Fostriecin-Mediated G2-M-Phase Growth Arrest Correlates With

[CANCER RESEARCH 58. 3611-3619, August 15, 1998] Fostriecin-mediated G2-M-phase Growth Arrest Correlates with Abnormal Centrosome Replication, the Formation of Aberrant Mitotic Spindles, and the Inhibition of Serine/Threonine Protein Phosphatase Activity1

Aiyang Cheng, Ron Balczon, Zhuang Zuo, Jamie S. Koons, AimÃ©eH. Walsh, and Richard E. Honkanen2

Departments of Biochemistry and Molecular Biology Â¡A.C, Z Z. J. S. K., A. H. W.. R. E. H.I ami Structural and Cellular Biology Â¡R.B.Â¡,College of Medicine. University of South Alabama. Mobile. Alabama 36688

ABSTRACT protein synthesis (7). Although these studies demonstrated that fos triecin affects a process essential for replication and transcription, no Fostriecin, a structurally unique phosphate ester, is presently under direct effect on DNA polymerase, RNA polymerase, or enzymes evaluation in clinical trials to determine its potential use as an antitumor within the purine or pyrimidine biosynthetic pathways has been noted drug in humans. Fostriecin has been reported as having inhibitory activity against DNA topoisomerase type II and protein phosphatases implicated (3, 7). Fostriecin was then shown to block cell growth in the G2 phase in cell-cycle control. However, the relative contribution of these mecha of the cell cycle and to have inhibitory activity against type II DNA nisms to the antitumor activity of Iosi ricchi has not yet been elucidated. In topoisomerase from Ehrlich ascites carcinomas (8), with 100 /J.M this study, after confirming that fostriecin is a potent inhibitor of serine/ fostriecin completely inhibiting the relaxation of supercoiled DNA by threonine protein phosphatase type 2A and a weak inhibitor of serine/ type II topoisomerase (IC50, 40 JLIM).Unlike other type II topoisomer threonine protein phosphatase type 1, we show that fostriecin inhibits ase inhibitors, however, fostriecin does not induce DNA strand â€”¿50%ofthe divalent cation independent serine/threonine protein phos breaks; instead, it seems to directly inhibit the catalytic activity of phatase (PPase) activity contained in whole cell homogenates of Chinese sensitive topoisomerases. In contrast, studies in CME or K562 leuke hamster ovary cells at concentrations associated with antitumor activity mia cell lines, exposed to the epipodophylotoxins teniposide (VM-26) (1-20 /UAH.Investigations into the cellular effects produced by fostriecin treatment reveal that 1-20 Â¿IMfostriecin induces a dose-dependent arrest or etoposide (VP-16) in combination with fostriecin, indicate that of cell growth during the G2-M phase of the cell cycle. Immunostaining of fostriecin does not have an inhibitory effect on topoisomerase II in treated cells indicates that growth arrest occurs before the completion of these cell models (9, 10). mitosis and that fostriecin-induced growth arrest is associated with the Recent studies suggest that the antitumor activity of fostriecin could aberrant amplification of centrosomes, which results in the formation of also originate from its ability to interfere with the reversible phos- abnormal mitotic spindles. The "mitotic block" induced by fostriecin is phorylation of proteins that are critical for progression through the cell reversible if treatment is discontinued in <24 h. However, after â€”¿24-30 cycle (11-13). At high concentrations (>125 /ÃœLM),fostriecininhibits h of continuous treatment, growth arrest is not reversible, and treated a mitotic entry checkpoint (11) and induces chromosome condensa cells die even when placed in fostriecin-free media. Correlative studies tion in interphase cells in a manner comparable to okadaic acid, a conducted with established PPase inhibitors reveal that, when applied at known inhibitor of PP13 and PP2A (11). Fostriecin-induced chromo concentrations that inhibit PPase activity to a comparable extent, both okadaic acid and cantharidin also induce aberrant centrosome replication, some condensation correlates with an increase in histone H2A and H3 the appearance of multiple aberrant mitotic spindles, and G2-M-phase phosphorylation in mouse mammary tumor cells, and fostriecin- growth arrest. These studies add additional support to the concept that induced hyperphosphorylation of vimentin correlates with the re PPase inhibition underlies the antitumor activity of fostriecin and suggest organization of intermediate filaments in hamster kidney cells (13). that other type-selective PPase inhibitors should be evaluated for potential In addition, inhibition studies conducted with PPases indicate that antitumor activity. fostriecin is a potent inhibitor of PP2A and a weaker inhibitor of PP1 (11, 14). Nonetheless, the concentration of fostriecin needed to INTRODUCTION achieve the above mentioned cell-cycle events (125-375 IJLM;Refs. 11-13) is substantially higher than that required for the inhibition of Fostriecin (2H-Pyran-2-one,5,6-dihydro-6-[3,6,13-trihydroxy-3- PPase activity (14), and the observed cellular effects were observed methyl-4-(phosphonooxy)-1,7,9, 11-tridecatetraenyl) is a structurally after a relatively brief treatment period (<6 h). In comparison, the novel phosphate ester antitumor antibiotic that contains an unsaturated studies that demonstrated the antitumor activity of fostriecin were lactone and a conjugated trine system (Fig. 1). Fostriecin is produced longer in term and conducted with lower concentrations (0.1-30 /XM) by Streptomyces pulveraceus (subspecies fostreus) and exhibits anti- of fostriecin (6). tumor activity against a wide spectrum of tumor cells in vitro (1-3). Recently, we have shown that fostriecin is capable of inhibiting Fostriecin also has excellent activity against L1210 lymphoid and approximately one-half of the divalent cation-independent PPase ac P388 lymphocytic leukemias in vivo (4, 5). Because of this marked tivity contained in RINmSF insulinoma cells at concentrations asso antitumor activity, fostriecin is being developed as an anticancer agent ciated with antitumor activity (14). This suggests that the inhibitory and is presently under evaluation in clinical trials (6). effects of fostriecin on specific PPases could result in antitumor The molecular mechanisms underlying the antitumor activity of activity and that further investigation into the possible mechanisms by fostriecin have been under investigation for several years. Initial which PPase inhibition results in antitumor activity is warranted. studies indicated that fostriecin rapidly inhibits RNA, DNA, and Therefore, in the present study we further characterize the inhibitory effect of fostriecin against PPases contained in CHO cell homoge Received 12/29/97; accepted 6/11/98. nates. We then determine the effects of fostriecin on cell cycle 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 progression in CHO cells after the administration of fostriecin at 18 U.S.C. Section 1734 solely to indicate this fact. concentrations associated with antitumor activity and compare the 1This work was supported by NIH Grant CA 60750 (to R. E H.) and American Cancer Society Grant CB-151 (to R. B.). 2 To whom requests for reprints should be addressed, at Department of Biochemistry 1The abbreviations used are: PP1, protein phosphatase type 1; PP2A, protein phos and Molecular Biology, MSB 2198, College of Medicine. University of South Alabama, phatase type 2A; PPase. serine/threonine protein phosphatase; PI. propidium iodide; CHO, Mobile. AL 36688. Chinese hamster ovary; FACS, fluorescence-activated cell-sorting. 3611

