[CANCER RESEARCH 46, 1063-1067, March 1986]

Biochemical Mode of Cytotoxic Action of Neplanocin A in L1210Leukemic Cells1

Makoto Inaba,2 Kyoko Nagashima, Shigeru Tsukagoshi, and Yoshio Sakurai

Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Kami-lkebukuro, Toshima-ku, Tokyo 170, Japan

ABSTRACT MATERIALS AND METHODS

Neplanocin A, a novel antitumor antibiotic, was investigatedto determine the biochemical mode(s) of its cytotoxic action. Themolecule is an adenosine analogue with a unique cyclopentanestructure in its ribose moiety. Both sublines of L1210 and P388leukemia resistant to neplanocin A were cross-resistant in vitroto bredinin and 9-/3-o-arabinofuranosyladenine, which have been

reported to be activated by adenosine kinase. The adenosinekinase activity was markedly reduced in the resistant sublinesas compared with that of the respective sensitive lines. Furthermore, neplanocin A competitively inhibited the phosphorylationreaction of adenosine in a cell-free system. The results indicate

that neplanocin A is activated by adenosine kinase. Regardingthe target site for neplanocin A, the antibiotic suppressed RNAsynthesis to a significantly greater extent than DMA synthesis.This RNA-preferential effect is unique among common antimet-

abolic antitumor agents.

INTRODUCTION

Neplanocin A is an antibiotic produced by the actinomyceteAmpullariella regularis A11079 (1) and possesses antitumor activity against murine leukemias such as L1210 and P388 (2). Inaddition, NPA3 exhibits significant antitumor activity on the

CD8F, mammary tumor, but is inactive against B16 melanomaand Lewis lung carcinoma.4 Despite the remarkable antitumor

activities of this drug, the mode of its cytotoxic action remainsunclear.

As shown in Fig. 1, NPA is an analogue of adenosine possessing a characteristic cyclopentane structure in its ribosemoiety (3). It is of interest to know what particular biochemicalfeature(s) this unique chemical structure confers upon this drug.In this regard, it has been reported that aristeromycin has acyclopentane structure instead of ribose and is phosphorylatedinto its nucleotide form by adenosine kinase, thereby inhibitingnot only an early step of de novo purine synthesis but also GMPkinase (4, 5). It is of interest to determine whether the 5'-

hydroxymethyl group attached to the double bond of cyclopentane can also be phosphorylated. The present paper describesstudies on the mode of activation and the major target site(s)responsible for the cytotoxic action of NPA.

Received 7/19/84; revised 7/24/85,11 /1 /85.; accepted 11/6/85.' Supported in part by Grants-in-Aid for Cancer Research from the Ministry of

Education, Science, and Culture, Japan.2To whom requests for reprints should be addressed.3The abbreviations used are: NPA, neplanocin A; dCF, 2'-deoxycoformycin;

ara-A, 9-ii-D-arabinofuranosyladenine; ara-C, 1-0-D-arabinofuranosylcytosine; 5-FUra, 5-fluorouracil; MTX, methotrexate; 6-MP, 6-mercaptopurine; L1210/NPA,L1210 cells resistant to neplanocin A.

4 M. Tsujino, S. Yaginuma, T. Fujii, K. Hayano, T. Matsuda, T. Watanabe, and

J. Abe, unpublished data.

Chemicals. Neplanocin A, bredinin, and 2'-deoxycoformycin kindly

were supplied by Toyo Jozo Co., Ltd. (Shizuoka, Japan); and 1-/3-D-

arabinofuranosylcytosine, by Asahi Chemical Industry Co., Ltd. (Tokyo,Japan). 5-Fluorouracil, 6-mercaptopurine, and 9-/3-o-arabinofuranosylad-

enine were purchased from Sigma Chemical Co. (St. Louis, MO), andmethotrexate from Lederle Japan, Ltd. (Tokyo, Japan). The radioactivecompounds [mef/jy/-3H]thymidine (43 Ci/mmol), deoxy[5-3H]cytidine (22Ci/mmol), and [5,6-3H]uridine (40 Ci/mmol) were purchased from Amer-sham International, pic (Amersham, England), and [2,8-3H]adenosine

