adenine phosphoribosyltransferase deficiency in cultured...

[CANCER RESEARCH 42. 4210-4214. October 1982]0008-5472/82/0042-OOOOS02.00

Adenine Phosphoribosyltransferase Deficiency in Cultured Mouse MammaryTumor FM3A Cells Resistant to 4-Carbamoylimidazolium 5-Olate'

Hideki Koyama2 and Hiro-aki Kodama

Department of Biochemistry, Cancer Institute, Japanese Foundation for Cancer Research, Kami-lkebukuro 1-37-1. Toshima-ku, Tokyo 170 [H. Koy.j. andDepartment of Physiology, Tohoku Dental University, Koriyama, Fukushima-ken 963 [H. Kod.]. Japan

ABSTRACT

4-Carbamoylimidazolium 5-olate (CIO), the aglycone of thenucleoside antibiotic, bredinin (4-carbamoyl-1-ÃŸ-D-ribofurano-sylimidazolium 5-olate), exhibited potent cytotoxic effects onsubclonal line F28-7 of C3H mouse mammary carcinoma FM3A

cells in culture. We isolated 11 cell lines resistant to CIO fromwild-type F28-7 cells mutagenized with /V-methyl-A/'-nitro-N-nitrosoguanidine. These resistant (cior) lines were 160- to 400-

fold less sensitive to CIO than were the wild-type cells and

inherited the resistant phenotypes during subculture for morethan 3 months in the drug-free medium. They were cross-resistant to an adenine analog, 2,6-diaminopurine, while 2,6-diaminopurine-resistant (dapr) lines, isolated independently,were cross-resistant to CIO. Neither of the ciÃ²1lines tested

were able to form colonies in agar medium containing azaserineand adenine, nor were they able to incorporate tritiated adenineinto the macromolecular fraction, indicating that they could notutilize exogenous adenine for growth. Enzyme assays usingcell-free extracts revealed that all the cior lines had undetect-

able levels of adenine Phosphoribosyltransferase (EC 2.4.2.7)activity, but they, except one, had normal levels of hypoxan-thine-guanine Phosphoribosyltransferase (EC 2.4.2.8) and

adenosine kinase (EC 2.7.1.20) activities. These results demonstrate that the CIO resistance in these lines is attributed todeficient adenine Phosphoribosyltransferase activity and therefore that CIO is activated by adenine Phosphoribosyltransferase to form a cytotoxic nucleotide within the drug-sensitive

cells.

INTRODUCTION

Both bredinin and CIO3 have marked cytotoxic effects on

mouse L5178Y or other murine tumor cells in in vitro culturesbut are not effective in preventing their in vivo growth (13, 18).Recently, Yoshida et al. (23) reported 2 chemically synthesizedderivatives of bredinin that possessed a potent antitumor activity against a wide variety of transplantable tumors. Moreover,these derivatives and CIO were found to have a collateralactivity against 6-mercaptopurine-resistant P388 and L1210

cells (6).Sakaguchi et al. (17-19) investigated the mechanism of

cytotoxicity of bredinin and CIO and indicated that bredinin

' This study was supported in part by a Grant-in-Aid for Cancer Research from

the Ministry of Education. Science and Culture, Japan.2 To whom requests for reprints should be addressed.3 The abbreviations used are: CIO, 4-carbamoylimidazolium 5-olate; APRT,

adenine Phosphoribosyltransferase; MNNG, /V-methyl-A/'-nitro-N-nitrosoguani-dine; FBS, fetal bovine serum; DAP. 2,6-diaminopurine; PRPP, 5-phosphorylri-bosyl 1-pyrophosphate; ED50.drug dosage at which the cell number was reducedby 50%; TCA, trichloroacetic acid; HGPRT, hypoxanthine-guanine Phosphoribosyltransferase; AK, adenosine kinase; ciÃ²', carbamoylimidazolium 5-olate resistant; dap'. 2,6-diaminopurine resistant.

Received January 19. 1982; accepted July 9, 1982.

was incorporated into cells and, without being metabolized,blocked their de novo purine synthesis by inhibiting the enzymatic steps which catalyze the conversion of IMP to GMP.They also showed that, after being converted to bredinin withinthe cells, CIO exhibited the same cytotoxic actions. In contrast,Fukui et al. (5) suggested from their enzymatic studies that CIOwas altered to an active nucleotide by APRT and that thisnucleotide itself inhibited IMP dehydrogenase, thus interferingwith the production of GMP.

