Characterization of Chromosome Aberrations

[CANCER RESEARCH 47, 5162-5170, October 1, 1987]
Characterization of Chromosome Aberrations Induced by Incubation at a
Restrictive Temperature in the Mouse Temperature-sensitive Mutant
tsFT20 Strain Containing Heat-labile DNA Polymerase a1
Toshihiko Eld,2 Takemi Enomoto, Yasufumi Murakami,3 Fumio Hanaoka, and Masa-atsu Yamada4
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
ABSTRACT
tsFT20 cells derived from a mouse mammary carcinoma cell line,
FM3A, which has temperature-sensitive DNA polymerase a activity (Y.
Murakami, (I. Yasuda, H. Miyazawa, F. Hanaoka, and M. Yamada,
Proc. Nati. Acad. Sci. USA, 82:1761-1765,1985) were rapidly commit
ted to death after temperature upshift to 39°C.tsFT20 cells synchronized
in S phase were more sensitive to the restrictive temperature than
exponentially growing cells. In order to gain insight into the processes
from the interruption of DNA synthesis to cell death, we analyzed
chromosome aberrations induced in tsFT20 cells which had been incu
bated for 2 or 4 h at the restrictive temperature and then cultured at the
permissive temperature. The majority of metaphase cells showed exten
sive chromosome aberrations such as chromatid gaps, breaks, and ex
changes; chromosome pulverizations; their mixed types; and ring chro
mosomes. Analyses with the use of cell synchronization and autoradiography revealed that chromosome aberrations were induced only in the
cells which synthesized DNA during incubation at 39°C.We classified
the chromosome aberrations into five types: gap or break type; exchange
type; pulverization type; complex type; and ring type. The temporal order
of the appearance of these types of chromosome aberrations was found
to be the above described order. It was further found that cycloheximide
dramatically repressed the induction of chromosome aberrations, and
metaphases with many chromosome aberrations exhibited a large number
of sister chromatid exchanges. These results indicate that abnormal
cessation of DNA replication in tsFT20 cells at the restrictive tempera
ture due to the inactivation of DNA polymerase a results in cell death
via induction of double-strand breaks which lead to chromosome aber
rations as well as sister chromatid exchanges.
directly participating in DNA replication are inhibited because
of the lack of specific inhibitors for DNA replication enzymes
except for aphidicolin and the unavailability of DNA ts mutants
having ts enzymes that participate in DNA replication.
In recent years, we have tried to isolate ts mutants related to
DNA replication in order to get a tool for the analysis of the
molecular mechanism of DNA replication in mammalian cells,
and we have isolated several such mutants (12-14). One of
these ts mutants, tsFT20, was found to have heat-labile DNA
polymerase «activity (12). Recently, we have succeeded in
proving that the DNA polymerase a molecule of tsFT20 cells
itself is heat-labile by using an ¡mmunoaffinity-purified en
zyme.6 tsFT20 cells are typical DNA ts mutants, which show
rapid decrease in DNA-synthesizing ability after temperature
upshift and are arrested in the S phase. It was found that the
rapid decrease in DNA-synthesizing ability correlated well with
the decrease in the intracellular level of DNA polymerase a
activity (15) and was due to the decrease in the frequency of
replicón initiation (16). In addition, it has been observed that
ts cells are rapidly committed to death after exposure to the
restrictive temperature.
In this study we have examined chromosome aberrations
induced in tsFT20 cells in order to gain insight into the proc
esses from the interruption of DNA replication, due to the
inactivation of the DNA polymerase a molecule, to cell death.
MATERIALS
INTRODUCTION
During recent years, it has become well known that besides
direct DNA lesions induced by X-rays or chemicals, the indirect
DNA lesions induced by imbalance of DNA precursor metab
olism also cause extensive chromosome instability in eukaryotic
cells. In addition, it is known that when de novo synthesis of
thymidylate is blocked, growing cells die. This phenomenon is
called thymineless death and has been initially found in prokaryotic cells (1). Thymineless death has been studied in eukar
yotic cells treated with drugs that block thymidylate metabolism
(2-6) and auxotrophic thy"5 mutants (7-9). Recent studies
using thy mutants have shown that thymidine starvation in
mammalian cells induces extensive chromosome aberrations
and results in thymineless death (10, 11). On the other hand,
little is known of what happens in cells in which enzymes
Received 3/3/87; revised 6/19/87; accepted 6/24/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by Grants-in-Aid for Scientific Research
and for Cancer Research from the Ministry of Education, Science and Culture,
Japan, and by the Naito Foundation.
2To whom requests for reprints should be addressed.
3 Present address: Riken Gene Bank, The Institute of Physical and Chemical
Research.
4 Present address: Hatano Research Institute, Food and Drug Safety Center.
5 The abbreviations used are: thy", thymidylate synthase-negative; CMF-PBS,
calcium- and magnesium-free phosphate-buffered saline; FdUrd, 5-fluoro-2'deoxyuridine; SCE, sister chromatid exchange; ts. temperature-sensitive.
AND METHODS
Cell Culture. tsFT20 cells (12) and FM3A clone 28 cells (wild-type)
which were originally established from a spontaneous mammary carci
noma in a C3H/He mouse (17) were incubated at 33°C(permissive
temperature) or at 39°C(restrictive temperature) in suspension culture
in RPMI 1640 (Flow Laboratories, England) supplemented with 10%
calf serum (Flow Laboratories, North Ryde, Australia). Cells used in
these experiments were free of Mycoplasma contamination.