(32.2 Ci/mmol) from New England Nuclear (Boston, MA).Tumors. Original L1210 and P388 leukemic lines were supplied by

the Mammalian Genetics and Animal Production Section, Division ofCancer Treatment, National Cancer Institute, NIH, Bethesda, MD. EachP388 subline resistant to 5-FUra, 6-MP, or MTX was developed in our

laboratory by an in vivo procedure (6). L1210 and P388 sublines resistantto NPA were developed recently for the present study by inoculatingmice with 2 x 106 L1210 or P388 leukemia cells and treating the animals

with NPA (3 mg/kg) daily for 5 days in each transplant generation.Complete resistance in vivo to such treatment was acquired on the thirdor fourth transplant generation. The resistance phenotype of somesubclones was stable even in the absence of NPA. Original cell lines andall drug-resistant sublines were passaged weekly through DBA/2 and

BALB/c x DBA/2 (CDF,) mice (Charles River Japan, Inc., Atsugi, Japan),respectively.

In Vitro Culture. RPMI 1640 (M. A. Byproducts, Walkersville, MD)supplemented with 10% fetal bovine serum (M. A. Byproducts), 10 ÕIM2-hydroxyethyldisulfide (Aldrich Chemical Co., Inc., Milwaukee, Wl), and

kanamycin, 100 /<g/rnl (Meiji Seika Kaisha, Ltd., Tokyo, Japan) was usedas culture medium. Cells were harvested from tumor-bearing mice 5 to

6 days after transplantation and suspended in the culture medium to aconcentration of 5 x 104 cells/ml. Various'concentrations of the drug

were added to the culture medium throughout the 48-h culture period.

For those cells exposed to the drug for 3 h, cell suspensions were dilutedwith 4 ml complete medium after incubation with the drug for 3 h andcentrifuged at 420 x g for 5 min. The cells were resuspended in freshmedium and cultured in a CO2 incubator at 37°Cfor 48 h. The number

of cells was counted in a Model ZBI Coulter Counter after a 5-min

incubation with 0.25% trypsin to disaggregate the cells.Assay of Adenosine Kinase. Adenosine kinase activity was assayed

according to the procedure of Rabin and Gottesman (7). Approximately108 cells harvested from the i.p. cavity of mice were suspended in 1 ml

of 20 mM sodium phosphate (pH 6.5) containing 0.5% Triton X-100,

stirred, and centrifuged at 20,000 x g for 30 min. The supernatant wasused as the enzyme source, and its protein content was determined bythe method of Lowry ef al. (8) with bovine serum albumin solution as astandard. The reaction mixture (80 ¡A)contained 50 HIM sodium phosphate (pH 6.5), 2.5 x 10~3 M ATP, 2.5 x 10"" M MgCI2, 2.5 x 10~5 M

[3H]adenosine (0.5 ^Ci), and cell-free extract. Unless otherwise stated,2.5 x 10~6 M 2'-deoxycoformycin was also present. The reaction was

carried out at 37°Cfor predetermined periods of time (usually up to 5min) and stopped by adding 1 x 1CT2M lanthanum chloride. The products

were collected on Whatman GF/C filters and counted with 10 ml Aquasol(New England Nuclear) in a Beckmann Model LS-355 scintillation counter.

The velocity of AMP production was determined by plotting the linear

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MODE OF ACTION OF NPA

relationship of AMP formed versus time of incubation.RNA and DNA Synthesis. Cells (2 x 105) were suspended in 1 ml

culture medium containing a given concentration of the various antitumordrugs and cultured in a CO2 incubator. For cultures containing NPA, ara-C, or 6-MP, 50 ¡Aof [5,6-3H]uridine (2 ^Ci), or [metf>y/-3H]thymidine (1

pC\) were added at predetermined times and incubated for pulse labeling.For cultures containing 5-FUra or MIX, 50 n\ of [5,6-3H]uridine or[5-3H]deoxycytidine (1 /¿Ci)were added. After exposure to the radiolabelfor 30 min the cells were immediately centrifuged at 1200 x g at 4°Cfor

5 min and suspended in 5 ml of 10% cold trichloroacetic acid solution.Trichloroacetic acid-insoluble fractions were collected on Whatman GF/

C filters and washed with both 5% cold trichloroacetic acid and salinesolution. After filters were kept in 1 ml of Protosol (New England Nuclear)overnight at room temperature, the radioactivity was counted with 10 mlof Econofluor (New England Nuclear) in a liquid scintillation counter.

