G2 Cell Cycle Arrest, Down-Regulation of Cyclin B, and Induction of Mitotic Catastrophe by the Flavoprotein Inhibitor Diphenyleneiodonium

Molecular Cancer Therapeutics 1229

G2 cell cycle arrest, down-regulation of cyclin B, and induction of mitotic catastrophe by the flavoprotein inhibitor diphenyleneiodonium

Robin M. Scaife is a key event in cell proliferation, whereby cytokinetic fission, following segregation of the condensed chromatin, Laboratory for Cancer Medicine, West Australian Institute for Medical Research, Centre for Medical Research, University of results in a doubling of cell numbers. Both the chromatin Western Australia, Perth, Western Australia, Australia segregation and the cytokinetic fission require assembly of specialized cytoskeletal structures composed of microtubules, microfilaments, and centrosomes (1, 2). Pharma- Abstract cologic perturbation of the microtubule cytoskeleton has hence been used to target cell proliferation in human Because proliferation of eukaryotic cells requires cell disease, and the microtubule stabilizing taxanes (such cycle–regulated chromatid separation by the mitotic as paclitaxel) have proven effective in the treatment of spindle, it is subject to regulation by mitotic checkpoints. cancers (3). To determine the mechanism of the antiproliferative In addition to mitosis, cell proliferation also requires activity of the flavoprotein-specific inhibitor diphenyl- mitogenic signaling to achieve DNA replication during eneiodonium (DPI), I have examined its effect on the cell S phase of the cell cycle. Among the broad range of mi- cycle and mitosis. Similar to paclitaxel, exposure to DPI causes an accumulation of cells with a 4N DNA content. togenic signaling components, reactive oxygen species However, unlike the paclitaxel-mediated mitotic block, (ROS) are emerging as important factors in cell prolifera- DPI-treated cells are arrested in the cell cycle prior to tion (4). By targeting the catalytic sites of phosphatases, mitosis. Although DPI-treated cells can arrest with fully ROS can influence a range of intracellular signaling events, separated centrosomes at opposite sides of the nucleus, including mitogenic signaling by mitogen-activated pro- these centrosomes fail to assemble mitotic spindle micro- tein kinases (MAPK; ref. 5). Indeed, inhibition of ROS pro- tubules and they do not accumulate the Thr288 phosphor- duction has been shown to inhibit cell proliferation (6, 7). ylated Aurora-A kinase marker of centrosome maturation. Intracellular ROS levels can be lowered by antioxidants In contrast with paclitaxel-arrested cells, DPI impairs such as N-acetyl-L-cysteine (NAC) or through inhibition of cyclin B1 accumulation. Release from DPI permits an the flavoprotein complex NAD(P)H oxidase by the flavo- accumulation of cyclin B1 and progression of the cells into protein-specific inhibitor diphenyleneiodonium (DPI; ref. mitosis. Conversely, exposure of paclitaxel-arrested mi- 8). As reported previously (7), I have found that NAC and totic cells to DPI causes a precipitous drop in cyclin B DPI profoundly inhibit cell proliferation. My results and Thr288 phosphorylated Aurora-A levels and leads to indicate that the effects of NAC and DPI on the cell cycle mitotic catastrophe in a range of cancerous and noncan- are fundamentally different. Indeed, I have found that, cerous cells. Hence, the antiproliferative activity of DPI whereas NAC delays cell cycle progression through G1, reflects a novel inhibitory mechanism of cell cycle DPI can prevent mitotic cell division by blocking the cell progression that can reverse spindle checkpoint-mediated cycle progression in G2. This antiproliferative effect of DPI 288 cell cycle arrest. [Mol Cancer Ther 2004;3(10):1229–37] correlates with its ability to impair cyclin B1 and Thr phosphorylated Aurora-A accumulation. Further, in mitot- ically arrested cells, DPI can induce down-regulation of Introduction cyclin B and Thr288 phosphorylated Aurora-A levels and a Cell proliferation requires a multitude of events to translate forced mitotic exit. I have therefore identified a novel a mitogenic signal into cell division. Mitotic division antiproliferative pharmacophore that seems to target mitotic cell cycle signaling components. In light of the inability of p53-compromised and retinoblastoma pro- Received 2/6/04; revised 7/21/04; accepted 8/11/04. tein–compromised cells to undergo tetraploidy-mediated Grant support: National Health and Medical Research Council (Canberra) cell cycle arrest following mitotic catastrophe (9), it is pos- grant 981649, West Australian Institute for Medical Research, and sible that iodonium-based pharmacophores (such as DPI) Royal Perth Hospital Medical Research Foundation. could increase the efficacy of spindle checkpoint-mediated The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked anticancer strategies. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Robin M. Scaife, Laboratory for Cancer Medicine, Materials and Methods West Australian Institute for Medical Research, Perth, Western Australia 6009, Australia. Phone: 61-8-9224-0338; Fax: 61-8-9224-0322. Cell Culture E-mail: [email protected] NIH 3T3, Rat1, HepG2 and MCF-7 cells were cultured in Copyright C 2004 American Association for Cancer Research. DMEM (Trace Biochemicals, Sydney, New South Wales,

