ICANCER RESEARCH54. 5160-5165. October 1. 1994@

ABSTRACT

Cyclocreatine (CCr), a substrate analogue of creatine kinase (CK),exhibits antitumor activity in vitro and in vivo. To address its mechanismof action, we have examined its effects on tumor cell proliferation, viability, and cell cycle progression. Complete inhibition of proliferation ofME-iSO cervical carcinoma cells was observed within S h of exposure toCCr and was characterized by an inhibition of progression out of allphases of the cell cycle. This initial effect was partially reversible on drugremovaL Increased cytotoxicity was observed after several days of drugexposure and was most specific to cells in S. Previous studies have shownthat CCr supports ATP regeneration throueji the CK system less efficiently than the natural substrate creatine and that CCr is active againsttumor cell lines with elevated levels of CL We propose here that thegeneral inhibition of cell cycle progression reflects an effect of CCr ontumor cell energy availability through CK and that impaired energyhomeostasis for several days leads to tumor cell death. Our results pointout the unique nature of CCr as an anticancer agent that inhibits progression out of all phases of' the cell cycle.

INTRODUCTION

CCr2 has been shown to act as an anticancer agent in a variety ofsystems. In vitro, CCr reduced the growth of 10 established solidtumor cell lines (1) but had no effect on three nontransformed lines(2). CCr also inhibited the in vitro growth of 20% of 51 freshlyisolated human tumor samples (3). In vivo, CCr inhibited the growthof human neuroblastoma and cervical carcinoma xenografts in nudemice and syngeneic tumors in rats, including a sarcoma and two breastcarcinomas (1, 4, 5). In these and other in vivo experiments, CCr wasnot associated with any specific toxicity (6). In combination therapy,CCr showed excellent synergistic activity when used with a widevariety of standard anticancer agents (5). The compound is currently

being evaluated for safety in Phase I clinical trials in cancer patients.CCr is a substrate analogue of CK, an enzyme suggested to play a role

in the process of tumorigenesis (3, 7). CK is overexpressed in manytumor types and is associated with metastatic disease (Ref. 3 and references therein, 8, 9). It is induced by several hormones (10—13),oncogenes (7), and other elements of signal transduction pathways (13—15).The creatine kinase/creatine phosphate system is involved in the maintenance of cellular energy homeostasis in tissues with large and flucftt

ating energy demands, such as skeletal muscle, heart, and brain (16). Thesystem functions as a spatial and temporal energy buffer in addition tomaintaining cellular pH, ATP:ADP ratios, and ADP levels. The role ofCK and its substrates creatine and creatine phosphate in cellular transformation is not yet fully understood.

It has been suggested that the phosphorylated form of CCr may actas an anticancer agent by impairing the functions of the creatinekinase/creatine phosphate system (1). CCr is phosphorylated by CK togenerate a new synthetic phosphagen, CCr-P, which is a poor substrate for CK and hence provides Al? less readily than creatine

Received 5/1 2/94; accepted 8/1/94.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I To whom requests for reprints should be addressed, at Amira, Inc., One KendallSquare, Building 700, Cambridge, MA 02139.

2 The abbreviations used are: CCr, cyclocreatine (1-carboxymethyl-2-iminoimidazolidine); FACS, fluorescence-activated cell scanning; PBS, phosphate-buffered saline;CCr-P, cyclocreatine phosphate;@ 50% inbibitory concentrations.

phosphate (17). Buildup of the synthetic phosphagen in tumor cellsmay modulate AlT-dependent processes such as signaling cascades,resulting in tumor growth inhibition. To contribute to our understanding of the mechanism of anticancer activity of CCr, we have investigated its effect on the proliferation, viability, and cell cycle of tumorcells. Our results emphasize the unique nature of CCr as an agent thatinhibits progression out of all phases of the cell cycle.

MATERIALS AND METHODS

Drugs, Cell Lines, and Cell Culture. CCr was chemically synthesized asdescribed (18). It was dissolved in the appropriate complete media at 56 mMby heating to 37°Cfor 15 mm, then rocking at room temperature for 1 h. TheME-180 cervical carcinoma and DU145 prostate tumor cell lines were obtamed from the American Type Culture Collection (Rockville, MD) and weregrown as suggested (19).

