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  1. 1. ICANCER RESEARCH54. 5160-5165. October 1. 1994@ Cell Cycle Studies of Cyclocreatine, a New Anticancer Agent Katherine J. Martin,' Elizabeth R. Winslow, and Rima Kaddurah-Daouk Amira. Inc., Cambridge. Massachusetts 02139 ABSTRACT phosphate (17). Buildup of the synthetic phosphagen in tumor cells may modulate AlT-dependent processes such as signaling cascades, Cyclocreatine (CCr), a substrate analogue of creatine kinase (CK), resulting in tumor growth inhibition. To contribute to our understand exhibits antitumor activity in vitro and in vivo. To address its mechanism ing of the mechanism of anticancer activity of CCr, we have inves of action, we have examined its effects on tumor cell proliferation, viabil ity, and cell cycle progression. Complete inhibition of proliferation of tigated its effect on the proliferation, viability, and cell cycle of tumor ME-iSO cervical carcinoma cells was observed within S h of exposure to cells. Our results emphasize the unique nature of CCr as an agent that CCr and was characterized by an inhibition of progression out of all inhibits progression out of all phases of the cell cycle. phases of the cell cycle. This initial effect was partially reversible on drug removaL Increased cytotoxicity was observed after several days of drug exposure and was most specific to cells in S. Previous studies have shown MATERIALS AND METHODS that CCr supports ATP regeneration throueji the CK system less effi Drugs, Cell Lines, and Cell Culture. CCr was chemically synthesized as ciently than the natural substrate creatine and that CCr is active against described (18). It was dissolved in the appropriate complete media at 56 mM tumor cell lines with elevated levels of CL We propose here that the by heating to 37°Cfor 15 mm, then rocking at room temperature for 1 h. The general inhibition of cell cycle progression reflects an effect of CCr on ME-180 cervical carcinoma and DU145 prostate tumor cell lines were ob tumor cell energy availability through CK and that impaired energy tamed from the American Type Culture Collection (Rockville, MD) and were homeostasis for several days leads to tumor cell death. Our results point grown as suggested (19). out the unique nature of CCr as an anticancer agent that inhibits pro Stem Cell Assays Cells were incubated in 77% Iscove's modified Dial gression out of all phases of' the cell cycle. becco's medium, 2 mML-glutamine,4 mMCad2, 2.3 g/liter NaC1,3 units/mI insulin, 0.5 mglml DEAE (diethylaminoethyl ether)-dextran, 1.5% bovine INTRODUCTION serum albumin, 10% fetal bovine serum, 10% horse serum, 2 mM sodium pyruvate, and 100 units/ml penicillin/streptomycin. The soft agar consisted of CCr2 has been shown to act as an anticancer agent in a variety of two layers: (a) a base feeder layer of 0.5% agar; and (b) a less solid top layer systems. In vitro, CCr reduced the growth of 10 established solid (0.3% agar) which contained the tumor cells. Cells were allowed to incubate in tumor cell lines (1) but had no effect on three nontransformed lines agar with continuous exposure to the drug for 21 days. Colonies were counted (2). CCr also inhibited the in vitro growth of 20% of 51 freshly after staining with p-iodonitrotetralium violet. IC50values were determined by isolated human tumor samples (3). In vivo, CCr inhibited the growth linear regression. of human neuroblastoma and cervical carcinoma xenografts in nude Growth Curves. Cells were plated and fed the following day with corn mice and syngeneic tumors in rats, including a sarcoma and two breast plete media containing CCr at the concentrations specified. After incubation carcinomas (1, 4, 5). In these and other in vivo experiments, CCr was for the specified time, cells were trypsinized, centrifuged, and resuspended in not associated with any specific toxicity (6). In combination therapy, 0.2% trypan blue in PBS. Viable cells were counted on a hemocytorneter. CCr showed excellent synergistic activity when used with a