RESULTS

To understand the mode of the cytotoxic action of NPA, NPA-

resistant cells were examined for resistance in vitro to bredininand ara-A. As illustrated in Fig. 2, the resistant cells had cross-resistance to both bredinin and ara-A. The degree of cross-

resistance to bredinin was almost equivalent to that of resistanceto NPA, whereas cross-resistance to ara-A was appreciablylower. Cross-resistance to bredinin and ara-A was examined also

in P388 leukemic cells sensitive and resistant to NPA, respectively, with nearly identical results (data not shown).

The slope of the dose-response curve of cells exposed to NPA

was significantly different from the slopes obtained when thecells were exposed to bredinin or ara-A. When growth inhibition

induced by these drugs was compared at the level of thatconcentration that inhibited 50% of in vitro cell growth for theoriginal sensitive cells, NPA was effective at a much lowerconcentration than bredinin or ara-A. In comparison with the

concentrations necessary for complete growth inhibition, however, NPA and bredinin possessed approximately the samepotency, whereas ara-A required a 10-fold greater concentration.

Using the same amount of supernatant protein (200 ¿¿9),adenosine kinase activities were compared in the presence orabsence of dCF between NPA-sensitive and -resistant lines ofboth L1210 and P388 leukemias (Fig. 3). The activity of adeno-

100

OH OH

50

0 \-lf-t-0 10

100

50

"o.

100

50

O IO'7IO'«IO"5IO'4

Bredinin(M)

' 10'°10''10'°10

NeplanocinA (M)

10"" 10"oM/<-i 1 1

O IO"7 IO"* IO'5 IO"4

ara-A (M)

Fig. 2. In vivo sensitivity of neplanocin A-sensitive and -resistant L1210 cells toneplanocin A, bredinin, and ara-A. Cells were cultured with various concentrationsof NPA (A), bredinin (B). or ara-A (C) for 48 h in a CO2 incubator, and number of

cells was determined by a Coulter Counter. •.original L1210; O, L1210/NPA.

300

O"..o

1234

Reaction time (rain)

1 2 3Reaction tint

Fig. 1. Chemical structure of neplanocin A.

Fig. 3. Adenosine kinase activities in L1210 (A) and P388 (B) leukemic cellssensitive or resistant to neplanocin A. The 20.000 x g supernatant (200 i¿gprotein)of NPA-sensitive (•,O) or -resistant :•D) of L1210 and P388 leukemia and 2.5x 10~5 M [2,8-3H]adenosine were used as enzyme source and substrate, respec

tively. The reaction was carried out in the presence (•,•)or absence (O, D) of 2.5x 10~" M dCF. For details of preparation, see "Materials and Methods".

sine kinase was markedly increased by the addition of dCF, aninhibitor of adenosine deaminase. Its potentiating activity seemedto be much greater in the parent cells of both leukemias ascompared to the respective NPA-resistant cells. In either the

presence or absence of dCF, however, the activity of adenosinekinase was remarkably reduced in the resistant cells as compared with that of the original sensitive cells.

Kinetic parameters of the adenosine kinase activity of crudeextracts from the sensitive and resistant cells were also compared, as shown in Table 1. In NPA-resistant cells of both L1210

and P388, one order lower Vmaxwas observed, while the Kmsuggested that affinity of the enzyme to its substrate wassomewhat higher in the resistant cells.

