Introduction

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Cell cycle checkpoints are important regulators of eukaryotic cell growth. Because loss of the G₁ checkpoint often occurs in malignant cells, synthetic, small molecule, modulators of the G₂/M checkpoint are of particular interest. Several clinically important anticancer compounds, such as the vinca alkaloids and taxanes, act as prominent inhibitors of G₂/M phase transition, thus validating the concept that disruption of G₂/M transition can be an effective mechanism for controlling some cancers. Nonetheless, all of these compounds disrupt tubulin directly and they have complex chemical structures that restrict chemical modification. In addition, several prominent disrupters of tubulin, such as nocodazole and colchicine, lack antitumor efficacy. Therefore, novel chemical structures that block G₂/M phase transition are valuable as pharmacological probes and represent possible lead structures for future therapeutic agents.

The naphthoquinone acetals, palmarumycins, diepoxins, and deoxypreussomerins (Fig. 1) are structurally unique fungal metabolites with both antifungal and antibacterial activities (Schlingmann et al., 1993; Krohn et al., 1994; Wipf and Jung, 1998) but their antiproliferative activity against malignant mammalian cells has not been extensively studied. Biological studies have been limited partly due to the extraordinary synthetic challenges associated with the extensive levels of oxygenation and the highly electrophilic functionality present in these spiroketal natural products. We have developed an efficient synthetic approach toward palmarumycins, diepoxins, and deoxypreussomerins (Wipf and Jung, 1999) and have generated a small focused library of analogs (Wipf et al., 2001). In a preliminary report we found that a number of these palmarumycins analogs had antiproliferative activity against human cancer cells (Wipf et al., 2001). We have now extended these studies by focusing on one of the most active members of the library, 8-(furan-3-ylmethoxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2'-naphtho[1",8"-de][1',3']dioxin, or SR-7, which retained the antiproliferative activity of palmarumycin CP₁ and diepoxin against the estrogen-receptor positive, p53 replete human MCF-7 breast cancer cells, the estrogen-receptor negative, p53 deficient human MDA-MB-231 breast cancer cells and virally transformed mouse fibroblasts.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Chemical structures of palmarumycin CP1, deoxypreussomerin A, diepoxin , and related unnatural analogs.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials and Chemicals. The synthesis and initial antiproliferative evaluation of the palmarumycin/diepoxin analogs have been described elsewhere (Wipf et al., 2001). The synthesis, and biochemical and cellular properties of the Cdc25 inhibitor SC-9 have also previously been published (Rice et al., 1997; Tamura et al., 1999). Curacin A was prepared as described previously (Wipf and Xu, 1996). tsFT210 cells, which contain a temperature-sensitive mutant form of Cdk1 allowing for convenient cell cycle synchronization, were a gift from Dr. Chris Norbury (Oxford University, Oxford, UK) and were maintained for no longer than 30 passages (Th'ng et al., 1990). Paclitaxel-resistant (1A9/PTX10, 1A9/PTX22) and parental 1A9 human ovarian carcinoma cells were gifts from Drs. Paraskevi Giannakakou and Tito Fojo of the National Cancer Institute (Bethesda, MD). The SV40 large T antigen transformed cells have been previously characterized (Vogt et al., 2000). Anti-Cdk1 (sc-54), anti-Cdc25, and anti-Bcl-2 (sc-509) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An agarose conjugate of anti-Cdk1 was used for immunoprecipitation. Histone H1 was obtained from Boehringer-Mannheim (Indianapolis, IN) and [-³²P]ATP (10 mCi/mmol) was from Amersham Life Science, Inc. (Arlington Heights, IL). Colchicine [ring C, methoxy-³H] (61.4 Ci/mmol, 2.3 TBq/mmol) was from NEN (Boston, MA). Paclitaxel was obtained from the Drug Synthesis Branch of the National Cancer Institute. All other reagents were from Sigma (St. Louis, MO) unless indicated otherwise.