wide Counts and each assay were repeated in triplicate. Results are reported as the mean of the assays. Repeated experiments gave comparable results. variety of standard anticancer agents (5). The compound is currently Reversal Colony Assays. Cells were plated at 1.5 X 10@ cells/25 cm2flask@ being evaluated for safety in Phase I clinical trials in cancer patients. The following day, complete media with CCr at the concentrations specified CCr is a substrate analogue of CK, an enzyme suggested to play a role were added to the exponentially growing cells. After treatment with CCr for in the process of tumorigenesis (3, 7). CK is overexpressed in many the specified time, cells were trypsinized, counted on a hemocytometer with tumor types and is associated with metastatic disease (Ref. 3 and refer trypan blue and plated in drug-free complete media into six 35-mm wells at a ences therein, 8, 9). It is induced by several hormones (10—13),onco range of densities from 500 to 5000 intact cells/well. For these experiments, genes (7), and other elements of signal transduction pathways (13—15). cells of all samples were counted and the same number of cells was plated for The creatine kinase/creatine phosphate system is involved in the main each control or drug treatment. Colonies were allowed to form for 7 days; they tenance of cellular energy homeostasis in tissues with large and flucftt were then stained with crystal violet and counted. Surviving fraction was calculated as the ratio of colonies formed after drug treatment to colonies ating energy demands, such as skeletal muscle, heart, and brain (16). The formed in untreated controls. Mean surviving fraction was calculated from at system functions as a spatial and temporal energy buffer in addition to least four replicate wells. The drug concentration resulting in 50% cell death maintaining cellular pH, ATP:ADP ratios, and ADP levels. The role of relative to untreated control was determined by linear regression. Each assay CK and its substrates creatine and creatine phosphate in cellular trans was repeated in triplicate and results are reported as the mean of three formation is not yet fully understood. experiments ± SE. the It has been suggested that the phosphorylated form of CCr may act FACS Analysis After the appropriatetreatment,cells were trypsinized, as an anticancer agent by impairing the functions of the creatine centrifuged, resuspended in PBS, and then gently vortexed while 95% ethanol kinase/creatine phosphate system (1). CCr is phosphorylated by CK to was slowly added to a final concentration of 70%. The fixed cells were stored generate a new synthetic phosphagen, CCr-P, which is a poor sub at — 20°C. prior to analysis by FACS, cells were centrifuged and resus Just strate for CK and hence provides Al? less readily than creatine pended to a concentration of 2 X 106cells/mI in 50 @.tWml propidium iodide in PBS. Samples were run through a FACSCan (Becton Dickinson). Results arc presented as the number of cells versus the amount of DNA as indicated by the Received 5/1 2/94; accepted 8/1/94. intensity of fluorescence. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with Cell Synchronization. To synchronizein S-phase,cells were treatedfor 24 18 U.S.C. Section 1734 solely to indicate this fact. h in complete media with 2 mM thymidine, then rinsed with PBS, and I To whom requests for reprints should be addressed, at Amira, Inc., One Kendall incubated for 8 h in media without thymidine. Media with thymidine was then Square, Building 700, Cambridge, MA 02139. added for an additional 24 h. To synchronize in mitosis, cells were treated for 2 The abbreviations used are: CCr, cyclocreatine (1-carboxymethyl-2-iminoimidazoli dine); FACS, fluorescence-activated cell scanning; PBS, phosphate-buffered saline; 24 h with 2 mM thymidine, followed by 8 h without thymidine and, then 4 h @ CCr-P, cyclocreatine phosphate; 50% inbibitory concentrations. with 0.06 @g/ml nocodazole. Cells were then trypsinized for 1 mm with 5160