Stem Cell Assays Cells were incubated in 77% Iscove's modified Dial

becco's medium, 2 mML-glutamine,4 mMCad2, 2.3 g/liter NaC1,3 units/mIinsulin, 0.5 mglml DEAE (diethylaminoethyl ether)-dextran, 1.5% bovineserum albumin, 10% fetal bovine serum, 10% horse serum, 2 mM sodiumpyruvate, and 100 units/ml penicillin/streptomycin.The soft agar consisted oftwo layers: (a) a base feeder layer of 0.5% agar; and (b) a less solid top layer(0.3% agar) which contained the tumor cells. Cells were allowed to incubate inagar with continuous exposure to the drug for 21 days. Colonies were countedafter staining with p-iodonitrotetraliumviolet. IC50values were determined bylinear regression.

Growth Curves. Cells were plated and fed the following day with cornplete media containing CCr at the concentrations specified. After incubationfor the specified time, cells were trypsinized, centrifuged, and resuspended in0.2% trypan blue in PBS. Viable cells were counted on a hemocytorneter.Counts and each assay were repeated in triplicate. Results are reported as themean of the assays. Repeated experiments gave comparable results.

Reversal Colony Assays. Cells were plated at 1.5 X 10@cells/25 cm2flask@The following day, complete media with CCr at the concentrations specifiedwere added to the exponentially growing cells. After treatment with CCr forthe specified time, cells were trypsinized, counted on a hemocytometer withtrypan blue and plated in drug-free complete media into six 35-mm wells at arange of densities from 500 to 5000 intact cells/well. For these experiments,cells of all samples were counted and the same number of cells was plated foreach control or drug treatment. Colonies were allowed to form for 7 days; theywere then stained with crystal violet and counted. Surviving fraction wascalculated as the ratio of colonies formed after drug treatment to colonies

formed in untreated controls. Mean surviving fraction was calculated from atleast four replicate wells. The drug concentration resulting in 50% cell deathrelative to untreated control was determined by linear regression. Each assaywas repeated in triplicate and results are reported as the mean of threeexperiments ±the SE.

FACS Analysis After the appropriatetreatment,cells were trypsinized,centrifuged, resuspended in PBS, and then gently vortexed while 95% ethanolwas slowly added to a final concentration of 70%. The fixed cells were storedat —20°C.Just prior to analysis by FACS, cells were centrifuged and resuspended to a concentration of 2 X 106cells/mI in 50 @.tWmlpropidium iodidein 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 theintensity of fluorescence.

Cell Synchronization. To synchronizein S-phase,cells weretreatedfor 24h in complete media with 2 mM thymidine, then rinsed with PBS, andincubated for 8 h in media without thymidine. Media with thymidine was thenadded for an additional 24 h. To synchronize in mitosis, cells were treated for

24 h with 2 mM thymidine, followed by 8 h without thymidine and, then 4 hwith 0.06 @g/ml nocodazole. Cells were then trypsinized for 1 mm with

5160

Cell Cycle Studies of Cyclocreatine, a New Anticancer Agent

Katherine J. Martin,' Elizabeth R. Winslow, and Rima Kaddurah-Daouk

Amira. Inc., Cambridge. Massachusetts 02139

CELL CYCLE S11JDIE5 OF CYCLOCREATINE

rocking and mitotic cells were collected, washed, and replated. To synchronizein G1, cells were synchronized in rnitosis then incubated for 8 h withoutthyrnidine or nocodazole.

CCr Uptake and Phosphorylation Assays. CCraccumulationin ME-180cells was assayed as described(18) with minor modifications@Briefly,subconfluent plates were incubatedwith CCr for the time indicated,washed with PBS, andthen fixed by adding 02 Mperchloricacid. Cells were scraped and resuspended,and an aliquot for protein determinationwas neutralized with NaOH. Proteincontent was measured by the method of Bradford (20). Remaining cells werecentrifuged for 2 mm at 13,000 X g. Free CCr in the supematant was measuredusing the chromogenic reagent Na3[Fe(CN)5NH3], which produces a blue color onbinding to CCr. Total CCr plus CCr-P was determinedfollowingconversionofCCr-P to free CCr by hearing to 65°Cfor 60 mm; CCr-P was calculated bysubtraction. Each assay was repeated in duplicate and the results were reported asthe mean of three experiments ±the SE

RESULTS

Effect of CCr on Colony Formation of ME-iSO Cervical Carcinoma Cells. Previous in vitro studies have shown that CCr inhibitsthe growth of a variety of established tumor cell lines with IC50 valuesin 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

which cells were seeded in soft agar and continuously exposed to thecompound during the 21-day period of colony formation. The cervicalcells were sensitive to CCr with a IC50 of 2.2 ±0.4 mt@.s.