Because the above results indirectly support the hypothesisthat NPA is phosphorylated by adenosine kinase in these tumorcells, the competitive influence of NPA on the phosphorylationreaction of adenosine in a cell-free system was examined. Theresulting Lineweaver-Burk plot shown in Fig. 4 provides direct

evidence that NPA is a competitive inhibitor of adenosine kinase;

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MODE OF ACTION OF NPA

Table 1

Kinetic parameters of adenosine kinase activities in L1210 and P388 leukemiccells sensitive or resistant to neplanocin A

The 20,000 x g supernatant (200 >ig as protein) of each of four cell lines and2.5 x 10~5 M [2,8-3H]adenosine were used as enzyme source and substrate,

respectively. The initial velocities of adenosine kinase activity were measured inthe presence of 2.5 x 10~* M dCF. According to LJneweaver-Burk plots thus

obtained, these kinetic parameters were estimated.

L1210 P388

Kinetic parameter Sensitive Resistant Sensitive Resistant

Vâ„¢.(pmol AMP formed/ 492 30 688 30mg protein/min)

Km(x10-«M) 15.8 9.6 19.7 13.1

2ECL

I,£

I

1

Fig. 4. LJneweaver-Burk plot of adenosine kinase activity in the presence orabsence of neplanocin A. The 20,000 x g supernatant of the original L1210 cellscontaining 20 ng protein and 2.5 x 10"* to 2.5 x 10~5 M [2,8-3H]adenosine were

used as enzyme source and substrate, respectively. The reaction mixture including2.5 x IO"* M dCF was incubated at 37°Cfor 5 min without NPA (O) or with 2.5 x10-5MNPA(»).

NPA itself is phosphorylated by adenosine kinase. Under thesame experimental conditions with a supernatant fraction of thecell homogenate of L1210, the relative affinities of adenosinekinase for adenosine and NPA were measured. From Fig. 4, theKm for adenosine was estimated to be 1.6 x 10~5 M, whereasthe K¡for NPA was found to be 3.1 x 10~5 M according to the

Dixon plot shown in Fig. 5. These results show that at least inthis experimental system adenosine kinase has a relativelystronger affinity for its natural substrate, adenosine, than forNPA, although not to any great extent.

To determine the target site(s) for NPA, the effect of thisantibiotic on DNA and RNA synthesis was examined. To beginwith, a dose-response relationship was measured by pulse la

beling with radioactive thymidine or undine for the last 30 min ofa 3-h exposure to various concentrations of NPA. A significantRNA-selective inhibitory effect was observed, in which RNAsynthesis inhibition gradually increased as the concentration ofNPA increased from 10~7 to 10~5 M, reaching a maximum inhi

bition of 50% (Fig. QA). Less than 20% inhibition was the maximum observed for DNA synthesis, even after exposure of thecells to an NPA concentration of 10~5 M, which is the concentra

tion that inhibited 90% of in vitro cell growth for NPA after 3 hexposure. The same experiment was performed with L1210/NPA cells, in which inhibition of RNA synthesis was found to besignificantly reduced, as compared with that in the sensitive cells,and no inhibition of DNA synthesis was observed (Fig. 66).

10Neplanocin A ( x10

Fig. 5. Dixon plot of adenosine kinase. The 20,000 x g supernatant of originalL1210 cells containing 20 i*g protein and 10~5 M (•)or 5 x 10"* M (O) [2,8-3H]adenosine were used as enzyme source and substrate, respectively. Thereaction mixture including 2.5 x 10~* M dCF was incubated at 37"C for 5 min with0, 1 x 10-5, 2 x 10-5, 5 x IO"5, or 1 x 10"4 M NPA. V, nmol AMP formed/mg

protein/min.

IO'4

(H)

Fig. 6. Inhibitory effect of neplanocin A on DNA and RNA synthesis in L1210cells sensitive and resistant to neplanocin A. Cells were incubated with variousconcentrations of NPA at 37°Cin complete culture medium. After 2.5 h, [methyl-3H]thymidine (O) or [5,6-3H]uridine (•)was added and incubated for an additional

30 min for pulse labeling of DNA or RNA, respectively. For comparison, the drugconcentration for 50% inhibition of in vitro cell growth of NPA was approximately10~5 M when L1210 cells were exposed to NPA for 3 h and then cultured for 45 h

in drug-free medium. A, original L1210; B, L1210/NPA.

