CYCLOCREATINE INHIBITS STIMULATED MOTILITY IN TUMOR CELLS POSSESSING CREATINE KINASE Peter T. MULVANEY 1 , Mary L. STRACKE 1 *, Suk Woo NAM 1 , Elisa WOODHOUSE 1 , Margaret O’KEEFE 2 , Timothy CLAIR 1 , Lance A. LIOTTA 1 , Rima KHADDURAH-DAOUK 2 and Elliott SCHIFFMANN 1 1 Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 2 AVICENA Group, Inc., Cambridge, MA, USA Cyclocreatine (1-carboxymethyl-2-iminoimidazolidine), an analog of creatine and a substrate for creatine kinase (EC 2.7.3.2), inhibits the stimulated motility of tumor cells which possess creatine kinase. A2058-055 human melanoma cells, transfected with a creatine kinase gene, showed an 80–90% reduction in chemotactic response to type IV collagen when incubated overnight in the presence of 10 mM cyclocreatine ( p F 0.0001 for n  8 experiments). T his inhibitory effect of cyclocreatine can be partially reversed by addition of creatine to the overnight cell treatment. N on-transfected cells, with very low levels of creatine kinase, were not significantly inhibited. Further experiments utilizing type IV collagen as attractant demonstrated that cyclocreatine inhibited the chemokinetic (91%) and the haptotactic (73%) responses and the in vitro invasion of A2058-055 cells through Matrigel- coated membranes (88%). In addition, motility stimulation of A2058-055 cells by either autotaxin or fibronectin was mark- edly inhibited by cyclocreatine. DU -145 prostatic tumor cells, which express endogenous creatine kinase, also have a re- duced motility response to either autotaxin or epidermal growth factor induced motility in the presence of cyclocre- atine. Int. J. Cancer 78:46–52, 1998.  1998 Wiley-Liss, Inc. ² Metastasis is a primary feature in tumor progression and a leading cause of death in cancer patients. Tumor cell motility is a major determinant of the metastatic cascade. In this regard, a variety of cytokines and extracellular matrix components have been shown to stimulate migration of tumor cells in vitro. These responses may be mediated by different signal-transduction path- ways, reflecting the diversity of metabolic mechanisms available to invasive tumor cells. While it has been known for some time that tumor cells rely on glycolysis as a major energy source for proliferation (Racker, 1956), it has recently been shown that the same pathway is used by malignant melanoma cells for migration (Beckner et al., 1990). The creatine kinase (CK) isoenzymes and their substrates, creatine (Cr) and phosphocreatine (Cr-P), are believed to play a pivotal role in energy transduction in tissues with large and fluctuating energy demands, such as skeletal muscle, heart and brain (Wallimann and Hemmer, 1994). The enzymes reversibly catalyze the transfer of a high-energy phosphoryl group from ATP to Cr. Several lines of evidence have suggested a role for this enzyme system in transformation. For example, colony formation of tumors in soft agar is markedly enhanced in cells with high levels of CK over that in cells with low or undetectable levels of the enzyme (Lillie et al., 1993). The brain isoform of CK (CK-BB) appears to be highly active in a variety of tumors, and in neurologic and neuroendocrine tumors high CK activity correlates with highly aggressive neoplasms and poor clinical prognosis (Ishiguro et al., 1990). Experiments reported by Kaddurah-Daouk et al. (1990) have demonstrated the regulation of expression of this brain isoform by an oncogenic product, E1a. Both the enzymatic activity and the mRNA levels were induced by over-expressed E1a. This induction appeared to correlate with the transformation properties of the oncogene. Later experiments revealed that expression of the brain isoform is inhibited by over-expression of the tumor- suppressor protein p53 (Zhao et al., 1994). The high levels of this enzyme in tumor cells along with its regulation by oncogenes and tumor-suppressor genes suggested a functional role for the enzyme system in processes of cellular transformation. A variety of analogs of Cr which impact the rate of ATP production through the CK reaction were synthesized and analyzed for their ability to inhibit tumor growth in soft agar (Martin et al., 1994a). Several were found to be active (Bergnes et al., 1996), with cyclocreatine (CCr) being the most active analog. CCr is phosphory- lated efficiently by CK both in vitro and in vivo, resulting in build-up of the synthetic phosphagen phosphocyclocreatine (CCr- P). However, comparison of the V max /K m ratios has shown that the phosphorylated CCr is 160-fold less efficient than Cr-P at sustain- ing ATP production through CK (Annesley and Walker, 1977). We now describe the effect of CCr on the motility of several tumor cell lines. We studied the effect of CCr on motility in a cell line transfected to over-express CK, a low-expressing transfectant, the parental cell line and a prostatic tumor cell line (DU-145). We found that CCr could inhibit motility of cells which express CK but not that of cells that expressed low or undetectable levels of this enzyme. MATERIAL AND METHODS Chemicals and media Cr, CCr and their phosphorylated forms were provided by AVICENA (Cambridge, MA). Type IV mouse EHS collagen and fibronectin were purchased from Collaborative Biomedical Prod- ucts (Bedford, MA), as was Matrigel for invasion assays. Epider- mal growth factor was purchased from Upstate Biotechnology (Lake Placid, NY). DMEM, EMEM, 100 penicillin/streptomy- cin, 200 mM glutamine and heat-inactivated FBS were purchased from Biofluids (Gaithersbug, MD). Autotaxin (ATX) was prepared as previously described (Clair et al., 1997). Cells A2058 cells and transfectants were maintained in DMEM supplemented by 2 mM glutamine, 1 penicillin/streptomycin and 10% (v/v) heat-inactivated FBS (complete DMEM). After reaching confluence, cells were detached with 0.05% trypsin and 0.02% EDTA and passaged (Aznavoorian et al., 1990). The parental A2058 cell line was transfected with the brain isoform of the CK gene to yield an over-expressing CK strain (A2058-055) and a low-expressing CK strain (A2058-032) (Lillie et al., 1993). These lines were obtained from AVICENA. DU-145 cells were purchased from the ATCC (Rockville, MD) and maintained in EMEM supplemented with 1 penicillin/ streptomycin and 10% FBS. Motility assays A2058 cells (both parental and transfected) were pre-incubated for 18 hr with or without agents in serum-free DMEM supple- mented with 0.1% (w/v) BSA (0.1% BSA-DMEM). Then, cells in *Correspondence to: Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Building 10/Room 2A33, Bethesda, MD 20892, USA. Fax: (301) 480-0853. E-mail: stracke@helix.nih.gov Received 15 December 1997; Revised 23 April 1998 Int. J. Cancer: 78, 46–52 (1998)  1998 Wiley-Liss, Inc. ² This article is a US Government work and, as such, is in the public domain in the United States of America. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer