[CANCER RESEARCH 33, 1829-1836, August 1973]
A High Susceptibility of Fanconi's Anemia to Chromosome
Breakage by DNA Cross-linking Agents1
Masao S. Sasaki and Akira Tonomura
Department of Human Cytogenetics, Institute for Medical Genetics, Tokyo Medical and Dental University, Yushima, Bunkyo-ku,
SUMMARY
Peripheral blood lymphocytes of patients with Fanconi's
anemia (FA) were tested for their susceptibility to chromo
some breakage by caffeine, chloramphenicol,
actinomy
cm D, methylmethanesulfonate,
nitrogen mustard, mito
mycin C, decarbamoyl mitomycin C, N-methyl-N'-nitro
N-nitrosoguanidine,
4-nitroquinoline
1-oxide, 8-methoxy
psoralen, and 60Co 7-ray and ultiviolet irradiations.
A
definitely abnormal response of the chromosomes of the
FA cells was found when they were treated with nitrogen
mustard and mitomycin C and after the irradiation with
long-wavelength
ultraviolet light in the presence of 8methoxypsoralen.
The specifically increased susceptibility
to these compounds,
which can introduce interstrand
cross-links into DNA, is interpreted as an indication that
the FA cells are defective in the repair mechanism to
tolerate the cross-links produced in their DNA. The im
pairment of the capacity to tolerate the lesions produced
in DNA may be implicated in the increased risk to develop
malignant neoplasms in this hereditary disorder.
Tokyo 113, Japan
to chromosome
breakage by ionizing irradiation. These
lines of experimental evidence are particularly interesting
in relation to the possible biological mechanisms under
lying the genesis of the high frequency of spontaneous
chromosome aberrations and the increased risk to the de
velopment of malignant neoplasms in this hereditary dis
order.
We studied the susceptibility of peripheral blood lympho
cytes of the FA patients to chromosome breakage by some
chromosome-breaking
agents and found that the chromo
somes of the FA patients were extremely sensitive to the
agents that could produce interstrand cross-links in DNA.
MATERIALS
AND METHODS
Materials. Heparinizedvenousbloodwasasepticallyob
tamed from 4 typical patients with FA (FA patients). The
levels of spontaneous chromosome aberrations in the FA
patients were compared with those in 3 children with aplas
tic anemia of unknown etiology (C patients).
Some
clinical findings of these patients are summarized in Table
I . In the FA patients, F-I and F-3, blood samples were
INTRODUCTION
available on 2 separate occasions, each being designed by
F-l(I), F-l(II), F-3(I), and F-3(II). The susceptibility of
FA,2 a rare hereditary disease possibly transmitted as an
blood lymphocytes from the FA patients to chromosome
autosomal recessive trait, is characterized by a progressive
breakage was compared with that of the lymphocytes from
hypoplastic pancytopenia associated with the combination
the C patients or from normal healthy adults.
of short stature, dark skin pigmentation
or café-au-lait
Leukocyte Cultures. Approximately 10 ml of freshly
spots, and diverse congenital anomalies. The development
drawn venous blood were allowed to stand at room tern
of leukemia and other malignant neoplasms are remark
perature to settle erythrocytes.
Supernatant
plasma to
ably frequent in the affected persons and in their relatives
gether with a small amount of erythrocytes (approximately
(28, 31). Pathogenesisof this disorder is at present ob 0.5 ml from the top phase of the sedimented erythrocyte
scure. Of particular interest in this regard is a recently
layer) was withdrawn. The cell suspension thus obtained
clearly established tendency of the affected persons toward
(usually 5 to 6 ml) was mixed with 5 to 7 volumes of cul
a high frequency of spontaneous chromosome aberrations
ture medium that consisted of NCTC-l09, fetal calf serum
(10). The etiology of these chromosome aberrations in the
(20%), and rehydrated PHA (type M, 2%). A series of
FA patients is not clear. However, recently Schuler et al. 4-ml cultures were set up and incubated at 37°for 72 hr
(29) found that chromosomes of an FA patient had an in
including the last 4 hr in the presence of colchicine.
creased sensitivity to an alkylating agent, tetramethane
Testing for Chromosomal Susceptibility. Test chemicals
sulfonil-d-mannit.
More recently, Higurashi and Conen
used in this study was caffeine (Iwaki and Co., Tokyo,
(13) found that the patients had an increased chromosomal
Japan), chloramphenicol
(Sankyo Co., Tokyo, Japan), ac
radiosensitivity
as manifested by an increased sensitivity
tinomycin D (Schwarz/Mann,
Orangeburg, N. Y.), MMS
(K and K Laboratories, Plainview, N. Y.), HN2 (a gift
from Dr. S. Tsukakoshi, Cancer Research Institute, Tokyo,
Japan), MMC (Kyowa Hakko Kogyo Co., Tokyo, Japan),
Japan.
DCMMC (a monofunctional derivative of MMC, supplied
by Kyowa Hakko Kogyo Co., Tokyo, Japan), MNNG
hemagglutinin;
M MS. methylmethanesulfonate;
HN2, nitrogen mustard;
Wis.), 4NQO
MMC, mitomycin C; DCMMC, decarbamoyl mitomycin C; MNNG, (Aldrich Chemical Co., Inc., Milwaukee,
(Tokyo Kasei Kogyo Co., Tokyo, Japan), and 8-methoxyp
N-methyl-N'-nitro-N-nitrosoguanidine;
4NQO, 4-nitroquinoline
I-oxide.
soralen (supplied by Taisho Pharmaceutical
Co., Tokyo,
Received December 27, 1972; accepted April 16, 1973.
I This
work
2 The
abbreviations
AUGUST
was
supported
by
used
are:
grants
FA,
from
Fanconi's
the
Ministry
anemia;
of
Education
PHA,
of
phyto
1973
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
1829
Masao S. Sasaki and Akira
Tonomura
Table I
Summary of cimica! traitsofpatientsCase
the FA and control
identificationPatientsAge
at
time of
examina
reflexiaControlC-IIF—+——
tion (yr)SexConsan
Infant
ation (dark
ity, hypoilism,
guinity
skin, café
plastic to
micros
anemiaPigment
between
au-lait
aplastic of
omy
parentsAplastic
spots)Deform- thumbMicro
mation
phthalmia,
cephalyMalfor of
strabismusHyper
kidneyMicro
?———?C-24F-+------C-32F-+-----FAF-l'6F-+++
+-+-+F-23F++++
+----F-34M++++
+----F-47M++++
±---a In Patient
F-I, erythrocyte
hexokinase
activity
was measured.
The activity
was at the normal
level as indicated
by the normal
rate ofglucose
6-phos
phate production.
Japan). Except for 8-methoxypsoralen,
the cultures were
treated with the test chemicals for the last 24 hr. At 48 hr
of culture time, the volume of culture was adjusted to 4.95
ml by supplementing NCTC-109, and then 0.05 ml of the
freshly prepared working solution of the test chemical was
added to give an appropriate final concentration.
For the
8-methoxypsoralen
treatment, ethanol solution of 8-methox
ypsoralen was serially diluted with Hanks' balanced salt
solution and finally with Medium TC-l99 in a concentra
tion of 0. 1 @g/ml. The cells cultured for 48 hr were col
lected by centrifugal sedimentation,
resuspended in the 8methoxypsoralen-containing
medium, incubated for 30
mm, and then irradiated in a glass culture flask with UV
with the wavelength of maximum emission of 355 nm
(FL2OS . BLB black-light
fluorescent
lamp;
Toshiba
Electric Co.) to give approximately
2 x l0@ ergs/sq mm.
After irradiation, the cells were washed and reincubated
with culture medium (NCTC-109 supplemented by 20%
fetal calf serum) for a further 24 hr. The experiment with 8methoxypsoralen
was carried out in the dark. In the separ
ate experiments using blood from normal healthy adults,
we obtained the dose-response
relations of chromosome
breaking effects of these test chemicals. The effects on the
chromosomes
of the FA and control cells were usually
evaluated at a single dose that produced a weak but sig
nificant rise of the frequency of chromosome aberrations
in blood cultures from normal healthy adults.
UV and -y-ray irradiations to the cultured lymphocytes
were made 18 hr prior to harvest. For the UV irradiation,
the cells in culture were collected by centrifugation, resus
pended in I ml culture medium, spread into a thin layer
in a 150-mm Petri dish, and allowed to settle for 30 mm
in the humidified atmosphere
with the aeration of 5%
CO2. then the excess medium was gently removed, and
the cells were exposed to 75 ergs/sq mm of 254 nm UV
(GL- 15 low-pressure germicidal lamp; Toshiba Electric
Co.). For the 7-ray irradiation, cells suspended in 1 ml
culture medium were transferred to a plastic tube and cx
posed to 100 rads of 60C0 -y-rays. The UV- and ‘y-ray
irradiated cells were resuspended in freshly prepared warm
I830
culture medium and reincubated for another 18 hr before
harvest.
In addition, in order to study the chromosomal
radio
sensitivity of the unstimulated lymphocytes, a I-mI aliquot
of plasma containing blood cells was transferred into 2
plastic tubes each containing about 0.5 ml. One of the 0.5ml fractions was directly exposed to 160 rads of 60Co ‘y
rays and then incubated with culture medium containing
PHA. In this experiment, the cultures were terminated at
50 hr of the total incubation time with the last 24 hr in the
presence of Colcemid. Another 0.5-mI fraction was used
as an unirradiated control culture, in which the cells were
not irradiated but otherwise processed in the same way.
Chromosome Analysis. Chromosome preparations were
made according to the standard air-drying method. Chrom
osome aberrations were scored recording dicentrics, rings,
abnormal monocentrics, chromatid breaks, chromatid gaps,
isochromatid gaps, and chromatid exchanges. Chromatid
breaks were stated only when the distal segments were dis
located from the chromosome axis. Because of the diffi
culty in distinguishing
between
them,
isochromatid
breaks of the NUpd type and acentric fragments of the
chromosomal
type were lumped toghether and scored as
acentric fragments. lsochromatid aberrations and chroma
tid exchanges were each scored as 1 aberration.
The susceptibility of chromosomes of the FA cells, RSCh,
was expressed by [X1 —XO1FA/[X@ —X0]C, where X
represents the frequency of chromosome
aberrations
in
treated (t) and untreated (0) conditions.
RESULTS
Spontaneous Chromosome Aberrations. The frequencies
of chromosome aberrations in untreated 72-hr cultures are
presented in Table 2. In both FA and control patients, virtu
ally most of the chromosome
aberrations
were of the
chromatid type, comprising gaps, breaks, and exchanges
CANCER RESEARCH
VOL. 33
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
Chromosomal
Susceptibility
in FA
Table 2
Chromsome aberrations in cultured lymphocytes from FA and control patients (72-hr culture)
Cultures were terminated at 72 hr. cdxa of the complete type and incomplete type were recorded separately. In the calculation
rations, F not associated with Dic and Ring were included as isochromatid breaks of the NUPd type.of
chromatid
aber
Chromosome aberrations
.
No.
of
Subjects
cells
tions/cellcompletetype)Control
@dx
(complete
type: in-
Cells
cdb
cdg
icdg
F
with
Dic
RingChromatid
aberra
chromo
some aberrations
(%)
patientsC-I
6.00.060C-2
200
0
2
10
0
0
0
0
3.00.030C-3
200
0
2
4
0
0
0
0
200
0
4
7
3
1
0
0
200
200
200
200
200
200
11:20
18:9
5:8
4:6
32:27
3:11
118
110
138
44
162
149
10
24
31
24
43
21
10
15
53
18
32
41
2
2
7
0
5
5
0
0
0
0
0
1 •
6.50.075FA
patientsF-l(I)
76.51.690F-l(II)
69.01.330F-2
91.02.275F-3(I)
54.00.900F-3(II)
81.52.140F-4
a cdx, chromatid
exchanges;
F, acentric
169
90
220
84
135
186
fragments;
Dic, dicentrics;
Ring,
centric
rings;
icdg,
isochromatid
87.02.035
gaps;
cdb, chromatid
breaks;
cdg, chro
matid gaps.
at the chromatid level. Chromosome
aberrations of the
chromosomal type, such as dicentrics and rings, were not
observed in the control patients and they were occasion
ally present in the FA patients. Since these chromosomal
type aberrations were not seen in the 50-hr cultures (see
below), in which all of the cells were in their 1st mitosis in
culture, they were assumed to be derived aberrations that
appeared in the 2nd or more advanced mitosis in culture,
and the levels of chromosome aberrations were compared
based on the frequency of chromatid aberrations includ
ing acentric fragments as isochromatid breaks of the NUpd
type. In the control patients, cells with chromatid aberra
tions were less than 10%. These values were at the same
level as in our previous data (25) and other published
data (9) on the blood cultures from normal healthy donors.