phosphorylated substrate, and the reaction was linear with respect to enzyme concentration and time. [32P]phosphate that was liberated by the enzymes was extracted as a phosphomolybdate complex and measured according to the methods of Killilea et al. (23). Inhibition of phosphatase activity by fostriecin, 'CH2OH okadaic acid, and cantharidin was determined by adding the inhibitors to the enzyme mixture 10 min before initiating the reaction with the addition of the substrate. Flow Cytometry. The cells were harvested and stained with PI; DNA content flow cytometry was then performed as described previously (24). Fostriecin Briefly, CHO cells in log phase growth were treated with the indicated amount Fig. I. Structure of fostriecin, C|9H27PO,Na. gram molecular weight 452.36 (70). of fostriecin. After 18 h, the cells were treated briefly with trypsin, separated from the flask by gentile tapping of the flask against the tissue culture hood, and collected by centrifugation. The cells were washed with PBS and resus effects produced by fostriecin with those produced by the addition of pended at a concentration of 1 X IO6 cells/ml in PBS containing PI (50 /Â¿g/ml). other PPase inhibitors at concentrations that inhibit CHO PPase ac The cells were placed in the dark for l h at 4Â°Cand then filtered through tivity to a similar extent (15-18). Our findings indicate that fostriecin 30-fim mesh. DNA content flow cytometry was then performed with a Becton is capable of inhibiting cellular PPase(s) that are necessary for the Dickinson FACS device as described previously (24). Indirect Immunofluorescence Microscopy. Cells were plated on 60-mm regulation of centrosome replication and that PPase inhibition results dishes in 6 ml of McCoy's 5A medium at a concentration of 0.5 X IO6 cells in the formation of aberrant mitotic spindles and G2-M-growth arrest. per dish and incubated in the absence or presence of the indicated amount of These findings suggest that the antitumor activity of fostriecin could fostriecin. Mitotic cells were harvested by "mitotic shake off and deposited by indeed result from the inhibition of PPase activity and support the centrifugation onto polylysine-coated coverslips. The cells were then fixed by concept that PPase inhibitors have the potential for development as immersion of the coverslips in -20Â°C methanol for 6-8 min and processed for antitumor drugs. immunofluorescence microscopy using previously published methods (25-27). Anti-a-tubulin (Sigma, St. Louis, MO) was used at 1:200 dilution and SPJ MATERIALS AND METHODS autoimmune human anticentrosome serum (26) was used at a dilution of 1:1000. FITC-labeled secondary antibodies (Boehringer Mannheim Biochemi- Materials. Phosphorylase kinase (EC 2.7.1.38), protein kinase A (3':5'- cals) were used at a 1:200 dilution. After processing, coverslips were mounted cyclic AMP dependent), phosphorylase b (EC 2.4.1.1), crude histone (type in PBS:glycerol ( 1:1) containing 25 /xg/ml HOECHST 33258 dye. Cells were 2AS), p-nitrophenyl phosphate, and mouse monoclonal anti-a-tubulin antibod observed using an Axiovert 35 M microscope (Carl Zeiss, Inc.) Images were ies (T-90260) were obtained from Sigma Chemical Company. FITC goat recorded using either T-Max 400, Ektachrome 400, or Gold 200 film (Eastman antimouse IgG was obtained from Boehringer Mannheim (Indianapolis, IN). Kodak, Inc.). ('(infocai Laser Scanning Microscopy. Samples were fixed, processed, Fostriecin was generously donated by the Division of Cancer Treatment, National Cancer Institute (Bethesda, MD) and Parke-Davis (Ann Arbor, MI). and labeled as described above. Samples were then examined with an ACAS Cell Culture. CHO cells were cultured in McCoy's 5A medium containing 570 image analyzer (Meridian Instruments, Okemos, MI) equipped with an 0.1 mM minimal essential amino acids, 2 mM glutamine, 1 mM sodium Innova 90-5 five-W argon ion laser (Coherent, Palo Alto, CA) and an pyruvate, and 10% fetal bovine serum in a 5% CO2 environment. Olympus IMT-2 inverted microscope. The 488-nm laser line was used to excite Preparation of Phosphoprotein Substrates. Histone (type 2AS from the FITC fluorochrome and emitted fluorescence was collected through an Sigma) was phosphorylated with cAMP-dependent protein kinase (protein Olympus Dapo 100 UV (NA = 1.3 oil) objective. Confocal images were kinase A) as described previously (18-20). Briefly, 20 mg of histone was collected at 1.0-p.M z intervals with a pinhole setting of 80 p.m as described incubated with 1 mg of protein kinase A, in a 20-mM Tris-Cl buffer (pH 7.4) previously (28). Twenty to 30 optical sections were analyzed per cell. containing 1 mCi [12P]ATP (150 /AMATP), 100 yM cAMP, 5 mM DTT, and 5 mM MgCl2 in a final volume of 4 ml. The reaction was allowed to continue RESULTS for 3.5-4 h at 30Â°Cand was terminated by the addition of 1.3 ml of 100% trichloroacetic acid (TCA). The precipitated phosphohistone was collected by Inhibition of PPase Activity by Fostriecin. When diluted below centrifugation at 3000 x g for 5 min. The supernatant was discarded, and the the titration end point (defined as the concentration of enzyme after pellet was redissolved in 4 ml of H,O. TCA was added to precipitate the which further dilution no longer affects the IC50; Refs. 14, 18), the phosphohistone, and this precipitation-resuspension wash was repeated 5 dephosphorylation of phosphohistone by the purified catalytic sub- times. The pellet produced upon the sixth TCA precipitation was washed 2 units of PP2A is potently inhibited by fostriecin (Fig. 2). Fostriecin times with 4 ml of ethanol:ethyl ether (1:4, v:v) and then 2 additional times inhibits the activity of PP2A in a dose-dependent manner, having an with acidified ethanol:ethyl ether (1:4; 0.1 N HC1). The washed histone was IC50 of 5.58 Â±0.41 nM. Fostriecin also inhibits the activity of PP1 in allowed to air-dry and was then resuspended in 5 mM Tris-Cl (pH 7.4). This procedure yields phosphohistone with a specific activity >4.5 x IO6 cpm/ a dose-dependent manner; however, Â»0,000-fold higher concentra tions of fostriecin are needed to affect the activity of PP1 (IC50, ~58 nMole incorporated phosphate. Purification of PP1 and PP2A. The catalytic subunit of PP1 was purified (MM).Under identical conditions, okadaic acid is a more potent inhib to apparent homogeneity, demonstrating a single band on SDS-PAGE and itor of both PP2A (IC50, =0.05 nM) and PP1 (IC50 =44 nM), whereas intense silver staining, according to the methods of Brautigan et al. (21), with cantharidin is a slightly less potent inhibitor of PP2A (IC50 =0.16 JUM) minor modifications (16-20). The catalytic subunit of PP2A was purified to and a more potent inhibitor of PP1 (IC50 =1.5 /AM).Still, fostriecin apparent homogeneity as described by Pallas et al. (22) using G-75 Sephadex demonstrates more selectivity against PP2A than either cantharidin or in the place of Ultrogel-AcA44 as reported previously (18, 21). okadaic acid. Determination of Phosphatase Activity. Phosphatase activity against phosphohistone was determined by the quantification of [32P] liberation from To determine whether fostriecin inhibits the activity of the native forms of PP1 and PP2A at concentrations comparable to those that phosphohistone (16-20). Assays, 80 Â¡i\total volume, containing 50 mM Tris-HCl (pH 7.4), 0.5 mM DTT, 1 mM EDTA, (assay buffer), and [32P]phos- affect the purified catalytic subunits, the effect of fostriecin on PPase phoprotein (1-2 fiM PO4), were conducted as described previously (16-20) activity contained in whole cell homogenates of CHO cells was using either the purified catalytic subunits of PP1 or PP2A or a dilute whole examined. Fig 2B shows the inhibitory dose-response of fostriecin in cell homogenate of cultured cells. Dephosphorylation reactions were routinely a homogenate of CHO cells diluted past the titration end point (â€”60 conducted for 10 min and initiated with the addition of the substrate. The jxg of protein/ml). Fostriecin inhibits the divalent cation-independent dephosphorylation of the substrate was kept to less than 10% of the total phosphatase activity in the whole-cell homogenate, with and an ap- 3612

