Classical Anticytokinins Do Not Interact with Cytokinin Receptors but Inhibit Cyclin-dependent Kinases
2007; Elsevier BV; Volume: 282; Issue: 19 Linguagem: Inglês
10.1074/jbc.m609750200
ISSN1083-351X
AutoresLukáš Spíchal, Vladimı́r Kryštof, Martina Paprskářová, René Lenobel, Jakub Stýskala, Pavla Binarová, Věra Cenklová, Lieven De Veylder, Dirk Inzé, George Kontopidis, Peter M. Fischer, Thomas Schmuölling, Miroslav Strnad,
Tópico(s)Histone Deacetylase Inhibitors Research
ResumoCytokinins are a class of plant hormones that regulate the cell cycle and diverse developmental and physiological processes. Several compounds have been identified that antagonize the effects of cytokinins. Based on structural similarities and competitive inhibition, it has been assumed that these anticytokinins act through a common cellular target, namely the cytokinin receptor. Here, we examined directly the possibility that various representative classical anticytokinins inhibit the Arabidopsis cytokinin receptors CRE1/AHK4 (cytokinin response 1/Arabidopsis histidine kinase 4) and AHK3 (Arabidopsis histidine kinase 3). We show that pyrrolo[2,3-d]pyrimidine and pyrazolo[4,3-d]pyrimidine anticytokinins do not act as competitors of cytokinins at the receptor level. Flow cytometry and microscopic analyses revealed that anticytokinins inhibit the cell cycle and cause disorganization of the microtubular cytoskeleton and apoptosis. This is consistent with the hypothesis that they inhibit regulatory cyclin-dependent kinase (CDK) enzymes. Biochemical studies demonstrated inhibition by selected anti-cytokinins of both Arabidopsis and human CDKs. X-ray determination of the crystal structure of a human CDK2-anticytokinin complex demonstrated that the antagonist occupies the ATP-binding site of CDK2. Finally, treatment of human cancer cell lines with anticytokinins demonstrated their ability to kill human cells with similar effectiveness as known CDK inhibitors. Cytokinins are a class of plant hormones that regulate the cell cycle and diverse developmental and physiological processes. Several compounds have been identified that antagonize the effects of cytokinins. Based on structural similarities and competitive inhibition, it has been assumed that these anticytokinins act through a common cellular target, namely the cytokinin receptor. Here, we examined directly the possibility that various representative classical anticytokinins inhibit the Arabidopsis cytokinin receptors CRE1/AHK4 (cytokinin response 1/Arabidopsis histidine kinase 4) and AHK3 (Arabidopsis histidine kinase 3). We show that pyrrolo[2,3-d]pyrimidine and pyrazolo[4,3-d]pyrimidine anticytokinins do not act as competitors of cytokinins at the receptor level. Flow cytometry and microscopic analyses revealed that anticytokinins inhibit the cell cycle and cause disorganization of the microtubular cytoskeleton and apoptosis. This is consistent with the hypothesis that they inhibit regulatory cyclin-dependent kinase (CDK) enzymes. Biochemical studies demonstrated inhibition by selected anti-cytokinins of both Arabidopsis and human CDKs. X-ray determination of the crystal structure of a human CDK2-anticytokinin complex demonstrated that the antagonist occupies the ATP-binding site of CDK2. Finally, treatment of human cancer cell lines with anticytokinins demonstrated their ability to kill human cells with similar effectiveness as known CDK inhibitors. Cytokinins are plant hormones that play essential roles in the regulation of various aspects of plant growth and development (1Mok D-W. Mok M.-C. