-h time point, compared with 46.1% of the control cells, indicating that DMA1/RNF8-depleted cells were compromised in their ability to maintain mitotic arrest (Fig. 4).
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FIGURE 4. Effect of DMA1/RNF8 depletion on mitotic progression as monitored by time-lapse microscopy. A. U2OS cells were transfected with control siRNA or siRNA specific for DMA1/RNF8 (DMA1-1), synchronized, and exposed to nocodazole 8 h after release from the G1-S arrest. The cells were monitored from 8 to 36 h after release from the G1-S arrest. The percentages of cells entering mitosis (Enter M) at any time during the observation period and the percentages of cells in mitosis (In M) at the 36-h time point are indicated. All percentages refer to the total number of cells monitored. Bars, SD. B. Images of a DMA1/RNF8-depleted cell that enters and exits mitosis during the observation period. Time is shown in minutes with time 0' being the time point when the cell becomes completely round and refractile.
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Yeast cells with mutant Dma1p escape from microtubule poison–induced mitotic arrest, and this escape is attributed to deregulated MEN/SIN activity because yeast Dma1p suppresses the MEN/SIN (10). By analogy, the escape from mitotic arrest induced by DMA1/RNF8 depletion might be dependent on the human MEN proteins LATS1 and MOB1A. Indeed, nocodazole-treated DMA1/RNF8-depleted cells did not exit mitosis when MOB1A was also depleted (Fig. 5
). This effect of MOB1A depletion was not due to a general inability of MOB1A-depleted cells to exit mitosis because MOB1A depletion did not inhibit the escape from mitotic arrest induced by depletion of the MAD2 spindle checkpoint protein (Fig. 5).
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FIGURE 5. Escape from mitotic arrest induced by depletion of DMA1/RNF8 requires MEN activity. A. U2OS cells were transfected with control siRNA or siRNA specific for DMA1/RNF8 (DMA1-1), MAD2, or MOB1A, or combinations thereof. Escape from mitotic arrest was monitored by flow cytometry analysis of cells with 4N DNA content for phosphorylation of histone H3 on Ser10. The peaks of cells staining negative and positive for histone H3 Ser10 phosphorylation are colored black and gray, respectively. Time points refer to the time of release from the G1-S block; nocodazole was added at 8 h. B. Immunoblot analysis of cells treated with control siRNA (c) or siRNA specific for DMA1/RNF8 (d), MAD2 (m), or MOB1A (b), or combinations thereof. *, a nonspecific band, just above MOB1A.
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DMA1/RNF8 Regulates Cyclin A and p27/KIP1 Protein Levels
To characterize the mitotic escape induced by DMA1/RNF8 depletion, synchronized control and DMA1/RNF8-silenced cells exposed to nocodazole were used to prepare cell extracts for analysis of various mitotic markers by immunoblotting. We monitored histone H3 phosphorylation on Ser10 (which had also been monitored by flow cytometry; Fig. 3C) and protein levels of cyclin B1, cyclin A, Aurora-A, Aurora-B, and p27/KIP1. We anticipated that DMA1/RNF8-silenced cells would have low levels of cyclin B1, cyclin A, Aurora-A, and Aurora-B because these proteins are substrates of the APC and their levels decrease when cells exit mitosis (1, 2). We also anticipated higher p27/KIP1 levels in the DMA1/RNF8-silenced cells because p27/KIP1 levels increase when cells exit mitosis and enter G1 (20). As predicted, the levels of cyclin A and Aurora-B and of histone H3 phosphorylation on Ser10 were lower, whereas the levels of p27/KIP1 were higher, in the DMA1/RNF8-depleted cells compared with the controls (Fig. 6A
). However, contrary to our predictions, cyclin B1 and Aurora-A levels did not differ in the control and DMA1/RNF8-depleted cells at all time points examined (Fig. 6A). This could indicate that not all the molecular pathways activated when cells normally exit mitosis are also activated when cells escape nocodazole-induced mitotic arrest by depleting DMA1/RNF8. Alternatively, because cyclin B1 and Aurora-A levels are high in G2 cells and fewer DMA1/RNF8-depleted cells enter mitosis than control cells, the high cyclin B1 and Aurora-A levels may reflect the somewhat increased G2 fraction of DMA1/RNF8-depleted cells (Fig. 4A). However, in this case, the levels of cyclin A and Aurora-B should also have been high.
