[CANCER RESEARCH 42. 4210-4214.
0008-5472/82/0042-OOOOS02.00
October
1982]
Adenine Phosphoribosyltransferase Deficiency in Cultured Mouse Mammary
Tumor 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
Department of Physiology, Tohoku Dental University, Koriyama, Fukushima-ken 963 [H. Kod.]. Japan
ABSTRACT
4-Carbamoylimidazolium
5-olate (CIO), the aglycone of the
nucleoside antibiotic, bredinin (4-carbamoyl-1-ß-D-ribofuranosylimidazolium 5-olate), exhibited potent cytotoxic effects on
subclonal line F28-7 of C3H mouse mammary carcinoma FM3A
cells in culture. We isolated 11 cell lines resistant to CIO from
wild-type F28-7 cells mutagenized with /V-methyl-A/'-nitro-Nnitrosoguanidine. These resistant (cior) lines were 160- to 400fold less sensitive to CIO than were the wild-type cells and
inherited the resistant phenotypes during subculture for more
than 3 months in the drug-free medium. They were crossresistant to an adenine analog, 2,6-diaminopurine, while 2,6diaminopurine-resistant
(dapr) lines, isolated independently,
were cross-resistant to CIO. Neither of the ciò1lines tested
were able to form colonies in agar medium containing azaserine
and adenine, nor were they able to incorporate tritiated adenine
into the macromolecular fraction, indicating that they could not
utilize exogenous adenine for growth. Enzyme assays using
cell-free extracts revealed that all the cior lines had undetectable levels of adenine Phosphoribosyltransferase
(EC 2.4.2.7)
activity, but they, except one, had normal levels of hypoxanthine-guanine
Phosphoribosyltransferase
(EC 2.4.2.8) and
adenosine kinase (EC 2.7.1.20) activities. These results dem
onstrate that the CIO resistance in these lines is attributed to
deficient adenine Phosphoribosyltransferase
activity and there
fore that CIO is activated by adenine Phosphoribosyltransfer
ase 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 cultures
but are not effective in preventing their in vivo growth (13, 18).
Recently, Yoshida et al. (23) reported 2 chemically synthesized
derivatives of bredinin that possessed a potent antitumor activ
ity against a wide variety of transplantable tumors. Moreover,
these derivatives and CIO were found to have a collateral
activity 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-nitrosoguanidine; FBS, fetal bovine serum; DAP. 2,6-diaminopurine;
PRPP, 5-phosphorylribosyl 1-pyrophosphate; ED50. drug dosage at which the cell number was reduced
by 50%; TCA, trichloroacetic acid; HGPRT, hypoxanthine-guanine
Phosphori
bosyltransferase; AK, adenosine kinase; ciò', carbamoylimidazolium
5-olate re
sistant; dap'. 2,6-diaminopurine resistant.
Received January 19. 1982; accepted July 9, 1982.
4210
1-37-1.
Toshima-ku,
Tokyo
170 [H. Koy.j.
and
was incorporated into cells and, without being metabolized,
blocked their de novo purine synthesis by inhibiting the enzy
matic steps which catalyze the conversion of IMP to GMP.
They also showed that, after being converted to bredinin within
the cells, CIO exhibited the same cytotoxic actions. In contrast,
Fukui et al. (5) suggested from their enzymatic studies that CIO
was altered to an active nucleotide by APRT and that this
nucleotide itself inhibited IMP dehydrogenase, thus interfering
with the production of GMP.
As an alternative approach to elucidate the activation and
cytotoxicity mechanisms of CIO, we took advantage of the
methodology of somatic cell genetics. We isolated 11 CIOresistant cell lines from MNNG-mutagenized mouse FM3A cells
and studied their resistance mechanisms. Our data show that
these resistant cell lines completely lack APRT activity and
thus demonstrate that CIO is phosphoribosylated
by the en
zyme to the active cytotoxic nucleotide which would then attack
IMP dehydrogenase and block the de novo synthesis of guanine nucleotides.