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 89-118Crossref PubMed Scopus (878) Google Scholar). They include a variety of chemicals with different degrees of structural similarity, some of which occur naturally in plants, and others that are known only as synthetic compounds. The natural cytokinins are adenine derivatives that can be classified according to the nature of their N6-side chain as either isoprenoid (zeatin) or aromatic (benzyladenine) cytokinins.Cytokinins are key regulators of the plant cell cycle, and the induction of cell division is considered diagnostic for this class of plant hormones. The molecular basis of this activity is only partially understood and may differ in different cell types. Cytokinins have been found to control tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase (2Zhang K. Letham D-S. John P.-C. Planta. 1996; 200: 2-12Crossref PubMed Scopus (225) Google Scholar), as well as the transcriptional activation of cyclin D3 (3Riou-Khamlichi C. Huntley R. Jacqmard A. Murray J.A. Science. 1999; 283: 1541-1544Crossref PubMed Scopus (629) Google Scholar). Some of the many physiological and developmental processes that are controlled by cytokinin, such as the formation and activity of shoot apical meristems, floral development, the breaking of bud dormancy, and seed germination (4Mok M-C. Mok D.-W.-S. Mok M.-C. Cytokinins. Chemistry, Activity and Function. 1994; (CRC Press, Boca Raton, FL): 155-166Google Scholar, 5Werner T. Motyka V. Laucou V. Smets R. Onckelen H.V. Schmuölling T. Plant Cell. 2003; 15: 2532-2550Crossref PubMed Scopus (1065) Google Scholar, 6Higuchi M. Pischke M-S. Mahonen A.-P. Miyawaki K. Hashimoto Y. Seki M. Kobayashi M. Shinozaki K. Kato T. Tabata S. Helariutta Y. Sussman M.-R. Kakimoto T. Proc Natl. Acad Sci. U. S. A. 2004; 101: 8821-8826Crossref PubMed Scopus (517) Google Scholar, 7Nishimura C. Ohashi Y. Sato S. Kato T. Tabata S. Ueguchi C. Plant Cell. 2004; 16: 1365-1377Crossref PubMed Scopus (461) Google Scholar, 8Riefler M. Novák O. Strnad M. Schmuölling T. Plant Cell. 2006; 18: 40-54Crossref PubMed Scopus (696) Google Scholar), are at least in part functionally linked to cell cycle control.Recently, several cytokinin receptors were identified in Arabidopsis (9Inoue T. Higuchi M. Hashimoto Y. Seki M. Kobayashi M. Kato T. Tabata S. Shinozaki K. Kakimoto T. Nature. 2001; 409: 1060-1063Crossref PubMed Scopus (693) Google Scholar, 10Suzuki T. Miwa K. Ishikawa K. Yamada H. Aiba H. Mizuno T. Plant Cell Physiol. 2001; 42: 107-113Crossref PubMed Scopus (303) Google Scholar, 11Ueguchi C. Sato S. Kato T. Tabata S. Plant Cell Physiol. 2001; 42: 751-755Crossref PubMed Scopus (166) Google Scholar, 12Yamada H. Suzuki T. Terada K. Takei K. Ishikawa K. Miwa K. Yamashino T. Mizuno T. Plant Cell Physiol. 2001; 42: 1017-1023Crossref PubMed Scopus (386) Google Scholar) and Zea mays (13Yonekura-Sakakibara K. Kojima M. Yamaya T. Sakakibara H. Plant Physiol. 2004; 134: 1654-1661Crossref PubMed Scopus (159) Google Scholar). To date, three cytokinin receptors have been identified in Arabidopsis, AHK2, 4The abbreviations used are: AHK2, Arabidopsis histidine kinase 2; AHK3, Arabidopsis histidine kinase 3; CRE1/AHK4, cytokinin response 1/Arabidopsis histidine kinase 4; CDK, cyclin-dependent kinase; tZ, trans-zeatin; MI, mitotic index; ANCYT1, 3-methyl-7-pentylaminopyrazolo(4,3-d)pyrimidine; ANCYT2, 4-(cyclopentylamino)-2-methylthiopyrrolo(2,3-d)pyrimidine; ANCYT3, 4-(cyclobutylamino)-2-methylpyrrolo(2,3-d)pyrimidine; HU, hydroxyurea. 