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Cell Type–Dependent Effects of Polo-Like Kinase 1 Inhibition Compared with Targeted Polo Box Interference in Cancer Cell Lines

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Cell Type–Dependent Effects of Polo-Like Kinase 1 Inhibition Compared with Targeted Polo Box Interference in Cancer Cell Lines 3189 Cell type–dependent effects of Polo-like kinase 1 inhibition compared with targeted polo box interference in cancer cell lines Jenny Fink, Karl Sanders, Alexandra Rippl, certain cell lines but highly contrasting effects in others. Sylvia Finkernagel, Thomas L. Beckers, This may point to subtle differences in the molecular and Mathias Schmidt machinery of mitosis regulation in cancer cells. [Mol Cancer Ther 2007;6(12):3189–97] Nycomed GmbH, Konstanz, Germany Introduction Abstract Polo-like kinase 1 (Plk1) has been identified as key player Multiple critical roles within mitosis have been assigned for G2-M transition and mitotic progression in both normal to Polo-like kinase 1 (Plk1), making it an attractive and tumor cells (1). Multiple roles have been assigned to candidate for mitotic targeting of cancer cells. Plk1 Plk1 at the entry into M phase, mitotic spindle formation, contains two domains amenable for targeted interference: condensation and separation of chromosomes, exit from a kinase domain responsible for the enzymatic function mitosis by activation of the anaphase-promoting complex, and a polo box domain necessary for substrate recogni- and in cytokinesis (reviewed in ref. 2). Moreover, recent tion and subcellular localization. Here, we compare two reports implicated an involvement of Plk1 in the resump- approaches for targeted interference with Plk1 function, tion of cell cycle reentry after checkpoint activation through either by a Plk1 small-molecule enzyme inhibitor or by DNA-damaging agents (3). It is therefore not surprising inducible overexpression of the polo box in human cancer that targeted interference with Plk1, primarily by anti- cell lines. Inducible expression of the Plk1 polo box bodies, antisense, or small interfering RNA technology but resulted in growth inhibition of RKOp27 human colon also with small-molecule inhibitors (4, 5), has been reported adenocarcinoma cells without obvious signs of mitotic to result in a blockade in mitosis with subsequent induction abnormalities. A Plk1 kinase inhibitor in the same cell line of cell death (6–9). Some of these reports even describe a arrested cells in mitosis with subsequent onset of differential response of nontransformed cells toward apoptosis. Similarly, PC-3 human prostate cancer cells interference with Plk1 function as compared with tumor were growth inhibited on expression of the polo box. cells. Moreover, overexpression of Plk1 has been observed Prolonged expression of the polo box in these cells in a wide variety of carcinomas of different pathologic resulted in the occurrence of binucleated or multinucleat- origin, including breast (10), ovary, colon, pancreas, lung, ed cells. In contrast, U2OS human osteosarcoma cells endometrium, brain, skin, head and neck, esophagus, responded to overexpression of the polo box with a gastric tract, and prostate (reviewed in ref. 11). Some of massive mitotic accumulation coinciding with the onset these studies correlated Plk1 overexpression with a poor of apoptosis. Comparison of spindle formation revealed patient prognosis even in multivariate analyses (12). very similar mitotic abnormalities in polo box–over- Human Plk1 is composed of two functional domains: The expressing U2OS cells compared with U2OS cells treated NH2-terminal kinase domain is highly conserved among with the Plk1 kinase inhibitor. We conclude that interfer- polo-like kinase family members. The canonical consensus ence with polo box function and inhibition of Plk1 kinase sequence for protein kinases in the ATP-binding subdo- activity can exert very similar phenotypic effects in main I (Gly-X-Gly-X-X-Gly-X-Val) is changed within the Plk1 kinase domain into Gly-X-Gly-Gly-X-Ala-X-C. The COOH-terminal region contains two polo boxes, each being 60 to 70 amino acids in length. Despite only a low amino acid sequence identity (12%), both polo boxdomains (PBD) Received 1/23/07; revised 8/16/07; accepted 10/9/07. form an intramolecular dimer with identical folds of a six- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked stranded h-sheet and an a-helix(13). Together with the advertisement in accordance with 18 U.S.C. Section 1734 solelyto region in between them and a linker between the kinase indicate this fact. domain and the first polo box, it functions as a phospho- Note: Current address for J. Fink: Institut Curie, Centre National de la Recherche Scientifique, Unite´Mixte de Recherche 144, 26 rue d’Ulm, serine/threonine binding module (13, 14). Using a phos- 75248 Paris, France. Current address for T.L. Beckers: Oncotest GmbH, pho-peptide library–based proteomic screen, the optimal am Flughafen 12-14, D-79108 Freiburg, Germany. recognition sequence has been identified as Ser-[pSer/ Requests for reprints: Mathias Schmidt, Nycomed GmbH, RPD/SO, pThr]-[Pro/X] (15). Many of the identified Plk1 substrates Byk-Gulden-Strasse 2, D-78467 Konstanz, Germany. Phone: 49-7531-842945; Fax: 49-7531-8492945. contain this consensus motif (16). The current polo box E-mail: [email protected] working model suggests that priming kinases such as Copyright C 2007 American Association for Cancer Research. mitogen-activated protein kinases, cyclin-dependent doi:10.1158/1535-7163.MCT-07-0048 kinases, and other mitotic kinases generate priming Mol Cancer Ther 2007;6(12). December 2007 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. 