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Research Articles: Therapeutics, Targets, and Development

Cantharidin-induced mitotic arrest is associated with the formation of aberrant mitotic spindles and lagging chromosomes resulting, in part, from the suppression of PP2A

Departments of ¹ Biochemistry and Molecular Biology, ² Pathology, and ³ Cell and Neurobiology, College of Medicine, University of South Alabama, Mobile, Alabama and ⁴ Department of Pharmacology, ISIS Pharmaceuticals, Carlsbad, California

Requests for reprints: Richard E. Honkanen, Department of Biochemistry and Molecular Biology, University of South Alabama, MSB 2362, Mobile, AL 36688. Phone: 251-460-6859; Fax: 251-460-6127. E-mail: rhonkanen{at}jaguar1.usouthal.edu

Abstract

Cantharidin, a natural vesicant, inhibits the activity of several PPP family phosphatases, displays antitumor activity, and induces apoptosis in many types of tumor cells. However, the molecular mechanisms underlying the antitumor activity of cantharidin are not clear. Here, dose-response studies confirm a strong correlation between the suppression of phosphatase activity and cell death. Flow cytometry analysis indicates that before apoptosis, cantharidin delays cell cycle progression following DNA replication with no apparent effect on G₁-S or S-G₂ phase progression. In contrast, studies with double thymidine-synchronized populations of cells indicate that cantharidin can rapidly arrest growth when added during G₂ or early M phase. Immunostaining indicates that cell cycle arrest occurs before the completion of mitosis and is associated with the appearance of aberrant mitotic spindles. Live cell imaging with time-lapse microscopy shows that cantharidin disrupts the metaphase alignment of chromosomes and produces a prolonged mitotic arrest, with the onset of apoptosis occurring before the onset of anaphase. To explore the contribution of individual phosphatases, antisense oligonucleotides and small interfering RNA were developed to suppress the expression of cantharidin-sensitive phosphatases. The suppression of PP2A, but not PP2Aß, is sufficient to induce metaphase arrest, during which time lagging chromosomes are observed moving between the spindle poles and the metaphase plate. Immunostaining revealed slightly abnormal, yet predominately bipolar, mitotic spindles. Nonetheless, after a 10- to 15-hour delay, the cells enter anaphase, suggesting that an additional cantharidin-sensitive phosphatase is involved in the progression from metaphase into anaphase or to prevent the onset of apoptosis in cells arrested during mitosis. [Mol Cancer Ther 2006;5(11):2727–36]

Introduction

Tablets dated to 180 AD from the Han Dynasty describe the use of Mylabris, a concoction produced from the Chinese blister beetle, for the treatment of abdominal mass (1). Therefore, Mylabris represents one of the oldest treatments for cancer in recorded history. In 1810, a French pharmacist (Robiquet) developed a procedure to produce an active constituent of Mylabris, cantharidin (exo, exo-2,3-dimethyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid anhydride; Fig. 1 ), which was followed by a period in which cantharidin was employed as an antitumor drug in Europe. By the early 1900s, however, cantharidin was considered by most physicians as too toxic for internal use and fell out of favor as a treatment for human cancer (2). Today, Mylabris is still employed in traditional Chinese medicine, and, recently, interest in the clinical use of cantharidin as an antitumor agent has re-emerged following reports that cantharidin and synthetic derivatives produce cytotoxic effects in a number of human tumor cell lines. Cantharidin also has cytotoxic activity against a number of primary tumor cells (3–7), and clinical testing revealed little myelosuppression (1, 3). Still, the clinical usefulness of cantharidin is limited by renal and mucous membrane toxicity (3, 8).

View larger version (16K): [in this window] [in a new window]   Figure 1. Cantharidin inhibits PPP family phosphatases and cell proliferation. A, effect of cantharidin (inset) on the divalent cation-independent phosphatase activity contained in a crude homogenate of A549 cells. Cells from a single 70% confluent 100-mm dish were rinsed with 4°C PBS, scraped, and sonicated in 1 mL Tris buffer [20 mmol/L Tris-HCl, 1 mmol/L EDTA, and 2 mmol/L DTT (pH 7.4)]. The homogenate was then diluted (65 µg protein/mL) and assayed for phosphohistone phosphatase activity in the presence of the indicated amount of cantharidin as described in Materials and Methods. B, effect of cantharidin on cell viability. A549 cells were plated in 96-well plates (5,000 per well) and allowed to grow. After 24 h, the indicated concentration of cantharidin was added, and the cells were incubated for the time indicated at 37°C. Cell viability was then measured using a Cell Titer 96 Aqueous One Solution Cell proliferation Assay according to the methods of the manufacturer (Promega). Points, mean of three independent experiments each conducted in triplicate; bars, SD.