4The abbreviations used are: AHK2, Arabidopsis histidine kinase 2; AHK3, Arabidopsis histidine kinase 3; CRE1/AHK4, cytokinin response 1/Arabidopsis histidine kinase 4; CDK, cyclin-dependent kinase; tZ, trans-zeatin; MI, mitotic index; ANCYT1, 3-methyl-7-pentylaminopyrazolo(4,3-d)pyrimidine; ANCYT2, 4-(cyclopentylamino)-2-methylthiopyrrolo(2,3-d)pyrimidine; ANCYT3, 4-(cyclobutylamino)-2-methylpyrrolo(2,3-d)pyrimidine; HU, hydroxyurea. AHK3, and CRE1/AHK4. All are membrane-located sensor histidine kinases with a predicted extracellular ligand-binding domain and cytoplasmic His kinase and receiver domains. It has been shown that the cytokinin signal is transmitted by a multistep phospho-relay system through a complex form of the two-component signaling pathway that has long been known in prokaryotes and lower eukaryotes. Among higher eukaryotes, the two-component signaling pathway is only found in plants (reviewed by Refs. 14Hwang I. Sheen J. Nature. 2001; 413: 383-389Crossref PubMed Scopus (713) Google Scholar, 15Heyl A. Schmuölling T. Curr. Opin. Plant Biol. 2003; 6: 480-488Crossref PubMed Scopus (183) Google Scholar, 16Ferreira F-J. Kieber J.-J. Curr. Opin. Plant Biol. 2005; 8: 518-525Crossref PubMed Scopus (216) Google Scholar, 17Heyl A. Werner T. Schmuölling T. Hedden P. Thomas S. Plant Hormone Signaling Annual Plant Reviews. 2006: 93-123Google Scholar).The development of agonists and antagonists of a particular physiological effect is useful in mechanism-of-action studies of biologically active natural products. The design of potential cytokinin antagonists has been based on the assumptions that 1) active cytokinins bind to one or more cellular receptor sites and 2) it should be possible to prepare compounds that have minimal cytokinin activity but retain sufficient structural similarity to the cytokinins to permit them to compete for available cytokinin receptor sites, thereby diminishing the biological activity of cytokinins. The potent naturally occurring cytokinin N6-isopentenyladenine served as the basis for initial structure-activity studies. Modification of the heterocyclic purine system yielded the first analogues with antagonistic activity that greatly reduced cytokinin activity in bioassays (18Skoog F. Armstrong D-J. Annu. Rev. Plant Physiol. 1970; 21: 359-384Crossref Google Scholar, 19Skoog F. Hamzi Q-H. Szweykowska A.-M. Leonard N.-J. Carraway K.-L. Fujii T. Helgeson J.-P. Loeppky R.-N. Phytochemistry. 1967; 6: 1169-1192Crossref Scopus (174) Google Scholar). Consequently, a number of substituted pyrrolo[2,3-d]pyrimidines, pyrazolo[4,3-d]pyrimidines, s-triazines, N-benzyl-N′-phenylureas, and N-arylcarbamates were subsequently prepared and tested for their ability to inhibit cytokinin-promoted processes in various bioassays, and a number of them were identified as potential anticytokinins (reviewed by 20). Because of their structural similarity to natural cytokinins and because their antagonistic effects were reversible by increasing the cytokinin concentration, it was hypothesized that these compounds work through interaction with a common cellular target, viz the cytokinin receptor (20Iwamura H. Mok D.-W.-S. Mok M.-C. Cytokinins: Chemistry, Activity and Function. 1994; (CRC Press, Boca Raton, FL): 43-55Google Scholar). However, until recently, direct proof that cytokinin receptors are the sites of cytokinin-anticytokinin interactions was lacking because no cytokinin receptors had been identified. Recent advances in our understanding of cytokinin signaling motivated us to re-examine anticytokinin modes of action.Here we show that representative anticytokinins are not competitive inhibitors of two Arabidopsis cytokinin receptors. Furthermore, using mainly the potent anticytokinin 3-methyl-7-pentylaminopyrazolo[4,3-d]pyrimidine (ANCYT1) as a representative example, we also show that anticytokinins inhibit cell cycle progression and cause cellular changes consistent with responses to known CDK inhibitors. We demonstrate CDK inhibition by anticytokinins in plants and humans and reveal the binding of ANCYT1 to the ATP-binding pocket of human CDK2. The observed activity of anticytokinins in human cancer cells makes them new candidates for drug research and development.EXPERIMENTAL PROCEDURESChemicals—trans-zeatin was obtained from Olchemim Ltd. (Olomouc, Czech Republic). The methods used to synthesize and characterize the anticytokinin analogues were as described previously (21Hecht S-M. Proc. Natl. Acad Sci. U. S. A. 1971; 68: 2608-2610Crossref PubMed Scopus (39) Google Scholar, 22Skoog F. Schmitz R-Y. Hecht S.