As an alternative approach to elucidate the activation andcytotoxicity mechanisms of CIO, we took advantage of themethodology of somatic cell genetics. We isolated 11 CIO-resistant cell lines from MNNG-mutagenized mouse FM3A cells

and studied their resistance mechanisms. Our data show thatthese resistant cell lines completely lack APRT activity andthus demonstrate that CIO is phosphoribosylated by the enzyme to the active cytotoxic nucleotide which would then attackIMP dehydrogenase and block the de novo synthesis of gua-

nine nucleotides.

MATERIALS AND METHODS

Culture Medium and Chemicals. For cell culture, a synthetic medium, designed by H. Koyama and designated ES medium, was obtained as a powdered form from Nissui Seiyaku Co., Tokyo, Japan. ESmedium consists of a modified autoclavable Eagle s minimal essentialmedium (22) enriched with 9 supplements: 0.2 mM concentrationseach of 7 nonessential amino acids (L-alanine, L-asparagine, u-asparticacid, L-glutamic acid, L-glycine, L-proline, and L-serine), 1 mM sodium

pyruvate, and vitamin B,2 (0.1 mg/liter). The antibiotic kanamycin (60mg/liter) was already included in it. Medium was routinely sterilized byautoclaving, except when filtration with 0.45-/im Sartorius membranefilters was used to prepare double-strength medium for agar plate

cultures. FBS was purchased from Grand Island Biological Co., GrandIsland, N. Y., and inactivated at 56Â° for 30 min before use. Dialyzed

FBS was prepared by dialyzing heat-inactivated FBS 3 times against

10 volumes of 0.9% NaCI solution and once against 10 volumes of ESmedium at 4Â°for 2 days and then by filtering as above. For colony

formation, agar medium which was composed of 95% ES medium,dialyzed 5% FBS, and 0.5 to 0.6% (w/v) agar [Special agar (Noble);Difco Laboratories, Detroit, Mich.] was prepared by the method ofKuroki(H).

Bredinin and CIO (SM-108) were kindly supplied by Sumitomo

Chemical Co., Ltd., Takarazuka, Japan. DAP hemisulfate, PRPP sodium salt, and DL-dithiothreitol were obtained from Sigma Chemical

Co., St. Louis, Mo.; adenine hydrochloride and ATP disodium salt wereobtained from Yamasa Shoyu Co., Ltd., Choshi, Japan; azaserine wasobtained from P-L Biochemicals, Inc., Milwaukee, Wis.; and MNNGwas obtained from Aldrich Chemical Co., Milwaukee, Wis. [2-3H]Ade-

nine (18 Ci/mmol) was obtained from The Radiochemical Centre,Amersham, England. [8-'4C]Adenine (55.6 mCi/mmol), [8-14C]adeno-sine (45.5 mCi/mmol), and [G-3H]hypoxanthine monohydrochloride

(3.8 Ci/mmol) were all purchased from New England Nuclear, Boston,Mass.

4210 CANCER RESEARCH VOL. 42

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APR T Deficiency in CIO-rÃ©sistant Mouse Tumor Cells

Cells and Culture Methods. The cell line used for wild-type cellswas subclonal line F28-7 of mouse mammary carcinoma FM3A cells(8). The 8-azaguanine-resistant FC-1 line (9) which had been isolatedfrom FM3A cells was also used. Both cell lines were maintained in 52-

mm plastic tissue culture dishes (Wako Pure Chemical Co., Ltd., Osaka,Japan) in 5 ml of ES medium supplemented with 2% fetal calf serum.They propagated in suspension with a population-doubling time ofabout 12 hr and reached saturation at a density of 2 x 106 cells/ml.