A key breakthrough in this area came from studies which showed that cantharidin binds with high affinity to a protein (12), which was thereafter identified as a protein phosphatase (13). Subsequent studies revealed that cantharidin functions as an inhibitor of several PPP family serine/threonine protein phosphatases (PPases), acting as a weak inhibitor of calcineurin (PP2B) and a potent inhibitor of three families (PP1, PP2A, and PP5) of structurally related PPases (14–16). Recent structural studies revealed that the cantharidin-sensitive PPases share a common catalytic mechanism (17), and structure/activity relationship studies indicate that cantharidin (15) and fostriecin (18), another natural product with antitumor activity, both act to inhibit the catalytic activity of the same PPP family phosphatases (15, 17, 18). Human genetic studies have identified four highly homologous isoforms of PP1 (PP1, PP1ß/, PP11, and PP12), two nearly identical isoforms of PP2A (PP2 and PP2Aß), and two PPases (designated PP4 and PP6) that, at the level of their primary amino acid sequence, share 54% and 60% identity with PP2A, respectively (for review, see ref. 15). Therefore, humans express at least nine cantharidin-sensitive PPases that are expressed ubiquitously.

To date, the inhibitory actions of cantharidin against purified phosphatases is well documented (15), and several studies indicate that cantharidin is capable of eliciting a "classic" apoptotic response. Nonetheless, little is known about the molecular mechanisms by which the inhibition of phosphatase activity induces or promotes a cytotoxic/antitumor effect, how the apoptotic/cytotoxic response is initiated, or even which of the nine cantharidin-sensitive phosphatases are involved. Here, we further characterize the biological activity of cantharidin in culture cells (A549 and HeLa) and develop small interfering RNA (siRNA) and antisense oligonucleotides capable of markedly suppressing the expression of several cantharidin-sensitive phosphatases. The studies presented indicate that cantharidin-induced apoptosis is preceded by the formation of aberrant mitotic spindles and metaphase growth arrest. Studies with antisense and siRNA indicate that PP2A is needed for proper chromosome segregation, and that the suppression of PP2A alone is sufficient to produce the prolonged mitotic arrest.

Materials and Methods

Cell Culture
A549 cells were obtained from the American Type Culture Collection (Manassas, VA), and HeLa-H2B-GFP cells were a generous gift from Dr. Kevin Sullivan (The Scripps Research Institute, La Jolla, CA). Both cell lines were cultured in DMEM supplemented with 10% fetal bovine serum and L-glutamine (4.0 mmol/L) at 37°C in 5% CO₂.

Phosphatase Assays
The production of phosphohistone and the measurement of phosphatase activity were conducted as described previously (14, 19).

Analysis of Cell Growth and Proliferation Assays
Cells were seeded in 12-well tissue culture plates at a density of 5 x 10⁴ per dish. On the next day, the cells were treated with PP2A-specific antisense oligonucleotides or the scrambled mismatch controls at a final concentration of 300 nmol/L as described above. On each of the next 5 days, the cell cultures were treated briefly with trypsin to detach the cells from the dish (three wells from each test group). The number of cells was then determined by counting using a hemacytometer. Cell viability was determined with trypan blue staining. The percentage of viable cells was calculated by dividing the number of cells excluding trypan blue by the total number of cells, and the results are reported as the mean ± SD of data collected from three independent experiments. The ability of cantharidin to inhibit the growth of cultured cells was determined using a tetrazolium-based proliferation assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and by counting the number of cells daily using a culture counter. Cell death was visualized by trypan blue exclusion and DNA staining using propidium iodide. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were done using Cell Titer (Promega, Madison, WI) using 96-well plates seeded with 5,000 cells per well in 80 µL of medium according to the methods of the manufacturer. Cantharidin (0.5–10 µmol/L) was added from concentrated stock solutions in N,N-dimethylformamide.