-M. Frye R.-B. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3508-3512Crossref PubMed Scopus (41) Google Scholar, 23Iwamura H. Masuda N. Koshimizu K. Matsubara S. Phytochemistry. 1979; 18: 217-222Crossref Scopus (41) Google Scholar). Radiolabeled trans-zeatin ([2-3H]zeatin) was obtained from Dr. Jan Hanuŝ (Isotope Laboratory, Institute of Experimental Botany, AS CR, Prague, Czech Republic).Bacterial Cytokinin Assay—Escherichia coli strains KMI001 harboring the plasmid pIN-III-AHK4 and pSTV28-AHK3, respectively, were described (10Suzuki T. Miwa K. Ishikawa K. Yamada H. Aiba H. Mizuno T. Plant Cell Physiol. 2001; 42: 107-113Crossref PubMed Scopus (303) Google Scholar, 12Yamada H. Suzuki T. Terada K. Takei K. Ishikawa K. Miwa K. Yamashino T. Mizuno T. Plant Cell Physiol. 2001; 42: 1017-1023Crossref PubMed Scopus (386) Google Scholar). Bacterial cytokinin assays were performed as described in Ref. 24Spíchal L. Rakova N-Y. Riefler M. Mizuno T. Romanov G.-A. Strnad M. Schmuölling T. Plant Cell Physiol. 2004; 45: 1299-1305Crossref PubMed Scopus (223) Google Scholar.Fractionation of E. coli and Binding Assay on Microsomes— CRE1/AHK4- and AHK3-expressing E. coli strains (10Suzuki T. Miwa K. Ishikawa K. Yamada H. Aiba H. Mizuno T. Plant Cell Physiol. 2001; 42: 107-113Crossref PubMed Scopus (303) Google Scholar, 12Yamada H. Suzuki T. Terada K. Takei K. Ishikawa K. Miwa K. Yamashino T. Mizuno T. Plant Cell Physiol. 2001; 42: 1017-1023Crossref PubMed Scopus (386) Google Scholar) were grown to A600 ∼ 1 at 25 °C and then fractionated into periplasmic, cytoplasmic, and membrane fractions. Fractionation and binding assays with E. coli membranes were carried out as described previously in Ref. 25Romanov G-A. Spíchal L. Lomin S.-N. Strnad M. Schmuölling T. Anal. Biochem. 2005; 347: 129-134Crossref PubMed Scopus (62) Google Scholar.Arabidopsis PARR5::GUS Reporter Gene Assay—Arabidopsis plants (Arabidopsis thaliana (L.) Heynh. accession Col-0) harboring PARR5::GUS gene reporter were described (26D'Agostino I.B. Deruere J. Kieber J.J. Plant Physiology. 2000; 124: 1706-1717Crossref PubMed Scopus (442) Google Scholar). The assay was carried out as described in Ref. 27Romanov G.A. Kieber J.J. Schmuölling T. FEBS. Lett. 2002; 515: 39-43Crossref PubMed Scopus (89) Google Scholar with slight modification. Seedlings were grown for 2-3 days (22 °C, 16 h light/8 h dark) in a 6-well plates (TPP, Switzerland), and then cytokinin and test compounds or solvent (Me2SO, final concentration 0.1%) were added as microaliquots to the desired final concentration. The seedlings were then incubated for 17 h at 22 °C in the dark.Protein Kinase Assays—The recombinant human protein kinases used for the selectivity screening of anticytokinins (see supplemental Table 1 and Fig. 5B) were produced and assayed as described in Refs. 28Wu S.-Y. McNae I. Kontopidis G. McClue S.-J. McInnes C. Stewart K.-J. Wang S. Zheleva D.-I. Marriage H. Lane D.-P. Taylor P. Fischer P.M. Walkinshaw M.D. Structure. 