Toxicity studies indicate that fostriecin rapidly inhibits cell prolifer 125 r ation (<24 h), with cytotoxic effects occurring <48-72 h after treatment. The apparent IC5() for 48 h cytotoxicity in CHO cells was estimated to be 5.5 /LIM,which is similar to the cytotoxic effects on 100 other cell types (for review, see Ref. 6). At high concentrations oC (> 100-300 /Â¿M)fostriecin killed >95% of the cells in <24 h. In o 75 contrast, treatment with low concentrations (^1 /J.M)for up to 72 h produces no visually apparent effects on CHO cell growth or mor 50 phology. Treatment with fostriecin at concentrations shown to have antitumor activity (1-20 Â¡JLM)resultsin growth arrest in <24 h, and 25 flow cytometry analysis of Pi-stained cells indicates that the number of cells containing twice the normal content of DNA increases in a dose-dependent manner 18 h after treatment with 1-25 /XMfostriecin OL (Fig. 3). This suggests that growth arrest occurs after DNA replication 12 11 10 9 8 7 6 5 4 3 2 is complete, but before the completion of anaphase (i.e., in the G2-M -Log [Inhibitor; (M)] phase of the cell cycle). Light microscopic analysis of the growth- arrested cells reveals that the treated cells are rounded in appearance, and the rounded cells easily detach from the flask. Nonetheless, after B treatment for periods up to 18 h, the arrested cells are viable (based on their ability to exclude trypan blue), and the rounded cells recover if they are plated in fresh media without fostriecin. If treatment is