Colony formation was carried out by agar plate culturing proceduresas described by Kuroki (11 ). Cells were plated on 10 ml of agar mediumin 100-mm bacterial plastic dishes (Wako Pure Chemical Co., Ltd.) andcultured for 7 to 10 days. All cultures were incubated at 37Â° in an

atmosphere at 5 to 10% CO? in air saturated with water.Isolation of Drug-resistant Cell Lines. The standard methods for

the isolation of somatic cell mutants were described previously (8).Briefly, logarithmic-phase cells of the wild-type F28-7 line were treated

with MNNG (0.5 fig/ml) for 2 hr, washed once with normal medium,and subcultured for 5 to 6 days in normal medium for any mutations tobe expressed. The cells were then harvested and counted with a ModelD Coulter Counter. Four dishes (100 mm in diameter) were plated with2.5 to 5 x 105 cells on 10 ml of agar medium containing either 10~5or 5 x 10~5 M CIO as selective agent. In addition, four 100-mm dishes

were plated with 100 cells on the drug-free, nonselective agar medium

to determine their plating efficiencies. These cultures were incubatedfor 7 to 10 days, and the number of resulting colonies with more than50 cells was counted. The frequencies of resistant cells were definedas the number of ClO-resistant colonies observed on the selective

medium divided by the number of cells plated after correction by theplating efficiencies on the nonselective medium. Some of the resistantcolonies were transferred with bamboo skewers to 35-mm plastic

dishes (Lux Scientific Corp., Newbury Park, Calif.) containing 1.5 ml ofthe drug-free growth medium and subcultured as reported previously(8). As controls, F28-7 cells not treated with MNNG were processed in

the same way to see if there were spontaneously occurring mutantsresistant to CIO.

Similarly, DAP-resistant cell lines were selected by plating MNNG-treated wild-type cells on agar medium containing 10~" M DAP and

used for the present study. The details about isolation and characterization of these lines will be reported elsewhere.

Growth Inhibition Assay. Logarithmic-phase cells were harvested,counted, and plated in duplicate at 10" cells/35-mm dish in 2 ml of ES

medium containing 2% dialyzed FBS and varying concentrations of thedrug to be assayed. These cultures were incubated for 72 Â±2 hr andcounted. The number of cells in an experimental dish was plotted as apercentage of the number of cells in the control dish. The degree ofdrug sensitivity in each cell line was expressed by the ED50.

Incorporation of [3H]Adenine into the Macromolecular Fraction.Cells were plated and incubated overnight at 105 cells/ml in 2 ml of

growth medium in 35-mm plastic dishes. Duplicate cultures were thenexposed to [3H]adenine (1 /iCi/ml) for 3 hr. The cells were subsequently

transferred to 2 ml of ice-cold 10% TCA, allowed to stand for approx

imately 15 min, and filtered on Whatman GF/C filters, these filterswere washed 5 times with 5 ml of cold 5% TCA and once with absolutealcohol, air-dried, placed in 5 ml of toluene-based scintillation fluid,

and counted for radioactivity in a Beckmann Model L8500 P scintillationcounter. At the time of labeling, cell counts were carried out usingduplicate cultures in order to calculate, on a per cell basis, the incorporation of tritiated adenine into the TCA-insoluble macromolecular

fraction.Enzyme Assay. The activities of APRT and HGPRT in cell-free

extracts were determined by the method of Wahl et al. (21). Logarithmic-phase cells were harvested, washed 3 times with Ca?+- and Mg? +-

free Dulbecco s phosphate-buffered saline (4), and stored frozen at-20Â° until use. At the time of assay, the cell pellet was thawed and

suspended in 0.2 to 0.5 ml of a medium composed of 10 rriM Tris-HCI(pH 7.4), 10 rriM MgCI?, 30 mM KCI, 1 mM DL-dithiothreitol, and 0.5%Triton X-100. After an occasional stirring with a vortex mixer for 20

min and centrifugation at 20,000 x g for 30 min, the supernatant wasused for both enzyme assays. Protein was determined by the methodof Lowry et al. (12) using bovine serum albumin as a standard.

The reaction mixture (50 /il) for the APRT assay contained: 50 mMTris-HCI (pH 7.4); 5 mM MgCI2; 0.1 mM EDTA; 1 mM PRPP; bovineserum albumin (2 mg/ml); 0.1 mM ['"CJadenine (0.28 /iCi); and cell

extract (2 to 10 fig of protein). For the HGPRT assay, the radioactiveadenine was replaced with 5 x 10~6 M [3H]hypoxanthine monohydro-

chloride (0.73 /Â¿Ci),and cell extract (1 to 5 fig of protein) was added toeach assay tube. The reaction was preincubated for 2 min at 37Â°,

started by addition of the cell extract, and continued for 15 or 30 min.Then, the reaction was stopped by adding 1 ml of 1.5 mM EDTA in 10mM Tris-HCI (pH 7.4). The reaction products were collected on Whatman DE81 filter papers and washed with 10 mM Tris-HCI (pH 7.4). The

filters were placed in vials containing 0.5 ml of 3% NaCI solution andcounted for radioactivity with 5 ml of toluene-Triton X-100-based

scintillation fluid as described above.The products were identified by the method of Jones and Sargent

