˛ ˛ Journal of Cellular Biochemistry 102:1405–1419 (2007) FAST TRACK A New, Unexpected Action of Olomoucine, a CDK Inhibitor, on Normal Human Cells: Up-Regulation of CLIMP-63, a Cytoskeleton-Linking Membrane Protein Jo ´ zefa Wesierska-Gadek, 1 * Marieta Gueorguieva, 1 Matthias P. Kramer, 1 Carmen Ranftler, 1 Bettina Sarg, 2 and Herbert Lindner 2 1 Cell Cycle Regulation Group, Department of Medicine I, Division: Institute of Cancer Research; Medical University of Vienna, Vienna, Austria 2 Division of Clinical Biochemistry, Biocenter, Medical University of Innsbruck, Innsbruck, Austria Abstract Inhibition of cyclin-dependent kinases (CDKs) is a novel strategy in the therapy of human malignancies. The pharmacological CDK inhibitors representing a few distinct classes of compounds exert different target specificity. Considering the fact that dividing and quiescent cells differ in their CDK activity and in the pattern of their expression, one might expect that anti-proliferative efficiency of the pharmacological CDK inhibitors would depend on the mitotic index of treated cells. The present article shows that olomoucine (OLO), a weak CDK2 inhibitor has new, unexpected activity. At concentrations up to 100 mM OLO did not inhibit proliferation of normal human cells, but arrested growth of human HL-60 leukemia cells. The anti-proliferative effect of OLO was clearly weaker than that of roscovitine (ROSC). Surprisingly, OLO at low doses strongly up-regulated a cellular protein with approximately 65 kDa in normal, but not in immortalized and cancer cells. By mass spectrometric analysis CLIMP-63, a cytoskeleton-linking membrane protein was identified as the major component of the up-regulated protein band. These results were subsequently confirmed by immunoblotting. Further experiments revealed that OLO, but not ROSC, strongly up-regulates CLIMP-63 in a dose- and time-dependent manner solely in senescent cells. J. Cell. Biochem. 102: 1405 – 1419, 2007. ß 2007 Wiley-Liss, Inc. Key words: CDK inhibitors; cell cycle arrest; quiescence; senescence; life-span; population doubling Cyclin-dependent kinases (CDKs) that regu- late sequential steps of the cell cycle in higher eukaryotes, received much attention of oncolo- gists during the past two decades [for reviews, see van den Heuvel and Harlow, 1993; Hunter and Pines, 1994; Vermeulen et al., 2003; Senderowicz, 2004]. CDKs, which are serine/ threonine kinases, form complexes with cyclins, their specific regulatory components. The acti- vation of CDKs is mediated by binding to their proper cyclin(s) and by their subsequent phos- phorylation at specific residues catalyzed by cyclin activating kinase (CAK). Most cancer cells harbor mutations in genes controlling the cell cycle resulting in an aberrant cell cycle progression [Kozar et al., 2004; Malumbres et al., 2004]. According to the current opinion CDK4/6 and CDK2 play a key role in the G 1 /S transition. Interestingly, abnormally elevated CDK4 activity is implicated in cancer primarily by mutations or alterations of its partner, cyclin D as well as by inactivation of its cellular inhibitor p16 INK4A [Rane et al., 1999; Sender- owicz, 2004]. On the other hand, CDK2 seems to be not directly affected by the enhancement of the activity or by mutations. Considering the increased activity of CDKs in malignant ß 2007 Wiley-Liss, Inc. Abbreviations used: APF, anti-proliferative factor; CDK, cyclin-dependent kinase; CDKI, cyclin-dependent kinase inhibitor; CKAP4/p63, cytoskeleton-associated protein 4/p63; CLIMP-63, cytoskeleton-linking membrane pro- tein-63; ER, endoplasmic reticulum; FACS, fluorescence- activated cell sorting; HIPK2, homeodomain protein kin- ase-2; LPC-1, late PDL cDNA-1; MTs, microtubules; OLO, olomoucine; PDL, population doubling level; PI, propidium iodide; PVDF, polyvinylidene difluoride; ROSC, roscovitine; WCL, whole cell lysate; MS/MS, tandem mass spectrometry. *Correspondence to: Jo ´zefa We ˛sierska-Ga ˛ dek, Department of Medicine I, Division: Institute of Cancer Research, Cell Cycle Regulation Group, Vienna Medical University, Borschkegasse 8 a, A-1090 Vienna, Austria. E-mail: jozefa.gadek-wesierski@meduniwien.ac.at Received 26 July 2007; Accepted 10 September 2007 DOI 10.1002/jcb.21596