Flow Cytometry
At the time indicated, cells were subjected to a brief treatment with trypsin, collected by centrifugation, resuspended in PBS, and stained with Vindelov's propidium iodide stain (50 µg/mL) containing RNase (200 µg/mL) at 4°C. DNA content flow cytometry analysis was done using routine methods with a Becton Dickinson fluorescence-activated cell sorter (FACS), gating to exclude cell debris as described previously (20).

Indirect Immunofluorescence Microscopy
Cells were plated onto 60-mm dishes containing 22 x 22 coverslips in 3 mL DMEM at a concentration of 300,000 per dish. At timed intervals, the coverslips were removed, washed with PBS (pH 7.4), fixed with methanol at –20°C, permeabilized with Triton X-100, and processed for immunofluorescence microscopy using previously published methods (21). Microtubules were visualized with anti--tubulin (Sigma, St. Louis, MO; primary, 1:100 dilution) and Alexa Fluor 488 antibodies (Invitrogen, Carlsbad, CA; secondary mouse monoclonal, 1:1,000). DNA was visualized with propidium iodide (50 µg/mL) or Draq5 (Biostatus Products, Shepshed, United Kingdom; 1:300). Centrosomes were visualized using anti-pericentrin (Covance, Berkeley, CA; primary rabbit polyclonal, 1:500) and Alexa Fluor 594 antibodies (Invitrogen; secondary goat anti-rabbit, 1:1,000). The samples were then examined using a Leica TCS SP2 confocal system equipped with argon, krypton, and He-Ne lasers. Confocal images were collected at 1.0-µm z intervals with a pinhole setting of 80 µm. Typically, 20 optical sections were analyzed per cell.

Live Cell Imaging and Time-lapse Video Microscopy
Cells were cultured in 60-mm dishes and incubated at 37°C in 5% CO₂ using a Neuve live cell chamber fitted into an Eclipse Nikon TE 2000-U microscope (Nikon Instruments, Inc., Melville, NY). Image acquisition was achieved using a COOLSNAP ES monochrome camera and processed with MetaMorph Premier software (Universal Imaging, Downingtown, PA).

Oligonucleotide Synthesis and Assay for Oligonucleotide Inhibition of PP2A Expression
Phosphorothioate deoxyoligonucleotides and 2'-O-methyl phosphorothioate deoxyoligonucleotides were synthesized and purified as described previously (22–24). The indicated cells were plated in 60-mm dished and cultured in DMEM containing 10% FCS. When the cells were 50% confluent, they were treated with oligonucleotides as previously described (22, 23). After treatment (4 hours), the cells were washed and cultured in fresh DMEM containing 10% FCS for 17 hours. The cells were then harvested, and total RNA was extracted with TRIzol Reagent (Life Technologies, Gaithersburg, MD) according to the methods of the manufacturer. Total RNA (20 µg) was fractionated on 1% agarose gels containing formaldehyde and transferred to DURLON-UV (Stratagene, Cedar Creek, TX) nylon membranes. Following UV cross-linking, the filters were hybridized with a [³²P] probe for human PP2A or PP2Aß as indicated. The human PP2A cDNA probes were generated from the full-length coding region of PP2A or PP2Aß, respectively, and [³²P] labeled with a DECAprime DNA Labeling kit (Ambion, Austin, TX) according to the manufacturer's protocol. Hybridization was done in the presence of 50% formamide at 42° C for 16 hours. Following hybridization, the membrane was subjected to two low-stringency washes (2 x SSC) at room temperature and then two high-stringency washes (0.1 x SSC/0.5% SDS) at 55°C. Hybridization was visualized by autoradiography, and quantification of hybridization signals was obtained using Fuji FLA-5000 imaging system. The membranes were then stripped and reprobed with a [³²P]-labeled glyceraldehyde-3-phosphate dehydrogenase cDNA probe to confirm that equal loading was achieved. Quantification of probe binding was achieved using a Fuji FL5000 imaging system.

Immunoblotting of PP2A
Western analysis was done essentially as described previously using polyclonal rabbit antibodies generated against a synthetic 15-amino-acid peptide identical to the COOH-terminal region of PP2A that does not discriminate between PP2A and PP2Aß (25).