Highly elevated levels of chromatid aberrations were ob
served in the FA patients, differing from those in the con
trol patients in the order of magnitude. More than 50%
of the cells contained chromatid aberrations. The ratio of
chromatid exchanges, chromatid breaks, chromatid gaps,
isochromatid
gaps, and acentric fragments was approxi
mately I :6:4: 1: I, which was not largely different among
6 samples from 4 FA patients.
Chromosomal Radiosensitivity.
Lymphocytes from the
FA patients were tested for their chromosomal radiosen
sitivity in the PHA-stimulated
and unstimulated
condi
tions. For the 1st part of the experiment, lymphocytes in
plasma were directly exposed to 160 rads of °Co7-rays
prior to the commencement of PHA culture, where most of
the cells were in G0 stage and irradiation-induced
chromo
some aberrations were expected to be of the chromosomal
type. The frequencies of chromosome
aberrations
were
studied in the 50-hr cultures. The results are shown in
Table 3, where the frequencies of chromosome aberrations
in the unirradiated
50-hr control cultures are also pre
sented. In the unirradiated control cultures, there was no
AUGUST
significant difference in the frequencies of chromatid aber
rations between 50- and 72-hr cultures. However, chromo
somal type aberrations were not found in the 50-hr cultures,
while these aberrations were present in substantial number
in the 72-hr cultures. In the cultures of the irradiated cells,
the frequencies of dicentrics plus rings were 0.245, 0.225,
and 0.210 per cell for 3 FA patients, F-I, F-2, and F-3,
respectively, and the frequencies of irradiation-induced
terminal deletions, as measured by the number of acentric
fragments not associated with dicentrics and centric rings
minus spontaneously occurring ones, were about 0. 1 per
cell in these patients. These values were in excellent agree
ment with our previous data (26), 0.223 ±0.026 dicentric
plus rings per cell and 0. 117 ±0.01 8 deletion per cell, in
cultures of lymphocytes given the same dose of 7-rays, for
control children of the similar age group.
For the 2nd part of the experiment, which was designed
to compare the susceptibility of cells in actively growing
phase (possibly during the DNA synthesis stage) to pro
duce chromatid aberrations by irradiations, lymphocytes
from Patient F-I and control patients, which were cultured
for 54 hr. were exposed either to 100 rads of ‘°Co
7-rays
or 75 ergs/sq mm of 254 nm UV and harvested 18 hr later.
The frequencies of chromosome aberrations are presented
in Table 4, where the susceptibilities are compared based
on the frequencies of chromatid
aberrations
jncluding
acentric fragments not associated with dicentrics and cen
tric rings. As seen in the table, the FA cells were not abnor
mally susceptible to chromosome breakage by 7-ray irradi
ation, but instead rather smaller number of chromosome
aberrations were observed in the irradiated FA cells. How
ever, the FA cells were slightly susceptible to UV irradia
tion. With UV dose of 75 ergs/sq mm, approximately
7
times more chromatid aberrations were newly produced in
the FA cells than in the control cells.
Effects of Antimetabolites.
The presence of 0.02 sg of
1973
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
1831
Masao S. Sasaki and Akira Tonomura
Table 3
Chromosome aberrations in lymphoctyes exposed to 160 rads of ‘y-rays
prior to PHA stimulation
(50-hr culture)
Plasma containing blood cells was exposed to y-rays and then cultured with PHA for 50 hr. Colcemid was added 24 hr before
harvest. Each experiment was accompanied by an unirradiated control culture. F' not associated with Dic and Ring were recorded
as del. The frequency of irradiation-induced del was obtained by subtracting the frequency in the unirradiated cells from that in
the irradiated cells.
aberrations.
of
cellsChromosome
NetcdxcdbcdgicdgF
CulturesNo.
(del)DicRingF-l
.