(7). For this, the reaction was terminated by adding 20 n\ of 0.2 MEDTA (pH 7.4) to each assay tube and chilling the tubes in ice. Four-Â¡j.\aliquots were spotted on cellulose thin-layer chromatography plates

(20 x 20 cm; Merck, Darmstadt, West Germany) with 0.02 fimol ofnonradioactive bases, nucleosides, and nucleotides as markers. Ascending chromatography was carried out for about 1.5 hr at roomtemperature either in 1 M ammonium acetate for the APRT assay or in5% Na2HPO4 for the HGPRT assay. The plates were dried, and themarker spots were located under a UV lamp, scraped, placed in vialscontaining 0.5 ml of distilled water, and counted for radioactivity asabove. This analysis revealed that more than 98% of the products byAPRT were found in the AMP marker spot while more than 96% ofthose by HGPRT were in the IMP marker spot.

AK activity was assayed by the method of Rabin and Gottesman(15). Cell pellets were suspended in 0.25 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 served as the enzymesource. Protein concentration was determined as described above.The reaction mixture (80 ill) contained: 50 mM sodium phosphate (pH6.5); 2.5 mM ATP; 0.25 mM MgCb; 2.5 x 10~" M [I4C]adenosine (0.45

iiCi); and cell-free extract (10 to 50 Â¿igof protein). The reaction wascarried out at 37Â° for 15 to 30 min and stopped by adding 0.1 M

lanthanum chloride. The products were collected on Whatman GF/Cfilters and counted with toluene scintillator as above.

Identification of the products was performed by immersing the incubated reaction mixture for 2 min into a boiling-water bath and chillingit in ice. Four-jul aliquots from each tube were spotted onto cellulose

plates, chromatographed (ascending) in distilled water as a solvent (1),and analyzed as described above. The radioactivity of the AMP markerspot accounted for nearly one-third of that found in the products.However, when 2.5 to 5.0 x 10~6 M coformycin, a potent adenosine

deaminase inhibitor (3), was added to the reaction mixture, the radioactivity of the inosine plus hypoxanthine spots was reduced to zerowhile the amount of AMP produced was not affected. These resultsindicate that adenosine deaminase activity existed in our cell-free

extracts but that it did not interfere with the adenosine kinase assay.The enzyme assays for APRT, HGPRT, and AK were linear with

protein concentration and incubation time for 40 min under all conditions used. One unit of enzyme was defined as the amount of enzymewhich yielded 1 nmol of the reaction products per min from theradioactive substrates. Specific activity (nmol/min/mg protein) wasalso calculated by dividing the enzyme units by the protein content ofthe extracts used for assay.

RESULTS

Selection of ClO-resistant Lines. Table 1 summarizes thefrequency of resistant colonies which appeared on agar plates

OCTOBER 1982 4211



H. Koyama and H. Kodama

Table 1Frequency of ClO-resistant cells in MNNG-treated and untreated wild-type cells

Treatment8Without

MNNGWith MNNGNo.

of cellsassayed1.8

x 10'7.2 x 10e4.6 X 106Concentra

tion ofCIO(M)io-51CT5

5 X IO'5No.

of resistant col

onies0

15428Frequency"<5.6

x 10~82.1 x 1CTS6.8 X 10~6

a Wild-type F28-7 cells were treated with or without MNNG (0.5 fig/ml) for 2

hr and assayed for the frequency of colonies resistant to CIO as described in"Materials and Methods.'

6 Defined in the text.

in the selective medium containing CIO. There were no resistantcolonies found in a control population of 1.8 x 107 wild-type

cells not treated with MNNG, showing that the frequency ofspontaneous mutations to CIO resistance was quite low (<5.6x 10~8). However, in the mutagen-treated population, resistantcolonies appeared at a frequency of as high as 2.1 x 10~5and 6.8 x 10~6 on agar plates containing 1CT5 and 5 x 10~5

M CIO, respectively. Five colonies from the former plates and6 from the latter were picked, transferred to drug-free medium,and established as ClO-resistant cell lines. These lines weredesignated as cior 1 to 11. They were subcultured for over 3

months (about 180 generations, with a doubling time of approximately 12 hr) in nonselective medium, over which time theClO-resistant phenotype was stably inherited.