Mol Cancer Ther 2004;3(10). October 2004

Australia) containing 10% FCS (Life Technologies) and ROS was determined following incubation of the cells 2 mmol/L L-glutamine (Trace Biochemicals) at 37jC and with 2V,7V-dichlorodihydrofluoresceindiacetate (Sigma 5% C02. Human mammary epithelial cells were obtained Chemical) for 30 minutes. The 2V,7V-dichlorofluorescein from Clonetics (Walkersville, MD) and cultured in fully and the PI signal were collected using CellQuest software supplemented MEGM (Clonetics). Cells were grown to on a FACSCalibur machine (Becton Dickinson, Mountain f50% confluency prior to overnight serum starvation. View, CA). Histograms of the 2V,7V-dichlorofluorescein or Fluorescence and Phase-Contrast Microscopy PI signal were obtained using FlowJo software (Tree Star, For immunofluorescence microscopy, cells were seeded Inc., Ashland, OR). onto coverslips coated with polylysine (0.1 mg/mL) prior Cell Lysis and Western Blotting to fixation in 4% paraformaldehyde-PBS. The fixed cells Cells were washed with PBS and lysed in ice-cold were then permeabilized for 1 minute with 0.2% Triton 50 mmol/L HEPES (pH 7.4) containing 150 mmol/L NaCl, X-100 in PBS containing 2.5 mg/mL bovine serum albu- 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10% min. Coverslips were rinsed with PBS and incubated with glycerol, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L 0.5 Ag/mL tetramethylrhodamine isothiocyanate-phalloidin Na3VO4, 10 mmol/L NaF, and protease inhibitors. Follow- (Sigma Chemical Co., St. Louis, MO), anti-h-tubulin anti- ing centrifugation at 10,000 Â g for 5 minutes, aliquots of bodies (10 Ag/mL, monoclonal antibody T-4026, Sigma cell lysates were subjected to SDS-PAGE and transferred Chemical), anti-g-tubulin antibodies (3.5 Ag/mL, monoclo- onto nitrocellulose membranes (Amersham, Piscataway, nal antibody T-6557, Sigma Chemical Co.), or anti–Thr288 NJ). Membranes were blocked with 10% nonfat milk phosphorylated Aurora-A antibodies (1:200 dilution, 3091, (Carnation, Las Vegas, NV)-5% bovine serum albumin Cell Signaling, Beverly, MA) at 37jC for 60 minutes in PBS (Boehringer, Indianapolis, IN) in TBS containing 0.5% containing 2.5 mg/mL bovine serum albumin. Following a Tween 20 and probed with antibodies directed against PBS wash, the coverslips were incubated with 5 Ag/mL either phospho–extracellular signal-regulated kinase (ERK) biotin-SP-conjugated goat anti-mouse (115-065-003, The MAPK (monoclonal antibody 7383, Santa Cruz Biotechnol- Jackson Laboratory, Bar Harbor, ME) or 7.5 Ag/mL ogy, Santa Cruz, CA), ERK MAPK (094, Santa Cruz biotin-conjugated goat anti-rabbit (BA-1000, Vector Labo- Biotechnology), Thr288 phosphorylated Aurora 2 (3091, Cell ratories, Burlingame, CA) at 37jC for 60 minutes in Signaling), cyclin B1 (monoclonal antibody GNS-1, 554176, PBS containing 2.5 mg/mL bovine serum albumin. Biotin- BD Biosciences, San Jose, CA), or cyclin A (571, Santa