  2. 2. CELL CYCLE S11JDIE5 OF CYCLOCREATINE rocking and mitotic cells were collected, washed, and replated. To synchronize in G1, cells were synchronized in rnitosis then incubated for 8 h without thyrnidine or nocodazole. CCr Uptake and Phosphorylation Assays. CCraccumulation ME-180 in —.—1 day cells was assayed as described(18) with minor modifications@ Briefly,subconflu —6--4 days ent plates were incubatedwith CCr for the time indicated,washed with PBS, and then fixed by adding 02 Mperchloricacid. Cells were scraped and resuspended, —•---7 days 0 and an aliquot for protein determination was neutralized with NaOH. Protein content was measured by the method of Bradford (20). Remaining cells were U) centrifuged for 2 mm at 13,000 X g. Free CCr in the supematant was measured a, C using the chromogenic reagent Na3[Fe(CN)5NH3], which produces a blue color on 0 binding to CCr. Total CCr plus CCr-P was determined following conversionof 0 0 CCr-P to free CCr by hearing to 65°C 60 mm; CCr-P was calculated by for subtraction. Each assay was repeated in duplicate and the results were reported as the mean of three experiments ± SE the 0 10 20 30 40 50 60 RESULTS Cyclocreatine (mM) Effect of CCr on Colony Formation of ME-iSO Cervical Car Fig. 2. Ability of ME-180 cervical carcinoma cells to resume normal growth after release from various times of exposure to cyclocreatine. Data are presented as percent of cinoma Cells. Previous in vitro studies have shown that CCr inhibits untreated controls. Points, mean of 3 experiments; bars, SE. the growth of a variety of established tumor cell lines with IC50 values in the low m@ range (1 to 6 mM) (1). To investigate the effect of CCr on the ME-180 cervical tumor line, we performed a stem-cell assay in prostate adenocarcinoma, the SiHa cervical carcinoma, and the which cells were seeded in soft agar and continuously exposed to the MCF-7 breast adenocarcinoma (data not shown). Results of this compound during the 21-day period of colony formation. The cervical experiment demonstrated that CCr inhibited cell proliferation and that cells were sensitive to CCr with a IC50 of 2.2 ±0.4 mt@.s. treated cells remained intact during exposure. It did not address, Effect of CCr on the Proliferation and Viabifity of ME-180 however, whether the arrested cells remained viable as defined by Cervical Carcinoma Celia. For most anticancer agents, cytotoxicity their ability to resume growth after drug removal. is measured using a standard colony assay following a brief drug To determine whether the CCr-arrested cells were viable, ME-180 exposure. However, an extended treatment time is required for the cells were treated with a range of concentrations of CCr for 1, 4, or 7 antitumor activity of CCr (1). Here we present results of experiments days, after which the drug was removed and the ability of cells to designed to differentiate cytotoxicity and cytostasis following long resume proliferation was measured. After drug removal, equal num periods of drug exposure. bers of intact treated or untreated cells were plated and colony Intact cells were counted following exposure to a range of CCr formation relative to untreated controls was determined. After treat concentrations over the course of 7 days. The resulting growth curves ment with 14 mM for 1 day, the activity of CCr was partially revers show that the drug had a dose-dependent effect on the rate of cell ible. Fifty % of the growth arrested cells were still viable as deter proliferation (Fig. 1). Within 24 h of the addition of 3.5 and 7 mr@i mined by their ability to form colonies after CCr removal (Fig. 2). CCr, ME-180 doubling times were reduced by 1.7- and 2.9-fold, When the drug treatment period was increased to several days, cell respectively. At 14 m@s,CCr completely arrested cell proliferation. viability was significantly reduced, with only 10—20%of arrested Under these conditions, cells remained intact as shown by their cells able to resume growth. In summary, the antitumor activity of continued ability to exclude