Resistance to CIO. We studied the cytotoxic effects of CIOon the growth of wild-type F28-7, FC-1, 2 ciÃ²' cell lines and 2dapr cell lines as a function of CIO concentration. As shown in

Chart 1, both F28-7 and FC-1 cells were very sensitive to CIO

and exhibited similar growth inhibtion curves with ED50 valuesof about 8 X 1CT7 M. On the other hand, cior3 and cio'8 lines

were much less sensitive than the above lines, and this wasalso the case for dap'5 and dapr28 lines. The ED50 values forthese resistant lines ranged from 1.3 x 10"" to 3.2 x 10~4

M, indicating that they were 160- to 400-fold more resistantthan the wild-type F28-7 cells. In addition, the data clearlyshow that the dap' lines were cross-resistant to CIO.

A similar result was obtained by testing the colony-formingability of these lines on agar plates containing 10~5 M CIO. Asshown in Table 2, 3 cior cell lines all grew and gave rise to

colonies either in the presence or absence (no addition) of thedrug with plating efficiencies comparable to that found in thewild-type cells.

We next examined whether or not cior lines showed cross-

resistance to DAP. These results are illustrated in Chart 2. Thegrowth of F28-7 and FC-1 cells was reduced to 50% in mediumcontaining 8.8 x 10~6 and 1.3 x 10~5 M DAP, respectively. Incontrast, both cior and dap' lines were 27- to 40-fold more

resistant to the adenine analog than were the wild-type F28-7cells. It is therefore evident that the ciÃ²' lines have cross-

resistance to DAP. These results suggest that ClO-resistantand DAP-resistant cells share the same resistance mechanism.DAP-resistant Chinese hamster (2, 20) or human cells (16) areknown to be defective in APRT activity. Thus, the present ciÃ²'as well as dap' mouse cells would be expected to lack the

enzyme activity.CIO is the aglycone of the antibiotic bredinin. We checked

whether these cell lines were resistant to bredinin. As shown inTable 3, 2 ciÃ²' and 2 dap' lines were less than 2-fold more

resistant to it than were the wild-type F28-7 cells, indicating no

cross-resistance of the mutants to the antibiotic. This finding

suggests that CIO and bredinin are activated by different mechanisms.

Utilization of Exogenous Adenine. We studied whether ciÃ²'

lines could utilize exogenously added adenine by plating andculturing them on agar medium containing 2 x 10~5 M azaser-ine and 10~4 M adenine (AA plate). Since azaserine inhibits

purine synthesis (14), only cells capable of phosphoribosylat-

100-

o

1o 50

10" 10' to'"

CIO(M)

io- io'

Chart 1. Effect of CIO on the growth of wild-type F28-7, FC-1, ciÃ²', and dap'

cell lines. Cells were cultured for 72 Â± 2 hr in medium containing varyingconcentrations of CIO and counted as described in "Materials and Methods."For each point, duplicate cultures were used. O, F28-7; * FC-1; G, cio'3; â€¢,cio'8; A, dap'5; A, dap'28.

Table 2Colony-forming ability of wild-type, do', and dap' cell lines on different agar

media

Three dishes (100 mm) were plated with 100 cells of each line on 10 ml ofagar medium containing CIO or azaserine plus adenine and cultured for 8 days.

Relative plating efficiency"

CelllineF28-7cio'3cio'6cio'8dap'28Noaddition1.00C0.880.930.971.04CIO(105M)00.960.900.880.97AA61.040000

Plating efficiencies relative to that observed for wild-type F28-7 cells(average of 2 determinations).

b AA. azaserine. 2 x 10~5 M; and adenine, 10~4 M.c The wild-type cells showed 98 colonies/dish.

100

oÂ°I 50

10' io- io" 10"'

DAP(M)

Chart 2. Effect of DAP on the growth of wild-type F28-7, FC-1, ciÃ²', and dap'

cell lines. The assay procedures and symbols were as described in the legend toChart 1.