Cruz labeled antigen-antibody complexes were then visualized Biotechnology) followed by horseradish peroxidase–con- by incubation for 60 minutes with PBS containing jugated secondary antibody (Silenus, Boronia, Victoria, 2.5 mg/mL bovine serum albumin and 2 Ag/mL Alexa Australia). Antigens were then visualized by enhanced 488–conjugated streptavidin (Molecular Probes, Eugene, chemiluminescence (Amersham) using Hyperfilm MP OR) and nuclei were stained with Hoescht 33342. For (Amersham). antibody double labeling, rabbit secondary antibody was MAPK Assays employed as described above, whereas mouse antigens Following lysis of Rat1 cells into 20 mmol/L HEPES (pH were detected using Cy3-conjugated donkey anti-mouse 7.7), 0.1 mmol/L EDTA, 100 mmol/L NaCl, 20 mmol/L serum (715-165-150, The Jackson Laboratory). Following a h-glycerophosphate, 100 Amol/L orthovanadate, 0.5 mmol/L PBS rinse, coverslips were mounted with SlowFade Light DTT, and 0.2% (w/v) Triton X-100 containing protease Antifade reagent (Molecular Probes). Images of represen- inhibitors, lysates were clarified by centrifugation at tative fields were obtained with Comos and Confocal 10,000 Â g for 5 minutes and incubated for 3 hours with Assistant software (Bio-Rad, Hercules, CA) following cap- Sepharose-bound GST-Elk. The washed Sepharose beads ture on a Diaphot 300 microscope (Nikon, Tokyo, Japan) were resuspended in 20 mmol/L HEPES (pH 7.6), h equipped for UV laser scanning confocal microscopy (Bio- 20 mmol/L MgCl2, 20 mmol/L -glycerophosphate, Rad MRC 1000/1024). Confocal microscope images of anti- 100 Amol/L orthovanadate, and 0.5 mmol/L DTT, and g-tubulin staining were used to determine intercentrosomal after incubation with 10 Amol/L g-[32P]ATP for 30 minutes distances by Optimas software–mediated measurement at 30jC, the samples were subjected to SDS-PAGE and of triplicate samples of at least 100 cells. Optimas software autoradiography. was similarly used to capture phase-contrast images of cells using a 6.3Â objective lens. Binding of Annexin V-Fluos (Roche, Indianapolis, IN) to cells was determined by fluo- Results rescence microscopy. The Antioxidant NAC Slows Cell Cycle Progression in Flow Cytometry G1 but Does Not Fully Block Mitotic Cell Division The DNA content of cells was determined following Because exposure of cells to NAC can decrease intracel- fixation of the cells in 90% methanol-PBS at À20jC after lular ROS levels, the effect of NAC on the cell cycle was trypsin-mediated detachment from the culture substrate. probed. Incubation of Rat1 fibroblasts for increasing After permeabilization with 0.2% Triton X-100 and addition periods with NAC led to a progressive accumulation of of 2 Ag/mL DNase-free RNase, the cells were stained with cells with 2N DNA content (Fig. 1A, red flow cytometry 2 Ag/mL propidium iodide (PI). The intracellular level of profiles). Exposure of these cells to NAC therefore seems to

