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CELLULAR AND MOLECULAR
Division of Pharmaceutical Biosciences, College of Pharmacy, Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea (Y.-M.H., K.-J.C., Y.-H.J., S.-K.L.); and Department of Medicinal Chemistry, College of Pharmacy, Seoul National University, Seoul, Korea (S.-Y.S., M.-W.C.)
Received September 3, 2003; accepted November 10, 2003.
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Abstract |
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-L-xylofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carboxamide) blocks cyclin-dependent kinase CDK1 and CDK2/cyclin A activity in vitro (IC50 1.4 and 61 nM, respectively) while minimally inhibiting the three other Ser/Thr protein kinases tested (IC50 21–86 µM). Reduced phosphorylated nucleolin and retinoblastoma protein levels showed it also efficiently inhibited cellular CDK1 and CDK2 activity (IC50 50–100 and 200–500 nM, respectively). Moreover, it blocked the functional activity of CDKs in tumor necrosis factor-related apoptosis-inducing ligand-induced SK-HEP-1 cell apoptosis 20 to 1000-fold more potently than olomoucine and roscovitine. Xylocydine is thus a novel and potent CDK inhibitor that could be used to interfere with cell cycle- and apoptosis-related CDK activity in various diseases.
; Li and Perrimon, 1997; Mufson, 1997; Okamoto et al., 1997; Ravitz and Wenner, 1997). Cyclin-dependent kinases (CDKs), which belong to this Ser/Thr kinase family, are regulators of eukaryotic cell cycle progression. These kinases phosphorylate various target proteins whose activities are crucial for cell cycle progression (Buchkovich et al., 1989). The cell cycle is controlled by mechanisms that operate at checkpoints in the G1 and G2 phases. These mechanisms involve complex interactions among many proteins, including CDKs (Sheaff and Roberts, 1998; Pines, 1999). CDK1 acts in concert with cyclin A and B to regulate the G2 to M phase transition. Specifically, CDK1 catalyzes phosphorylation events that are necessary for the initiation of and progression through the M phase. The substrates for CDK1/cyclin B include nuclear proteins such as histone H1, nucleolin, lamins, and condensin, as well as caldesmon, vimentin, src tyrosine kinase, and kinesin-related motor proteins (Moreno and Nurse, 1990; Nigg, 1993). This diversity of substrates highlights the fact that CDK1/cyclin B plays a central role in the M phase in that this complex induces nuclear lamina disassembly, chromatin condensation/segregation, spindle formation, and cytoskeletal reorganization. CDK2 and cyclin E are responsible for the G1/S transition, while CDK2 and cyclin A regulate the progression through the S phase. CDK2 regulates the phosphorylation of the retinoblastoma protein (Rb) at Thr821 and Thr826. The phosphorylation of Rb allows the cell to enter the S and G2/M phases and causes the cell cycle to proceed (Zarkowska and Mittnacht, 1997; Mittnacht, 1998; Harbour et al., 1999).
Achieving cell cycle control is an important goal in the treatment of diseases characterized by uncontrolled cell proliferation that results from some fault in the cell cycle. Consequently, the CDKs could be important targets for such therapeutic intervention (Sielecki et al., 2000). Inhibiting CDK activity may be particularly useful in the treatment of cancer (Meijer et al., 1999; Senderowicz and Sausville, 2000). Supporting this notion is that 90% of human tumors contain a fault in some component of the Rb pathway (Sellers and Kaelin, 1997). Many small molecular inhibitors of CDKs have been described. These include flavopiridol (Senderowicz et al., 1998), olomoucine (Abraham et al., 1995; Havlicek et al., 1997), roscovitine (Pippin et al., 1997; Wang et al., 2001), and microxine (Killday et al., 2001). They are thought to inhibit CDK activity by interacting with the CDKs at their ATP-binding sites (Vesely et al., 1994; De Azevedo et al., 1997; Meijer et al., 1997; Legraverend et al., 1998).