MATERIALS
AND METHODS
Culture Medium and Chemicals. For cell culture, a synthetic me
dium, designed by H. Koyama and designated ES medium, was ob
tained as a powdered form from Nissui Seiyaku Co., Tokyo, Japan. ES
medium consists of a modified autoclavable Eagle s minimal essential
medium (22) enriched with 9 supplements: 0.2 mM concentrations
each of 7 nonessential amino acids (L-alanine, L-asparagine, u-aspartic
acid, 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 (60
mg/liter) was already included in it. Medium was routinely sterilized by
autoclaving, except when filtration with 0.45-/im Sartorius membrane
filters was used to prepare double-strength
medium for agar plate
cultures. FBS was purchased from Grand Island Biological Co., Grand
Island, N. Y., and inactivated at 56° 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 ES
medium at 4°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 of
Kuroki(H).
Bredinin and CIO (SM-108) were kindly supplied by Sumitomo
Chemical Co., Ltd., Takarazuka, Japan. DAP hemisulfate, PRPP so
dium salt, and DL-dithiothreitol were obtained from Sigma Chemical
Co., St. Louis, Mo.; adenine hydrochloride and ATP disodium salt were
obtained from Yamasa Shoyu Co., Ltd., Choshi, Japan; azaserine was
obtained from P-L Biochemicals, Inc., Milwaukee, Wis.; and MNNG
was obtained from Aldrich Chemical Co., Milwaukee, Wis. [2-3H]Adenine (18 Ci/mmol) was obtained from The Radiochemical
Centre,
Amersham, England. [8-'4C]Adenine (55.6 mCi/mmol), [8-14C]adenosine (45.5 mCi/mmol), and [G-3H]hypoxanthine
monohydrochloride
(3.8 Ci/mmol)
Mass.
were all purchased from New England Nuclear, Boston,
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research.
VOL. 42
APR T Deficiency
Cells and Culture Methods. The cell line used for wild-type cells
was subclonal line F28-7 of mouse mammary carcinoma FM3A cells
(8). The 8-azaguanine-resistant
FC-1 line (9) which had been isolated
from FM3A cells was also used. Both cell lines were maintained in 52mm 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 of
about 12 hr and reached saturation at a density of 2 x 106 cells/ml.
Colony formation was carried out by agar plate culturing procedures
as described by Kuroki (11 ). Cells were plated on 10 ml of agar medium
in 100-mm bacterial plastic dishes (Wako Pure Chemical Co., Ltd.) and
cultured for 7 to 10 days. All cultures were incubated at 37° in an
in CIO-résistant Mouse
Tumor Cells
min and centrifugation at 20,000 x g for 30 min, the supernatant was
used for both enzyme assays. Protein was determined by the method
of Lowry et al. (12) using bovine serum albumin as a standard.
The reaction mixture (50 /il) for the APRT assay contained: 50 mM
Tris-HCI (pH 7.4); 5 mM MgCI2; 0.1 mM EDTA; 1 mM PRPP; bovine
serum 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 radioactive
adenine was replaced with 5 x 10~6 M [3H]hypoxanthine monohydrochloride (0.73 /¿Ci),and cell extract (1 to 5 fig of protein) was added to
each assay tube. The reaction was preincubated for 2 min at 37°,
atmosphere at 5 to 10% CO? in air saturated with water.
Isolation of Drug-resistant Cell Lines. The standard methods for
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 10
mM Tris-HCI (pH 7.4). The reaction products were collected on What
man DE81 filter papers and washed with 10 mM Tris-HCI (pH 7.4). The
the isolation of somatic cell mutants were described previously (8).
Briefly, logarithmic-phase cells of the wild-type F28-7 line were treated
filters were placed in vials containing 0.5 ml of 3% NaCI solution and
counted for radioactivity with 5 ml of toluene-Triton
X-100-based
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 to
be expressed. The cells were then harvested and counted with a Model
D Coulter Counter. Four dishes (100 mm in diameter) were plated with
2.5 to 5 x 105 cells on 10 ml of agar medium containing either 10~5
or 5 x 10~5 M CIO as selective agent. In addition, four 100-mm dishes
scintillation fluid as described above.