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(Fig. 1B). A quantitative analysis of the anti-h-tubulin and Hoescht-stained mitotic cells confirmed that NAC reduced but did not fully block the paclitaxel-mediated accumulation of cells in mitosis (Fig. 1C). Inhibition of Cell Proliferation by Decreased ROS Levels Does Not Correlate with Apoptosis ROS levels can be lowered by antioxidants such as NAC as well as by inhibitors of the flavoprotein NAD(P)H oxidases such as DPI. Intracellular levels of ROS can be assayed by measuring the intracellular oxidation of non- fluorescent 2V,7V-dichlorodihydrofluoresceindiacetate to the fluorescent 2V,7V-dichlorofluorescein (6). Flow cytometry of 2V,7V-dichlorodihydrofluoresceindiacetate–treated Rat1 cells revealed that exposure of these cells to 25 mmol/L NAC or 5 Amol/L DPI led to a comparable decrease in 2V,7V- dichlorofluorescein signal relative to cells incubated with 2V,7V-dichlorodihydrofluoresceindiacetate alone (Fig. 2A). As reported previously with both normal and trans- formed cells (6, 7), quantitative evaluation of Rat1 and NIH 3T3 cell proliferation with a range of DPI concen- trations indicated that it has a potent antiproliferative activity. Proliferation of either Rat1 or NIH 3T3 cells was greatly reduced by 2.5 Amol/L DPI, whereas a concentration of 8 Amol/L DPI fully blocked proliferation (Fig. 2B). To determine whether the DPI-mediated suppression of cell proliferation could be due to a loss of cells by DPI- mediated apoptosis, cells were stained with fluorescent Annexin V. Whereas exposure of the cells to UV light resulted in numerous Annexin V–stained cells (Fig. 2C, 3), the number of apoptotic cells arising from exposure to DPI was quite low (Fig. 2C, 2). Indeed, 9 hours after addition of DPI to the culture medium, there was essentially no sign of apoptosis. This contrasts with the high proportion of Figure 1. The antioxidant NAC slows cell cycle progression in G1 but Annexin V binding cells that arise following exposure to does not fully block mitotic cell division. A, Rat1 cells were incubated without (red)orwith(blue)1Amol/L paclitaxel for 6 hours following UV light (Fig. 2D). As reported previously (6), prolonged treatment without (top) or with 25 mmol/L NAC for 6 (middle)or20 exposure to DPI did however lead to a modest degree of (bottom) hours. Cells were then processed for PI flow cytometry. B, Rat1 apoptosis (Fig. 2D). cells were incubated with 1 Amol/L paclitaxel after exposure to 25 mmol/L NAC for 6 hours. The cells were then processed for anti-h-tubulin (green) Inhibition of Cell Proliferation by Decreased ROS and Hoescht fluorescence microscopy (blue). Bar, 100 Am. C, Rat1 cells Levels Does Not Correlate with MAPK Inhibition were either left untreated or incubated with 1 Amol/L paclitaxel with and Because ROS have been implicated in MAPK-mediated without prior exposure to 25 mmol/L NAC for 6 and 20 hours. Columns, percentage of mitotic cells following fluorescence microscopy of anti-h- mitogenic signal transduction pathways (4, 10), the loss of tubulin and Hoescht staining. cell proliferation in response to DPI or NAC treatment has been interpreted as being due to antioxidant-mediated lowering of intracellular ROS levels (6, 7, 11). The effect of NAC and DPI on mitogenic signaling was hence examined inhibit cell cycle progression, resulting in an accumulation by Western blotting with phosphospecific MAPK anti- of cells in the G1 phase of the cell cycle. Inhibition of cell bodies (Fig. 3A) and by in vitro ERK kinase assays (Fig. 3B). cycle progression by NAC was confirmed by treating the Interestingly, whereas prolonged exposure of Rat1 cells to cells with paclitaxel (Fig. 1A, blue flow cytometry profiles). NAC significantly reduced ERK MAPK (p42 and p44 By arresting cells in mitosis, paclitaxel leads to an MAPK) phosphorylation (Fig. 3A, lane 6), exposure to DPI accumulation of cells with 4N DNA content (Fig. 1A, top). caused a pronounced increase in the levels of these Exposure of the cells for increasing periods to NAC prior phosphorylated MAPK as well as ERK activation (Fig. 3A to the paclitaxel treatment led to a progressive reduction and B, lane 3). Conversely, whereas NAC did not affect the in the accumulation of cells with a 4N DNA content (Fig. levels of phosphorylated p38 MAPK, exposure to either 1A, middle and bottom). However, NAC did not lead to a DPI or the nitric oxide synthetase inhibitor L-NAME (which complete arrest of cell cycle progression. Indeed, anti-h- does not seem to appreciably affect Rat1 cell proliferation) tubulin and Hoescht staining revealed significant numbers substantially reduced the phosphorylation of this MAPK of mitotic cells in the presence of both NAC and paclitaxel (data not shown).