o O Fostriecin

256

O 256

12 11 10 9 8 7 6 5 4 3 2 256 -Log [Inhibitor; (M)] 256 Fig. 2. Comparison of the inhibitory effect of fostriecin with that of other known PPase inhibitors. A. inhibitory effect of fostriecin (A. A), okadaic acid ( *. 0 ). cantharidin (â€¢, O), and Taxol â€¢¿D) on the activity of the purified catalytic subunils of PP1 (filled symbols) and PP2A (open symbols). B, effect of fostriecin (A), okadaic acid (*), cantharidin (â€¢),and Taxol (â€¢)on the divalent cation-independent PPase activity con tained in dilute whole-cell homogenates of CHO cells. Phosphatase activity was deter mined using a standard PPase assay and phosphohistone as the substrate. Assays were initiated by the addition of the substrate, and the [32P]phosphate liberated by the enzymes was extracted as a phosphomolybdate complex (see experimental procedures for details). Phosphohistone phosphatase activity was 334 Â± 8.1 nmol of Pi/min/mg protein, B 2,100 Â±244 nmol of Pi/min/mg protein, and 432 Â±9.6 pmol of Pi/min/mg protein for PP1. PP2A, and the CHO homogenate, respectively. Each point represents the mean Â±SD (n = 6). 50 nM parent IC50 of ~1 /IM. Nonlinear regression analysis of the dose- 30 nM inhibition curves indicates that the inhibition of PPase activity in the CHO cell homogenate best fits a multisite model, which is consistent with predicted values for a homogenate composed of both PP1 and PP2A. However, Northern analysis indicates that CHO cells also contain mRNA that encodes two structurally related PPases, PP4, and PP5 (not shown), which, if sensitive to fostriecin, may contribute to the observed PPase activity against phosphohistone. The presence of additional PPase activity is further suggested by the shape of the inhibition curve produced with dilute CHO cell homogenates, which should have a more pronounced plateau between IO"8 and IO"6 M if 02 PP1 and PP2A are the only PPases contained in the homogenate. Fig. 3. DNA content flow cytometric histograms of fostriecin and okadaic acid and Alternatively, the native PPases may associate with regulatory pro cantharidin/Taxol-treated CHO cells indicate that all three PPase inhibitors and Taxol arrest cell growth in the G,-M phase of the cell cycle. Logarithmically growing CHO cells teins or other cellular components that alter the sensitivity of PP1 were treated with fostriecin (A) or okadaic acid (B} at the concentrations indicated for and/or PP2A to fostriecin. 18 h. Controls (0 /IM, OnM) were treated with solvent alone. The cells were harvested and Fostriecin Treatment Increases the Proportion of Cells in G2-M stained with PI (10 Â¿Ag/ml):DNA content flow cytomelry was then performed. DNA content is measured on the X-axis as fluorescence, and GÂ¡(2 N) and G2-M (4 N) Phase of the Cell Cycle. The effects of fostriecin on CHO cells populations are indicated. The numbers on the x-axis represent flow cyotometric channel varies, depending on the amount of fostriecin added to the cell culture. units, and the results shown are representative of three independent experiments. 3613