The products were identified by the method
(7). For this, the reaction was terminated by
EDTA (pH 7.4) to each assay tube and chilling
¡j.\
aliquots were spotted on cellulose thin-layer
were plated with 100 cells on the drug-free,
nonselective
agar medium
to determine their plating efficiencies. These cultures were incubated
for 7 to 10 days, and the number of resulting colonies with more than
50 cells was counted. The frequencies of resistant cells were defined
as the number of ClO-resistant colonies observed on the selective
medium divided by the number of cells plated after correction by the
plating efficiencies on the nonselective medium. Some of the resistant
colonies were transferred with bamboo skewers to 35-mm plastic
dishes (Lux Scientific Corp., Newbury Park, Calif.) containing 1.5 ml of
the 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 mutants
resistant to CIO.
Similarly, DAP-resistant cell lines were selected by plating MNNGtreated wild-type cells on agar medium containing 10~" M DAP and
used for the present
zation of these lines
Growth Inhibition
counted, and plated
study. The details about isolation and characteri
will be reported elsewhere.
Assay. Logarithmic-phase cells were harvested,
in duplicate at 10" cells/35-mm dish in 2 ml of ES
medium containing 2% dialyzed FBS and varying concentrations of the
drug to be assayed. These cultures were incubated for 72 ±2 hr and
counted. The number of cells in an experimental dish was plotted as a
percentage of the number of cells in the control dish. The degree of
drug 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 then
exposed 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 filters
were washed 5 times with 5 ml of cold 5% TCA and once with absolute
alcohol, air-dried, placed in 5 ml of toluene-based scintillation fluid,
and counted for radioactivity in a Beckmann Model L8500 P scintillation
counter. At the time of labeling, cell counts were carried out using
duplicate cultures in order to calculate, on a per cell basis, the incor
poration 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). Logarith
mic-phase cells were harvested, washed 3 times with Ca?+- and Mg? +free Dulbecco s phosphate-buffered
saline (4), and stored frozen at
-20° 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
OCTOBER
1982
of Jones and Sargent
adding 20 n\ of 0.2 M
the tubes in ice. Fourchromatography plates
(20 x 20 cm; Merck, Darmstadt, West Germany) with 0.02 fimol of
nonradioactive bases, nucleosides, and nucleotides as markers. As
cending chromatography
was carried out for about 1.5 hr at room
temperature either in 1 M ammonium acetate for the APRT assay or in
5% Na2HPO4 for the HGPRT assay. The plates were dried, and the
marker spots were located under a UV lamp, scraped, placed in vials
containing 0.5 ml of distilled water, and counted for radioactivity as
above. This analysis revealed that more than 98% of the products by
APRT were found in the AMP marker spot while more than 96% of
those 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 phos
phate (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 enzyme
source. Protein concentration was determined as described above.
The reaction mixture (80 ill) contained: 50 mM sodium phosphate (pH
6.5); 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 ¿igof protein). The reaction was
carried out at 37° for 15 to 30 min and stopped by adding 0.1 M
lanthanum chloride. The products were collected on Whatman GF/C
filters and counted with toluene scintillator as above.
Identification of the products was performed by immersing the in
cubated reaction mixture for 2 min into a boiling-water bath and chilling
it 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 marker
spot 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 radio
activity of the inosine plus hypoxanthine spots was reduced to zero
while the amount of AMP produced was not affected. These results
indicate 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 condi
tions used. One unit of enzyme was defined as the amount of enzyme
which yielded 1 nmol of the reaction products per min from the
radioactive substrates. Specific activity (nmol/min/mg
protein) was
also calculated by dividing the enzyme units by the protein content of
the extracts used for assay.
RESULTS
Selection of ClO-resistant Lines. Table 1 summarizes the
frequency of resistant colonies which appeared on agar plates
4211
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research.
H. Koyama and H. Kodama
Frequency of ClO-resistant
Table 1
cells in MNNG-treated
and untreated wild-type cells
of re
of cells
tion CIO(M)io-51CT5
of
sistant col
assayed1.8
onies0
x 10~8
x 10'
MNNG
7.2 x 10e
2.1 x 1CTS
With MNNGNo.
154
5 X IO'5No.
6.8 X 10~6
4.6 X 106Concentra
28Frequency"<5.6
a Wild-type F28-7 cells were treated with or without MNNG (0.5 fig/ml) for 2
Treatment8Without
hr and assayed for the frequency
"Materials and Methods.'