Effect of CCr on the Proliferation and Viabifity of ME-180Cervical Carcinoma Celia. For most anticancer agents, cytotoxicityis measured using a standard colony assay following a brief drugexposure. However, an extended treatment time is required for theantitumor activity of CCr (1). Here we present results of experiments

designed to differentiate cytotoxicity and cytostasis following longperiods of drug exposure.

Intact cells were counted following exposure to a range of CCr

concentrations over the course of 7 days. The resulting growth curves

show that the drug had a dose-dependent effect on the rate of cellproliferation (Fig. 1). Within 24 h of the addition of 3.5 and 7 mr@iCCr, ME-180 doubling times were reduced by 1.7- and 2.9-fold,respectively. At 14 m@s,CCr completely arrested cell proliferation.Under these conditions, cells remained intact as shown by theircontinued ability to exclude trypan blue. Time-lapse photography andvideography showed that this effect was not due to a balance betweencell division and death (data not shown). Similar dose-dependentgrowth inhibition was observed in CCr-sensitive tumor cell lines otherthan ME-180, including the HT-29 colon carcinoma, the DU145

(I)

a,00

a,.0Ez

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.Points,meanof 3replicates; bars, SD.

—.—1day—6--4 days

—•---7days0

U)a,C000

0 10 20 30 40 50 60

Cyclocreatine (mM)

Fig. 2. Ability of ME-180 cervical carcinoma cells to resume normal growth afterrelease from various times of exposure to cyclocreatine. Data are presented as percent ofuntreated controls. Points, mean of 3 experiments; bars, SE.

prostate adenocarcinoma, the SiHa cervical carcinoma, and theMCF-7 breast adenocarcinoma (data not shown). Results of this

experiment demonstrated that CCr inhibited cell proliferation and thattreated cells remained intact during exposure. It did not address,

however, whether the arrested cells remained viable as defined by

their ability to resume growth after drug removal.To determine whether the CCr-arrested cells were viable, ME-180

cells were treated with a range of concentrations of CCr for 1, 4, or 7days, after which the drug was removed and the ability of cells toresume proliferation was measured. After drug removal, equal numbers of intact treated or untreated cells were plated and colonyformation relative to untreated controls was determined. After treatment with 14 mM for 1 day, the activity of CCr was partially revers

ible. Fifty % of the growth arrested cells were still viable as determined by their ability to form colonies after CCr removal (Fig. 2).When the drug treatment period was increased to several days, cellviability was significantly reduced, with only 10—20%of arrestedcells able to resume growth. In summary, the antitumor activity ofCCr is due to both cytostatic and cytotoxic effects.

Growth curves of cells treated with CCr for an extended period oftime support the conclusion that CCr irreversibly damages cells.ME-180 cervical carcinoma cells were treated for 28 days with 14 m@iCCr. 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 saturationvalue at high doses of CCr (Fig. 2). This is consistent with phasespecific cytotoxicity or with the presence of a subpopulation of drugresistant cells. To examine subpopulations we isolated 12 single cellclones by plating the parent line into 96-well plates. Cells in wellswith a single colony were expanded and assayed using the reversalcolony assay. Results revealed no evidence of resistant clones (datanot shown). Further experiments that address phase-specific cytotoxicity are presented later in the manuscript.

Effect of CCr on ME-iSO Cell-Cycle Progression. ME-180 cellswere treated with a range of CCr concentrations and the cell cycledistribution 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 rateswere reduced (Fig. 1). No major alterations in the cell cycle distributions were seen (Fig. 3; Table 1). A minor, 2-fold accumulation inG2-M was seen after 16 h but was not sustained. The absence of amajor accumulation of cells in any specific phase of the cycle

CCr treatment time (days)

5161

G1SG2-M3.5

mat0 h24 h96h68.0

55.668.715.4

20.619.516.6

23.811.87.0

m@0 h8 h16 h24 h96 h68.0

55.249.956.671.715.4

18.920.919.818.216.6

25.929.223.5

10.114

mat0 h8 h16 h24 h96 h68.0

58.151.652.960.415.4

15.813.519.221.016.6

26.234.927.918.5

CELL CYCLE STUDIES OF CYCLOCREATINE

the timing of cell cycle inhibition. CCr and CCr-P accumulatedsteadily in the cervical tumor cells, reaching one-half of the maximumlevels after about 8 h and maximum levels after 48 h (Fig. 6). Thus,the timing of CCr and CCr-P accumulation corresponds to the timingof the block to cell cycle progression.