Table I Abnormal cÃ©ntrasemereplication and milotic spindle formation correlates spindle microtubule formation was examined by immunostaining with with the inhibition of PPase activity in CHO cells Cells (0.25 X 10*) were plated on 60-mm dishes and treated with solvent alone an antibody to a tubulin. (control). Taxol, or the indicated amount of each PPase inhibitor. Four sets of dishes for Immunostaining with antitubulin antibodies revealed marked dif each treatment group were then: (Â«)immunostained to visualize microtubules (Figs. 4 and ferences between the control and fostriecin-, okadaic acid-, canthari 5); (b) immunostained to visulalize centrosomes (Fig: 5); (c) assayed for PPase activity; din-, or Taxol-treated cells (Figs. 4 and 5). At mitosis, >96% of the and (if) stained with PI and (e) analyzed by FACs analysis (Fig 3). The cultures treated with fostriecin. cantharidin. and okadaic acid but not Taxolor solvent alone (control), had control cells contain a typical array of antiparallel microtubules char decreased PPase activity, which correlated with aberrant centrosome duplication, the acteristic of a normal bipolar mitotic spindle (Fig. 4). In contrast, the appearance of cells containing multiple aberrant spindles, and C2 M-phase growth arrest. fostriecin-treated cells often contained aberrant spindle apparatus PPase activity composed of multiple poles (Figs. 4C and 5B), and the percentage of (pmoles/min/mg protein); FACS analysis cells containing multiple spindle poles increased in a dose-dependent Treatment mean Â±SE (Â«) (ratio of cells, G,:G2-M) manner (data not shown). Treatment with okadaic acid produced ControlCantaridin (18)44.3Â±11.1 (Ã•M)Fostriecin(5 Â±9.3(12)36.3 similar effects to those observed with fostriecin (Figs. 4E and 5D), UM)Okadaic(10 Â±3.8(9)33.1 whereas Taxol treatment resulted in the appearance of cells containing nM)Taxolacid (30 Â±4.2(12)175.5 aggregates of loosely organized microtubules (Figs. 4G and 5E). On (10/iM)184.6 Â±12.5(9)2.620.490.560.220.18 the basis of observations of mitotic structures by confocal fluores cence microscopy, it seems that treatment with fostriecin, okadaic continued for >24 h, however, most of the rounded cells cannot acid, or cantharidin does not prevent the polymerization of microtu recover when the fostriecin is removed, and the cells proceed to die bule structures in general; rather, treatment disrupts the number and with characteristics suggesting that death is induced via apoptosis organization of the spindle poles (Fig. 5). As reported previously, (29-31). PPase inhibition studies conducted in parallel with these treatment with Taxol seems to stabilize polymerized microtubules growth inhibition studies indicate that treated cells contain ~30-40% (35-36), resulting in the formation of larger microtubule aggregates. of the divalent cation-independent PPase activity observed in the Immunostaining with anticentrosome antibodies suggests that the corresponding controls (Fig. 2; Table 1). formation of aberrant spindle poles is associated with aberrant cen Okadaic Acid and Cantharidin Can Mimic the Cellular Effects trosome replication (Fig. 5). As typically observed in mammalian Produced by Fostriecin. Treatment of CHO cells with two other cells, the bipolar mitotic spindle pole is organized by a single centro PPase inhibitors, okadaic acid and cantharidin, at concentrations that some pair in >96% of control mitotic CHO cells. In marked contrast, inhibit PPase activity to an amount comparable to that observed after cells treated with fostriecin at concentrations that produced the aber treatment with fostriecin reveal many similarities. Like fostriecin, rant mitotic spindles contained more than two centrosomes (3 to when okadaic acid is added to cell cultures at concentrations that >10), and, again, similar effects were produced by treatment with inhibit PP2A activity completely and PP1 activity by >50 (i.e., >100 okadaic acid or cantharidin at concentrations that inhibited PPase nM), okadaic acid is toxic, killing >95% of the cells in <24 h. activity to a comparable extent. The abnormally amplified centro Similarly, the addition of 2 nM okadaic acid for up to 72 h, which somes in cells treated with any of the PPase inhibitors tested seem to inhibits the activity of PP2A completely and has little, if any, effect on retain microtubule-nucleating activity (Fig. 5 A-D), and the presence PP1, produces no visually apparent affects on CHO cell growth or of multiple centrosomes had profound effects on chromosome segre morphology. Treatment with 4-50 nM okadaic acid, which inhibits gation. In most instances, treated cells accumulate with the presence CHO PPase activity to an extent comparable to that produced by 1-50 of multiple spindle microtubules and condensed chromosomes that /AMfostriecin, results in growth arrest. Flow cytometry analysis of lack metaphase alignment (Fig. 4). When large numbers of centro Pi-stained cells after treatment with 10-50 nM okadaic acid indicates somes (S4) are present, aberrant multipolar spindles form, with that the number of cells containing twice the normal content of DNA chromosomes randomly distributed between the spindle poles. These increases in a dose-dependent manner (Fig. 3), and the G2-M-phase cells fail to proceed to anaphase, and cell death ensues in â€”¿24-48h. When the "mitotic cells" are washed and replated in media that is free block produced by okadaic acid is slightly more pronounced than that produced by fostriecin. Furthermore, okadaic acid growth-arrested of inhibitors, most cells recover and complete anaphase after a 3-6 h cells are easily detached from the flask and rounded in appearance. In delay. Thus, the block is reversible. However, when the duration of addition, like the fostriecin-arrested cells, the okadaic acid growth- treatment is >24 h, most of the cells cannot recover, and cell death arrested cells can recover after treatment with <40 nM okadaic acid ensues with the characteristics of apoptosis (data not shown). Taxol- for up to 18 h, whereas treatment for >24 h results in apoptotic cell treated cells contain a normal complement of centrosomes, which fail death (32-34). Similar observations were also made with cantharidin to migrate to opposite ends of the cell. (data not shown), and comparison of the PPase activities of fostriecin-, Fostriecin, Okadaic Acid, or Cantharidin Treatment Has No cantharidin-, or okadaic acid-treated cell cultures indicates that there is a Apparent Effect on the Amount of p53 mRNA in CHO Cells. The strong correlation between PPase inhibition and the accumulation of cells loss of the p53 tumor suppressor protein has been linked to abnormal in G2-M phase of the cell cycle (Figs. 2 and 3; Table 1). centrosome replication and aberrant chromosomal separation in Fostriecin Induces Abnormal Centrosome Replication Associ mouse fibroblasts (38-40), and okadaic acid treatment of A549 cells ated with Aberrant Spindle Formation. To further characterize the results in decreased p53 mRNA levels (41). Thus, we performed mechanisms associated with fostriecin-induced growth arrest, we ex Northern blot analysis to determine whether treatment with fostriecin amined the effects of fostriecin on centrosome and microtubule be or okadaic acid affects the expression of p53, which could, in turn, havior. Then, as above, we compared the effects produced by fostrie result in aberrant centrosome replication. Neither fostriecin, okadaic cin with those produced by okadaic acid and cantharidin under acid, or cantharidin had an effect on the amount of mRNA detected by conditions that inhibited PPase activity to a comparable extent. As an a p53 specific probe in CHO cells (data not shown), suggesting that additional control, we treated the cells with Taxol, a mitotic blocking aberrant centrosome replication does not result entirely from the agent that stabilizes microtubules (35, 36) and has no apparent effect ability of PPase inhibitors to inhibit the expression of p53. However, on PPase activity (Fig. 2). Centrosomes were identified by immuno- these studies do not preclude the possibility that PPase inhibitors staining with the well-characterized SPJ human autoimmune anticen- affect the functions of the p53 protein, which is phosphorylated on trosome antiserum (26, 37), and the ability of centrosomes to nucleate several sites. 3614