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FIGURE 6. DMA1/RNF8 regulates the protein levels of specific APC substrates. A. Escape from mitotic arrest induced by DMA1/RNF8 depletion is associated with low levels of cyclin A and increased levels of p27/KIP1. Synchronized U2OS cells treated with control (c) or DMA1/RNF8-specific (DMA1-1; d) siRNA were exposed to nocodazole 8 h after release from the G1-S block. Cell extracts were prepared at various time points (relative to the release from the G1-S block) and immunoblotted for histone H3 phosphorylation on Ser10, cyclins A and B1, Aurora-A and Aurora-B, and p27/KIP1 (p27). B. Escape from mitotic arrest induced by MAD2 depletion is associated with low levels of cyclin B1. Synchronized U2OS cells were treated as in A. m, MAD2-specific siRNA; M and NM, mitotic and nonmitotic forms of Cdc25C, respectively; *, nonspecific bands. C. Low cyclin A and high p27/KIP1 levels in DMA1/RNF8-depleted cells escaping mitotic arrest require MOB1A. Synchronized U2OS cells were treated as in A. b, MOB1A-specific siRNA; db, combination of both DMA1/RNF8- and MOB1A-specific siRNAs. D. Model for the mechanism by which DMA1/RNF8 and MAD2 promote mitotic arrest. DMA1/RNF8 regulates cyclin A and p27/KIP1 protein levels by inhibiting the MEN, whereas MAD2 regulates cyclin B1 levels. Human CDC14A/B may be implicated in the mechanism by which the MEN regulates cyclin A and p27/KIP1 levels.
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We subsequently compared cyclin A, cyclin B1, and p27/KIP1 levels in cells that escaped mitotic arrest by depleting DMA1/RNF8 versus MAD2. In both cases, there was a shift in Cdc25C electrophoretic migration, which indicates that the cells had entered mitosis (21), and a comparable decrease in histone H3 phosphorylation, indicating a similar inability to maintain mitotic arrest (Fig. 6B). However, cyclin A levels were lower and p27/KIP1 levels were higher in DMA1/RNF8-depleted cells compared with MAD2-depleted cells (Fig. 6B), indicating that the decrease in cyclin A levels and the increase in p27/KIP1 levels are characteristic of cells that escape mitotic arrest when DMA1/RNF8 is depleted.
Finally, we asked whether the decrease in cyclin A levels and the increase in p27/KIP1 levels observed in DMA1/RNF8-depleted cells were dependent on the human MEN. Cells in which both DMA1/RNF8 and MOB1A were depleted did not escape mitotic arrest (Fig. 5) and also failed to show the changes in cyclin A and p27/KIP1 levels observed in DMA1/RNF8-depleted cells (Fig. 6C). Thus, the regulation of cyclin A and p27/KIP1 levels by DMA1/RNF8 is mediated by the human MEN.
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Discussion
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We propose here that the previously partially characterized gene RNF8 (here referred to as DMA1/RNF8) is the human orthologue of the S. pombe DMA1 and S. cerevisiae DMA1 and DMA2 mitotic checkpoint genes. Several observations support this functional assignment. First, the DMA1/RNF8 protein has a similar domain organization comprising NH2-terminal FHA and COOH-terminal RING domains as the yeast DMA proteins (Fig. 1). Second, DMA1/RNF8 localized to the midbody, which is consistent with the localization of S. pombe Dma1p at the division site (10). Third, depletion of DMA1/RNF8 compromised mitotic arrest in cells exposed to nocodazole, similar to the defective mitotic arrest phenotype that gave S. pombe Dma1p its name (9). Fourth, silencing of DMA1/RNF8 leads to increased levels of the CDK inhibitor p27/KIP1, consistent with increased levels of the CDK inhibitor Sic1p in S. cerevisiae cells when the MEN becomes activated (22). And fifth, DMA1/RNF8 and the human MEN kinase LATS1/MOB1A function in the same molecular pathway (Fig. 6D), as reported for their S. pombe counterparts (10). We further note that, while this article was under review, another group also reported that DMA1/RNF8 regulates mitotic exit (23).