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Figure 2. Inhibition of cell proliferation by reduction in ROS levels does not correlate with apoptosis. A, Rat1 cells were either left untreated (red)or incubated with 5 Amol/L DPI (orange), 25 mmol/L NAC (green), or no drug (blue) for 6 hours prior to addition of 50 Amol/L 2V,7V- dichlorodihydrofluoresceindiacetate (DCF-DA) for 30 minutes. The 2V,7V-dichlorofluorescein fluorescence was then measured by flow cytometry following detachment of the cells using trypsin-EDTA. B, Rat1 (blue) and NIH 3T3 (red) cells were counted 24 hours after plating (t = 0). Following overnight exposure of the cells to 0 (no addn), 0.8, 2.5, or 8 Amol/L DPI, the cell proliferation was quantified by determination of the number of cells for each DPI concentration. C, Rat1 cells were either left untreated (1), incubated with 10 Amol/L DPI for 16 hours (2), or briefly exposed to UV light followed by a 16-hour incubation (3). The cells were visualized by fluorescence microscopy following incubation with fluorescently tagged Annexin V and Hoescht. Bar, 50 Am. D, Columns, percentage of Annexin V-Fluos – stained cells for the indicated conditions.

Inhibition of Cell Cycle Progression by DPI determined by treating cells with both DPI and paclitaxel. To further examine the antiproliferative activity of DPI, The ability of paclitaxel-treated cells to reach mitosis in the its effect on cell cycle progression was examined by flow absence or presence of DPI was assayed by anti-h-tubulin cytometry of PI-stained cells. Exposure of Rat1 fibroblasts and Hoescht fluorescence microscopy. Paclitaxel-treated and NIH 3T3 cells to DPI led to a dose-sensitive increase in cells were able to form partially assembled mitotic spindles the number of cells with 4N DNA content (Fig. 4A; data not (as evidenced by the anti-h-tubulin staining) and could shown). In the absence of treatment, there were 62% 2N condense their chromatin (as evidenced by the Hoescht and 22% 4N Rat1 cells, whereas at 4 Amol/L DPI this staining; Fig. 5A, 3). Following 8 hours of exposure to distribution shifted to 36% 2N and 48% 4N DNA content paclitaxel, over 30% of the cells entered mitosis (Fig. 5B). (Fig. 4B). Accumulation of cells with 4N DNA content, as By contrast, cells treated with paclitaxel and DPI failed determined by PI flow cytometry, suggests that DPI affects to either condense their chromatin or rearrange their cell cycle progression. interphase microtubule array into a mitotic spindle DPI Blocks Entry into Mitosis alignment (Fig. 5A, 4). Paclitaxel-mediated mitotic arrest was reduced f6-fold by exposure to 5 Amol/L DPI, Mitotic cells were clearly detectable by anti-h-tubulin A and Hoescht fluorescence microscopy of untreated Rat1 whereas 10 mol/L DPI fully abolished the entry into cells (Fig. 5A, 1). However, essentially no mitotic cells mitosis (Fig. 5B). This indicates that DPI can fully block were discernable following DPI treatment (Fig. 5A, 2). mitotic cell division and that this occurs by a DPI-mediated Similar to other microtubule perturbing compounds, such cell cycle arrest prior to mitosis. as paclitaxel, the antiproliferative activity of DPI also DPI Treatment Results in Centrosome Separation correlates with an accumulation of cells with 4N DNA Failure of DPI-treated cells to reach mitosis indicates that content. The ability of DPI to block mitosis was therefore they have become arrested in the cell cycle prior to M phase.

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Thr288 phosphorylation of the Aurora-A kinase (13). Whereas Thr288 phosphorylated Aurora-A was localized to mitotic spindle poles from prophase to metaphase (Fig. 7A, 1-3), no Thr288 phosphorylated Aurora-A was detected, by immunofluorescence microscopy, on the fully separated centrosomes of DPI-treated cells (Fig. 7A, 4-6). Similarly, Western blots revealed that, whereas paclitaxel treatment caused an enrichment in Thr288 phosphorylated Aurora-A, exposure to DPI did not result in an increase in cellular levels of Thr288 phosphorylated Aurora-A (Fig. 7B). Failure of Thr288 phosphorylated Aurora-A to accumulate on the centrosomes of DPI-treated cells hence seems to be due to a DPI-mediated block in the cell cycle prior to Aurora-A Thr288 phosphorylation. DPI Reversibly Impairs Accumulation of Cyclin B1, Aurora-A Thr288 Phosphorylation, and Mitotic Entry

Prior to mitotic centrosome activation at the G2-M transition, cell cycle progression through G2 to mitosis is accompanied by an accumulation of cyclin B1. Hence, anti– cyclin B1 Western blotting indicated that, essentially in parallel with mitotic arrest, paclitaxel treatment led to a pronounced increase in intracellular levels of cyclin B1