Fig. 4. Abnormal spindle formation and chromo some condensation in cells treated with PPase inhib itors correlates with G2-M-phase growth arrest. CHO cells treated with solvent alone (A and fl), with 10 /J.M fostriecin (C and D), with 30 nM okadaic acid (E and F), or with 10 (Â¿MTaxol(G and tf) were collected by mitotic shake-off 18 h after treatment. The mitotic cells were then collected and fixed on polylysine coated coverslips. Tubulin was visualized by immunofluorescence after treatment with anti-a-tubulin (primary) and FITC-labeled (secondary) antibodies (A, C. E, and G). DNA was visualized by staining with HOECHST 33258 stain (B. D, F, and //). G2-M- growth arrest was confirmed with flow cytometry from cells treated with PPase inhibitors as described above, and PPase activity was measured in samples of the same cells visualized by immunostaining. Similar results were obtained with 24 independent experi ments (Table 1).

DISCUSSION also characterized the effects of fostriecin on the PPases contained in dilute whole cell homogenates of CHO cells, which contain the Although numerous studies have demonstrated that fostriecin (CI920, "native forms" of PP1 and PP2A and possibly other fostriecin sensi PD110140, NSC 339638) has considerable antitumor activity both in tive PPases (see "Results"). With both the purified catalytic subunits vitro and in vivo (1-6), the mechanism underlying the antitumor activity and the dilute CHO cell homogenate, fostriecin was found to have of fostriecin is not fully understood. In cultured cells, fostriecin inhibits cell cycle progression in the G2-M phase of cell growth, and fostriecin inhibitory activity against both PP1 (IC50,58 /XM)and PP2A (IC50,5.6 nM), which is consistent with the effects of fostriecin on the divalent has inhibitory activity against type II DNA topoisomerase in vitro (8). cation-independent PPase activity contained in homogenates of Because type II DNA topoisomerase activity is necessary for DNA replication, the ability of fostriecin to inhibit DNA topoisomerase activity RINmSF insulinoma cells (14). is consistent with its ability to induce G2-M-phase growth arrest. More Although the data from the human clinical trials are not yet avail able, numerous preclinical studies indicate that â€”¿0.5-15Â¿IMfostriecin recent studies, however, suggest that fostriecin also has inhibitory activity against PPases, which may also function to regulate cell cycle progres is capable of producing antitumor activity in both cultured human sion through the G2-M phase of the cell cycle (Refs. 11 and 14; for cells and animals (3, 5, 6, 43). This correlates well with the IC50 ~ 1 review, see Ref. 42). /XM)for fostriecin-mediated inhibition of the divalent cation-indepen To investigate the cellular effects of fostriecin further, in this study dent PPase activity contained in the CHO (Fig. 2) and RINmFS (14) we characterized the inhibitory activity of fostriecin against two cell homogenates. Studies conducted with purified PP1 and PP2A PPases implicated in cell-cycle control, PP1 and PP2A (for review, indicate that 15 p.Mfostriecin completely inhibits the activity of PP2A see Ref. 42), using the purified catalytic subunits of these PPases. We and â€”¿30%ofthe activity of PP1. However, because the IC50 for 3615

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0 1 2 3 4 >5 1 2 3 4 >5

ww5004003002001000â€”-- 125 100 75 50 25 0 1 2 3 4 >5 1 2 3 4 >5 Spindle Poles Centrosomes

Fig. 5. Abnormal spindle formation correlates with the amplification of centrosomes in cells treated with PPase inhibitors. CHO cells treated with solvent alone (A), with IO /J.M fostriecin (B). with 5 Â¡IMcantharidin (O, with 30 HMokadaic acid (D), or with 10 /AMTaxol (E) were collected by mitotic shake-off 18 h after drug treatment and immunostained with either anti-a-tubulin (/) or anticentrosome (Ili) antibodies. Antibody-antigen complexes were then detected with FITC-conjugated antirabbit IgG and were visualized in 2-^im confocal slices by fluorescence microscopy. Arrows indicate centrosomes, and the actual number of spindle poles (If) and centrosomes (IV) per cell were scored using data from five independent experiments in which microtubules and centrosomes were visualized by indirect immunofluorescence microscopy as described in Fig. 4. G2-M-phase growth arrest was confirmed with FACS analysis of cells treated with PPase inhibitors as described above, and PPase activity was measured in samples taken from the same set of cells visualized by immunostaining. Similar results were obtained with ^4 independent experiments (Table 1). 3616