Effect of CCr on DU14S Cell Cycle Progression. To determinewhether CCr has similar effects on other cell lines, DU145 prostateadenocarcinoma cells were treated with the drug for 4 days, and then

fixed, stained with propidium iodide, and analyzed on a FACSCan. Forcomparison, ME-180 cervical carcinoma cells were treated in parallel.The concentration of CCr used was the minimum required to completely block cell proliferation (data not shown). A lower CCr concentration that reduced the proliferation rate by approximately 70%was also included. DNA histograms showed essentially no change incell cycle distributions of the two cell lines following CCr treatment(Fig. 7). At the concentrations that arrested proliferation, unalteredcell cycle distributions indicate that CCr blocked progression out ofall phases of the cell cycle in both cell lines.

DISCUSSION

We have investigated the effects of CCr on proliferation, viability,and cell cycle progression of a representative CCr-sensitive tumor cellline. Cyclocreatine demonstrated components of both cytostatic andcytotoxic activity and caused a general block of progression out of allphases of the cell cycle.

Inhibition of cell cycle progression out of all phases is unusual foran anticancer agent. Such agents generally block at a specific phase(reviewed in Ref. 21). For example, the Vinca alkaloids, which inhibitthe assembly of microtubules, block cell cycle progression in G2-M.Inhibitors of DNA synthesis, such as hydroxyurea and 1-f3-D-arabinofuranosyl cytosine, block cell cycle progression specifically at theG1-S border. We propose that the general cell cycle block of CCr

reflects an effect of the compound on tumor cell energy availabilitywhich would be detrimental to many processes of the cell cycle.Compounds with anticancer activity that have been reported to blockgeneral cell cycle progression in some cell lines include interferon ‘r(22) and genestein, a tyrosine kinase inhibitor (23). Both of thesecompounds act through cell signaling pathways and are likely to havemany effects on tumor cells.

We have noted that CCr also induced a relatively minor (2-fold)accumulation of cells in the G2-M. This effect occurred early (within

24 h of exposure to CCr) and may reflect an effect of the drug on a

Table 1 Cell cycle distribution of MEI8O cells after treatment with cyclocreatineME-l80 cells were treated as for Fig. 3. Data are given as the percentage of the total

number of cells.

3.5 mM CCr 7 mM CCr 14 mM CCr

Oh@@ @Jc@ J@

8h

16h

5162

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Fig. 3. Representative DNA histograms of ME-180 cervical carcinoma cells treatedwith 3.5, 7, and 14 mM cyclocreatine for 0, 8, 16, or 24 h. Largest peak, cells in G1; peakto the right, cells in G2 and M; area between the peaks, cells in S.

indicates that the predominant effect of the drug was to block pro

gression out of all phases of the cell cycle.To further analyze this apparent block of all phases of the cell cycle,

we looked at progression of synchronized ME-180 cells out of G1, S.or mitosis in the presence or absence of CCr. After 0, 8, 24, 48, 72,and 96 h the cell cycle distribution was analyzed. Progression out ofeach phase was significantly reduced relative to the control within thefirst 8 h of treatment with CCr (Fig. 4). With continued treatment,progression was blocked. We note that in some cases the number ofcells with a DNA content corresponding to S seemed to decrease.Since growth curves showed no decrease in cell number over this timecourse, this change may indicate a loss of DNA from S cells.

Phase-specific Cytotoxicity. To determine whether CCr is cytotoxic to cells during a specific phase of the cell cycle, ME-180 cells

were blocked in G1, S, or M as described. The synchronizing agentwas removed and cells were grown in the presence or absence of CCrfor 4 days. Equal numbers of intact cells were then plated and allowedto form colonies. FACS analysis of the cell cycle distribution wasperformed immediately after synchronization and at several timepoints during CCr treatment (Fig. 4; Table 2). This analysis showedthat some cell cycle progression did occur after the synchronizingagent was removed and before CCr blocked cell cycle progression.This progression was for the most part limited to the first 8 h of CCr

exposure.Results of the reversal colony assays showed that CCr was more

toxic to cells that were in G1-S for the majority ofthe treatment period(Fig. 5, Column B) than to cells that remained predominantly in G1(Fig. 5; Column A). It was most toxic to cells that were in S and G2for the majority of the treatment period (Fig. 5; Column C). Thispopulation of cells spent more time in S while exposed to CCr thandid the other two groups. Thus, we conclude that CCr is a phasespecific cytotoxic agent that kills cells in S following several days ofexposure.