Antiproliferative Assay. The proliferation of MCF-7, MDA-MB-231 and SV40 transformed mouse embryonic fibroblasts was measured by a previously described colorimetric assay (Vogt et al., 1998; Wipf et al., 2001). Briefly, we seeded 4000 to 6000 cells/well in microtiter plates. Cells were allowed to attach overnight and treated with vehicle or compounds for 72 h, after which the medium was replaced with serum-free medium containing 0.1% 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide. Plates were incubated for 3 h in the dark and total cell number was determined spectrophotometrically at 540 nm as previously described (Vogt et al., 1998). The growth inhibition of 1A9, 1A9/PTX10, and 1A9/PTX22 cells was also measured with a slightly different colorimetric assay. Cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum and the paclitaxel-resistant cells also contained 17 nM paclitaxel and 10 µM verapamil. Cells were plated (2000/well) in 96-well plates and allowed to attach and grow for 72 h (paclitaxel and verapamil were removed from resistant cell medium 2 weeks before this plating). They were then treated with 0.5% DMSO vehicle control or 0.08 to 10 µM compound. The number of cells was determined spectrophotometrically at 490 nm minus absorbance at 630 nm after exposure to 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and N-methylphenazine methyl sulfate.

Flow Cytometry Analysis. tsFT210 cells were seeded at 2 × 10⁵ cells/ml and maintained at 32.0°C as previously described (Th'ng et al., 1990; Tamura et al., 1999). Cell proliferation was blocked at G₂ phase by incubation at 39.4°C for 17 h. The synchronized cells were then released by reincubating at 32.0°C and treated immediately with 0 to 10 µM SR-7, 1 µM nocodazole, or 100 µM SC-9, respectively, to probe for G₂/M arrest. Cells were treated 6 h after G₂/M release to determine G₁ arrest. We used 100 µM SC-9 and 50 µM roscovitine as positive control compounds for G₁ arrest. A final concentration of 0.5% DMSO was used for all compounds and as a negative control. For both G₂/M and G₁ blockage studies, treated cells were incubated at 32.0°C for an additional 6 h after each drug exposure, and then harvested with phosphate-buffered saline at 5 × 10⁵ cells/ml. The harvested cells were stained with a solution containing 50 µg/ml propidium iodide and 250 µg/ml RNase A. Flow cytometry analysis was conducted with a Becton Dickinson FACS Star (Franklin Lakes, NJ). Cell cycle distribution was statistically analyzed using a one-tailed Student's t test assuming unequal variances.

Western Blotting and Cdk1 Assays. tsFT210 cells were harvested using the same procedure for cell synchronizing and drug exposure as described above for the G₂/M flow cytometric analysis. The protein lysates were analyzed by Western blotting for Cdk1 as described previously (Tamura et al., 2000). We used Cdk1 isolated from human MCF-7 cells to assay for in vitro inhibition of enzyme activity because of available antibodies and convenience. Asynchronous cells grown at 37°C in 5% CO₂ in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum were treated with lysis buffer and harvested as previously described (Vogt et al., 1998). Cdk1 kinase activity assay was performed as previously described (Yu et al., 1998). Briefly, 2 mg of the protein lysates was incubated with anti-Cdk1 antibody agarose conjugate for 2 h at 4°C. The immunoprecipitates were treated in vitro with DMSO vehicle, 300 nM flavopiridol, or 10 µM SR-7 for 20 min at 30°C. The treated immunoprecipitates were reincubated in 20 µl of kinase reaction buffer (Yu et al., 1998) for an additional 20 min at 30°C with 3 µg of histone H1, 20 mM Tris-HCl, 10 mM MgCl₂, 5 µM cold ATP, and 10 µCi of [-³²P]ATP. Histone H1 was separated from other proteins by SDS-PAGE and analyzed for incorporation of radioactive phosphate with a Molecular Dynamics (Sunnyvale, CA) STORM 860 PhosphoImager.

Tubulin Polymerization. Tubulin without microtubule-associated proteins was isolated from fresh bovine brains (Hamel and Lin, 1984). Inhibition of assembly reactions was carried out as described previously (Verdier-Pinard et al., 1998). Tubulin (1 mg/ml) was preincubated for 15 min at 30°C with compounds, 4% DMSO, and 0.8 M monosodium glutamate. The reaction mixtures were cooled to 0°C, adjusted to 0.4 mM GTP, and transferred to cuvettes in a Beckman-Coulter 7400 spectrophotometer with a six-cuvette holder on a motorized stage reading absorbance at 340 nm. Baselines were established at 0°C and the temperature was raised to 30°C in approximately 1 min with an electronically controlled Peltier temperature controller. The change in absorbance 20 min after samples reached 30°C was used to calculate the extent of polymerization. The change in absorbance for vehicle without GTP was considered 100% assembly inhibition, whereas the change in absorbance for GTP-containing reaction mixtures with DMSO vehicle was used for 0% inhibition. Each series of determinations included positive and negative control samples.