trypan blue. Time-lapse photography and CCr is due to both cytostatic and cytotoxic effects. videography showed that this effect was not due to a balance between Growth curves of cells treated with CCr for an extended period of cell division and death (data not shown). Similar dose-dependent time support the conclusion that CCr irreversibly damages cells. growth inhibition was observed in CCr-sensitive tumor cell lines other ME-180 cervical carcinoma cells were treated for 28 days with 14 m@i than ME-180, including the HT-29 colon carcinoma, the DU145 CCr. The number of intact, dye-excluding cells decreased by 50% after 15 days and by 1 log after 28 days (data not shown). Dose-survival curves for CCr decreased to a constant saturation value at high doses of CCr (Fig. 2). This is consistent with phase specific cytotoxicity or with the presence of a subpopulation of drug resistant cells. To examine subpopulations we isolated 12 single cell clones by plating the parent line into 96-well plates. Cells in wells (I) a, with a single colony were expanded and assayed using the reversal 0 colony assay. Results revealed no evidence of resistant clones (data 0 a, not shown). Further experiments that address phase-specific cytotox .0 E icity are presented later in the manuscript. z Effect of CCr on ME-iSO Cell-Cycle Progression. ME-180 cells were treated with a range of CCr concentrations and the cell cycle distribution was examined after 0, 8, 16, and 24 h of drug treatment. The highest concentration used was that at which growth was arrested, and lower concentrations represent doses where proliferation rates CCr treatment time (days) were reduced (Fig. 1). No major alterations in the cell cycle distribu tions were seen (Fig. 3; Table 1). A minor, 2-fold accumulation in Fig. 1. Growth curves of ME-180 cervical cells. Cells were continuously exposed toO (•), 3.5mM(0), 7 mM(L@), 14mM(0), or56inst(A)cyclocreatine. mean 3 Points, of G2-M was seen after 16 h but was not sustained. The absence of a replicates; bars, SD. major accumulation of cells in any specific phase of the cycle 5161
  3. 3. CELL CYCLE STUDIES OF CYCLOCREATINE 3.5 mM CCr 7 mM CCr 14 mM CCr the timing of cell cycle inhibition. CCr and CCr-P accumulated steadily in the cervical tumor cells, reaching one-half of the maximum levels after about 8 h and maximum levels after 48 h (Fig. 6). Thus, @ Oh @Jc@ J@ the timing of CCr and CCr-P accumulation corresponds to the timing of the block to cell cycle progression. Effect of CCr on DU14S Cell Cycle Progression. To determine whether CCr has similar effects on other cell lines, DU145 prostate 8h adenocarcinoma cells were treated with the drug for 4 days, and then fixed, stained with propidium iodide, and analyzed on a FACSCan. For comparison, ME-180 cervical carcinoma cells were treated in parallel. The concentration of CCr used was the minimum required to com 16h pletely block cell proliferation (data not shown). A lower CCr con LJk@L@ L@JLA@ .@ centration that reduced the proliferation rate by approximately 70% was also included. DNA histograms showed essentially no change in cell cycle distributions of the two cell lines following CCr treatment (Fig. 7). At the concentrations that arrested proliferation, unaltered cell cycle distributions indicate that CCr blocked progression out of @ 24h/L .... @, , all phases of the cell cycle in both cell lines. Fig. 3. Representative DNA histograms of ME-180 cervical carcinoma cells treated with 3.5, 7, and 14 mM cyclocreatine for 0, 8, 16, or 24 h. Largest peak, cells in G1; peak to the right, cells in G2 and M; area between the peaks, cells in S. DISCUSSION We have investigated the effects of CCr on proliferation, viability, indicates that the predominant effect of the drug was to block pro and cell cycle progression of a representative CCr-sensitive tumor cell gression out of all phases of the cell cycle. line. Cyclocreatine demonstrated components of both cytostatic and To further analyze this apparent block of all phases of the cell