6 Defined in the text.
of colonies resistant to CIO as described
lines could utilize exogenously added adenine by plating and
culturing them on agar medium containing 2 x 10~5 M azaserine and 10~4 M adenine (AA plate). Since azaserine inhibits
purine synthesis (14), only cells capable of phosphoribosylat-
in
100-
in the selective medium containing CIO. There were no resistant
colonies found in a control population of 1.8 x 107 wild-type
cells not treated with MNNG, showing that the frequency of
spontaneous mutations to CIO resistance was quite low (<5.6
x 10~8). However, in the mutagen-treated population, resistant
colonies appeared at a frequency of as high as 2.1 x 10~5
and 6.8 x 10~6 on agar plates containing 1CT5 and 5 x 10~5
M CIO, respectively. Five colonies from the former plates and
6 from the latter were picked, transferred to drug-free medium,
and established as ClO-resistant cell lines. These lines were
designated as cior 1 to 11. They were subcultured for over 3
months (about 180 generations, with a doubling time of ap
proximately 12 hr) in nonselective medium, over which time the
ClO-resistant phenotype was stably inherited.
Resistance
on the growth
dapr cell lines
Chart 1, both
and exhibited
of about 8 X
cross-resistance of the mutants to the antibiotic. This finding
suggests that CIO and bredinin are activated by different mech
anisms.
Utilization of Exogenous Adenine. We studied whether ciò'
to CIO. We studied the cytotoxic effects of CIO
of wild-type F28-7, FC-1, 2 ciò' cell lines and 2
as a function of CIO concentration. As shown in
F28-7 and FC-1 cells were very sensitive to CIO
similar growth inhibtion curves with ED50 values
1CT7 M. On the other hand, cior3 and cio'8 lines
were much less sensitive than the above lines, and this was
also the case for dap'5 and dapr28 lines. The ED50 values for
these resistant lines ranged from 1.3 x 10"" to 3.2 x 10~4
o
1
o
50
10"
to'"
10'
io'
io-
CIO(M)
Chart 1. Effect of CIO on the growth of wild-type F28-7, FC-1, ciò', and dap'
cell lines. Cells were cultured for 72 ± 2 hr in medium containing varying
concentrations 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; •,
cio'8; A, dap'5; A, dap'28.
Table 2
ability of wild-type, do', and dap' cell lines on different agar
Colony-forming
media
Three dishes (100 mm) were plated with 100 cells of each line on 10 ml of
agar medium containing CIO or azaserine plus adenine and cultured for 8 days.
Relative plating efficiency"
5M)00.960.900.880.97AA61.040000
(10
Cell
lineF28-7cio'3cio'6cio'8dap'28No
addition1.00C0.880.930.971.04CIO
M, indicating that they were 160- to 400-fold more resistant
than the wild-type F28-7 cells. In addition, the data clearly
show that the dap' lines were cross-resistant to CIO.
A similar result was obtained by testing the colony-forming
ability of these lines on agar plates containing 10~5 M CIO. As
shown in Table 2, 3 cior cell lines all grew and gave rise to
colonies either in the presence or absence (no addition) of the
drug with plating efficiencies comparable to that found in the
wild-type cells.
We next examined whether or not cior lines showed crossresistance to DAP. These results are illustrated in Chart 2. The
growth of F28-7 and FC-1 cells was reduced to 50% in medium
containing 8.8 x 10~6 and 1.3 x 10~5 M DAP, respectively. In
contrast, both cior and dap' lines were 27- to 40-fold more
resistant to the adenine analog than were the wild-type F28-7
cells. It is therefore evident that the ciò' lines have crossresistance to DAP. These results suggest that ClO-resistant
and DAP-resistant cells share the same resistance mechanism.
DAP-resistant Chinese hamster (2, 20) or human cells (16) are
known to be defective in APRT activity. Thus, the present ciò'
as well as dap' mouse cells would be expected to lack the
enzyme activity.
CIO is the aglycone of the antibiotic bredinin.
whether these cell lines were resistant to bredinin.
Table 3, 2 ciò' and 2 dap' lines were less than
resistant to it than were the wild-type F28-7 cells,
4212
Plating efficiencies relative to that observed for wild-type
(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.
cells
100
o
°
I
50
10'
We checked
As shown in
2-fold more
indicating no
F28-7
io-
io"
10"'
DAP(M)
Chart 2. Effect of DAP on the growth of wild-type F28-7, FC-1, ciò', and dap'
cell lines. The assay procedures and symbols were as described in the legend to
Chart 1.