Inhibition of [³H]Colchicine Binding. Using methods described previously (Verdier-Pinard et al., 1998), we incubated 5 µM [³H]colchicine with either 5% DMSO vehicle or compound (5 or 50 µM) at 37°C for 15 min with 1 µM tubulin in the presence of 1 M monosodium glutamate, 0.1 M glucose-1-phosphate, 1 mM MgCl₂, 1 mM GTP, and 0.5 mg/ml bovine serum albumin. The solutions were filtered through two stacks of DEAE-cellulose filters and the radioactivity in the filtrate was determined by liquid scintillation spectrometry. Each series of determinations included positive controls of 5 and 50 µM curacin A.

Phosphatase Assays. We measured the activities of the GST-fusion proteins Cdc25B₂, vaccinia H1-related phosphatase, and human recombinant PTP1B with the assay conditions that have previously been described (Rice et al., 1997). Fluorescence emission from the product of the substrate, O-methyl fluorescein phosphate (Molecular Probes, Inc., Eugene, OR), was measured over a 20- to 60-min reaction period at ambient temperature with a multiwell plate reader (PerSeptive Biosystems Cytofluor II; Framingham, MA; excitation filter/band width, 485/20; emission filter/band width, 530/30). All enzyme reactions were linear over the incubation time and were directly proportional to both the enzyme and substrate concentration.

Bcl-2 Phosphorylation. Attempts to determine the phosphorylation status of Bcl-2 in tsFT210 cells using two different antibodies were unsuccessful due to the antibodies' inability to detect mouse Bcl-2 or the lack of specificity. Therefore, phosphorylated and nonphosphorylated Bcl-2 was detected in lysates from human MCF-7 cells (American Type Culture Collection, Manassas, VA) treated with microtubule-perturbing agents. Equal amounts of protein were separated by electrophoresis on 15% SDS-PAGE followed by immunoblotting with an anti-human Bcl-2 antibody (sc-509; Santa Cruz Biotechnology). Positive antibody reactions were visualized using peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) and an enhanced chemiluminescence detection system (Renaissance; NEN) according to manufacturer's instructions.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Cytotoxicity and G₂/M Phase Inhibition by SR-7. MCF-7 cells showed similar sensitivity to the antiproliferative actions of palmarumycin CP₁, diepoxin , and SR-7 with IC₅₀ values of 0.96, 1.64, and 1.13 µM, respectively (Fig. 2A). In contrast, the close structural analog SR-4 had considerably less antiproliferative activity against these cells with an IC₅₀ of 11.2 µM (Fig. 2A). Because of the potential importance of the tumor suppressor gene p53 and the estrogen receptor in controlling the cellular response to cytotoxic agents, we also examined the sensitivity of MDA-MB-231 cells, which lack functional p53 and estrogen receptors. As indicated in Fig. 2B, MDA-MB-231 cells were equally sensitive to palmarumycin CP₁, diepoxin , and SR-7, with IC₅₀ values of 2.61, 2.01, and 2.44 µM, respectively. In contrast, the IC₅₀ for SR-4 was 10.3 µM, revealing the importance of the 2-furyl moiety in the C8 position of SR-7. As might be expected, these p53 and estrogen receptor-deficient cells were generally less sensitive to both natural products and analogs compared with MCF-7 cells. The differential cytotoxicity of the SR-7:SR-4 pair was confirmed when we tested mouse embryonic fibroblasts transformed with SV40 large T antigen; we found 3 µM SR-7, palmarumycin CP₁, and diepoxin  were required to inhibit growth by 50%, whereas no significant inhibition was seen with 10 µM SR-4, the highest concentration tested (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Growth inhibition of human breast cancer cells by palmarumycin CP1, diepoxin , SR-7, and SR-4. Exponentially growing MCF-7 (A) or MDA-MB-231 (B) cells were exposed to various concentrations of palmarumycin CP1 (), diepoxin  (), SR-7 (), or SR-4 () for 72 h and the cell number determined using 0.1% 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide as described under Experimental Procedures. Control values were vehicle-treated cells. N = 8; bars, S.E.M. *p < 0.01.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Inhibition of G2/M cell cycle progression by SR-7. tsFT210 cells were cultured at the permissive temperature of 32.0°C (A) and then incubated for 17 h at the nonpermissive temperature of 39.4°C (B). Cells were released from cycle arrest by shifting to the 32.0°C medium. The cells were then incubated for 6 h in the presence of DMSO vehicle (C), 1 µM nocodazole (D), 2.5 µM SR-7 (E), 5 µM SR-7 (F), 7.5 µM SR-7 (G), 10 µM SR-7 (H), or 100 µM SC-9 (I). Fluorescence corresponding to 2C and 4C DNA content is represented by vertical bars. These results are representative of three independent experiments that are quantified in Table 1.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Failure of SR-7 to inhibit G1 cell cycle progression. tsFT210 cells were cultured at the permissive temperature of 32.0°C (A) and then incubated for 17 h at the nonpermissive temperature of 39.4°C (B). Cells were released from the G2/M block by incubation at 32.0°C for 6 h (C) and then incubated for an additional 6 h in the presence of various agents. These were DMSO vehicle (D), 50 µM roscovitine (E), 5 µM SR-7 (F), 10 µM SR-7 (G), or 100 µM SC-9 (H). Fluorescence corresponding to 2C and 4C DNA contents is represented by vertical bars. These results were replicated in a second independent experiment and both are quantified in Table 2.