2003; 11: 399-410Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar and 29Wang S. Meades C. Wood G. Osnowski A. Anderson S. Yuill R. Thomas M. Mezna M. Jackson W. Midgley C. Griffiths G. Fleming I. Green S. McNae I. Wu S.Y. McInnes C. Zheleva D. Walkinshaw M.D. Fischer P.M. J. Med. Chem. 2004; 47: 1662-1675Crossref PubMed Scopus (154) Google Scholar. Protein extraction and purification of Arabidopsis CDKs by binding to p13suc1 beads or immunoprecipitation with antibodies specific to Arabidopsis CDKA;1 and CDKB1;1 and protein kinase activity measurements were carried out as described in Refs. 30Boögre L. Zwerger K. Meskiene I. Binarová P. Csizmadia V. Planck C. Wagner E. Hirt H. Heberle-Bors E. Plant Physiol. 1997; 113: 841-852Crossref PubMed Scopus (47) Google Scholar and 31Verkest A. de O. Manes C.-L. Vercruysse S. Maes S. Van Der Schueren E. Beeckman T. Genschik P. Kuiper M. Inze D. De Veylder L. Plant Cell. 2005; 17: 1723-1736Crossref PubMed Scopus (216) Google Scholar, respectively.Protein X-ray Crystallography—Expression, purification, and crystallization of monomeric human CDK2, as well as ligand introduction, data collection, processing, structure solution, and refinement, were all carried out using methods analogous to those previously described for complex structures with non-cytokinin CDK2 ligands (28Wu S.-Y. McNae I. Kontopidis G. McClue S.-J. McInnes C. Stewart K.-J. Wang S. Zheleva D.-I. Marriage H. Lane D.-P. Taylor P. Fischer P.M. Walkinshaw M.D. Structure. 2003; 11: 399-410Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 29Wang S. Meades C. Wood G. Osnowski A. Anderson S. Yuill R. Thomas M. Mezna M. Jackson W. Midgley C. Griffiths G. Fleming I. Green S. McNae I. Wu S.Y. McInnes C. Zheleva D. Walkinshaw M.D. Fischer P.M. J. Med. Chem. 2004; 47: 1662-1675Crossref PubMed Scopus (154) Google Scholar). Data collection and refinement statistics for the CDK2-ANCYT1 complex are presented in Table S2 (supplemental Table 2).Cell Cycle and Apoptosis Study—Root tip meristems of Vicia faba were synchronized as described previously in Ref. 32Doležel J. Číhalíková i J. Lucretti S. Planta. 1992; 188: 93-98Crossref PubMed Scopus (129) Google Scholar, and the relative DNA contents of V. faba nuclei isolated from root tips were analyzed by flow cytometry analysis as described in Ref. 33Lucretti S. Doležel J. Galbraith D-V.-V. Bourgue D.-P. Bohneret H.-J. Methods in Cell Biology. 1995; (Academic Press, New York): 61-83Crossref PubMed Scopus (22) Google Scholar. The frequencies of prophase and metaphase cells, the mitotic index (MI), were determined in squash preparations and stained according to the standard Feulgen procedure. The percentage of MI was obtained from randomly chosen samples of 1,000-2,000 cells from each treated variants and from the control cells.Immunofluorescence Staining of Microtubules—Root tips or cultured cells were fixed for 1 h in 3.7% paraformaldehyde and processed for immunofluorescence as described in Ref. 34Binarová P. Čihalíková J. Doležel J. Cell Biol. Int. 1993; 9: 847-856Crossref Scopus (25) Google Scholar.Testing of Cytotoxicity—The human breast carcinoma MCF-7, human chronic myelogenous leukemia K-562, and human osteogenic sarcoma HOS cell lines (obtained from ATCC, Rockville, MD) were used for cytotoxicity determinations of the tested anticytokinins using a calcein AM assay as described in Ref. 35Moravcová D. Kryŝtof V. Havlíček L. Moravec J. Lenobel R. Strnad M. Bioorg. Med. Chem. Lett. 2003; 13: 2989-2992Crossref PubMed Scopus (29) Google Scholar.RESULTSFig. 1 shows the chemical structures of the three anticytokinins ANCYT1, ANCYT2 (4-(cyclopentylamino)-2-methylthiopyrrolo[2,3-d]pyrimidine), and ANCYT3 (4-(cyclobutyl-amino)-2-methylpyrrolo[2,3-d]pyrimidine), which were selected for this study as being the most active compounds known in their respective substance class (20Iwamura H. Mok D.-W.-S. Mok M.-C. Cytokinins: Chemistry, Activity and Function. 