Interestingly, depletion of DMA1/RNF8 compromised the ability of nocodazole-treated cells to maintain mitotic arrest in prometaphase/metaphase, although the MEN, which is inhibited by DMA1/RNF8, is thought to be activated later in mitosis. In S. cerevisiae the MEN is activated in late anaphase, when one spindle pole enters the bud, whereas in S. pombe the SIN initiates septum formation, which is a cytokinesis event (6, 8). Our results, however, imply that in nocodazole-treated human cells in prometaphase/metaphase, the MEN can be active and that its activity needs to be suppressed by DMA1/RNF8 to maintain a mitotic arrest. The same may also be true in yeast exposed to microtubule poisons because deletion of DMA1 compromises mitotic arrest in both S. cerevisiae and S. pombe (9, 11).
The mechanism by which DMA1/RNF8 enforces mitotic arrest may involve inhibition of degradation of specific APC substrates. In cells that escaped mitotic arrest due to depletion of DMA1/RNF8, the protein levels of cyclin A and Aurora-B were lower compared with control cells, whereas in cells that escaped mitotic arrest by MAD2 depletion, cyclin B1 levels were lower (24), suggesting that DMA1/RNF8 and MAD2 inhibit degradation of distinct APC substrates (Fig. 6D). Previous studies had also suggested that APC substrates are differentially degraded. For example, degradation of cyclin A precedes degradation of cyclin B1 in cells going through mitosis and the spindle checkpoint inhibits degradation of cyclin B1 and securin but not cyclin A (25-28).
A second mechanism by which DMA1/RNF8 enforces mitotic arrest may involve maintaining low levels of p27/KIP1 (Fig. 6D). p27/KIP1 levels were high throughout the cell cycle in DMA1/RNF8-depleted cells (Fig. 3C). Because p27/KIP1 is a CDK inhibitor, its high levels in DMA1/RNF8-depleted cells may facilitate mitotic exit by compromising CDK activity (29). Inhibition of CDK activity by high levels of p27/KIP1 in DMA1/RNF8-depleted cells may also explain the small decrease in mitotic entry (Fig. 4A).
The regulation of cyclin A and p27/KIP1 levels by DMA1/RNF8 is not direct but involves the human MEN (Fig. 6C). In turn, how the human MEN regulates cyclin A and p27/KIP1 is not clear. The yeast MEN/SIN activates the Cdc14 phosphatase (S. cerevisiae terminology), which dephosphorylates CDK substrates (6, 8, 22). Two putative human orthologues of S. cerevisiae Cdc14 have been identified, CDC14 A and CDC14B. Interestingly, CDC14A can dephosphorylate and activate the CDH1-associated pool of the APC, suggesting that it could regulate degradation of specific APC substrates (30).
In conclusion, we have identified DMA1/RNF8 as a modulator of the human MEN and showed that it regulates the protein levels of specific APC substrates. These studies pave the way for understanding the link between cancer and deregulation of pathways that control mitosis because several MEN genes, including LATS1 and MOB1A, are tumor suppressors (31-35).