3190 Targeted Plk1 Interference in Cancer Cell Lines phosphorylation sites, which are bound by the polo boxof concentration twice as high as the final concentration in the Plk1. Thus, Plk1 is brought into close proximity to its test. RKOp27 and PC-3 cells were seeded into 96-well flat- substrate, which facilitates subsequent phosphorylation bottomed plates at a density of 5,000 per well in a volume of (17–19). It has to be noted that a priming phosphorylation 50 AL/well. To determine the effect on arrested RKOp27 of Plk1 substrates is not absolutely mandatory because cells, these cells were seeded at a density of 15,000 per well in bacterially expressed unphosphorylated substrates of Plk1 medium containing 10 Amol/L ponasterone A. Twenty-four can also be phosphorylated by Plk1 (20). Several studies hours after seeding, 50 AL of each compound dilution in have shown that this motif recognition model is also crucial DMEM were added per well with 1% DMSO–treated cells as for the targeting of Plk1 to specific subcellular locations a control. After incubation with the compounds for 72 h at (21–23). Interestingly, Sak, the fourth member of the polo- 37jC, 10 AL of Alamar Blue solution (Biosource) were added like kinase family, contains only one polo box. Crystal and the fluorescence was measured (excitation, 544 nm; structure analyses of the polo boxmotif have shown that emission, 590 nm). For calculation of cell viability, the the Sak polo boxforms a homodimer in vitro and in vivo emission value from untreated cells was set as 100% viability and localizes to centrosomes and the cleavage furrow and the emission rates of treated cells were set in relation to during cytokinesis (24). the values of untreated cells. The corresponding IC50 values Due to its structural hallmarks, its overexpression in were determined from the concentration-effect curves. various cancer types, and the intrinsic dependence of Flow Cytometric Analysis tumor cells on its activity in mitosis, Plk1 represents an After completion of the desired treatment, cells were attractive candidate for molecular targeted cancer therapy. harvested by trypsinization, and an aliquot of 1 Â 106 cells It is conceivable that specific inhibitors of polo-like kinases was washed once with cold PBS and then fixed with cold may be developed through targeting either the kinase 70% ethanol. The DNA was stained with a solution con- domain or the PBD. taining 25 Ag/mL propidium iodide and 10 Ag/mL RNase In this study, we compared the physiologic consequences A in PBS for 6 h. Cell cycle distribution was analyzed with of Plk1 kinase inhibition with a small-molecule inhibitor (4) a FACScan flow cytometer (Becton Dickinson) at an exci- and the disruption of polo boxfunction through inducible tation wavelength of 488 nm. ectopic overexpression of the polo box in three different Cell Staining cancer cell lines. We observed that interference with polo On completion of the desired treatment, cells were boxfunction and inhibition of Plk1 kinase activity can exert harvested by trypsinization. An aliquot (5 Â 103 cells) very similar phenotypic effects in certain cancer cells. Yet was spun onto glass slides using a cytospin and subse- distinct differences were observed with respect to respon- quently fixed and stained with the Hemacolor kit according siveness toward these two forms of targeted Plk1 interfer- to the manufacturer’s instructions. For the staining of ence in other cell types. microtubules, cells were fixed with 4% paraformaldehyde in PBS for 5 min, washed with PBS, and permeabilized with 0.2% Triton X-100 for 5 min. After blocking with 2% bovine Materials and Methods serum albumin for 30 min, the cells were incubated with an Materials and all other reagents were purchased from antibody against a-tubulin for 1 h at 37jC. Binding was Sigma Chemical unless otherwise specified. The Hemacolor visualized by incubation with a Cy3-labeled antimouse staining kit was obtained from Merck; antibodies were antibody (Dianova). The slides were washed with PBS obtained from various suppliers: Plk1 (Santa Cruz Biotech- between each antibody incubation step and DNA was nology or Cell Signaling Technology); h-actin and a-tubulin counterstained for 5 min with 100 ng/mL Hoechst 33342 (Sigma); Myt-1, cleaved caspase-3, and phospho-histone (Invitrogen) followed by final rinsing with deionized water. H3 (Cell Signaling Technology); and V5 (Invitrogen).
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