1994; (CRC Press, Boca Raton, FL): 43-55Google Scholar).We initially tested the activity of the compounds in the classical tobacco callus growth assay for cytokinins, using an experimental design similar to that described by Hecht (21Hecht S-M. Proc. Natl. Acad Sci. U. S. A. 1971; 68: 2608-2610Crossref PubMed Scopus (39) Google Scholar) and Skoog et al. (36Skoog F. Schmitz R-Y. Bock R.-M. Hecht S.-M. Phytochemistry. 1973; 12: 25-37Crossref Scopus (42) Google Scholar). Callus growth increased with increasing cytokinin (trans-zeatin) concentration, reaching a maximum at 0.5-1 μm (see supplemental Fig. S1). Growth was inhibited by increasing concentrations of anticytokinin, and ANCYT1 almost completely inhibited callus growth at a concentration of 10 μm (see supplemental Fig. S1).To investigate whether or not the growth inhibitory effect of anticytokinins results from the blocking of cytokinin receptors, we studied their interactions with the CRE1/AHK4 and AHK3 receptors of Arabidopsis. For this, we used E. coli reporter strains expressing single cytokinin receptors and the cytokinin-activated reporter gene cps::lacZ (10Suzuki T. Miwa K. Ishikawa K. Yamada H. Aiba H. Mizuno T. Plant Cell Physiol. 2001; 42: 107-113Crossref PubMed Scopus (303) Google Scholar, 12Yamada H. Suzuki T. Terada K. Takei K. Ishikawa K. Miwa K. Yamashino T. Mizuno T. Plant Cell Physiol. 2001; 42: 1017-1023Crossref PubMed Scopus (386) Google Scholar, 24Spíchal L. Rakova N-Y. Riefler M. Mizuno T. Romanov G.-A. Strnad M. Schmuölling T. Plant Cell Physiol. 2004; 45: 1299-1305Crossref PubMed Scopus (223) Google Scholar). Data presented in Fig. 2, A and B, show that none of these anticytokinins was able to activate the receptors, even at a concentration 500-fold greater than that required for receptor activation by trans-zeatin.FIGURE 2Cytokinin receptor studies. A and B, comparison of the sensitivity of CRE1/AHK4 (A) and AHK3 (B) to 1 μm ANCYT1, ANCYT2, and ANCYT3, adenine (Ade, negative control) and trans-zeatin (tZ, positive control) in the bacterial assay. The activity of non-induced strains is indicated by the dotted line. Insets show activation of the cytokinin receptors by the compounds in a dose-dependent manner. Error bars show S.D. (n = 3). C and D, competitive binding assay with CRE1/AHK4-(C) and AHK3-containing (D) E. coli membranes. Binding of 2 nm [2-3H]zeatin (3HtZ) was assayed together with a 1,000-fold higher concentration of ANCYT1, ANCYT2, and ANCYT3 with adenine as negative control and unlabeled tZ as positive control. Error bars show S.D. (n = 2). E, effect of anticytokinins on induction of the PARR5::GUS gene by cytokinin. PARR5::GUS transgenic Arabidopsis seedlings were incubated with 1 μm benzyladenine (BA) in the presence or absence of 1 and 10 μm concentration of ANCYT1, ANCYT2, and ANCYT3; Me2SO (DMSO) (0.1%) was tested as solvent control. Error bars show S.D. (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Competitive inhibition of in vitro binding of trans-zeatin to CRE1/AHK4 and AHK3 by the proposed antagonists was investigated. After fractionation of E. coli cells, the presence of the CRE1/AHK4 and AHK3 proteins in isolated membranes, but not in the periplasm and cytoplasm fractions, was verified by equilibrium dialysis using [2-3H]zeatin (not shown). A competitive binding assay of the representative anticytokinins was then carried out, employing unlabeled trans-zeatin and adenine as positive and negative controls, respectively. Binding of radioactively labeled trans-zeatin to CRE1/AHK4 and AHK3 was inhibited competitively by unlabeled trans-zeatin (Fig. 2, C and D). In contrast, neither adenine nor any of the anticytokinins competed with trans-zeatin for receptor binding, even at 1,000-fold excess.To support our observation that representative anticytokinins do not compete with cytokinin for binding to the cytokinin receptors, we next determined whether the anticytokinins are able to block cytokinin primary signal transduction. ARR5 is a member of the type-A response regulators identified as cytokinin primary response genes (26D'Agostino I.B. Deruere J. Kieber J.J. Plant Physiology. 