CANCER RESEARCH VOL. 42
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research.
APRT Deficiency
ing adenine by the action of APRT would multiply on the AA
plates. As shown in Table 2, column 4, 3 ciò' lines tested failed
to give colonies. This indicates that they were not able to utilize
exogenous adenine. dap'28 cells also revealed the same
growth property.
These results were further confirmed by testing the ability of
ciò' lines to incorporate tritiated adenine into the 5% TCAinsoluble cell fraction (Table 4). cio'3, cio'4, and cio'10 cells
exhibited greatly reduced levels of [3H]adenine uptake, while
cior8 cells could incorporate it at a little higher rate, supporting
the above data that these mutant lines were unable to utilize
exogenous adenine. These results strongly suggest that CIO
resistance 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 the
metabolism of CIO. Table 5 summarizes the results which are
expressed as specific activities. Wild-type F28-7 and FC-1
cells had high levels of APRT activity, whereas 11 cior mutants
showed undetectable amounts, or less than 1% of the activity
found in the F28-7 cell extract. In addition, the activity in dapr5
cells was low (10% of the F28-7 cell activity), while dap'28
cells had no activity. Assay of mixtures of wild-type cell extracts
and extracts from cior or dap' cell lines gave activity interme
diate between these 2 extracts alone, thus ruling out the
possibility of a diffusible APRT inhibitor (data not shown).
On the other hand, 10 of 11 ciò' lines (except for cio'4) and
2 dap' lines had levels of HGPRT activity similar to that of the
wild-type F28-7 cells. The cio'4 line could result from a double
mutation. FC-1 cells totally lacked HGPRT because of 8-azaguanine resistance (9). Furthermore, all the cell lines listed in
Table 5 possessed the same levels of AK activity.
These data demonstrate that the phenotype of CIO resist
ance in these cell lines results from a defect in APRT enzyme
activity and that CIO is activated by APRT to form a cytotoxic
nucleotide. This nucleotide would probably block guanine nuTable 3
Effect 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.
Cell
lineF28-7FC-1cio'3cio'8dap'5dap'28EDMa (M)5.4
10~4.3
X
10"8.8
X
10~8.8
X
10~1.0
X
10"1.0
x
x 10~
Average of 2 determinations.
Table 4
Ability of wild-type, do', and dap' cell lines to incorporate [2H]adenine into the
macromolecular fraction
Cells were labeled for 3 hr with [3H]adenine (1 ¿iCi/ml), and the radioactivity
incorporated into the 5% TCA-insoluble macromolecular fraction was counted as
described in "Materials and Methods."
Cell line
F28-7
cio'3
cio'4
cio'8
ciò'10
dap'28
Incorporation
of [3H]adeninea (%)
100
2.0
1.9
7.2
0.5
0.7
Expressed as a percentage of the activity found with wild-type F28-7 cells
(average of 2 to 3 determinations).
OCTOBER 1982
in ClO-resistant
Mouse
Tumor Cells
Table 5
Enzyme activities in wild-type do', and dap' cell lines
The procedures for preparation of cell-free extracts and enzyme assays were
described in "Materials and Methods."
Specific activity (nmol/min/mg
protein)
Cell
lineF28-7FC-1cio'1cio'2cio'3cio'4cio'5cio'6cio'7cio'8cio'9cio'10cio'1
0.3a1.8
±
±0.2<0.010.93
±0.10.01
0.0100.01
±
0.010<0.010.02
±
0.241.0
±
±0.00.95
0.23<0.010.98
±
0.151.2
±
±0.30.95
0.000.02
±
0.020.98
±
0.00<0.01<0.01<0.010.02
±
1dap'5dap'28APRT2.3
0.020.22
±
0.02<0.01HGPRT1.0
±
0.030.92
±
0.010.87
±
0.060.92
±
0.020.75
±
0.131.0
±
±0.1AK3.0
0.11.6
±
0.02.2
±
0.22.3
±
0.32.7
±
0.02.5
±
0.42.4
±
0.22.8
±
0.23.0
±
0.52.5
±
0.03.0
±
0.32.4
±
0.12.5
±
0.2ND62.3
±
±0.2
Mean ±S.D. of 2 to 3 determinations.