PP2A inhibition by fostriecin (5.6 nM) is substantially lower than the Immunostaining with anticentrosome antibodies indicates that the amount associated with antitumor activity, it seems that the inhibition aberrant spindle formation is associated with the deregulation of of PP2A alone is not sufficient to produce antitumor activity. Simi normal centrosome regulation, and there is an excellent correlation larly, as observed for the inhibition of DNA topoisomerase II activity, between the appearance of cells with multiple centrosomes and cells the IC50 for the inhibition of PP1 (â€”58/XM)is greater than the IC50 with aberrant spindles (Fig. 5). In contrast, treatment with Taxol for antitumor activity. Thus, because many signaling cascades that produces a mitotic block without affecting centrosome regulation. regulate cell cycle progression require the sequential activation of These results are similar to the effects observed after okadaic acid several different protein kinases, the antitumor activity of fostriecin treatment of LLC-PK cells (40 nM; Ref. 57), GH4 cells (10 nM; Ref. could require the complete inhibition of PP2A, which may be neces 58), and A549 cells (20 nM; data not shown), all of which arrest in a sary to inhibit one branch of the process, in combination with the metaphase-like mitotic state and contain aberrant multipolar spindles. partial inhibition of PP1, which may be necessary for the inhibition of Similarly, both PP1- and PP2A-like PPases have been implicated in a second branch of the process or a sequential step in the cascade (42). the control of microtubule dynamics during mitosis in Xenopus egg Alternatively, because the inhibition studies conducted with the CHO extracts (59); and, in Drosophila, PP2A deficiency leads to death in cell homogenate suggest that there are additional fostriecin-sensitive embryogenesis around the time of cellularization, with PP2A-defi- PPases, the antitumor activity of fostriecin may arise from the inhi cient embryos exhibiting over-condensed chromatin, a block in mito bition of one of these PPases. This concept is tentatively supported by sis, and multiple centrosomes in cellularized embryos (60). Thus, the molecular cloning studies that have identified several PPases struc ability of fostriecin to induce the formation of aberrant mitotic cells turally related to PP2A [i.e., PP4 (44, 45), PP5 (46), PP6 (47), or PP7 has many characteristics that are also produced by other PPase inhib (48)], and once methods for purifying these PPases have been estab itors when applied at a concentration that produces a comparable lished, it will be interesting to determine whether they are indeed decrease in PPase activity. sensitive to fostriecin with kinetic properties that correlate more Because many signaling cascades leading to cell growth are stim ulated by phosphorylation-mediated events, it has been assumed that precisely with the data obtained from the studies on antitumor activity. Of course, once absorbed by a cell, fostriecin could undergo rapid PPases are negative regulators of cellular growth. This concept has degradation or inactivation, which may make the effective intracellu- been perpetuated by studies indicating that okadaic acid has tumor- lar concentration of fostriecin lower than that which is added to the promoting activity in the mouse skin model (61, 62). In contrast, the data presented in this study suggest that the inhibition of PPase incubation media. activity leads to antitumor activity. Closer examination of the data Additional evidence implying that PPase inhibition contributes to presented in the mouse skin model, however, reveals that okadaic acid the antitumor activity of fostriecin comes from the comparison of the was applied at a concentration of 123 JJ.M(61). At concentrations of effects produced by fostriecin with those produced by the treatment >1-10 fxM,okadaic acid completely inhibits the activity of both PP1 with other well-characterized PPase inhibitors. As reported previously and PP2A as well as all of the divalent cation-independent PPase with L1210-cells (8), fostriecin produces a dose-dependent arrest of activity contained in dilute whole-cell homogenates (14-20). In com cell growth in the G2-M phase of the cell cycle when added to CHO parison, the present study used okadaic acid at a much lower concen cell cultures at concentrations associated with antitumor activity (Fig. tration (1-50 nM), which does not inhibit the activity of PP1 com 3). Treatment of CHO cells with either okadaic acid or cantharidin at pletely. This suggests that the antitumor activity of PPase inhibitors concentrations that inhibit CHO PPase activity to a comparable extent occurs after the exposure to PPase inhibitors at concentrations that also induces G2-M-phase growth arrest in a dose-dependent manner, inhibit PP2A without completely inhibiting the activity of PP1. Sim and neither okadaic acid (100 nM) nor cantharidin (50 /IM) has an apparent effect on DNA topoisomerase activity.4 Thus, although the ilar findings were observed after the treatment of transformed NIH 3T3 cells with 8 nM okadaic acid, which causes the cells to revert to PPases that are involved clearly need further characterization, the a flat phenotype and to lose their ability to form colonies in soft agar induction of G2-M-phase growth arrest seems to arise as a conse (63). Neoplastic transformation due to the sequential treatment with quence of decreased PPase activity. benzo(a)pyrene and 12-O-tetradecanoyl-phorbol-13 acetate is also In mammalian cells the phosphorylation of centrosomal proteins is inhibited by the simultaneous treatment with 1.2 or 12.5 nM okadaic associated with the initiation of microtubule nucleation from mitotic acid in CHO cells (64), and treatment of C3H/10T1/2 mouse fibro centrosomes, an event necessary for the formation of the bipolar blasts with 6-10 nM okadaic acid inhibits the ability of 3-methylchol- spindle, the balanced separation of chromosomes, and the progression anthrene and 12-O-tetradecanoyl-phorbol-13 acetate to produce trans through M phase of the cell cycle (49). Similarly, in lower eukaryotes, formed colonies in a two-stage transformation assay (65). mutations affecting homologues of mammalian PPase (sds21+, dis2+/ bwsl+, and bimG) are associated with mitotic defects in the estab Furthermore, 10 nM okadaic acid arrests GH4 (58) and myeloid leukemia (66) cell growth with a corresponding increase in the pro lishment of cell polarity and the separation of sister chromatids portion of cells present in the G-.-M phase of the cell cycle, and (50-54). Genetic experiments in Drosophila (55) and studies with treatment with cantharidin, or structural derivatives of cantharidin, has PPase neutralizing antibodies in rat embryo fibroblasts also indicate been shown to have antitumor activity against KB cells in culture and that PPases are required for the completion of mitosis (56). We, on reticulocell sarcoma and ascites hepatoma in mice (67, 68). Thus, the therefore, investigated whether the growth arrest induced by fostriecin ability of a PPase inhibitor to act as a tumor promoter or an antitumor affects microtubule nucleation or normal centrosome function. In antibiotic could reflect the PPases that are affected. Alternatively, because addition, to assess whether the observed effects are due to PPase the inhibition of both PP1 and PP2A is toxic to cells in culture, it is inhibition, we again tested okadaic acid and cantharidin at concentra possible that the promotion by okadaic acid of tumor formation in mouse tions that produce a comparable amount of PPase inhibition. As seen skin may arise from a nonspecific consequence of wound healing, an in Figs. 4 and 5, all three PPase inhibitors have a pronounced effect on effective promoter of mouse skin carcinogenesis (69). mitotic spindle formation, characterized by the appearance of multiple In summary, the data presented in this study confirm that fostriecin aberrant spindle poles that are randomly distributed inside the cell. has inhibitory activity against both purified PP1 and PP2A as well as the divalent cation-independent PPases contained in crude CHO cell 4 X. Huang and R. E. Honkanen. unpublished observations. homogenates. Comparative studies conducted with other well-charac- 3617