Results

Cantharidin-Induced Cytotoxicity Correlates with the Inhibition of Divalent Cation-Independent Phosphatase Activity
Using a [³²P]phosphohistone-based phosphatase assay, the divalent cation-independent serine/threonine phosphatase activity contained in dilute human cell homogenates represents the activity of PP1, PP2A, PP4, PP5, and possibly PP6 (15). The IC₅₀ (concentration inhibiting 50% activity) for cantharidin-mediated inhibition of divalent cation-independent PPase activity in A549 cell homogenates was estimated at 0.5 µmol/L (Fig. 1A). To compare PPase inhibition with growth suppression and cytotoxicity, dose-response studies were conducted in which replicate plates were treated with the indicated concentrations of cantharidin. At timed intervals, cell proliferation and viability were assessed using a metabolic assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], a cell proliferation assay (counting the number of cells using a culture counter), and a trypan blue exclusion assay (counting the number of live cells based on their ability to exclude trypan blue). All three of these methods produced complementary results (Fig. 1B). At high concentrations (>10 µmol/L), cantharidin rapidly inhibits proliferation, and trypan blue exclusion studies indicate that >95% of the cells were killed in <24 hours. In contrast, treatment with low concentrations (0.1 µmol/L) for up to 72 hours had no apparent effects on cell viability, growth, or morphology. At concentrations sufficient to inhibit the activity of PP1-PP5 (1–5 µmol/L), cell-counting studies indicate that cantharidin rapidly (24 hours) inhibits cell proliferation in a dose-dependent manner. Viability studies indicate that by 48 to 72 hours, most of the cells treated with >2 to 5 µmol/L cantharidin die (Fig. 1B). For A549 cells, the apparent LC₅₀ (concentration killing 50% of the cells) for continuous exposure at 48 hours was 2.0 µmol/L, which correlates with the near complete inhibition of divalent cation-independent PPase activity (i.e., PP1–PP5).

Cantharidin Treatment Increases the Proportion of Cells in the G₂-M Phase of the Cell Cycle
To further characterize the growth arrest induced by cantharidin, A549 cells were treated with the indicated amount of cantharidin (0–5 µmol/L) and then subjected to FACS analysis. FACS analysis of propidium iodide–stained cells indicates that the number of cells containing twice the G₁ content of DNA (4N) increases in a dose-dependent manner 24 hours after treatment with 2 to 5 µmol/L cantharidin (Fig. 2A ). To further test the effects of cantharidin on the cell cycle progression, A549 cell cultures that were synchronized by double thymidine treatment were treated with cantharidin immediately after release from thymidine-induced growth arrest. As seen in Fig. 2B, following release from the second thymidine block, control cells rapidly progress into S phase, and cells with 4N DNA are observed after 6 to 7 hours. The cells then divide, and <12 hours after release, a G₁ population of cells is again observed. When the double thymidine arrested cells were treated with cantharidin immediately after release from growth suppression, the progression of cells into S phase was not delayed (compare Fig. 2B and C; 4 HR). However, following DNA replication, the majority of the cantharidin-treated cells arrest, most still containing 4N DNA 16 hours after treatment (compare Fig. 2B and C; 12 and 16 HR). When cantharidin was added to double thymidine-arrested cell cultures at timed intervals (2, 4, 6, and 7 hours after release from G₁ growth suppression), FACS analysis showed that all treatment groups contained 4N DNA at 16 hours after release (Fig. 2D). These studies suggest that cantharidin-induced growth arrest occurs after DNA replication is complete but before cell division (i.e., G₂-M phase).