Cdk1 Dephosphorylation in the Presence of SR-7. The major controlling molecule for G₂/M transition is the cyclin-dependent kinase Cdk1, whose cellular activity is tightly regulated by phosphorylation (Hunter and Pines, 1994; Pines, 1999). Therefore, we performed Western blotting on tsFT210 cell extracts to determine the Cdk1 phosphorylation level in the presence or absence of SR-7. Protein lysates of tsFT210 cells arrested at the G₂/M boundary were harvested and analyzed by SDS-PAGE. Similar to our previous observations (Tamura et al., 1999), approximately 50% of Cdk1 was in the mitotic-inactive hyperphosphorylated form as reflected by a slower migrating Cdk1 (Fig. 5, lane 1). The phosphorylation of Cdk1 decreased gradually after cells were released from G₂/M block, and most of the Cdk1 was dephosphorylated, and thus activated, 6 h after G₂/M release, even in the presence of the DMSO vehicle (Fig. 5, lanes 2-4). When we incubated cells with 1 µM nocodazole, which caused a G₂/M arrest, no hyperphosphorylation of Cdk1 was seen, consistent with its proposed inhibitory activity after Cdk1 activation (Fig. 5, lane 5). Similarly, Cdk1 was completely dephosphorylated in the presence of either 10 or 20 µM SR-7 (Fig. 5, lanes 6 and 7). In contrast, treatment with 100 µM SC-9, which also causes G₂/M block (Tamura et al., 1999), yielded a hyperphosphorylated Cdk1 (Fig. 5, lane 8). In vitro studies confirmed that SR-7 and SR-4 at 30 µM caused no inhibition of recombinant Cdc25B₂, vaccinia H1-related phosphatase, or PTP1B activity; even at 100 µM we found 16% inhibition (data not shown). We also examined the ability of SR-7 to directly inhibit Cdk1 kinase activity. Cellular Cdk1 was immunoprecipitated with an anti-Cdk1 antibody and the resulting protein treated with DMSO vehicle, 0.3 µM flavopiridol, or 30 µM SR-7 for 20 min. Exposure to 0.3 µM flavopiridol, a known Cdk1 inhibitor, completely blocked the ability of the immunoprecipitated Cdk1 to phosphorylate histone H1, whereas treatment of the immunoprecipitate with 30 µM SR-7 did not inhibit activity (Fig. 6). We are uncertain whether the apparent increase in Cdk1 activity seen in Fig. 6 was biologically important. Nonetheless, the G₂/M blockage produced by SR-7 was clearly not associated with hyperphosphorylation or direct inhibition of Cdk1 or inhibition of Cdc25 enzyme activity.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Cdk1 dephosphorylation and activation by SR-7 in synchronous tsFT210 cells. G2/M synchronous tsFT210 cells were treated with vehicle or various compounds and permitted to reenter the cell cycle by culturing at 32.0°C. We isolated protein lysates from cells that were not incubated (0 h) or from cells incubated for 2 to 6 h at the permissive temperature in the presence of a compound or vehicle. The protein lysates were analyzed by Western blotting for Cdk1 content and phosphorylation status as described under Experimental Procedures. DMSO control, 0 to 6 h (lanes 1-4). Nocodazole, 1 µM for 6 h (lane 5), SR-7, 10 and 20 µM for 6 h (lanes 6 and 7), and SC-9, 50 µM for 6 h (lane 8). These results were confirmed in a second independent experiment.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Cdk1 kinase activity after SR-7 treatment. Lysates from asynchronous MCF-7 cells were immunoprecipitated with anti-Cdk1 antibody coupled to agarose and the resulting immunoprecipitate treated with DMSO vehicle, 300 nM flavopiridol, or 30 µM SR-7 for 20 min. The resulting immunocomplexes were tested for their ability to phosphorylate histone H1 using [-32P]ATP. A, phosphorylation of histone H1. B, total Cdk1 protein level as measured with an anti-Cdk1 antibody. C, quantification of the intensity of histone H1 phosphorylation normalized to the total Cdk1 amount. The columns in C correspond to lanes 1, 2, and 3 in A and B.