Dic + Ring/_________________
cellDel/cell
Observed
(I)
Unirradiated
0.1300.085F-2
Irradiated200
20026
29119
13641
4817
139(9) 72(26)0
400
90
0.2450.045
Unirradiated
0.2100.110F-3
Irradiated200
2008
II230
24995
12527
84(42)0
2920(20)
340
110
0.2250.100
20013
4671
18358
1479
324(4) 73(33)0
340
80
(II)
Unirradiated
Irradiated60
a F, acentric
fragments;
Dic, dicentrics;
Ring,
centric
rings;
del, deletions;
cdx, chromatid
0.2100.0670.1650.098
exchanges;
cdb, chromatid
breaks;
cdg, chromatid gaps; icdg, isochromatid gaps.
Table 4
Chromosome aberrations in cultured lymphocytes exposed to 7-rays and U V 18 hr prior to harvest (72-hr culture)
The cells cultured for 54 hr were irradiated, and chromosome aberrations were scored after 18 hr of postirradiation incubation time.
with
aberrationsCells
of X,RScdx'cdbcdg.
cellsChromosome
lrradiationsSubjectsNo.
(del)DicRing‘y-rays(lOOrads)C-2
chromosome
aberrations
icdgF
aberrations/
cellX,
—
(%)Chromatid
0.080<1254nmUV(75
F-I(I)100 2000
557
17310 863
1312(9)
29(27)3
21
129.0
84.50.290
1.7700.260
ergs/sqmm)C-I
F-I(I)200 2006
325
29514 1262
150(0)17(16)0
20
010.5
91.00.135
2.4200.1050.730'@7.0
a cdx, chromatid
exchanges;
cdb, chromatid
breaks;
cdg, chromatid
gaps;
icdg, isochromatid
gaps;
F (del),
acentric
fragments
recorded
as deletions;
Dic, dicentrics; Ring, centric rings.
b RSCh
C The
‘the
susceptibility
frequency
is
significantly
of
the
FA
higher
cells
to
than
the
chromosome
simple
additive
breakage.
value
(p
<
0.05).
actinomycin D per ml produced a small but significant
change type, whereas such exchange aberrations were only
amount of chromatid aberrations in cultures of the control
1.3 to 4.2% in the treated cells.
cells, and with the same concentration
the frequency of
Effects of DN A-attacking Chemicals. Among the alkyl
chromosome aberrations in the FA cells was slightly dc
ating agents tested, a definitely abnormal response of the
vated but not significantly different from the simple addi
FA cells was found after treatment with HN2 and MMC.
tive value (Table 5). Rather abnormal response was found With these difunctional alkylating agents at a concentration
of 0.01 sg/ml, which was a dose to produce only about 0.2
in the FA cells treated with caffeine and chloramphenicol.
The effects of caffeine were evaluated at doses of 50 and chromatid aberration per cell in the control cultures, an
extensive amount of chromosome aberrations, mostly of the
500 zg/ml. With these doses of caffeine, the frequencies
chromatid type, was produced in the FA cells. The abnor
of chromosome aberrations in the FA cells were signifi
mal response of the FA cells to these difunctional alkylat
cantly higher than the simple additive values; approxi
mately 5 times more aberrations were newly produced in ing agents is clearly seen in Chart I , where the distribution
profiles of chromatid aberrations are presented. The shift
the FA cells than in the control cells. Differential respon
siveness of the FA cells to chromosome breakage
was of the distribution profile was remarkable in the FA cells
treated with HN2 and MMC. The number of chromatid
more pronounced when they were treated with chloram
phenicol. With 0. 1 mg of chloramphenicol per ml, the ef aberrations involved in a cell varied widely and in some of
the FA cells chromosome aberrations were too extensive
ficiency to produce chromosome aberrations was approxi
under the microscope. The suscepti
mately 12 times higher in the FA cells than in the control to be enumerated
cells. Of significant feature in the FA cells treated with bilities of the FA cells to chromosome breakage by HN2
these antimetabolites
was a largely decreased number of and MMC varied among patients, ranging from about
exchange aberrations; in the untreated FA cells approxi
33-fold in the lowest case to more than 100-fold in the high
mately 10% of the chromatid aberrations were of the cx est case over the control cells.