Figure 3. Inhibition of cell proliferation by reduction in ROS levels does not correlate with inhibition of MAPK. Rat1 cells were either serum starved for 24 hours and then treated with platelet-derived growth factor (PDGF; lane 1) or cultured in 10% FCS in the absence of drug (lane 2), 10 Amol/L DPI for 3 (lane 3) and 16 (lane 4) hours, or 25 mmol/L NAC for 3 (lane 5) and 16 (lane 6) hours. The cells were then lysed and either subjected to SDS-PAGE/Western blotting using anti-p-MAPK and anti-MAPK antibodies (A) or ERK kinase assay using GST-Elk as substrate (B).

To further characterize this cell cycle arrest, DPI-treated Rat1 and NIH 3T3 cells were stained with anti-g-tubulin to label centrosomes. During most of the cell cycle, centrosomes remain relatively near to each other and centrosome disjunction usually only occurs toward the end of G2 phase prior to the onset of mitosis (12). Anti-g-tubulin immunofluorescence staining of cells revealed that, whereas the centrosomes remained unseparated in the majority of untreated cells (Fig. 6A, 1), in most of the DPI-treated cells the centrosomes seemed to have separated (Fig. 6A, 2). Indeed, in the presence of DPI, centrosomes of Rat1 cells, and NIH 3T3 cells in particular, were frequently aligned at opposite sides of the nucleus as evidenced by the frequent occurrence of intercentrosomal distances approximating the mean nuclear diameter (Fig. 6B; data not shown). This suggests that in the presence of DPI the cell cycle may continue up to late G2, at which point further cell cycle progression is blocked. Activation of the Centrosome-Associated Aurora-A Kinase Does Not Occur in DPI-Treated Cells

Indicative of a G2-M arrest in the cell cycle, exposure to DPI leads to the accumulation of cells with 4N DNA content and fully separated centrosomes. Failure of these cells Figure 4. Inhibition of cell cycle progression by DPI. A, Rat1 fibroblasts to progress into mitosis suggests that DPI may impair mi- were exposed overnight to 0, 1, 2, or 4 Amol/L DPI and then processed for PI flow cytometry. B, proportion of cells with 2N (blue), 2N-4N (gray), totic functions of the centrosomes. This was probed by and 4N (red) DNA content following overnight incubation with 0, 1, 2, or assaying for mitotic activation of the centrosome through 4 Amol/L DPI.

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DPI Decreases the Levels of Cyclin B1 and Thr288 Phosphorylated Aurora-A in Paclitaxel-Arrested Cells To further probe the effect of DPI on mitotic events, cells were treated with paclitaxel prior to exposure to DPI. The paclitaxel caused mitotic arrest and a pronounced accumulation of cyclin B1 (Fig. 9A). Exposure of these paclitaxel- treated cells to DPI caused a substantial loss of cyclin B1, reaching levels comparable with (or less than) untreated cells 6 hours after the addition of DPI to the culture medium (Fig. 9A). Exposure of the paclitaxel-treated cells to DPI similarly led to a decrease in the levels of Thr288 phosphorylated Aurora-A (Fig. 9B). Loss of these mitotic protein markers by exposure to both paclitaxel and DPI was not due to progression of the cells from M to G1 phase because flow cytometric analysis of PI-stained cells revealed that they continued to have a mostly 4N DNA content in the presence of paclitaxel alone or in combination with DPI (Fig. 9C). DPI Causes Mitotic Catastrophe in Paclitaxel- Arrested Cells Phase-contrast microscopy of cells exposed to either paclitaxel alone (Fig. 10A, 1 and 5) or paclitaxel followed by the addition of DPI (Fig. 10A, 2 and 6) indicated that, in addition to the loss of cyclin B1 and Thr288 phosphorylated Aurora-A, exposure to DPI also caused a striking reversal of the rounded and translucent mitotic phenotype. By fluorescence microscopy of Hoescht staining, it was

Figure 5. DPI blocks entry into mitosis. A, Rat1 fibroblasts were either left untreated (1) or treated with 8 Amol/L DPI (2), 1 Amol/L paclitaxel (3), or both paclitaxel and DPI (4) for 8 hours. The cells were then processed for fluorescence microscopy of anti-h-tubulin (green) and Hoescht (blue). Bar, 50 Am. B, Rat1 cells were either left untreated or incubated with 1 Amol/L paclitaxel for 30 minutes prior to exposure to 5 and 10 Amol/L DPI for 8 hours. The cells were then visualized by Hoescht fluorescence microscopy and the percentage of mitotic cells was determined by the number of cells with condensed chromatin.