0 ND. not determined.
cleotide production
sensitive cells.
by inhibiting IMP dehydrogenase
in CIO-
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 able
to incorporate tritiated adenine into the macromolecular frac
tion; and (e) deficient in APRT activity.
These lines of evidence demonstrate that the mechanism of
CIO resistance involves a deficiency in APRT enzyme activity
and that CIO is activated by the enzyme to exert its cytotoxic
effects on cells. Since APRT catalyzes phosphoribosylation of
the nitrogen atom at position 9 of adenine by PRPP to form
AMP, CIO will be converted to 4-carbamoylimidazolium
5-olate1-ribosyl-5'-monophosphate
(bredinin 5'-monophosphate).
In
earlier works, Sakaguchi ef al. (18) could not identify this
nucleotide 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 that
bredinin was the activated form of CIO within the cell. They
also indicated, by studies on the reversal effects of many purine
compounds on CIO or bredinin cytotoxicity,
that bredinin
blocked the de novo purine synthesis by preventing the con
version of IMP to GMP. However, Fukui ef al. (5) studied the
activation mechanism of CIO with cell-free extracts prepared
from Ehrlich ascites tumor cells and showed that CIO was
converted to the above nucleotide form and that the chemically
synthesized nucleotide was a stronger competitive inhibitor for
IMP dehydrogenase than was either CIO or bredinin. Therefore,
they suggested that the nucleotide was the active metabolite
producing the cytotoxicity. Our present results support and
extend their observations.
Unmutagenized populations of F28-7 cells gave no resistant
colonies, showing that the frequency of appearance of spon
taneously resistant mutants is quite low (Table 1). Since the
APRT locus resides on chromosome 8 (10), the genes on both
chromosomes of this pair are ordinally functioning. Since the
APRT defect behaves as a recessive trait (7), the expression of
4213
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research.
H. Koyama and H. Kodama
CIO-résistant phenotypes would require either a double muta
tion or a single mutation followed by some chromosomal rear
rangement which would lead to the homozygous state of a
mutated APRT gene. This may be the reason for the low
frequency of appearance of spontaneous mutants. This finding
may be favorable for CIO or its derivatives as cancer chemotherapeutics.
In addition, our data indicate the usefulness of CIO for the
field of somatic cell genetics as an effective selecting agent for
isolating APRT-deficient mutants from cultured mammalian
cells.
ACKNOWLEDGMENTS
We would like to thank Drs. M. Inaba and M. Fukui for their helpful suggestions
concerning these studies.
REFERENCES
1. Chan. T.-S.. Creagan, R. P.. and Reardon, M. P. Adenosine kinase as a new
selective marker in somatic cell genetics: isolation of adenosine kinasedeficient mouse cell lines and human-mouse hybrid cell lines containing
adenosine kinase. Somatic Cell Genet., 4: 1-12, 1978.
2. Chasin, L. A. Mutation affecting adenine phosphoribosyltransferase
activity
in Chinese hamster cells. Cell, 2: 37-41. 1974.
3. Debatisse, M., Berry, M., and Buttin, G. The potentiation of adenine toxicity
to Chinese hamster cells by coformycin: suppression in mutants with altered
regulation 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 with
poliomyelitis viruses. J. Exp. Med., 99: 167-182, 1954.
5. Fukui, M., Inaba, M., Tsukagoshi. S., and Sakurai, Y. New antitumor imidazol
derivative, 5-carbamoyl-1H-imidazol-4-yl
piperonylate. as an inhibitor of
purine 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. Collateral
sensitivity of 6-mercaptopurine-resistant
sublines of P388 and L1210 leu
kemia to the new purine antagonists, 5-carbamoyl-1 H-imidazol-4-yl piperon
ylate and 4-carbamoylimidazolium
5-olate. Cancer Res., 42: 1103-1106,
1982.
7. Jones, G. E., and Sargent, P. A. Mutants of cultured Chinese hamster cells
deficient in adenine phosphoribosyltransferase.
Cell. 2. 43-54, 1974.