FACS analyses showed no evidence for apoptotic cell death in

response to treatment with CCr for up to 4 days. Apoptosis is characterized by extensive DNA degradation, which causes the appearance of a peak to the left of the G1 peak. Further studies are necessaryto confirm this observation.

Uptake and Phosphorylation of CCr. Uptake and phosphorylation of CCr in the ME-180 cell line were measured for comparison to

Table 2 Cell cycle distribution of cells at time of release from blotreatment with cyclocrearine

Data are given as the percentage of the total number of cellsck

and after 8 hofG1

SG2-MA

0 h 0.6 6.08 h 77.9 15.493.56.6B

Oh 86.3 6.38 h 40.1 56.14.82.4C

0 h 0.9 91.88 h 1.0 45.66.3 48.4

CELL CYCLESTUDIES OF CYCLOCREATINE

b. C.

@k@HJL@

:@ Ii@II@ 1jL@

a.

Oh

8h

24 h

48h

72 h

96h

@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.

used in combination with a number of different standard chemotherapy agents that function through a variety of mechanisms (5).

The activities reported here required 3—14mr@iof CCr. Comparablelevels of CCr have been shown previously to actively accumulate intissues of mice, rats, and chicks (reviewed in Ref. 6). Levels of 20—30mM CCr have been achieved in tissues with high CK activity such as

heart and skeletal muscle (29). CCr accumulated in Ehrlich ascitestumor cells in mice to 11 mM (30, 31) and in solid tumor tissues to at

specific mitotic event. Since CCr reduces AlT availability throughCK, we note that CK has been reported to localize to the mitoticspindle (24, 25) and has been implicated in the process of providingenergy during mitosis (26).

CCr demonstrated cytotoxicity that appeared to be specific for cellsin S. Anticancer agents with a number of different mechanisms ofaction have also been shown to be cytotoxic in S (27). Thus, it isdifficult to gain insight into the mechanism of CCr-induced cytotoxicity based on its S specificity. We note that other compounds that

reversibly inhibit cell cycle progression have been found to kill tumorcells after several days of exposure, e.g., bleomycin at lower concentrations (28).

Cell cycle effects of anticancer agents are often used to predicteffective combination treatments. Additive anticancer activity generally requires that two drugs have different effects on the cell cycle,indicative of different and complementary mechanisms of activity.Since CCr is unusual in its ability to prevent progression out of allphases of the cell cycle, it follows that it could be effective when usedin combination with a wide variety of standard chemotherapeutics.Indeed, CCr has shown remarkable synergy in vitro and in vivo when

5163

0.7

0.6

C 0.500i@ 0.4a)

.@ Q3

Cl) 0.2

0.1

A B CFig. 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,(B) G@, and (C) 5, at which time cyclocreatine was added. As in the experiment of Fig.

4, the cell cycle progressed for about 8 h after removal of synchronizing agent and wasthen blocked for the remainder of the 4-day treatment period by cyclocreatine in (A) G@,(B) G1 and 5, or (C) S and M. Cell cycle distributions after 0 and 8 h of cyclocreatine

treatment are presented in Table 2. Columns, mean of 6 replicates; bars, SD.

50

90 -@0C,0

80@0

.@. REFERENCES

0 1 2 3 4 5Cyclocreatine treatment time (days)

CELLCYCLE5TUDIE5OF CYCLOCREATINE

We thank Dr. Ed Greenfield (Repligen Corp.) for FACSCaE analyses,Dalton Chemical (Toronto, Ontario, Canada) for the synthesis of CCr, VrindaKhandekar for stem cell assays, and David Shaw for assays of CCr uptake andphosphorylation.

a,C

!@

0 0.

@,a)

0-lb(u@@50

E@

Fig. 6. Uptake and phosphorylation of cyclocreatine in ME-180 cervical carcinomacells. Points, mean of 3 experiments; bars, SE.