Changes in the DNA and RNA synthesis of L1210 cells exposed continuously to 10~5 M NPA were observed after pulse

labeling for 30 min with radioactive precursors at various timepoints. RNA synthesis inhibition increased and reached a maximum 12 h after the initiation of incubation with NPA. On theother hand, inhibition of DNA synthesis seemed to succeed thatof RNA synthesis for incubation periods up to 15 h (Fig. 7).

DISCUSSION

Because the use of resistant sublines often aids in the elucidation of the cytotoxic action of a given compound, we initiallyexamined NPA-resistant sublines of L1210 and P388 leukemiafor cross-resistance in vitro to bredinin and ara-A. Both com

pounds are known to be activated by adenosine kinase (9, 10).From its chemical structure, one would expect NPA to be phos-

phorylated by adenosine kinase. Our results indicate that thesesublines have cross-resistance to both bredinin and ara-A (Fig.2). The lower degree of cross-resistance to ara-A may be ex-

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MODE OF ACTION OF NPA

O 5 10 15

Incubation time (hr)

Fig. 7. Kinetics of DNA and RNA synthesis inhibition by neplanocin A in L1210cells. Cells were incubated without or with 10~5 M NPA at 37°Cin complete culture

medium. DNA or RNA synthesis was measured every 3 h by adding [methyl-3H]thymidine (O) or [5,6-3H]uridine (•),respectively, 30 min before the end of each

observation period.

50

'S 0\-tt-L

\r-v

0 10"°10"310

5-FU (M)

O IO" 10"' IO"

6-MP (M)

Fig. 8. Comparison of DNA and RNA synthesis inhibition by typical antimetabolicantitumor agents in L1210 cells. Cells were incubated with various concentrationsof ara-C (A), MTX (8), 5-FUra (5-FU) (C), or 6-MP (D) at 37°Cin complete culturemedium. After 2.5 h [mett>y/-3H]thymidine or [5-3H]deoxycytidine in the case ofMTX and 5-FUra (O) or [5,6-3H]uridine (•)was added and the cells were incubated

for an additional 30 min for pulse labeling of DNA and RNA, respectively. Forcomparison, the values for the drug concentration for 90% inhibition of in vitro cellgrowth were approximately 3 x 10"*, 2 x 10"*, 3 x 10~5, and 3 x 10~3 M for ara-

C, MTX, 5-FUra, and 6-MP, respectively, when L1210 cells were exposed to eachof these agents for 3 h and then cultured for 45 h in drug-free medium.

plained by the fact that not only adenosine kinase but alsodeoxycytidine kinase is involved in the phosphorylation of ara-A

(9). In subsequent experiments we found that the adenosinekinase activity of the resistant cells was significantly decreasedas compared with that of sensitive cells in both L1210 and P388systems (Fig. 2; Table 1). These findings strongly suggest thatNPA is activated by adenosine kinase, regardless of the cyclo-

pentane structure in its ribose moiety.The competitive action of NPA on the phosphorylation reaction

of adenosine in a cell-free system provides direct evidence foran activation mode of NPA. Comparison of the Kmfor adenosineand the K, for NPA revealed that adenosine kinase has a slightlylower affinity for NPA rather than for its natural substrate, adenosine (Figs. 4 and 5), although the Kms are significantly higher

than the authentic value (11), probably because of the use ofcell-free extracts as an enzyme source. Very recently, Saunders

et al. (12) reported that Chinese hamster ovary cells deficient inadenosine kinase failed to phosphorylate NPA.