View larger version (30K): [in this window] [in a new window]   Figure 2. DNA content flow cytometric histograms of propidium iodide–stained A549 cells. A, cantharidin dose-response. Logarithmically growing A549 cells were treated with cantharidin at the concentrations indicated for 24 h. Controls were treated with solvent alone. At the time points indicated, the cells were harvested, and DNA content flow cytometry analysis was done as described in Materials and Methods. DNA content shown (X-axis) as fluorescence, and populations of cells containing 2N and 4N DNA are shaded with dark gray (2N and 4N populations, arrowheads). S-phase cells are shaded with diagonal lines. The sub-G1 populations (dead and apoptotic cells) are shaded as light gray. B and C, FACS analysis of A549 cell cultures following release from double thymidine-induced G1 growth arrest. Double thymidine treatments were used to arrest A549 cell cultures in late G1 phase of the cell cycle. The cells were then treated with solvent (B) or cantharidin (C) and released from growth suppression. At the times indicated, the cells were fixed and processed for DNA content FACS analysis as described above. D, FACS analysis of A549 cells treated with cantharidin at timed intervals after release from growth suppression. A549 cells were arrested with double thymidine treatment and then released from growth suppression as above. Cantharidin was added to the culture at the times indicated. The cells were then fixed 16 h after they were released from double thymidine growth arrest. Data shown are representative of at least three independent experiments each conducted in triplicate.

View larger version (19K): [in this window] [in a new window]   Figure 3. Abnormal spindle formation in cells treated with cantharidin. Representative A549 cells treated with solvent alone (A) or cantharidin (B–E). A549 cells were treated with 2 µmol/L cantharidin and fixed on coverslips 24 h later. Microtubules (green) were then visualized by immunofluorescence following treatment with anti--tubulin (primary) and Alexa Fluor 488–labeled (secondary) antibodies. DNA (red) was visualized by staining with propidium iodide. E, serial z sections obtained by confocal microscopy of cantharidin-treated cells stained for anti--tubulin antibodies as above. F, effect of cantharidin on spindle formation. A549 cells were treated with the indicated amount of cantharidin. After 24 h, the cells were fixed. Microtubules and DNA were then visualized as described above. Metaphase cells (300 for each concentration of cantharidin) were then scored as containing normal or aberrant spindles. Percentage containing normal bipolar spindles (white columns) or aberrant spindles (filled columns). A to F, similar results were obtained with four or more independent experiments.

View larger version (20K): [in this window] [in a new window]   Figure 4. Inhibition of PP2A mRNA and protein expression by treatment with antisense oligodeoxynucleotides. A, relative positioning of the predicted hybridization sites within the human PP2A or PP2Aß mRNA of 43 antisense oligodeoxynucleotides that were evaluated for their ability to inhibit PP2A expression in cultured A549 cells. Antisense oligodeoxynucleotides that inhibit the expression of PP2A and PP2Aß mRNA were identified by treating A549 cells with the indicated oligodeoxynucleotides at a concentration of 300 nmol/L. mRNA was prepared 24 h later and analyzed for PP2A, PP2Aß, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA levels by Northern blot analysis. B, inhibition of PP2Aß mRNA levels by ISIS 110179, ISIS 110181, ISIS 110186, or ISIS 110189. C, inhibition of PP2A by ISIS 110159 or ISIS 110163. D, specificity of oligos targeting PP2A (ISIS 110159) and PP2Aß (ISIS 110181). A549 cells were treated with increasing concentrations (0–300 nmol/L) of the indicated antisense oligodeoxynucleotides or corresponding mismatched control analogues (MM) that contain 13 base changes (mismatches) within the sequence of the indicated target. Total mRNA was prepared 24 h later and analyzed for PP2A, PP2Aß, and G3PDH mRNA levels by Northern blot analysis. IC50s were estimated from plots produced by quantification of PP2A mRNA levels after normalization to G3PDH mRNA in A549 cells following treatment with increasing concentrations of oligodeoxynucleotides (illustrated for ISIS 110159). E, Western blots of PP2A protein levels in A549 cells. Cells were treated with the mismatch control oligodeoxynucleotides, ISIS 110159 (targeting PP2A) or ISIS 110181 (targeting PP2Aß), and protein extracts were prepared 48 or 76 h later. Each lane contained 40 µg protein. F, target-specific inhibition of ISIS 110159 and ISIS 110181. A549 cells were treated with 200 or 400 nmol/L antisense oligodeoxynucleotides targeting the indicated isoform of PP2A. Total protein was prepared 48 or 76 h later and analyzed for PP4 and PP6 by Western blot analysis.