Fig. 7. RepresentativeDNA histograms of ME-180 cervical and DU145 prostate tumorcell lines (a) untreated or treated with levels of cyclocreatine that cause (b) a slowing ofgrowth or (c) 100% growth inhibition.

ME18O DU145

a.

b.

C.

least 5 mM.3 In the latter study, this level of accumulation corresponded to that at which in vivo tumor growth inhibition was observed.3 In in vivo experiments, CCr has been well tolerated whenadministered at high doses such as 1% of the feed (1, 4, 6) or 1gm/kg/day i.p. or i.v. (5). Thus, levels at which we saw cell cycleeffects in vitro are safely achieved and are effective in reducing tumorgrowth in animal tissues.

In conclusion, we summarize the unique properties of CCr as ananticancer agent. The compound is capable of inhibiting tumor growthin a variety of systems without adverse side effects (1, 3—5)and actssynergistically when used in combination with many different chemotherapeutic agents that act through a variety of mechanisms (5).Here we show that CCr exhibits components of both cytostatic andcytotoxic activity. It is unusual in its ability to prevent cell cycleprogression out of all phases of the cycle. Thus, CCr has the potentialto be an effective addition to anticancer chemotherapies.

3 L Schimmel and R. Kaddurah-Daouk, personal communication.

5164

100 ACKNOWLEDGMENTS

1. Lillie, J. W., O'Keefe, M., Valinski, H., Hamlin, A., Varban, M. L, and KaddurahDaouk, R. Cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) inhibits thegrowth 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) inhibitsthe replication of human herpes viruses. Antiviral Res., 23: 203—218,1994.

3. Martin, K. J., Chen, S-F., aark, 0. M., Degen, D., Wajima, M., Von Hoff, D. D., andKaddurah-Daouk, R. Evaluation of creatine analogs as a new class of anticanceragents using freshly explanted human tumor cells. J. Nail. Cancer Inst., 86: 608-613,1994.

4. Miller, E. E., Evans, A. E., and Cohn, M. Inhibition of tumor growth by creatine andcyclocreatine. Proc. Nail. Aced. Sci. USA, 90: 3304-3308, 1993.

5. Teicher, B. A., Menon, K, Northey, D., Liu, J., Kufe, D. W., and Kaddurah-Daouk,R. Cyclocreatine in cancer chemotherapy. Cancer Chemother. PharmacoL, in press,1994.

6. Walker, J. B. Creatine: biosynthesis, regulation, and function. Adv. Enzymol., 50:177-241,1979.

7. Kaddurah-Daouk, R., Lillie, J. W., Daouk, 0. H., Green, M. R., Kingston, R., andSchimmel, P. Induction of a cellular enzyme for energy metabolism by transformingdomains of adenovirus Eta. Mol. Cell. Biol., 10: 1476—1483, 1990.

8. Gazdar, A. F., Zweig, M. H., Carney, D. N., Van Steirteghen, A. C., Baylin, S. B., andMinna, J. D. Levels of creatine kinase and its BB isoenzyme in lung cancer specimensand cultures. Cancer Rca., 41: 2773—2777,1981.

9. Ishiguro, Y., Kato, K., Akatsuka, H., and Ito, T. The diagnostic and prognostic valueof pretreatment serum creatine kinase BB levels in patients with neuroblastoma.Cancer (Phila.), 65: 2014—2019,1990.

10. Reins, N. A., and Kaye, A. M. Identification of the major component of estrogeninduced protein of rat uterus as the BB isozyme of creatine kinase. J. Biol. Chem.,256: 23—26,1981.

11. Somjen, D., weisman, Y., Hard, A., Berger, E., and Kaye, A. M. Direct andsex-specific stimulation by sex steroids of creatine kinase activity and DNA synthesisin rat bone. Proc. Nati. Acad. Sci. USA, 86: 3361—3365,1989.

12. Binderman, I., Hard, S., Earon, Y., Tomer, A., Weisman, Y., Kaye, A. M., andSomjen, D. Acute stimulation of creatine kinase activity by vitamin D metabolites inthe developing cerebellum. Biochim. Biophys. Acts, 972: 9—16,1988.

13. Somjen, D., Zor, U., Kaye, A. M., Hard, k, and Binderman, I. Parathyroid hormoneinduction of creatine kinase activity and DNA synthesis is mimicked by phospholipase C, diacylglycerol and phorbol ester. Biochim. Biophys. Acts, 931: 215-223,1987.