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Materials and Methods
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Cell Culture and Cell Cycle Synchronization
U20S osteosarcoma cells were cultured according to established procedures. Clones stably expressing GFP-tagged DMA1/RNF8 were prepared using a pIRESN2 bicistronic plasmid (Clontech Laboratories). For synchronization, the cells were arrested in G1-S by feeding with medium containing 1.3 mmol/L thymidine (Calbiochem) for 20 h. Then (0-h time point) the cells were released into the cell cycle by exchanging the medium with medium containing 250 µmol/L thymidine and 250 µmol/L 2-deoxycytidine (Sigma-Aldrich). After 8 h, the medium was exchanged again with medium containing 1.3 mmol/L thymidine to prevent the cells from going through multiple cell cycles. Nocodazole (1 µmol/L; Calbiochem) was also added at the 8-h time point, as indicated. For time-lapse microscopy, cells were synchronized as described above, placed on the microscope stage 8 h after release from the G1-S block, and observed using a phase-contrast lens. The experiment was done in duplicate and, for each condition, a minimum of 150 cells were followed throughout the observation period.
Intracellular Localization of DMA1/RNF8
U20S cells expressing GFP-tagged DMA1/RNF8 were examined live after staining the genomic DNA with Hoechst dye 33342 (Sigma-Aldrich). Alternatively, the cells were fixed in 4% paraformaldehyde for 10 min and then stained with an antibody against Aurora-B (BD Transduction Laboratories) and with 4',6-diamidino-2-phenylindole.
siRNA Transfections
All siRNA transfections were done by incubating cells seeded in 6-cm-diameter dishes with mixtures of Oligofectamine (Invitrogen Life Technologies) and 240 pmol of control (luciferase), MAD2- (UCCGUUCAGUGAUCAGACAdTdT), or DMA1-specific (GGUGAAGUGGCCAGUACACdTdT or GUCACAGAGGUUCAUCAUGdTdT; DMA1-1 and DMA1-2, respectively) siRNAs (Dharmacon). Cell synchronization was initiated 24 h after the siRNA transfection.
Preparation of Whole-Cell Extracts and Immunoblotting
Cells were lysed in a buffer consisting of 50 mmol/L Tris (pH 8.0), 120 mmol/L NaCl, 0.5% NP40, 1 mmol/L DTT, 0.4 µg/mL Pefabloc SC, 2 µg/mL pepstatin, 0.1 µmol/L staurosporine, 1 mmol/L sodium vanadate, and 15 mmol/L sodium fluoride. After lysis, the DNA was digested by adding 0.1 units/µL DNase (Roche) and 10 mmol/L MgCl2 for 2 to 3 h at 16°C. The lysates were then cleared by centrifugation, resolved by SDS-PAGE, and immunoblotted with antibodies against hemagglutinin (Santa Cruz Biotechnology), histone H3 phosphorylated on Ser10 (Upstate), cyclin A (Upstate), cyclin B1 (PharMingen), p27/Kip1 (BD Transduction Laboratories), Cdc25C (Upstate), MAD2 (Babco), and DMA1/RNF8 (a rabbit polyclonal antibody prepared against residues 302-448 of the human protein). Equal protein loading of cell extracts was monitored by immunoblotting for actin (Upstate).
Flow Cytometry Analysis
Cells fixed in 70% ethanol for a minimum of 24 h were extracted on ice with 0.25% Triton X-100/PBS for 5 min, washed with PBS, and then incubated with 1 µg of an antibody that recognizes histone H3 phosphorylated on Ser10 diluted in 1% bovine serum albumin/PBS for 1 h. The cells were then washed with PBS, incubated for 30 min with 2 µg of Alexa-Fluor 488–conjugated antirabbit antibody (Molecular Probes) diluted in 1% bovine serum albumin/PBS, washed again, and resuspended in PBS supplemented with 5 µg/mL RNase (Roche) and propidium iodide. After a minimum of 20 min, the cells were analyzed by flow cytometry (Becton Dickinson).
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Acknowledgements
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We thank Dan McCollum for helpful discussions and the Wistar Institute Nucleic Acid and Hybridoma Facilities for DNA sequencing analysis and generating the MOB1A monoclonal antibody, respectively.
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Notes
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Grant support: National Cancer Institute grant CA105160 (T.D. Halazonetis) and National Cancer Institute Training Grants CA09171 (R.L. Tuttle) and CA09677 (J. Bothos and M.K. Summers).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8/31/07;
revised 9/19/07;
accepted 9/26/07.
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Abstract
Introduction