We previously have shown that sangivamycin and toyocamycin, which were isolated from microorganisms, inhibit CDK activity (Park et al., 1996). To search for an inhibitor that specifically inhibits the CDK1 kinase and has minimal side effects on other Ser/Thr protein kinases and whose solubility is better than that of other known inhibitors, we synthesized L-derivatives of sangivamycin (Chun et al., 1999). One of these is xylocydine, which is 4-amino-6-bromo-7-(-L-xylofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carboxamide. The chemical structure of xylocydine is shown in Fig. 1. In this study, we show that xylocydine selectively inhibits CDK1 and CDK2 kinase activity in vitro. Assays with cultured cells examining the effect of xylocydine on intracellular levels of the phosphorylated forms of Rb or nucleolin, which are the respective target substrates of CDK1 and CDK2, showed that xylocydine also strongly inhibits the intracellular kinase activity of these CDKs. In addition, we provide evidence that this CDK inhibitor prevents the apoptotic progression of TRAIL-treated cells by blocking cell death-associated CDK activation.
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Materials and Methods |
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Reagents. Olomoucine, roscovitine, and nocodazole were purchased from Calbiochem (San Diego, CA), dissolved in dimethyl sulfoxide, and stored at –-20°C as 10 mM stock. TRAIL was purchased from Calbiochem and stored at –70°C.
In Vitro Kinase Assay. The kinase assays were performed at 37°C for 15 min in reaction buffer with a final volume of 50 µl that contains a synthetic peptide that serves as a specific substrate for each kinase. For the CDK1 kinase assay, various doses of extracts from HeLa cells that had been arrested in M phase by treatment with nocodazole (Promega, Madison, WI) for 20 h were added to a reaction buffer containing 50 mM Tris (pH 7.4), 2 mM DTT, 10 mM MgCl2, 1 mM EGTA, 40 mM -glycerophosphate, 0.1 mM Na3VO4, 1.25 mM ATP, 1 µCi of [
-32P]ATP, and 50 µM H1 peptide (PKTPKKAKKL) (Promega). For the protein kinase A (PKA) assay, various units of PKA (Promega) were added to a reaction buffer containing 30 µM dibutyryl cAMP and 1 mM kemptide (LRRASLG). For the casein kinase II (CKII) assay, various units of CKII (Promega) were added to a reaction buffer containing 0.1 mM ATP and 600 µM peptide substrate (RRREEETEEE). For the protein kinase C (PKC) assay, PKC (Promega) was added to a reaction buffer containing 20 mM Tris (pH 7.4), 10 mM MgCl2, 0.25 mM EGTA, 0.4 mM CaCl2, 40 mg/ml bovine serum albumin, 0.3 mg/ml phosphatidylserine, 0.03 mg/ml diacylglycerol, 2.5 mM ATP, 2.5 µCi of [-32P]ATP, and 50 µM neurogranin (AAKIQASFRGHMARKK). The reaction was stopped by adding trichloroacetic acid and then trapped on P-81 paper. The paper was washed with 2% H3PO4 and dried. Radioactivity trapped on the P-81 paper was measured by a liquid scintillation counter (BAS-2500). The procedures used in these assays are detailed by the manufacturers of the kinases.
For the histone H1 kinase assay (CDK2 kinase assay), 200 µg of protein extract of SK-HEP-1 cells were immunoprecipitated overnight at 4°C with an antibody specific for cyclin A. Protein A-agarose beads (50 µl) were added to the immunoprecipitates and incubated for 2 h. The beads were washed three times with lysis buffer and twice with kinase assay buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 20 mM EGTA, 1 mM DTT, 50 mM -glycerophosphate, 25 mM NaF, 0.1 mM Na3VO4, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml antipain, and 1 mM phenylmethylsulfonyl fluoride. Kinase activity was assayed by incubating the complex for 15 min at 30°C in 50 µl of kinase assay buffer supplemented with 5 µg of histone H1, 10 µCi of [-32P]ATP, 5 µM protein kinase inhibitor, and 10 µM ATP. It was analyzed by 12% SDS-polyacrylamide gel electrophoresis followed by autoradiography. The intensity of the bands was analyzed by phosphorimaging (WALLAC-1409).