cycle, cytotoxic activity and caused a general block of progression out of all we looked at progression of synchronized ME-180 cells out of G1, S. phases of the cell cycle. or mitosis in the presence or absence of CCr. After 0, 8, 24, 48, 72, Inhibition of cell cycle progression out of all phases is unusual for and 96 h the cell cycle distribution was analyzed. Progression out of an anticancer agent. Such agents generally block at a specific phase each phase was significantly reduced relative to the control within the (reviewed in Ref. 21). For example, the Vinca alkaloids, which inhibit first 8 h of treatment with CCr (Fig. 4). With continued treatment, the assembly of microtubules, block cell cycle progression in G2-M. progression was blocked. We note that in some cases the number of Inhibitors of DNA synthesis, such as hydroxyurea and 1-f3-D-arabino cells with a DNA content corresponding to S seemed to decrease. furanosyl cytosine, block cell cycle progression specifically at the Since growth curves showed no decrease in cell number over this time G1-S border. We propose that the general cell cycle block of CCr course, this change may indicate a loss of DNA from S cells. reflects an effect of the compound on tumor cell energy availability Phase-specific Cytotoxicity. To determine whether CCr is cyto which would be detrimental to many processes of the cell cycle. toxic to cells during a specific phase of the cell cycle, ME-180 cells Compounds with anticancer activity that have been reported to block were blocked in G1, S, or M as described. The synchronizing agent general cell cycle progression in some cell lines include interferon ‘r was removed and cells were grown in the presence or absence of CCr (22) and genestein, a tyrosine kinase inhibitor (23). Both of these for 4 days. Equal numbers of intact cells were then plated and allowed compounds act through cell signaling pathways and are likely to have to form colonies. FACS analysis of the cell cycle distribution was many effects on tumor cells. performed immediately after synchronization and at several time We have noted that CCr also induced a relatively minor (2-fold) points during CCr treatment (Fig. 4; Table 2). This analysis showed accumulation of cells in the G2-M. This effect occurred early (within that some cell cycle progression did occur after the synchronizing 24 h of exposure to CCr) and may reflect an effect of the drug on a agent was removed and before CCr blocked cell cycle progression. This progression was for the most part limited to the first 8 h of CCr Table 1 Cell cycle distribution of MEI8O cells after treatment with cyclocreatine exposure. ME-l80 cells were treated as for Fig. 3. Data are given as the percentage of the total Results of the reversal colony assays showed that CCr was more number of cells. toxic to cells that were in G1-S for the majority ofthe treatment period G1SG2-M3.5 (Fig. 5, Column B) than to cells that remained predominantly in G1 mat (Fig. 5; Column A). It was most toxic to cells that were in S and G2 0h for the majority of the treatment period (Fig. 5; Column C). This 24 h 55.6 20.6 23.8 11.87.0 96h68.0 68.715.4 19.516.6 population of cells spent more time in S while exposed to CCr than did the other two groups. Thus, we conclude that CCr is a phase m@ specific cytotoxic agent that kills cells in S following several days of 0h 8h 55.2 18.9 25.9 exposure. 16 h 49.9 20.9 29.2 FACS analyses showed no evidence for apoptotic cell death in 24 h 56.6 19.8 23.5 10.114 96 h68.0 71.715.4 18.216.6 response to treatment with CCr for up to 4 days. Apoptosis is char acterized by extensive DNA degradation, which causes the appear mat ance of a peak to the left of the G1 peak. Further studies are necessary 0h 8h 58.1 15.8 26.2 to confirm this observation. 16 h 51.6 13.5 34.9 Uptake and Phosphorylation of CCr. Uptake and phosphoryla 24 h 52.9 19.2 27.9 tion of CCr in the ME-180 cell line were measured for comparison to 96 h68.0 60.415.4 21.016.6 18.5 5162