4214
8. Koyama. H.. Ayusawa, D., Okawa, M., Takatsuki, A., and Tamura. G.
Tunicamycin-resistant
mutations in mouse FM3A cells. Mutât.Res., in press,
1982.
9. Koyama, H., Yatabe, I., and Ono, T Isolation and characterization of hybrids
between mouse and Chinese hamster cell lines. Exp. Cell Res., 62. 455463, 1970.
10. Kzak, C., Nichols, E., and Ruddle, F. H. Gene linkage analysis in the mouse
by somatic cell hybridization: assignment of adenine phosphoribosyltrans
ferase to chromosome 8 and a-galactosidase to the X chromosome. Somatic
Cell Genet., 7. 371-382, 1975.
11. Kuroki, T. Agar plate culture and Lederberg-style replica plating of mam
malian cells. Methods Cell Biol.. 9: 157-178, 1975.
12. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein
measurement with the Polin phenol reagent. J. Biol. Chem., 193: 265-275.
1951.
13. Mizuno. K., Tsujino, M., Takada, M.. Hayashi, M., Atsumi. K., Asano, K.,
and Matsuda, T. Studies on bredinin 1. Isolation, characterization
and
biological properties. J. Antibiot. (Tokyo), 27: 775-782, 1974.
14. Patterson, D. Biochemical genetics of Chinese hamster cell mutants with
deviant purine metabolism: biochemical analysis of eight mutants. Somatic
Cell Genet., 1: 91-110, 1975.
15. Rabin. M.S., and Gottesman, M. M. High frequency of mutation to tubercidin
resistance in CHO cells. Somatic Cell Genet., 5: 571-583, 1979.
16. Rappaport, H., and DeMars, R. Diaminopurine-resistant
mutants of cultured
diploid human fibroblasts. Genetics, 75 335-345, 1973.
17. Sakaguchi, K., Tsujino, M., Hayano, M., Kawai, K., Mizuno, K., and Hayano.
K. Mode of action of bredinin with guanylic acid on L5178Y mouse leukemia
cells. J. Antibiot. (Tokyo), 29. 1320-1327,
1976.
18. Sakaguchi, K., Tsujino, M., Mizuno, K.. Hayano, K., and Ishida, N. Effect of
bredinin and its aglycone on L5178Y cells. J. Antibiot. (Tokyo), 28. 798803, 1975.
19. Sakaguchi, K , Tsujino, M., Yoshizawa, M., Mizuno, K., and Hayano, K.
Action of bredinin on mammalian cells. Cancer Res., 35. 1643-1648,
1975.
20. Taylor, M. W., Pipkorn, J. H.. Tokito, M. K., and Pozzatti, R. O., Jr. Purine
mutants of mammalian cell lines: III Control of purine biosynthesis in adenine
phosphoribosyltransferase
mutants of CHO cells. Somatic Cell Genet., 3.
195-206, 1977.
21. Wahl, G. M., Hughes, S. H., and Capecchi, M. R. Immunological character
ization of hypoxanthine-guanine
phosphoribosyltransferase
mutants of
mouse L cells: evidence for mutations at different loci in the HGPRT gene.
J. Cell. Physiol., 85. 307-320, 1975.
22. Yamane, I., Matsuya, Y., and Jimbo, K. An autoclavable powdered culture
medium for mammalian cells. Proc. Soc. Exp. Biol. Med., Õ27. 335-336,
1968.
23. Yoshida, N., Kiyohara, T., Fukui, M., Atsumi, T., Ogino, S., Inaba, M.,
Tsukagoshi, S., and Sakurai, Y. Antitumor activities of newly synthesized 5carbamoyl-1 H-imidazol-4-yl
1-adamantanecarboxylate
and 5-carbamoyl1H-imidazol-4-yl piperonylate. Cancer Res.. 40: 3810-3814,
1980.
CANCER
RESEARCH
VOL.
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research.
42
Adenine Phosphoribosyltransferase Deficiency Tumor FM3A
Cells Resistant to 4-Carbamoylimidazolium 5-Olate
Hideki Koyama and Hiro-aki Kodama
Cancer Res 1982;42:4210-4214.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/42/10/4210
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1982 American Association for Cancer Research.