Western Blot Analysis. Western blots were performed according to standard methods. Briefly, cell pellets were lysed in lysis buffer (0.5% Triton X-100, 20 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM DTT, 1 mM EGTA, 50 mM -glycerophosphate, 25 mM NaF, 1 mM Na3VO4, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml antipain) for 1 h at 4°C. Lysates were subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Gelman Instrument Co., Ann Arbor, MI). The membranes were incubated with the following antibodies, which were diluted 1:1000: monoclonal mouse anti--actin from Sigma-Aldrich (St. Louis, MO), polyclonal goat anti-phospho-Rb, polyclonal rabbit anti-nucleolin, and polyclonal rabbit anti-PARP (all from Santa Cruz Biochemicals, Santa Cruz, CA). The bands were visualized with horseradish peroxidase-conjugated antibodies and the ECL system (Amersham Biosciences Inc., Piscataway, NJ).
MTT Tetrazolium Dye Assay. Cell viability was measured by the MTT assay. SK-HEP-1 cells in the logarithmic phase of cell growth were aliquotted into 48-well plates at a cell density of 3 x 104 cells/well and then exposed to various concentrations of xylocydine, roscovitine, or olomoucine along with 10 µg/ml TRAIL. At the end of the period, the MTT reagent (5 mg/ml in phosphate-buffered saline) (Sigma-Aldrich) was added to each well. After 3 h of incubation at 37°C, the supernatants were removed, and the precipitated formazan crystals were dissolved in dimethyl sulfoxide. The absorbance, which was proportional to the degree of cell viability, was determined by an enzyme-linked immunosorbent assay reader.
Caspase-3 Activity Assay. The caspase-3 substrate Ac-DEVD-AFC (BD PharMingen, San Diego, CA) was used in caspase-3 assays according to a procedure based on the manufacturer's instructions. 20 µg of cell lysates were added to the reaction buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM DTT, 0.1% CHAPS, 10% sucrose) containing 25 µM Ac-DEVD-AFC in a 96-well plate. Lysates were incubated at 37°C for 1 h. Fluorescence of the cleavage product was measured using a SpectraFluor F129003 (Tecan US, Durham, NC) at excitation and emission wavelengths of 405 and 465 nm, respectively.
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Results |
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We then examined whether xylocydine also inhibits CDK activity within SK-HEP-1 cells. It was not possible to directly determine the inhibitory effect of the chemical inhibitor within the cells by using immunocomplex kinase assays (see Discussion), and thus we measured this effect indirectly by examining the levels of the phosphoforms of Rb and nucleolin after xylocydine treatment. These two proteins are the specific cellular substrate proteins of CDK2 and CDK1, respectively. We found that xylocydine reduces the levels of the Rb form that is phosphorylated at Thr821/826 in an inhibitor dose-dependent manner (Fig. 3A). The Thr821/826 residues of Rb are known to be specifically phosphorylated by CDK2 (Knudsen and Wang, 1996; Zarkowska and Mittnacht, 1997). Our data indicate that xylocydine inhibits the phosphorylation of Rb and hence CDK2 activity in cell cultures with an approximate IC50 value of 
200 to 500 nM. We also showed with the same system that olomoucine and roscovitine, which are well established CDK inhibitors, inhibited the phosphorylation of Rb with IC50 values of 140 and 30 µM, respectively (Fig. 3, B and C). Thus, xylocydine can inhibit CDK2 activity in cell cultures with 350- and 75-fold higher potency than olomoucine and roscovitine, respectively.