Initially, it seemed very difficult to identify the activation enzymeof NPA because adenosine was not able to reverse the growth-

inhibitory action of NPA on L1210 cells grown in vitro. Thepresence of 1(T4 M adenosine did not prevent the growth-inhibitory effect on cells by 10~5 M NPA in the presence of 10~4

M dCF (data not shown). Yoshida ef al. (13) also found similarresults after culturing L5178Y cells in vitro. On the other hand,Sakaguchi et al. (14) and Koyama and Tsuji (10) failed to reversethe cytotoxic action of bredinin, which has been reported to beactivated by adenosine kinase, by addition of adenosine in theculture medium for L5178Y and FM3A cells, respectively. Theseresults suggest general difficulty in reversing the cytotoxic actionof adenosine kinase-activated drugs by exogenous adenosine.

This difficulty may be the result of limited intracellular utilizationof exogenous adenosine as compared with other bases ornucleosides, probably due to the abundant endogenous adenosine pool. In spite of the absence of any reversal effect byadenosine, however, our findings indicate that adenosine kinaseis the activation enzyme of NPA. Identification of an assumedactive metabolite of NPA on a chromatogram is difficult stillbecause of the unavailability of radioactive NPA.

In general, the nature of the activation enzyme of a givencompound is integrally associated with the pattern of cross-resistance. Most probably cross-resistance occurs as a result of

the sharing of activation and/or degradation enzymes betweendrugs. In terms of its activation enzyme, NPA appears to beunique among currently available antitumor agents because thereis no major clinically available antitumor drug activated by adenosine kinase. Interestingly, P388 leukemia cells resistant to 5-FUra, ara-C, 6-MP, or MTX are not cross-resistant to NPA either

in vitro or in vivo (data not shown).In terms of the target site(s) for the action on NPA, it has been

recently reported that NPA is metabolized to S-neplanocylme-thionine, which inhibits S-adenosylhomocysteine hydrolase (15,

16). In addition, the same workers showed that NPA was apotent inhibitor of vaccinia virus multiplication in cultured mouseL-cells. At a concentration of 10~6 M, NPA induced almost

complete inhibition of both plaque formation of the virus andhydrolase activity in the infected L-cells but only relatively mildinhibition of growth in the L-cells themselves. These resultssuggest that the inhibitory effect of NPA on S-adenosylhomo

cysteine hydrolase is not the primary mechanism by which thisantibiotic exerts toxicity against mammalian cells. In this connection, another group also found that NPA specifically inhibitedRNA methylation because S-neplanocylmethionine was a poor

methyl donor for RNA methyltransferase (17).In our preliminary study on the target site for NPA, we found

that NPA selectively inhibited RNA synthesis (Fig. 6). Althoughthe maximal inhibition of the rate of RNA synthesis was 50 to60% even at 10~5 to 10~4 M NPA and incubation periods up to 3

h, prolongation of the exposure time caused almost completeinhibition of not only RNA but also of DNA synthesis (Fig. 7). Forcomparison, we examined the relative inhibitory activities ofseveral typical antimetabolic antitumor agents against RNA andDNA synthesis. With ara-C and MTX, a DNA-specific inhibitionwas observed. On the other hand, 5-FUra exerted a more potent

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MODE OF ACTION OF NPA

inhibitory effect on synthesis of DMA rather than RNA, and 6-MP

suppressed both syntheses similarly (Fig. 8). Comparing theactions of NPA with each of these antitumor agents, we find theRNA-preferential action of NPA to be unique. We speculate thatNPA is a poor substrate for ribonucleotide reducíasebecause ofits characteristic cyclopentane structure. If this proves to be thecase, the chemical uniqueness of the ribose moiety may provideNPA with distinctive biochemical features. The precise nature ofthe inhibitory action of NPA on RNA synthesis remains to be

determined.

REFERENCES

1. Yaginuma, S., Muto, N., Tsujino, M., Sudate, Y., Hayashi, M., and Otani, M.Studies on Neplanocin A, new antitumor antibiotic. I. Producing organism,isolation and characterization. J. Antibiot. (Tokyo),34: 359-366,1981.