SR-7 Effects on Tubulin. We next examined the ability of SR-7 to alter tubulin polymerization or depolymerization in vitro. Addition of 0.4 mM GTP to isolated bovine brain tubulin produced robust polymerization that began to plateau approximately 20 min after microtubule assembly commenced (Fig. 7A). Inclusion of 5 µM curacin A completely inhibited GTP-induced tubulin assembly, whereas 1 µM curacin A caused a 50% inhibition. In contrast, SR-7 even at 40 µM caused only moderate inhibition of tubulin assembly (Fig. 7A). Furthermore, we found no evidence that SR-7 could bind to the colchicine site of tubulin. As indicated in Table 3, at 5 µM SR-7 failed to significantly inhibit colchicine binding, whereas the positive control curacin A caused almost 90% inhibition at 5 µM. At 50 µM, SR-7 actually enhanced [³H]colchicine binding by an unknown mechanism. Thus, we concluded SR-7 did not compete for the most common small-molecule target on tubulin, the colchicine binding site, although we have not formally excluded the possibility that SR-7 could bind to some other site, such as that used by the vinca alkaloids.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Perturbation of tubulin assembly in vitro. A, tubulin assembly inhibition assay. Compounds (predissolved in DMSO) were preincubated with tubulin-containing monosodium glutamate at 30°C for 15 min. Samples were cooled to 0°C and GTP was added. Samples were placed in a temperature-controlled multicuvette holder of a spectrophotometer held at 0°C. Baselines were established and temperature was rapidly raised to 30°C. Turbidity development in the cuvettes was measured at 350 nm. B, microtubule stabilization/hypernucleation assay in the absence of GTP. Drugs were added to the tubulin plus monosodium glutamate mixture at 0°C, placed in the spectrophotometer, and temperature was raised to 30°C.

Effect of SR-7 on Bcl-2 Phosphorylation. All known microtubule-disrupting compounds cause hyperphosphorylation of the antiapoptotic protein Bcl-2 (Haldar et al., 1995; Basu and Haldar, 1998). Thus, we examined the phosphorylation status of Bcl-2 in cells treated with various compounds, including SR-7. Although paclitaxel, nocodazole, and vincristine caused prominent phosphorylation of Bcl-2, SR-7 at either 3 or 10 µM failed to generate obvious hyperphosphorylated Bcl-2 (Fig. 8). Densitometric evaluation revealed 40% of the Bcl-2 was phosphorylated after nocodazole treatment, whereas <2% of the Bcl-2 was phosphorylated in control or SR-7-treated cells. These results strongly suggested SR-7 did not directly disrupt tubulin in whole cells.