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To determine CDK1 activity in cells after treatment with xylocydine, we utilized the C23 antibody that specifically recognizes nucleolin that has been phosphorylated by CDK1/cyclin B (Fig. 4A). In SK-HEP-1 cells that had been arrested in M-phase by treatment with 0.5 µg/ml nocodazole for 24 h, nucleolin was detected as a hyperphosphorylated form. Treatment of the cells with increasing doses of xylocydine decreased the protein levels of hyperphosphorylated nucleolin in an inhibitor dose-dependent fashion. Thus, xylocydine can inhibit the phosphorylation of nucleolin and hence it blocks CDK1 activity in cells with an approximate IC50 value of 50 to 100 nM (Fig. 4A). In contrast, olomoucine and roscovitine inhibited the phosphorylation of nucleolin in SK-HEP-1 cells with approximate IC50 values of 50 to 100 and 1 to 10 µM, respectively (Fig. 4, B and C). Thus, xylocydine can inhibit CDK1 activity in cell cultures with approximately 1000- and 200-fold higher potency than olomoucine and roscovitine, respectively.
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Xylocydine Blocks Apoptotic Progression in TRAIL-Treated SK-HEP-1 Cells. Our earlier results together with other reports showed that cyclin-dependent kinases are upregulated during apoptotic cell death, and these apoptotic cell deaths can be prevented by selectively inhibiting these apoptosis-associated CDK activity. We also observed that in SK-HEP-1 cells expressing dominant negative CDK2 (DN-CDK2), the progression of TRAIL-induced apoptosis is prevented (data not shown). Thus, CDK2 activity in TRAIL-induced apoptosis is thought to be essentially involved in apoptotic progression. To assess whether xylocydine can block the functional activity of CDKs within cells, we examined the effect of xylocydine on the progression of apoptosis in TRAIL-treated SK-HEP-1 cells. We first examined the effect of xylocydine on TRAIL-induced apoptotic cell morphology, namely cell rounding, membrane blebbing, and cell shrinkage. We found that treating the cells with TRAIL together with xylocydine (4 nM), olomoucine (5 µM), or roscovitine (0.7 µM) almost completely abolished the morphological features of TRAIL-induced apoptosis (Fig. 5A). We then assessed whether TRAIL-induced cell death, as measured by examining cell viability, can be blocked by treating the cells with xylocydine. We found that xylocydine (4 nM), olomoucine (5 µM), and roscovitine (0.07–0.7 µM) reduced the TRAIL-induced cell death in an inhibitor dose-dependent fashion to the levels seen in SK-HEP-1 cells that have not been treated with TRAIL (Fig. 5B). To assess if this cytotoxicity is related with apoptosis, we further examined whether xylocydine prevents TRAIL-induced caspase-3 activity, a biochemical marker enzyme of apoptosis. The results showed that xylocydine markedly inhibited activation of caspase-3 activity in a time-dependent manner (Fig. 5C) and also prevented proteolytic cleavage of PARP, a cellular substrate of caspase-3, in the cells that have been treated with TRAIL (Fig. 5C). These results indicated that xylocydine can block the cell death-associated CDK/cyclin activity in TRAIL-induced cells.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-L-xylofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carboxamide) is a novel and selective inhibitor of both CDK1 and CDK2. The inhibitory activity of this chemical on CDK kinase activity (IC50 1.4 and 61 nM, respectively for CDK1 and CDK2) is about four orders of magnitude higher than its ability to inhibit the PKA, PKC, and CKII Ser/Thr protein kinases. Thus, xylocydine can inhibit CDK1 and CDK2 kinase activity with a very high selectivity over other types of Ser/Thr protein kinases in vitro.
Interestingly, the in vitro kinase assays showed that xylocydine inhibits CDK1 activity 40-fold better (IC50 1.4 nM) than it blocks CDK2 activity (IC50 61 nM) (Fig. 2). This suggests that this chemical inhibitor preferentially inhibits CDK1 activity over CDK2 activity. In contrast, the two known inhibitors olomoucine and roscovitine inhibit the in vitro activity of CDK1 and CDK2 to similar extents, and much less strongly than xylocydine (IC50 values of 7 µM and 0.45–0.7 µM, respectively) (Fig. 1A) (Knockaert et al., 2002). Thus, xylocydine can inhibit the in vitro activity of CDK1 with a 300- and 5000-fold greater specificity than roscovitine and olomoucine.