2000; 124: 1706-1717Crossref PubMed Scopus (442) Google Scholar). We used transgenic Arabidopsis seedlings harboring the PARR5::GUS reporter (26D'Agostino I.B. Deruere J. Kieber J.J. Plant Physiology. 2000; 124: 1706-1717Crossref PubMed Scopus (442) Google Scholar, 27Romanov G.A. Kieber J.J. Schmuölling T. FEBS. Lett. 2002; 515: 39-43Crossref PubMed Scopus (89) Google Scholar) to test the effects of ANCYT1, ANCYT2, and ANCYT3 on induction of ARR5 triggered by the cytokinin benzyladenine. Data presented in Fig. 2E show that none of the anticytokinins was able to reduce the level of ARR5::GUS.The activity that is measured by most cytokinin bioassays is the induction of cell division. To explore the activity of anticytokinins on this process, we measured their inhibitory activity on the cell cycle directly, choosing ANCYT1 as a typical example. The effect of ANCYT1 on cell division was studied in Arabidopsis cell suspension cultures and V. faba root meristems. In several independent experiments, the MI was about 5-7% in the control Arabidopsis cells, whereas a significant decrease in MI to 1.5% was observed after treatment with 100 μm ANCYT1. No significant mitotic activity was detected in cells treated with a higher concentration (200 μm) of ANCYT1.In asynchronous root meristems of V. faba, the MI decreased from 8% in the control to 2% after a 12-h treatment with 400 μm ANCYT1. To characterize further the inhibitory effect of ANCYT1 on cell cycling, root meristem cells of V. faba were synchronized with hydroxyurea (HU). Synchronization of root meristems increased MI from 8 to 55% as counted 7 h after HU removal in control cells. ANCYT1 was applied immediately after HU removal. Flow cytometric analysis showed that the proportion of cells in G1 increased 10 h after release from the HU block in the untreated control, indicating that control cells progressed completely through mitosis (Fig. 3A); in contrast, a significantly larger proportion of the cells treated with 400 μm ANCYT1 still retained the G2/M DNA content at this time point (Fig. 3B). Together with the observed decrease in MI, these data indicate that ANCYT1 treatment inhibited the G2/M transition.FIGURE 3Flow cytometric analysis of nuclear DNA content in control and ANCYT1-treated cells. V. faba root meristem cells were synchronized with HU. ANCYT1 (400 μm) was applied immediately after HU removal, and 10 h later, the DNA content of both control cells (A) and ANCYT1-treated cells (B) was measured.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Microscopic observation of both Arabidopsis and V. faba cells revealed that aberrant mitotic chromosome arrangements rather than regular metaphases were frequently present in mitotic cells that had been treated with ANCYT1. Immunofluorescent labeling of tubulin showed that abnormalities of cell cycle-specific arrays of microtubules were induced by ANCYT1 in Arabidopsis cells after treatment with a dose of 100 μm and were more pronounced at 200 μm (Fig. 4, A-F). Normal mitotic microtubule arrays typical of metaphase spindles, anaphase spindles, and cytokinetic apparatus phragmoplasts were observed in control Arabidopsis cells (Fig. 4, A-C, respectively). In contrast, ANCYT1-treated Arabidopsis cultures contained cells in pre-prophase, with microtubules randomly arranged in the nuclei and with