Specificity of Antisense Inhibition
To examine the specificity of oligonucleotides targeting PP2A (ISIS 110159 and ISIS 110163) and PP2Aß (ISIS 110179, ISIS 110181, ISIS 110186, and ISIS 110189), the effects of each oligonucleotide and appropriate mismatch control analogues (designated ISIS 110159 MM1, etc.) were tested. The mismatch control analogues contain the same base composition as the corresponding antisense oligonucleotide but with sequences noncomplementary to the target mRNA. As seen in Fig. 4B to E, treatment of A459 cells with the indicated oligonucleotides produced a dose-dependent reduction of the appropriate PP2A mRNA levels with the most potent antisense oligonucleotides having IC₅₀s < 50 nmol/L. No effect was observed following treatment with the mismatch controls (Fig. 4D), even when applied at concentrations 10 times that of the IC₅₀ for highly active oligonucleotides. Blots were stripped and reprobed with a glyceraldehyde-3-phosphate dehydrogenase cDNA probe to show equal RNA loading. Studies with additional mismatch controls and different cell types produced similar results (data not shown).

Although the differences in codon usage allowed for the production of isoform-specific probes suitable for Northern analysis, PP2A and PP2Aß are 97% identical at the level of their primary amino acid sequences. Therefore, it was not possible to generate isoform-specific antibodies. Nonetheless, Western analysis using an antibody that recognizes both isoforms of PP2A revealed that treatment with ISIS 110159 (targeting PP2A) decreased PP2A levels by 80% after 48 hours (Fig. 4E). The suppression of PP2Aß with ISIS 110181 produced an 20% decrease. Treatment with 200 nmol/L of ISIS 110159 in combination with 200 nmol/L ISIS 110181 produced a marked suppression of PP2A after 48 hours. These observations are consistent with reports studying the expression of PP2A isoforms, in which PP2A mRNA levels were observed at approximately four times the level of PP2Aß.

To further test the specificity of the antisense oligonucleotides targeting PP2A mRNA, the effect of ISIS 110159 on expression of two structurally related PPases (PP4 and PP6) was tested by Western analysis using antibodies generated against peptides unique to the indicated PPases. As seen in Fig. 4F, ISIS 110159 has no effect on the expression of PP4 or PP6, even at a concentration (400 nmol/L) that is >20 times the IC₅₀ (17.8 nmol/L) for PP2A. Because the sequence targeted by ISIS 110159 is not contained in PP4 or PP6, ISIS 110159 would not be expected to inhibit the expression of these proteins if ISIS 110159 is inhibiting the expression of PP2A mRNA via an RNase H-medicated antisense mechanism.

Reduction of PP2A Expression with ISIS 110159 Leads to Failure of Various Mitotic Events
FACS analysis of propidium iodide–stained A549 cells revealed that the suppression of PP2A with ISIS 110159 produced an increase in cells containing 4N DNA that was comparable with cells 24 hours after treatment with 2 µmol/L cantharidin. In contrast, FACS analysis of cells treated with oligonucleotides targeting PP2Aß or PP5 failed to show an increase in cells containing 4N DNA (data not shown). Immunostaining with anti--tubulin antibodies revealed differences in the appearance of the mitotic spindles between control and cantharidin or ISIS 110159 (targeting PP2A)–treated cells (compare Figs. 3 and 5 ). As observed following treatment with cantharidin, cells treated with ISIS 110159 contained condensed chromosomes, and immunostaining with antibodies targeting pericentrin revealed the normal complement of two centrosomes in control, cantharidin-treated (data not shown) and ISIS 110159–treated cells A549 cells (Fig. 5B). In addition, the mitotic spindles in ISIS 11059–treated metaphase cells were not well organized. However, most cells still produced a somewhat bipolar spindle apparatus (Fig. 5E). As observed with cantharidin, the microtubules in nondividing cells with suppressed PP2A expression were similar in appearance to untreated or mismatched controls (data not shown).

View larger version (12K): [in this window] [in a new window]   Figure 5. Mitotic abnormalities associated with the suppression of PP2A. Representative confocal microscopic images of A549 cells treated with ISIS 110159. A549 cells were treated with 300 nmol/L ISIS 110159 and fixed on coverslips 24 h later. A, microtubules (green) were then visualized by immunofluorescence following treatment with anti--tubulin and Alexa Fluor 488–labeled antibodies as described in Fig. 3. B, centrosomes were visualized by treatment with pericentrin. C, DNA (blue) was visualized by staining with Draq5. D, computer-assisted merge of images (A), (B), and (C). E, representative images of serial z sections of ISIS 110519–treated mitotic cells stained for anti--tubulin. Similar results were obtained with five or more independent experiments.