14. Chida, K@,Kasahara, K, Tsuneaga, M., Kohno, Y., Yamada, S., Ohmi, S., andKuroki, T. Purification and identification of creatine phosphokinase B as a substrateof protein kinase C in mouse skin in vivo. Biochem. Biophys. Res. Commun., 173:351—357,1990.

15. Chida, K., Tsuneaga, M., Kasahara, IC, Kohno, Y., and Kuroki, T. Regulation ofcreatine phosphokinase B activity by protein kinase C. Biochem. Biophys. Rca.Commun., 173: 346—350,1990.

16. Wallimann, T., Wyss, M., Brdiczka, D., Nicolay, K, and Eppenberger, H. M.Intracellular compartmentation, structure and function of creatine kinase isoenzymesin tissues with high and fluctuating energy demands: the “phosphocreatinecircuit―forcellular energy homeostasis. Biochem. J., 281: 21—40,1992.

17. Annesley, T. M., and Walker, J. B. Cyclocreatine phosphate as a substitute forcreatine phosphate in vertebrate tissues. Energetic considerations. Biochem. Biophys.Rca. Commun., 74: 185-190, 1977.

18. Griffiths, G. R., and Walker, J. B. Accumulation of analog of phosphocreatine inmuscle of chicks fed 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine). J. BioLChem., 251: 2049—54,1976.

19. Hay, R., Caputo, J., Chen, T. R., Macy, M., McClintoch, P., and Reid, Y. (ads.).American Type Culture Collection Catalog of Cell Lines and Hybridomas, Ed. 7.Rockville, MD: American Type Culture Collection, 1992.

20. Bradford, M. M. A rapid and sensitive method for the quantitiation of microgramquantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.,72: 248—254,1976.

21. Charcosset, J-Y. Effects of antineoplastic agents on the cell cycle progression. Biol.Cell, 58: 135—138,1986.

22. Jernberg-Wilklind, H., Patterson, M., and Nilsson, K. Recombinant interferon-ginhibits the growth of IL-6-dependent human multiple myeloma cell lines in vitro.Eur. J. Haematol., 46: 231—239,1991.

23. Usui, T., Yoshida, M., Abe, K, Osada, H., Isono, IL and Beppu, T. Uncoupled cellcycle without mitosis induced by a protein kinase inhibitor, K-252a. J. Cell Biol., 115:1275—1282,1991.

24. Koons, S. J., Eckert, B. S., and Zobel, C. R. Immunofluorescenceand inhibitor studieson creatine kinase and mitosis Exp. Cell Res., 140: 401-409, 1982.

25. Fuseler, J. W., Eckert, B. S., Koons, S. J., and Shay, J. W. The association of creatinephosphokinase with the mitotic spindle. In: R. M. Dowben, and J. W. Shay (ads.),

CELLCYCLESTUDl@OF CYCLOCREATINE

Cell and Muscle Motility, Vol. 2, pp. 103—119.New York: Plenum, 1982. 29. Turner, D. M., and Walker, I. B. Enhanced ability of skeletal muscle containing26. Cande, w. z. Creatine kinase role in anaphase chromosome movement. Nature cyclocreatine phosphate to sustain AlP levels during ischemia following @-adrener

(Land.), 304: 557—558,1983. gic stimulation. J. Biol. Chem., 262: 6605—6609, 1987.27. Bhuyan, B. K., Scheidt, L G., and Fraser, T. J. Cell cycle phase-specificity of 30. Ohira, Y., Ishine, S., Inoue, N., and Yunoki, K. Reduced growth of Ehrlich ascites

antitumor agents. Cancer Res., 32: 398—407, 1972. tumor cells in creatine-depleted mice fed @-guanidinopropionic acid. Biochem. Bin28. Tounekti, 0., Pron, 0., Belehradek, J., Jr., and Mir, L M. Bleomycin, an apoptosis- phys. Acta, 1097: 117—122,1991.

mimetic drug that induces two types of cell death depending on the number of 31. Becker, S., and Schneider, F. Investigations on the function of creatine kinase inmolecules internalized. Cancer Res., 53: 5462—5469,1993. Ehrlich ascites tumor cells. Biol. Chem. Hoppe-Seyler, 370: 357—365,1989.

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