To test whether xylocydine can inhibit CDK activity within mammalian cells, we determined the levels of the phosphoforms of Rb and nucleolin in SK-HEP-1 cells that have been treated with xylocydine. Rb and nucleolin are the specific cellular substrate proteins of CDK2 and CDK1, respectively. This assay only indirectly assesses CDK activity within the cells but we could not use the more direct assay, namely the immunocomplex kinase assay, because a preliminary study showed that the inhibitory effect of xylocydine on CDK activity is significantly influenced by the washing conditions used during the immunoprecipitation of CDK complexes (data not shown). This observation suggests that the chemical inhibitor bound to the active site of the CDKs tends to dissociate from the complex during the preparation of the immunocomplexes. Thus, we indirectly determined the levels of the Rb and nucleolin phosphoforms after treating the cells with the chemical inhibitor. Our data indicate that xylocydine inhibits the phosphorylation of Rb and hence CDK2 activity with an approximate IC50 value of 200 to 500 nM in cell cultures. It also suppresses CDK1 activity as represented by the protein levels of phospho-nucleolin with an IC50 value of 50 to 100 nM. These data are in line with the in vitro kinase assay data in that xylocydine can more selectively inhibit CDK1 activity than CDK2 activity. However, the IC50 values obtained by the in vitro assay differ from those measured by the cell assay by two orders of magnitude. This discrepancy may be due to limited cell membrane permeability of this chemical inhibitor (Fig. 4).
A growing body of evidence suggests that CDK activity is essential for the progression of apoptosis (Jin et al., 2000; Kim et al., 2001). Recent studies also showed that CDK activity is involved in the TRAIL-induced apoptosis of various cell lines, including neutrophil granulocytes (Renshaw et al., 2003), murine renal cancer cells (Seki et al., 2003), leukemia cells (Uno et al., 2003), and SK-HEP-1 cells (data not shown). To provide further evidence that xylocydine is able to selectively inhibit functional CDK activity in cells, we tested whether the TRAIL-induced apoptotic death of SK-HEP-1 cells can be prevented by treating the cells with xylocydine. Interestingly, xylocydine suppresses the TRAIL-induced apoptotic cell death in a dose-dependent manner down to the levels of apoptosis seen in the SK-HEP-1 control cells that had not been treated with TRAIL (Fig. 5B). Thus, xylocydine at a concentration of 4 nM prevents the apoptotic cell death of more than 90% of the TRAIL-treated SK-HEP-1 cells. Olomoucine and roscovitine also prevent the apoptotic cell death in a dose-dependent fashion, but higher doses of 5 and 0.07–0.7 µM, respectively, are necessary. Notably, these chemical inhibitors including xylocydine suppress apoptotic cell death at low doses but these suppressive effects are attenuated at higher concentrations (Fig. 5B). We also found that the viability of the cells that have been cotreated with TRAIL and a high dose of the inhibitors is significantly higher than that of cells treated with TRAIL alone. Thus, these observations support the notion that CDK activity is involved in the apoptotic cell death induced by treatment with TRAIL. They also show that xylocydine can block the functional effects of the CDKs within cells.
Collectively, our results with the cell culture and in vitro assay systems used here suggest that xylocydine is a selective and potent CDK inhibitor. This chemical inhibitor can also act as a very potent anti-apoptotic agent by selectively blocking cell death-associated CDK activation within cells that has been induced by treatment with TRAIL. Thus, we propose that xylocydine could serve as an anti-apoptotic reagent that could be useful in preventing certain types of apoptotic cell death, for example, that induced by pathogens.
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Footnotes |
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This work was supported by the National Research Laboratory Fund (M10104000129-02J000005910), the Ministry of Science and Technology, and by Grant R01-2000-000-00113-0 from the Basic Research Program of the Korea Science and Engineering Foundation.
Address correspondence to: Dr. Seung-Ki Lee, Division of Pharmaceutical Biosciences, College of Pharmacy, Seoul National University, San 56–1, Shillim-Dong, Kwanak-Gu, Seoul, 151–742, Korea. E-mail: sklcrs{at}plaza.snu.ac.kr
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