View larger version (42K): [in this window] [in a new window]   Figure 6. Localization of H2B-GFP protein in HeLa cells. Representative fluorescent and corresponding differential interference contrast microscopic images of live HeLa cells expressing H2B-GFP in various phases of the cell cycle. A, representative images selected from time-lapse studies of control cells illustrating prophase (0 min), early metaphase (20 min), late metaphase (70 min), anaphase (80 min), and telophase (86 min). B, cantharidin treatment of prophase cells prevents the alignment of chromosomes at the metaphase plate. H2B-GFP–expressing HeLa cells were treated at time 0 with 4 µmol/L cantharidin, and time-lapse live cell imaging studies were conducted for 24 h. Representative images taken at the times indicated. C, cantharidin treatment of metaphase cells delays anaphase and disrupts the metaphase alignment of condensed chromosomes. H2B-GFP–expressing HeLa cells were treated with cantharidin, and time-lapse live cell imaging was conducted for 24 h. Representative images taken at the times indicated. D, representative fluorescent and differential interference contrast images from time-lapse studies monitoring cell cycle progression and H2B-GFP expression in cells treated with ISIS 110159. H2B-GFP–expressing HeLa cells were treated with ISIS 110159, and 30 h later, time-lapse imaging studies were conducted as described above. E, enlarged images highlighting differences in control, cantharidin-treated, and ISIS 110159–treated cells. Arrows indicate the fluorescence of lagging sister chromatids.

When HeLa-H2B-GFP cells were treated with ISIS 110159, as observed with other human cell lines, 30 hours was required for PP2A protein levels to become suppressed. Before this time, cell division occurs normally. When PP2A levels were suppressed, time-lapse fluorescence microscopy indicated that in prometaphase or metaphase cells, most of the chromosomes are condensed and eventually align at the metaphase plate. However, in nearly all instances, anaphase was delayed for >15 hours (Fig. 6D). ISIS 110159–treated cells also exhibited small populations of chromosomes localized at various points between the metaphase plate and the spindle pole (Fig. 6E, arrows). In addition, whereas the cantharidin-arrested cells fail to enter anaphase and become apoptotic, the ISIS 110159–treated cells eventually enter anaphase and divide after the prolonged metaphase arrest. Still, it should be noted that 48 to 72 hours after treatment with ISIS 111059, numerous apoptotic cells were observed.

Discussion

Here, as observed in T24 cells (7), with A549 and HeLa cells, we observed acute toxicity by cantharidin at high concentrations (10 µmol/L) and apoptosis at slightly lower concentrations (2–5 µmol/L). As observed in other studies, dose-response studies revealed a strong correlation between the inhibition of divalent cation-independent serine/threonine phosphatase activity (i.e., PP1–PP5) and cell death. In addition, this study shows that before the onset of apoptosis in dividing cells, cantharidin induces a prolonged mitotic arrest that is associated with the formation of aberrant mitotic spindles, the failure of condensed chromosomes to align at the metaphase plate, and the failure of cells to enter anaphase (Figs. 1–3). Time-lapse microscopy studies also indicate that the cells that arrest in metaphase become apoptotic after a prolonged (>10 hours) delay. In contrast, the nondividing cells are similar in appearance to controls.

To gain insight into which of the cantharidin-sensitive PPases affect cell cycle progression and survival, we developed siRNA and antisense oligonucleotides capable of suppressing the expression of PP11 (20), PP5 (24, 28, 30), and, here, PP2A and PP2Aß. FACS analysis of propidium iodide–stained A549 cells treated with siRNA or antisense oligonucleotides targeting each of these PPase individually revealed that the suppression of PP5 expression results in a G₁ growth arrest with the concomitant increase in the expression of p21^Waf1/cip1 (24). Subsequently, this response was shown to be dependent on the presence of both glucocorticoid receptors and the p53 tumor suppressor protein (24). Moreover, the suppression of PP5 unveiled a link between GR activation, the phosphorylation of p53 at Ser¹⁵, and p21-mediated G₁ growth arrest (28). Because Ser¹⁵ phosphorylation of p53 is known to aid p21 expression, part of the cantharidin-mediated cellular responses can be mimicked by the suppression of PP5 expression. Nonetheless, in cells lines lacking p53 or GRs, the suppression of PP5 expression with antisense or siRNA did not result in an extensive mitotic arrest or prolonged progression into anaphase, suggesting that the inhibition of PP5 alone is not sufficient to induce a cantharidin-like mitotic delay.