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1998 American Association for Cancer Research. FOSTRIECIN-MEDIATED G,-M-PHASE GROWTH ARREST terized PPase inhibitors and fostriecin suggest that the ability of 19. Critz, S. D.. and Honkanen. R. E. Protein phosphatases and their role in the regulation fostriecin to block cell growth in the G2-M phase of the cell cycle of membrane currents in sensory neurons of Aplysia. Neuroprotocols: a companion to methods in neurosciences. 6: 78-83, 1995. results from the inhibition of PPases that function to regulate centro- 20. Honkanen, R. E., Dukelow. M.. Moore, R. E.. Zwiller, J.. Khatra. B. S.. and Boynton. some replication and the formation of a bipolar mitotic spindle. A. L. Cyanobacterial nodularin is a potent inhibitor of Type 1 and Type 2A protein Because the PPase inhibitor-treated cells can recover after the appear phosphatases. Mol. Pharmacol. 40: 577-583. 1991. 21. Brautigan. D. L.. Schriner, C. L., and Gruppuso. P. A. Phosphorylase phosphatase ance of multiple spindles and because Taxol treatment also produces catalytic subunit. Evidence that the A/r = 33,000 enzyme fragment is derived from a a mitotic block leading to cell death without inhibiting PPase activity, native protein of M, = 70,000. J. Biol. Chem.. 260: 4295-4302. 1985. the "trigger" leading to cell death seems to arise as a consequence of 22. Pallas, D. C.. Shahrink. L. K.. Martin. B. L.. Jaspers, S.. Miller. T. B.. Brautigan. D. L., and Roberts. T. M. Polyoma small and middle T antigens and SV40 small t the inability to proceed through the cell-cycle rather than a direct antigen form stable complexes with protein phosphatase 2A. Cell. 60: 167-176. 1990. effect on PPase activity. Still, a direct effect on PPase activity cannot 23. Killilea, S. D., Aylward, J. H., Mellgren, R. L., and Lee, E. Y. C. Purification and properties of bovine myocardial phosphorylase phosphatase (protein phosphatase C). be ruled out. Therefore, these studies support the concept that the Arch. Biochem. Biophys.. 191: 638-646. 1978. antitumor activity of fostriecin may indeed arise from its inhibitory 24. Funkhouser. J. D.. Cheshire. L. B.. Read. R. J.. Hester. R. B., and Peterson. R. D. activity against cellular PPases and suggests that other PPase inhibi Monoclonal antibody isolation of type II pneumocytes. Cytometry. S: 321-326. 1987. 25. Balczon. R., and Brinkley. B. R. Tubulin interaction with kinetochore proteins: tors should be evaluated for antitumor activity. analysis by in vitro assembly and chemical cross-linking. J. Cell Biol., 705: 855-862, 1987. 26. Balczon, R.. and West. K. The identification of mammalian cenlrosomal antigens using human autoimmune anticentrosome antisera. Cell Motil. Cytoskeleton. 20: ACKNOWLEDGMENTS 121-135, 1991. 27. Balczon. R.. Bao, L., and Zimmer. W. E. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1998 American Association for Cancer Research. Fostriecin-mediated G2-M-phase Growth Arrest Correlates with Abnormal Centrosome Replication, the Formation of Aberrant Mitotic Spindles, and the Inhibition of Serine/Threonine Protein Phosphatase Activity

Aiyang Cheng, Ron Balczon, Zhuang Zuo, et al.

Cancer Res 1998;58:3611-3619.

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