(Fig. 8A). To examine how DPI may block cell cycle progression from G2 to M phase, its effect on the accumulation of cyclin B1 was examined. Whereas DPI led to a progressive increase in the number of cells with 4N DNA content (see Fig. 4A and B), the accumulation of cyclin B1 was impaired (Fig. 8A). Following removal of the DPI, cyclin B1 and Thr288 phosphorylated Aurora-A could accumulate (Fig. 8B) and the cells could also enter into mitosis as determined by Hoescht staining of the condensed chromatin (Fig. 8C). The Figure 6. DPI treatment results in centrosome separation. A, NIH 3T3 effects of DPI on cyclin B1 levels and cell cycle progression cells were either left untreated (1) or treated overnight with 4 Amol/L DPI are hence not due to an irreversible nonspecific toxicity. (2). Cells were processed for immunofluorescence microscopy with anti-g- tubulin. Bar, 50 Am. B, intercentrosomal distance determined by anti-g- Curiously, DPI did not appreciably affect the levels of tubulin immunofluorescence staining (light gray, no addition; dark gray, cyclin A (Fig. 8B). DPI). The mean nuclear diameter of the cells was 18.5 Am.

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it leads to an accumulation of cells with 4N DNA content. Additional differences between NAC and DPI are apparent as NAC, but not DPI, suppresses MAPK activity. The antiproliferative effects of NAC and DPI therefore seem to be mechanistically distinct and may not necessarily be due to effects on MAPK.

Figure 7. Activation of the centrosome-associated Aurora-A kinase does not occur in DPI-treated cells. A, Rat1 fibroblasts were either left untreated (1-3) or exposed for 8 hours to 10 Amol/L DPI (4-6) and processed for fluorescence microscopy of anti-g-tubulin (1 and 4), Thr288 phosphorylated Aurora-A (2 and 5), and Hoescht (3 and 6). Bar, 20 Am. B, Rat1 fibroblasts were left untreated (lane 1), serum starved (lane 8), or exposed to either 10 Amol/L DPI (lanes 2-4)or1Amol/L paclitaxel (Tx; lanes 5-7) for 2, 4, and 8 hours. The cells were then lysed and subjected to SDS-PAGE/Western blot using anti – Thr288 phosphorylated Aurora-A and anti-MAPK antibodies (as a loading control). determined that, characteristic of mitotic catastrophe, the loss of the rounded and translucent mitotic phenotype was accompanied by chromatin decondensation and micro- nucleation (Fig. 10A, 4 and 8). By counting the number of paclitaxel-arrested cells with condensed chromatin prior to and following exposure to DPI, the DPI-induced mitotic exit seems to be very pronounced (Fig. 10B).

Discussion The results presented here show that the flavoprotein inhibitor DPI can inhibit G2 cell cycle progression. A range of antioxidants, including DPI, have been reported to inhibit proliferation of both untransformed Figure 8. DPI reversibly impairs accumulation of cyclin B1, Aurora-A and cancerous cells (6, 7), and this inhibition is thought Thr288 phosphorylation, and mitotic entry. A, Rat1 fibroblasts were either to occur by reduction in intracellular ROS levels. By left untreated (lane 1), serum starved (lane 8), or exposed to either 10 Amol/L DPI (lanes 2-4)or1Amol/L paclitaxel (lanes 5-7) for 2, 4, and targeting catalytic cysteine residues, ROS can inactivate 8 hours. The cells were then lysed and subjected to SDS-PAGE/Western phosphatases and thereby affect numerous cell signaling blot using anti – cyclin B1 and anti-MAPK antibodies (as a loading control). pathways (4). In accordance with previous reports (14, 15), B, Rat1 fibroblasts were left untreated (lane 1) or were exposed to either 10 Amol/L DPI for 8 hours (lane 2) followed by release from the DPI for 3 the results presented here show that the antioxidant and 6 hours (lanes 3 and 4)or1Amol/L paclitaxel 4 and 8 hours (lanes 5 NAC may inhibit mitotic cell division by decreasing and 6). The cells were then lysed and subjected to SDS-PAGE/Western mitogenic signaling by MAPK. This leads to an accumu- blot using anti – cyclin B1, anti – Thr288 phosphorylated Aurora-A, anti – lation of cells in the G phase (2N DNA content) of the cell cyclin A, and anti-MAPK antibodies (as a loading control). C, Rat1 1 fibroblasts were exposed for 8 hours to 10 Amol/L DPI and processed for cycle and a slowing of cell cycle progression. By contrast, fluorescence microscopy of Hoescht staining prior to (1) and following (2) although DPI has a comparable antioxidative potential, a 6-hour release from the DPI. Bar, 50 Am.