APRT Deficiency in ClO-resistant Mouse Tumor Cells

ing adenine by the action of APRT would multiply on the AAplates. As shown in Table 2, column 4, 3 ciÃ²' lines tested failed

to give colonies. This indicates that they were not able to utilizeexogenous adenine. dap'28 cells also revealed the same

growth property.These results were further confirmed by testing the ability of

ciÃ²' lines to incorporate tritiated adenine into the 5% TCA-insoluble cell fraction (Table 4). cio'3, cio'4, and cio'10 cellsexhibited greatly reduced levels of [3H]adenine uptake, whilecior8 cells could incorporate it at a little higher rate, supporting

the above data that these mutant lines were unable to utilizeexogenous adenine. These results strongly suggest that CIOresistance may result from deficiency in APRT activity.

Lack of APRT Activity. Using cell-free extracts prepared

from 15 different cell lines, we assayed 3 kinds of enzymes,APRT, HGPRT, and AK, all of which might be involved in themetabolism of CIO. Table 5 summarizes the results which areexpressed as specific activities. Wild-type F28-7 and FC-1cells had high levels of APRT activity, whereas 11 cior mutants

showed undetectable amounts, or less than 1% of the activityfound in the F28-7 cell extract. In addition, the activity in dapr5cells was low (10% of the F28-7 cell activity), while dap'28

cells had no activity. Assay of mixtures of wild-type cell extractsand extracts from cior or dap' cell lines gave activity interme

diate between these 2 extracts alone, thus ruling out thepossibility of a diffusible APRT inhibitor (data not shown).

On the other hand, 10 of 11 ciÃ²' lines (except for cio'4) and2 dap' lines had levels of HGPRT activity similar to that of thewild-type F28-7 cells. The cio'4 line could result from a double

mutation. FC-1 cells totally lacked HGPRT because of 8-aza-

guanine resistance (9). Furthermore, all the cell lines listed inTable 5 possessed the same levels of AK activity.

These data demonstrate that the phenotype of CIO resistance in these cell lines results from a defect in APRT enzymeactivity and that CIO is activated by APRT to form a cytotoxicnucleotide. This nucleotide would probably block guanine nu-

Table 3Effect of bredinin on the growth of wild-type, do', and dap' cell lines

The assay procedures were as described in the legend to Chart 1.

CelllineF28-7FC-1cio'3cio'8dap'5dap'28EDMa(M)5.4

X10~4.3X10"8.8X10~8.8X10~1.0x10"1.0x 10~

Average of 2 determinations.

Table 4Ability of wild-type, do', and dap' cell lines to incorporate [2H]adenine into the

macromolecular fractionCells were labeled for 3 hr with [3H]adenine (1 Â¿iCi/ml), and the radioactivity

incorporated into the 5% TCA-insoluble macromolecular fraction was counted asdescribed in "Materials and Methods."

Cell line Incorporation of [3H]adeninea (%)

F28-7cio'3cio'4cio'8ciÃ²'10dap'28

1002.01.97.20.50.7

Table 5Enzyme activities in wild-type do', and dap' cell lines

The procedures for preparation of cell-free extracts and enzyme assays weredescribed in "Materials and Methods."

Specific activity (nmol/min/mg protein)

CelllineF28-7FC-1cio'1cio'2cio'3cio'4cio'5cio'6cio'7cio'8cio'9cio'10cio'1

1dap'5dap'28APRT2.3

Â±0.3a1.8

Â±0.10.01Â±0.0100.01

Â±0.010<0.010.02

Â±0.000.02Â±0.00<0.01<0.01<0.010.02

Â±0.020.22Â±0.02<0.01HGPRT1.0

Â±0.2<0.010.93

Â±0.241.0Â±0.00.95

Â±0.23<0.010.98

Â±0.151.2Â±0.30.95

Â±0.020.98Â±0.030.92Â±0.010.87Â±0.060.92Â±0.020.75Â±0.131.0Â±0.1AK3.0

Â±0.11.6Â±0.02.2Â±0.22.3Â±0.32.7Â±0.02.5Â±0.42.4Â±0.22.8Â±0.23.0Â±0.52.5Â±0.03.0Â±0.32.4Â±0.12.5Â±0.2ND62.3

Â±0.2

Expressed as a percentage of the activity found with wild-type F28-7 cells(average of 2 to 3 determinations).

Mean Â±S.D. of 2 to 3 determinations.0 ND. not determined.

cleotide production by inhibiting IMP dehydrogenase in CIO-

sensitive cells.