1832
CANCER RESEARCH
VOL. 33
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
Chromosomal
Susceptibility
in FA
Table 5
Chromosome aberrations after treatment with some chromsome-breaking
chemicals
The test chemicals were present for the last 24 hr in the 72-hr culture. The cells with more than 50 aberrations were recorded as C@@@-cells,
and in the
calculation of the frequency of chromatid aberrations such cells were scored as the cells with 50 aberrations. F' not associated with Dic and Ring were
scored as isochromatid breaks of the NUpd type. A-I, A-2, and A-3 are blood cultures from normal healthy adults as controls, in which X0's are 0.06,
0.03, and 0.08, respectively.
with
of
ChemicalsSubjectsNo.
chromosome
aberrations/
aberrations(%)Chromatid cellX@
X,RSchbcdxcdbcdg
cellsC@@1cellsChromosomeaberrationsCells
icdgFDicRingCaffeine
—
200
F-I (I)
A-I
100
F-3(II)200 2000
0
0
00
240
6
168
0
45
51
536 79732 403
4
36
8
280
34
3
1320
4
0
150
0
0
317.0
D
0.4402.7Chloramphenicol
(0.02@g/ml)C-3
F-I (I)100 2000
00
176 25717 130
1
100
120
00
022.0
85.00.240
2.1300.165
F-I (I)100 2000
00
184 42212 148
3
241
160
00
019.0
93.00.200
3.1400.125
(0.01@zg/ml)C-I, 2,3
200
F-I (I)
F-3(I)600 2000
33.6MMS
8
96
135
636063
0
00
0
020.2
98.5
91.50.253
0
0
8
45
3
25
158
89
13
13737 113
20
22
112
519
4870
6226
I
1
0
00
0
0
0
015.8
72.0
77.5
100
1000.178
1.480
—0.210
1.565
—0.470
20.620
20.590
14.9800.14313.290<1
0
5
0
10
0
0
0
024.0
97.0
100
93.0
97.00.282
11.385
9.695c
28.640
26.365'@ 113.6
8.450
7.550C 32.5
17.6100.232l5.575'@41.8
50@g/m1
500@eg/mlC-I
4.8Actinomycin
l.450c11.6HN2
(0.1mg/mI)C-3
l@eg/ml
200
F-I (I)
F-4
200
A-2
50
F-I (I)400 500
5@zg/mlC-l,2
<1MMC
2, 3
F-I (I)
F-2
F-3(I)
F-4600
67.1DCMMC(2@g/ml)A-2
17
1694
103160
(0.01 zg/ml)C-I,
200
200
200
2000
10
56
10
3225
F-3(II)200 2000
l.l70c6.0MNNG(l@ig/ml)A-3
02
—0.165<14NQO(l0'M)A-3
F-I (II)100 2000
01
F-l(II)100 2000
01
F-I (II)200 2000
52
0.120<18MOP
(0.1 ag/mI)
+ 355nm UV
II
210
16
1629
2863
1137
182850
148
65
53
946459
88.0
59.0
1000.210
2.420
0.730'@
1.070
1.010
7.0150.150 4.875c4.9
11.145
9•455C
7.7200.203 6.820'@46.6
17314 23923 181
4
332
390
40
017.5
92.50.225
3.3100.195
206
6517 Ill
2
241
130
10
023.0
63.50.270
1.1650.190
312
10617 113
2
231
170
10
020.0
66.50.230
1.4500.150
10
026.0
97.50.280
4
181110
2001831
<1
10.0550.2008.725c43.6
(2 x 10' ergs/
sq mm)A-3
a F, acentric fragments; Dic, dicentrics; Ring, centric rings; icdg, isochromatid gaps; cdx, chromatid exchanges; cdb, chromatid breaks; cdg, chroma
tid gaps.
0 RSCh,
C The
the
frequency
susceptibility
is
of
significantly
the
FA
higher
cell
than
to
chromosome
the
simple
breakage.
additive
value
(p
Neither the treatment with 8-methoxypsoralen
alone nor
irradiation with 355 nm UV alone were effective enough
to produce chromosome aberrations in the FA and control
cells. However, in the FA cells exposed to 2 x 10' ergs/
sq mm of 355 nm UV in the presence of 0. 1 .tg of 8-methox
ypsoralen per ml, an extensive amount of chromatid aber
rations was produced. As seen in Table 5, the FA cells were
43.6 times more susceptible to chromosome breakage than
the control cells, the value being very comparable to those
for HN2 and MMC.