highly condensed chromatin and persistent nuclear envelopes. These results are consistent with our observation that ANCYT1 blocked or delayed the G2/M transition (Fig. 4D). ANCYT1 also affected the organization of mitotic microtubules, causing a collapse of the microtubular cytoskeleton, accompanied by a strong affinity of the randomly arranged microtubules for chromatin and by the formation of irregular microtubule arrays, such as circles in the cytoplasm (Fig. 4E). The microtubules were also clustered randomly around newly forming daughter nuclei in telophase of ANCYT1-treated cells (Fig. 4F). Further significant cellular effects induced by ANCYT1 included apoptotic nuclear changes. Although in the control Arabidosis cells, only 1.5-2% of cells showed apoptotic DNA double strand breaks, up to 30% of the cells were apoptotic following a 3-h treatment with 50-100 μm ANCYT1 (Fig. 4, G and H). Similar changes in microtubule organization and induction of apoptosis were also observed after treatment of V. faba root meristems with ANCYT1 (not shown).FIGURE 4Changes at the cellular level after ANCYT1 treatment. Immunofluorescent visualization of the microtubular cytoskeleton in A. thaliana cells treated with ANCYT1 for 6 his shown. α-Tubulin was visualized with fluorescein isothiocyanate (green) and chromatin with 4′,6-diamidino-2-phenylindole (blue). A-C, control Arabidopsis cells showing a metaphase spindle (A), an anaphase spindle (B), and a phragmoplast (C). D-F, Arabidopsis cells treated with 100 μm ANCYT1: cell arrested in G2/M with microtubules randomly arranged around the nucleus (D), collapsed interphase microtubules forming circles in cytoplasm (E), and late telophase cell with aberrant microtubules (F). Bar = 10 μm. G and H, apoptotic changes in ANCYT1-treated cells. Immunofluorescent labeling of double strand DNA breaks in Arabidopsis suspension cells is shown. double-stranded DNA breaks were visualized after bromodeoxyuridine incorporation with mouse anti-bromodeoxyuridine antibody and anti-mouse-fluorescein isothiocyanate-conjugated secondary antibody (green). Chromatin was visualized with 4′,6-diamidino-2-phenylindole (blue). G, control Arabidopsis cells. H, Arabidopsis cells treated with 100μm of ANCYT1 for 3 h.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The cellular abnormalities observed after anticytokinin treatment are reminiscent of changes caused by roscovitine, a known inhibitor of the pivotal mammalian cell cycle regulator CDK2 (37Binarová P. Doležel J. Heberle-Bors E. Strnad M. Boögre L. Plant J. 1998; 16: 697-707Crossref PubMed Google Scholar). We therefore tested the influence of anticytokinins on CDK activity of a key plant cell cycle regulator, CDKA;1. CDKs were purified from an Arabidopsis suspension cell culture by two different methods, viz by immunoprecipitation with an anti-CDKA;1 antibody and by affinity chromatography with p13suc1-Sepharose. Fig. 5A shows that all three anticytokinins significantly inhibited phosphorylation of histone H1 by immunopurified CDKs of Arabidopsis at concentrations between 10 and 100 μm. However, ANCYT3 was less effective and showed inhibitory activity only at 100 μm (Fig. 5A). Interestingly, G2/M kinase CDKB1;1, immunoprecipitated with a specific antibody, proved to be much less sensitive to roscovitine and ANCYT1 (data not shown). Similar results were also obtained with human CDKs, where mitotic CDK1 is less sensitive to roscovitine and ANCYT3 than CDK2 (see below and Ref. 38Schulze-Gahmen U. Brandsen J. Jones H-D. Morgan D.-O. Meijer L. Veselý J. Kim S.-H. Proteins Struct. Funct. Genet. 1995; 22: 378-391Crossref PubMed Scopus (282) Google Scholar). In
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