DISCUSSION

In this study, we isolated 11 ClO-resistant cell lines from

mouse FM3A cells mutagenized with MNNG. These lines were:(a) much less sensitive to CIO; (b) cross-resistant to DAP; (c)

unable to utilize exogenous adenine for growth; (d) hardly ableto incorporate tritiated adenine into the macromolecular fraction; and (e) deficient in APRT activity.

These lines of evidence demonstrate that the mechanism ofCIO resistance involves a deficiency in APRT enzyme activityand that CIO is activated by the enzyme to exert its cytotoxiceffects on cells. Since APRT catalyzes phosphoribosylation ofthe nitrogen atom at position 9 of adenine by PRPP to formAMP, CIO will be converted to 4-carbamoylimidazolium 5-olate-1-ribosyl-5'-monophosphate (bredinin 5'-monophosphate). In

earlier works, Sakaguchi ef al. (18) could not identify thisnucleotide in L5178Y cells cultured with 14C-labeled CIO or in

serum and urine of rats given the radioactive drug P.O.; instead,they found radioactive bredinin. Thus, they concluded thatbredinin was the activated form of CIO within the cell. Theyalso indicated, by studies on the reversal effects of many purinecompounds on CIO or bredinin cytotoxicity, that bredininblocked the de novo purine synthesis by preventing the conversion of IMP to GMP. However, Fukui ef al. (5) studied theactivation mechanism of CIO with cell-free extracts prepared

from Ehrlich ascites tumor cells and showed that CIO wasconverted to the above nucleotide form and that the chemicallysynthesized nucleotide was a stronger competitive inhibitor forIMP dehydrogenase than was either CIO or bredinin. Therefore,they suggested that the nucleotide was the active metaboliteproducing the cytotoxicity. Our present results support andextend their observations.

Unmutagenized populations of F28-7 cells gave no resistant

colonies, showing that the frequency of appearance of spontaneously resistant mutants is quite low (Table 1). Since theAPRT locus resides on chromosome 8 (10), the genes on bothchromosomes of this pair are ordinally functioning. Since theAPRT defect behaves as a recessive trait (7), the expression of

OCTOBER 1982 4213



H. Koyama and H. Kodama

CIO-rÃ©sistant phenotypes would require either a double muta

tion or a single mutation followed by some chromosomal rearrangement which would lead to the homozygous state of amutated APRT gene. This may be the reason for the lowfrequency of appearance of spontaneous mutants. This findingmay be favorable for CIO or its derivatives as cancer chemo-therapeutics.

In addition, our data indicate the usefulness of CIO for thefield of somatic cell genetics as an effective selecting agent forisolating APRT-deficient mutants from cultured mammalian

cells.

ACKNOWLEDGMENTS

We would like to thank Drs. M. Inaba and M. Fukui for their helpful suggestionsconcerning these studies.

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2. Chasin, L. A. Mutation affecting adenine phosphoribosyltransferase activityin Chinese hamster cells. Cell, 2: 37-41. 1974.

3. Debatisse, M., Berry, M., and Buttin, G. The potentiation of adenine toxicityto Chinese hamster cells by coformycin: suppression in mutants with alteredregulation of purine biosynthesis or increased adenylate deaminase activity.J. Cell. Physiol., 106: 1-11, 1981.

4. Dulbecco, R.. and Vogt, M. Plaque formation and isolation of pure lines withpoliomyelitis viruses. J. Exp. Med., 99: 167-182, 1954.

5. Fukui, M., Inaba, M., Tsukagoshi. S., and Sakurai, Y. New antitumor imidazolderivative, 5-carbamoyl-1H-imidazol-4-yl piperonylate. as an inhibitor ofpurine synthesis and its activation by adenine phosphoribosyltransferase.Cancer Res., 42: 1098-1102, 1982.

6. Inaba. M., Fukui, M., Yoshida, N., Tsukagoshi, S., and Sakurai. Y. Collateralsensitivity of 6-mercaptopurine-resistant sublines of P388 and L1210 leukemia to the new purine antagonists, 5-carbamoyl-1 H-imidazol-4-yl piperonylate and 4-carbamoylimidazolium 5-olate. Cancer Res., 42: 1103-1106,1982.

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1982;42:4210-4214. Cancer Res Hideki Koyama and Hiro-aki Kodama Cells Resistant to 4-Carbamoylimidazolium 5-OlateAdenine Phosphoribosyltransferase Deficiency Tumor FM3A

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