In contrast to these difunctional alkylating agents and
8-methoxypsoralen,
the FA cells were not abnormally sen
AUGUST
<
0.05).
sitive to the other chemicals, MMS, DCMMC, MNNG,
and 4NQO. The susceptibility to chromosome breakage
by MMS was evaluated in cultures treated in concentra
tions of I and 5 @sg/m1.The effects of MNNG and 4NQO
were studied in cultures treated at I @ig/ml and l0_ M,
respectively. As in the case of 7-ray irradiation,
the
treatments
with these chemicals yielded fewer chromo
some aberrations in the FA cells than in the control cells.
The susceptibility of the FA cells to DCMMC, a mono
functional derivative of MMC, was 6 times higher than
that of the control cells as measured in cultures treated
in a concentration of 2 .tg/ml. However, the effectiveness
1973
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
1833
Masao S. Sasaki and Akira
70
Tonomura
F-I(I)
F- 1(1)
AdP@_..
Untreated
some aberrations was found in cultured lymphocytes from
our FA patients. The etiology of these spontaneously oc
curring chromosome aberrations in the FA patients is at
present obscure. The experimental evidence recently pre
sented by Higurashi and Conen (I 3) is suggestive of the in
creased lability of chromosomes
as indicated by the in
creased sensitivity to chromosome breakage by ionizing
irradiation.
However, using an essentially similar tech
nique, it was not fully substantiated by the results obtained
in our present experiments; the chromosomes of lympho
cytes from the FA patients responded normally to 7-ray
irradiation. In some FA patients, a lowered level of ATP
has been found as a consequence of low hexokinase activity
or due to the increased ATPase (I I, 19). Thus, the empha
sis has been placed on the possibility that the hexokinase
deficiency or diminished ATP level may be causally related
to the increased level of spontaneous chromosome aberra
tions in this syndrome, since ATP is known to be necessary
for the normal functioning of the repair of chromosome
breakage or breakage induced in DNA (18, 20, 21, 32).
However, in other cases in which biochemical analysis
has been made, including our own, no disturbance in carbo
hydrate metabolism or any sign of diminished ATP level
has been disclosed (2, 27, 29). Yet, in all cases chromo
some aberrations were present.
- Untreated
60
L
50
40
@
@\.@@.Actinomycin
D
.
,,,..Caffeine
Uv
30
20
I0
0
@
0
2
4
6
70
@
@
60
@;@@#__. Untreated
I
C,.)
F-I(I)
40
@
..
MMS
o 30
@
\...@MMC
@20
HN2
@l0
@
0'
)
2
4
6
70
@
@
@
@
@
60
1@/
50
Untreated
8
tO
2
50
F—I(II)
40
\@,,MNNG
*\
I
4NQO
16
8
2022
2426@
Unt at d
@..ga@'•re e
30
20
40
30
4
It seems
probable
that an important
finding
may be a
definitely
abnormal
susceptibility
ofthe
FA cells to chromo
some breakage
by difunctional
alkylating
agents.
Recently,
F-3(II)
Schuler
etal.(29)reported
asimilarfindingthatthefre
O,,,DCMMC
d
I0
0 I
0
quency ofchromosome
aberrations
from FA patients was synergized
\
2
‘.
@,
i i t@—i_4
6
8
0
polyfunctional
sulfonil-d-mannit.
age by other
MMS,
MNNG,
2
@\@—8MOP+355nmUV
@
of normal
@a—•,,a
‘ ‘ ‘ I
0
12
4
6
8
20
22
24 26@
functional
alkylating
agent,
I , 2 , 5 , 6-tetramethane
The susceptibility
to chromosome
break
monofunctionally
reacting
agents
such
as
4NQO,
and DCMMC
was within
a limit
level. These
-@-@_j are specifically
(or
in cultured lymphocytes
by the treatment
with a
findings
indicate
susceptible
to chromosome
polyfunctional)
alkylating
that
the FA cells
breakage by di
agents.