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Figure 9. DPI decreases the levels of cyclin B1 and Thr288 phosphorylated Aurora-A in paclitaxel-arrested cells. MCF-7 and HepG2 cells were either left untreated (lanes 1 and 4) or treated with 1 Amol/L paclitaxel for 8 hours followed by an additional incubation for 6 hours in the absence (lanes 2 and 5) or presence (lanes 3 and 6)of10Amol/L DPI. The cells were then lysed and subjected to SDS-PAGE/Western blot using anti – cyclin B1 (A) or Thr288 phosphorylated Aurora-A (B). Anti-MAPK antibodies are presented as loading controls. Following overnight treatment with 0.5 Amol/L paclitaxel, MCF-7 and human mammary epithelial cells (HMEC) were incubated for 6 more hours in the absence (blue)or presence (green)of10Amol/L DPI. The paclitaxel/DPI-treated cells and untreated cells (red) were then processed for PI flow cytometry (C).

Interestingly, unlike cells arrested by microtubule drugs, the DPI-treated cells fail to enter mitosis. Indeed, even in the presence of paclitaxel, DPI can fully block cell cycle progression to mitotic prophase. The block of DPI- mediated cell cycle progression hence seems to be due to failure of the cells to undergo the transition from G2 to Figure 10. DPI causes mitotic catastrophe in paclitaxel-arrested cells. M phase. A, MCF-7 (1-4) and human mammary epithelial cells (5-8) were treated A striking consequence of DPI treatment is the appear- overnight with 0.5 Amol/L paclitaxel and then incubated for an additional 6 hours in the absence (1, 3, 5, and 7) or presence (2, 4, 6, and 8)of ance of cells with fully separated centrosomes. Following 10 Amol/L DPI. The cells were then visualized by phase-contrast their duplication during S phase, centrosomes usually microscopy (1, 2, 5, and 6) and fluorescence microscopy of Hoescht remain tethered near to each other until the cells approach staining (3, 4, 7, and 8). Bar, 50 Am for the phase-contrast images and the G -M transition prior to mitotic entry (12). Hence, 25 Am for the fluorescence images. B, percentage of mitotic paclitaxel- 2 arrested Rat1, MCF-7, and human mammary epithelial cells was usually only a relatively small fraction of the interphase determined (by the number of cells with condensed chromatin) following cells have fully separated centrosomes because this is a 6-hour incubation with and without 10 Amol/L DPI.

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usually followed by assembly of the mitotic spindle and References cell division. Centrosome separation is affected by a range 1. Casenghi M, Meraldi P, Weinhart U, Duncan PI, Korner R, Nigg EA. Polo-like kinase 1 regulates Nlp, a centrosome protein involved in of kinases (16, 17), and their mitotic activation is under the microtubule nucleation. Dev Cell 2003;5:113 – 25. control of several cell cycle–regulated kinases (18). The 2. Piel M, Nordberg J, Euteneur U, Bornens M. Centrosome-dependent NIMA kinase Nek1 is involved in centrosome separation exit of cytokinesis in animal cells. Science 2001;291:1550 – 3. (19), whereas Aurora-A and the polo-like kinase Plk1 3. Wood K, Cornwell WD, Jackson JR. Past and future of the mitotic spindle as an oncology target. Curr Opin Pharmacol 2001;1:370 – 7. regulate the centrosome-mediated G2-M transition and assembly of mitotic spindle microtubules, respectively (1). 4. Chiarugi P, Cirri P. 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Mol Cancer Ther 2004;3(10). October 2004

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