  4. 4. CELL CYCLESTUDIES OF CYCLOCREATINE a. b. C. Oh 8h @k@HJL@ 24 h 48h 72 h 96h :@ Ii @II@ 1jL@ @i-CCr -CCr +CCr -CCr +CCr -CCr Fig. 4. DNA histograms of ME-180 cells treated for the times indicated with 0 or 14 mat cyclocreatine after release from (a) G@, b) 5, or (c) M. ( Table 2 Cell cycle distribution of cells at time of release from blo ofG1 and after 8 h used in combination with a number of different standard chemother treatment with cyclocrearine apy agents that function through a variety of mechanisms (5). Data are given as the percentage of the total number of cellsck The activities reported here required 3—14 of CCr. Comparable mr@i SG2-MA levels of CCr have been shown previously to actively accumulate in 0 h 0.6 6.0 6.6B 8h 77.9 15.493.5 tissues of mice, rats, and chicks (reviewed in Ref. 6). Levels of 20—30 mM CCr have been achieved in tissues with high CK activity such as Oh 86.3 6.3 2.4C 8h 40.1 56.14.8 heart and skeletal muscle (29). CCr accumulated in Ehrlich ascites 0 h 0.9 91.8 tumor cells in mice to 11 mM (30, 31) and in solid tumor tissues to at 8 h 1.0 45.66.3 48.4 specific mitotic event. Since CCr reduces AlT availability through 0.7 CK, we note that CK has been reported to localize to the mitotic spindle (24, 25) and has been implicated in the process of providing 0.6 energy during mitosis (26). CCr demonstrated cytotoxicity that appeared to be specific for cells C 0.5 0 in S. Anticancer agents with a number of different mechanisms of 0 action have also been shown to be cytotoxic in S (27). Thus, it is i@ 0.4 difficult to gain insight into the mechanism of CCr-induced cytotox a) .@ Q3 icity based on its S specificity. We note that other compounds that reversibly inhibit cell cycle progression have been found to kill tumor Cl) 0.2 cells after several days of exposure, e.g., bleomycin at lower concen trations (28). 0.1 Cell cycle effects of anticancer agents are often used to predict effective combination treatments. Additive anticancer activity gener ally requires that two drugs have different effects on the cell cycle, A B C indicative of different and complementary mechanisms of activity. Fig. 5. Survival of synchronized ME-180 cervical carcinoma cells after treatment with 14 mat cyclocreatine for 4 days. Cells were synchronized and then released from (A) M, Since CCr is unusual in its ability to prevent progression out of all (B) G@, and (C) 5, at which time cyclocreatine was added. As in the experiment of Fig. phases of the cell cycle, it follows that it could be effective when used 4, the cell cycle progressed for about 8 h after removal of synchronizing agent and was then blocked for the remainder of the 4-day treatment period by cyclocreatine in (A) G@, in combination with a wide variety of standard chemotherapeutics. (B) G1 and 5, or (C) S and M. Cell cycle distributions after 0 and 8 h of cyclocreatine Indeed, CCr has shown remarkable synergy in vitro and in vivo when treatment are presented in Table 2. Columns, mean of 6 replicates; bars, SD. 5163
  5. 5. CELLCYCLE5TUDIE5OF CYCLOCREATINE 1 00 ACKNOWLEDGMENTS We thank Dr. Ed Greenfield (Repligen Corp.) for FACSCaE analyses, a, Dalton Chemical (Toronto, Ontario, Canada) for the synthesis of CCr, Vrinda C 90 -@ 0 Khandekar for stem cell assays, and David Shaw for assays of CCr uptake and @ ! C, 0 phosphorylation. @ 0 0. 80 @,a) 0 0-lb .@. REFERENCES (u@ @50 E@ 1. Lillie, J. W., O'Keefe, M., Valinski, H., Hamlin, A., Varban, M. L, and Kaddurah Daouk, R. Cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) inhibits the growth of a broad spectrum of cancer cells derived from solid tumors. Cancer Rca., 53: 3172-3178, 1993. 2. Lillie, J. W., Smee, D. F., Huffman, J. H., Hansen, L J., Sidwell, R. W., and Kaddurah-Daouk, R. Cyclocreatine (1-cathoxyrnethyl-2-imnoimidazolidine) inhibits 50 the replication of human herpes viruses. Antiviral Res., 23: 203—218, 1994. 0 1 2 3 4 5 3. Martin, K. J., Chen, S-F., aark, 0. M., Degen, D., Wajima, M., Von Hoff, D. D., and Kaddurah-Daouk, R. 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