Although
NUMBER OF CHROMATID ABERRATIONS
the mechnisms involved in the formation of chromosome
as a consequence of the primary alkylation
Chart I. Distributions of chromatid aberrations among FA cells. The aberrations
treatments were made at doses that were low enough to produce less than lesions produced in DNA are not precisely known, all
0.3 aberration per cell in cultures from normal healthy adults, i.e., 50 @zg available evidence suggests that the observed differential
of caffeine per ml, 0.02 @gof actinomycin D per ml, 0.1 mg of chloram
susceptibility of the FA cells to chromosome breakage by
phenicol per ml, 75 ergs/sq mm of 254 nm UV, 100 rads of -y-rays, 0.01 difunctional
alkylating agents may be ascribed to the dif
ferential responsiveness to the difunctional alkylation Ic
lLg of DCMMC per ml, I @gof MNNG per ml, l0' M 4NQO, and 2 x sions, possibly interstrand
cross-links, which can be pro
10' ergs/sq mm of 355 nm UV in the presence of 0.1 @gof 8-methoxy
duced in their DNA. Of interest in this regard is that the
psoralen (8MOP) per ml. Distributions of chromatid aberrations in un
of the FA cells are cross-sensitive
to 8treated FA cultures are also presented. In each culture, 200 cells were chromosomes
methoxypsoralen,
as
indicated
by
the
similarly
increased
counted. With these doses of the test agents, the abnormal susceptibility
susceptibility to chromsome breakage, which is known to
of the FA cells to chromosome breakage can be readily seen by the discern
cross-links in DNA when the treatment
ible shift of the distribution profile. For the methods of treatment, see form interstrand
is followed by the irradiation with long-wavelength UV (7).
Tables 4 and 5.
The results obtained from the present experiment are
was apparently less marked when compared with that of
still too premature to allow us to pinpoint the location of
the difunctional MMC.
the biological defect inherent in the FA cells. However, as
suming a central role of the lesions produced in DNA in
the development of chromosome aberrations, it is tempting
DISCUSSION
to correlated the high susceptibility to chromosome break
As previously noted, in the chromosomes
of this syn age by these DNA cross-linking agents with the impair
ment in the repair or bypassing mechanisms to tolerate
drome, a highly increased level of spontaneous chromo
@Lg of
I834
HN2
per
ml,
l
@g of
MMS
per
ml,
0.01
ig
of
MMC
per
ml,
2
CANCER RESEARCH
VOL. 33
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1973 American Association for Cancer Research.
Chromosomal
Susceptibility
in FA
another repair mechanism such as recombination
repair
the interstrand DNA cross-links. There is evidence in bac
(24). However, it has been shown in bacteria that the re
teria that the HN2- and sulfur mustard-induced
DNA
cross-links are efficiently removed possibly by an excision
tation yield.
mechanism
similar to excision repair of UV-induced
pyrimidine dimers (14, 16, 17). The recovery from the
cross-links has also been demonstrated
in cultured mam
malian cells (8, 23). Stimulated by these lines of evidence,
ACKNOWLEDGMENTS
a preliminary experiment has been made to see whether
We are indebted to Dr. A. Ozawa (Tokyo Medical and Dental Univer
the FA cells are handicapped in the excision repair of the
alkylation lesions produced in their DNA. However, the sity), Dr. N. Taguchi (National Children's HÃ spital), and Dr. S. Shike
(Toho University), for their cooperation and clinical information concern
level of unscheduled DNA synthesis was not distinguish
able from that of the normal cells as indicated by the ing the patients, and to Dr. K. Sasazuki (Tokyo Medical and Dental
University) for determining hexokinase activity. We also thank to Dr. S.
MMC- and HN2-stimulated
incorporation
of thymidine
Tsukakoshi (Cancer Research Institute) for his generous gift of nitrogen
3H in cultured lymphocytes and skin fibroblasts obtained
mustard. We are grateful to Dr. S. Kondo (Osaka University Faculty of
from the FA patients (M. S. Sasaki, unpublished data).
Medicine), for his valuable suggestions and comments.
Recently, Regan et a!. (22) found that the excision of UV
induced pyrimidine dimers was normally functioning in
the FA cells. Moreover, with regard to the differential
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CANCER RESEARCH
VOL. 33
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A High Susceptibility of Fanconi's Anemia to Chromosome
Breakage by DNA Cross-linking Agents
Masao S. Sasaki and Akira Tonomura
Cancer Res 1973;33:1829-1836.
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