View larger version (32K): [in this window] [in a new window]   Fig. 8.   Phosphorylation status of Bcl-2 after treatment with SR-7. Proteins from lysates of MCF-7 cells treated with 0.5 µM paclitaxel, 1 µM nocodozole, 1 µM vincristine, and 3 or 10 µM SR-7 were separated by electrophoresis on 15% SDS-PAGE followed by immunoblotting with an antibody to Bcl-2. The phosphorylated form of Bcl-2 (P) appeared as the upper bands and the underphosphorylated form (U) was the lower band. These results were confirmed in a second independent experiment.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The unique chemical structure of the natural products palmarumycins and diepoxins and their potential for functionalization suggest their biological activity should be examined in greater detail. Our cell culture results demonstrated compounds of this general class are potent inhibitors of cell division. Interestingly, the preservation of antiproliferative activity against murine and human malignant cells with different p53 and estrogen receptor status by both palmarumycin CP₁ and SR-7 indicated that the epoxide moieties of diepoxin  are not essential for cell growth inhibition. Furthermore, the retention of antiproliferative activity by SR-7 revealed that the C8 position of the palmarumycin/diepoxin structure could be modified to form unnatural analogs but the loss of activity with SR-4 demonstrated that the nature of this substitution was important. Based on this study and others (Wipf et al., 2001), we have concluded that allylic -systems present in heterocyclic or aromatic ethers lead to a significant increase mammalian cell growth inhibition.

When we examined SR-7 in greater detail, we found it blocked tsFT210 cells at a G₂/M checkpoint. Cell cycle checkpoints are critical regulators of genome integrity and faithful cell replication. One of the main abnormalities in human tumor cells is the loss of the G₁-phase checkpoint, which not only permits cellular replication but also encourages genomic instability. Consequently, enforcement of the G₂/M checkpoint represents an attractive mode of action for new antineoplastic agents. G₂/M progression is tightly regulated by several cellular macromolecules, including tubulins, and microtubule-associated proteins and motor proteins, such as kinesins and dynesins. An additional essential regulator is the maturation/M-phase promoting factor comprising Cdk1/cyclin B. Cdk1/cyclin B itself is regulated by a complex group of positive and negative regulating kinases. In mammalian cells, these include wee1, myt1, cyclin-activating kinase, Chk1, and cds1 kinases. In addition, Cdc25 phosphatases, which are also regulated by other kinases and phosphatases, are responsible for the activation of Cdk1.

Inhibitors of tubulin polymerization or depolymerization are widely available but only a few disrupters of other regulators of G₂/M progression have been identified. For example, we have identified several novel small-molecule inhibitors of Cdc25 that block G₂/M progression but they also affect G₁ transition (Tamura et al., 1999, 2000). Others have recently isolated a novel mitotic blocker that appears to act as a specific inhibitor of a mitotic kinesin (Mayer et al., 1999). Nonetheless, there continues to be a great need for pharmacologically distinct agents both to investigate the G₂/M progression process and as new pharmacophores for drug design strategies.

One of the most attractive cellular systems for identifying novel agents that inhibit cell cycle progression at the G₂/M phase boundary has been the mouse Cdk1 mutant cell line tsFT210. This cell line has two point mutations in Cdk1, an Ile52Val change in the PSTAIR region and Pro272Ser change in the C-terminal domain of the protein (Th'ng et al., 1990). These mutations produce a thermolabile Cdk1 that is inactive when cells are incubated at 39.4°C, leading to arrest in the mid-to-late G₂ phase. These cells are not only useful for identifying inhibitors of Cdk1 but also for highlighting agents that disrupt any of the other factors essential for G₂/M progression (Osada et al., 1997). In the current study, we have used tsFT210 cells to identify a novel small molecule that effectively blocks the G₂/M transition without functioning through any well established mechanism. Although the cell cycle block has some biochemical features that are similar to those seen with nocodazole, our in vitro studies do not support a direct role for SR-7 with tubulin. Furthermore, cells resistant to paclitaxel are not markedly resistant to SR-7. Important also was our failure to see hyperphosphorylation of Bcl-2, which is a hallmark of tubulin interactive agents. Thus, SR-7 appears to exert its antimitotic effects by a novel mechanism that does not primarily involve microtubule perturbations; possible candidate targets for SR-7 action, such as microtubule-associated proteins or motor proteins, warrant future investigation.