The suppression of PP2Aß had no apparent effect on cell cycle progression, whereas the suppression of either PP11 or PP2A resulted in the accumulation of 4N cells. Studies with antisense targeting PP11 suggest that it is not needed for mitotic spindle formation or progression from metaphase to anaphase. Rather, PP11 is needed in late-stage cytokinesis and the formation of dikaryons (20). To date, the molecular events affected by PP11 are not clear. Nonetheless, because cantharidin-treated cells arrest in metaphase and become apoptotic before any indications of cytokinesis, PP11 does not seem to contribute to the "cantharidin phenotype" observed in the cell lines studied. In contrast, the data obtained with ISIS 110159 indicate that PP2A elicits the failure of several mitotic events that are also induced by 2 to 5 µmol/L cantharidin (a concentration sufficient to completely inhibit PP2A activity; ref. 14). Notably, treatment with ISIS 110159 produces a prolonged mitotic arrest that is associated with aberrant mitotic structures. Still, PP2A suppression does not entirely mimic the actions of cantharidin. In comparison with cells treated with ISIS 110159, the spindles of the cantharidin-treated cells are less organized, and the condensed chromosomes do not align at the metaphase plate. In addition, after a 10- to 12-hour delay, the ISIS 110159–treated cells progress into anaphase and divide, whereas the cantharidin-treated cells do not enter anaphase and become apoptotic.

During mitosis, the sister chromatids (replicated copies of each chromosome) are physically linked before their segregation. This association, called cohesion, must be sustained during the G₂ phase of the cell cycle and during most of metaphase because the kinetochores of each sister chromatid must bind microtubules from opposite spindle poles to ensure segregation away from each other at anaphase (31). Sister chromatid cohesion is mediated by a complex of proteins called cohesin. In mammals, the phosphorylation of centromeric cohesin during mitosis promotes its dissociation from the chromatids, which is an important part of the "trigger" for chromosome segregation. Therefore, it is critical for cells to guard centromeric cohesion from the actions of mitotic kinases before the onset of anaphase. Recently, PP2A has been shown to associate with Shugosin (Sgo1; Drosophila, MEI-S332), one of the proteins that is needed for the protection of centromeric cohesion in animals (32, 33). In addition, the association of PP2A with Sgo1 has been shown to be necessary for the centromeric localization of Sgo1 and proper chromosome segregation in human cells (32, 33). The data shown here are consistent with the role of PP2A in the protection of chromosome segregation, for cantharidin treatment of mitotic cells at concentrations that inhibit the activity of PP2A results in the dissociation of the chromosomes aligned on the metaphase plate in a random and unorganized manner. Furthermore, because treatment with ISIS 110159 results in the formation of lagging sister chromatids, our data indicate the involvement of the isoform of PP2A is needed for proper chromosome segregation. Still, additional studies will be needed to elucidate details of how the suppression of PP2A results in arrest during metaphase.

In conclusion, PP2A is needed for proper chromosome segregation, and the suppression of PP2A with ISIS 110159 is sufficient to induce a prolonged metaphase arrest. However, whereas cantharidin produced a similar metaphase arrest that resulted in cell death before anaphase, the suppression of PP2A expression did not ultimately prevent cells from entering anaphase. In addition, the mitotic spindles of the cantharidin-treated cells were generally multipolar and less organized than the cells treated with ISIS 110159. Therefore, additional cantharidin-sensitive PPases are likely to also contribute to the cytotoxic actions of cantharidin.

Footnotes

Grant support: NIH/National Cancer Institute grants CA-60750 (R.E. Honkanen) and NIH/HL 77769 (S. Ofori-Acquah). This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program grant C06 RR11174 from the National Center for Research Resources.

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 5/12/06; revised 8/ 9/06; accepted 9/25/06.

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