2. Tsujino, M., Yaginuma, S., Fujii, T., Hayano, K., Matsuda, T., Watanabe, T.,and Abe, J. Neplanocins,new antitumor agents: biological activities. In: J. 0.Nelson and C. Grassi (eds.), Current Chemotherapy and Infectious Disease,Proceedingsof the 11th InternationalCongressof Chemotherapy(October 1-5,1979, Boston, MA),pp. 1559-1561. Washington,DC:The AmericanSocietyfor Microbiology, 1980.

3. Hayashi,M., Yaginuma, S., Yoshioka, H., and Nakatsu, K. Studies on neplan-ocin A, new antitumor antibiotic. II. Structure determination. J. Antibiot. (Tokyo), 34: 675-680, 1981.

4. Bennett, L. L, Jr., Allan,P. W., and Hill,D. L. Metabolicstudieswith carbocyclicanalogs of purine nucleosides. Mol. Pharmacol.,4: 208-217,1968.

5. Hill, D. L., Straight, S., Allan, P. W., and Bennett, L. L., Jr. Inhibitionof guaninemetabolism of mammaliantumor cells by the carbocyclic analogue of adeno-sine. Mol. Pharmacol.,7: 375-380,1971.

6. Inaba, M., Fujikura, R., and Sakurai, Y. Comparative study on in vivo development of resistanceto various classesof antitumor agents ¡nP388 leukemia.Gann, 70:607-613,1979.

7. Rabin, M. S., and Gottesman, M. M. High frequency of mutation to tubercidinresistance in CHO cells. Somatic Cell Genet., 5: 571-583,1979.

8. Lowry, O. H., Rosebrough, N. J., Fair, A. L., and Randall, R. J. Proteinmeasurement with the Folin phenol reagent. J. Biol. Chem., 793: 265-275,1951.

9. Brockman, R. W., Cheng, Y-C., Schabel, F. M., Jr., and Montgomery, J. A.Metabolism and chemotherapeutic activity of 9-n-arabinofuranosyl-2-tluoroa-denine against murine leukemia L1210 and evidence for its phosphorylationby deoxycytidine kinase. Cancer Res., 40: 3610-3615,1980.

10. Koyama, H., and Tsuji, M. Genetic and biochemicalstudies on the activationand cytotoxic mechanismof bredinin,a potent inhibitor of purme biosynthesisin mammaliancells. Biochem. Pharmacol.,32: 3547-3553,1983.

11. Chang, C-H., Brockman, R. W., and Bennett, L. L., Jr. Adenosinekinase fromL1210 cells—purificationand some properties of the enzyme. J. Biol. Chem.,255. 2366-2371,1980.

12. Saunders, P. P., Tan M-T., and Robins, R. K. Metabolism and action ofneplanocinA in Chinesehamster ovary cells. Biochem. Pharmacol.,34: 2749-2754, 1985.

13. Yoshida, M., Hoshi, A., and Kuretani, K. Effect of Neplanocin A and itsderivatives on the growth of L5178Y cells. Proceedings of the JapaneseCancer Association, 41st Annual Meeting, No. 794,1982.

14. Sakaguchi,K., Tsujino, M., Yoshizawa,M., Mizuno, K., and Hayano,K. Actionof bredininon mammaliancells. Cancer Res., 35:1643-1648,1975.

15. Keller, B. T., and Borchardt, R. T. Metabolic conversion of neplanocinA to S-neplanocylmethioninebymouseL929 cells. Biochem.Biophys. Res.Commun.,720: 131-137, 1984.

16. Borchardt, R. T., Keller, B. T., and Patel-Thombre,U. NeplanocinA. A potentinhibitor of S-adenosylhomocysteinehydrolase and of vaccinia virus multiplication in mouse L929 cells. J. Biol. Chem., 259: 4353-4358, 1984.

17. Glazer, R. I., and Knode, M. C. Neplanocin A—acyclopentenyl analog ofadenosinewith specificity for inhibiting RNA methylation. J. Biol. Chem., 259:12964-12969,1984.

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1986;46:1063-1067. Cancer Res   Makoto Inaba, Kyoko Nagashima, Shigeru Tsukagoshi, et al.   Leukemic CellsBiochemical Mode of Cytotoxic Action of Neplanocin A in L1210

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