Assay for Colony-forming Ability. Exponentially growing tsFT20
cells and wild-type cells or tsFT20 cells synchronized at Gi-S boundary
(approximately 1.5 x IO6) were inoculated in glass tubes (1.5 x 10.5
cm) containing 5 ml of the growth medium. The cells were incubated
at 39°Cfor indicated periods in a water bath and then collected by
centrifugation at 1400 x g for 5 min at 4°C.After a washing with icecold CMF-PBS, the cells were suspended in and diluted with the growth
medium to a cell concentration of 100 and 300/ml. One volume of the
diluted cell suspension was mixed with 2 volumes of 0.5% agar solution
(agar Noble; Difco Laboratories, Detroit, MI) dissolved in RPMI 1640
containing 10% calf serum and antibiotics (100 //g/nil streptomycin
sulfate and 100 units/ml penicillin G potassium), and 3 ml of the
mixture were poured on 4 ml of 0.5% underlayer agar in a glass dish
(60 mm in diameter). After incubation at 33°Cfor 2 weeks in CO2
incubator, the number of colonies in the soft agar was counted (4
dishes/point).
Cell Synchronization. Exponentially growing cells were inoculated
into the growth medium containing 10% dialyzed calf serum and the
antibiotics at a concentration of 1.5 x IO5 cells/ml. The cells were
incubated at 33°Cfor 1 day and FdUrd was added at a final concentra" R. Takayama, S. Tada, F. Hanaoka, and M. Ui, unpublished data.
5162
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research.
ANALYSIS OF CHROMOSOME
INSTABILITY
tion of 3 x 10~8 M. The cells were incubated at 33°Cfor 14 h to be
arrested at d-S boundary. The cells were released from the FdUrd
block by two washings with ice-cold CMF-PBS and suspended in warm
growth medium containing the antibiotics. Over 80% of the population
thus synchronized was arrested at the d-S boundary and most of the
remainder was arrested in S phase according to cytofluorometric anal
ysis (15).
Assays for Chromosome Aberrations and Mitotic Index. The cells (3
x IO5) from exponentially growing cultures or synchronized cultures
were inoculated into a glass dish (diameter 30 mm) with 2 ml of the
growth medium. After incubation for indicated periods, Colcemid was
added to the culture at a final concentration of 0.1 ¿ig/ml,and the cells
were incubated at 33 °Cfor indicated periods in a CC>2incubator. The
cells were chilled in an ice-water bath and transferred into a glass tube.
The cells were collected by centrifugation and washed with ice-cold
CMF-PBS. The pellet was resuspended in 1 ml of 0.5% sodium citrate
and incubated at 37°Cfor 5 min with shaking, and then 5 ml of the
fixative (methanol:acetic acid, 3:1) were added. After standing at room
temperature for 5 min, the suspension was centrifuged, and the pellet
was washed once with the fixative. The resultant pellet was suspended
in a small volume of the fixative. The suspension was dropped on a
glass slide and air dried. Cells fixed on the glass were stained with 3%
Giemsa solution for 15 min. Mitotic index was determined by exam
ining about 500 cells. As for chromosome aberrations, about 200
metaphases were examined at x 400. Chromosome aberrations are
classified as follows. "Gap or break type" is defined as the aberration
exhibiting a chromosome or chromatid gap or break observed in a
metaphase which contains less than six aberrations. "Exchange type"
is defined as the aberration exhibiting a chromatid interchange or a
chromosome/chromatid
interchange observed in a metaphase which
contains less than six aberrations. "Complex type" is an aberration
consisting of more than six aberrations within a metaphase including
chromatid gaps, breaks, exchange types, and the fragmented chromo
somes as shown in Fig. 2, b-d. "Pulverization type" is represented by
pulverized chromosomes as shown in Fig. le. "Ring type" is the
aberration exhibiting a centric ring or a ring derived from a chromosome-chromatid interchange. The frequencies of gap or break type,
exchange type, and ring type are expressed as the number of sites
exhibiting aberrations per 100 metaphases by examining about 200
metaphases. The frequencies of complex type and pulverization type
are expressed as the number of metaphases exhibiting the aberration
per 100 metaphases by examining about 200 metaphases.
Autoradiographic Analysis. Exponentially growing cells (5 x 10s)
were inoculated into a culture flask (25 cm2) containing 5 ml of the
growth medium supplemented with 10% dialyzed calf serum and the
antibiotics. The cells were incubated at 33"C for 2 days and then
incubated at 39°Cfor 4 h. The cells were pulse-labeled with 4 ^Ci/ml
[3H]thymidine (20 Ci/mmol; Amersham, United Kingdom) for 10 min.
The labeled cells were collected by centrifugation and washed twice
with ice-cold CMF-PBS. The cells were suspended at a concentration
of 1.5 x 10' cells/ml in the growth medium containing 1 x IO"5 M
thymidine and 0. l j/g/ml Colcemid. After incubation at 33°Cfor 24 h,
IN tsFT20 CELLS
15 min, washed with water, and soaked in Mcllvaine's buffer (196 mM
disodium hydrogen phosphate-2 HIMcitric acid, pH 8.25). The glass
slide was covered with a cover glass and sealed with colorless nail polish
to hold the Mcllvaine's buffer between the glass slide and the coverglass.
The glass slide was exposed to UV at a 5-cm distance from a germicida!
lamp (Toshiba GL15) for 10 min on a hot plate set at 60"( , washed
with water, and stained with 2% Giemsa solution. The stained meta
phases were examined at x 1000.
RESULTS
Decrease in Colony-forming Ability during Incubation at the
Restrictive Temperature. As reported previously (12, 15, 16),
tsFT20 cells have heat-labile DNA polymerase a activity. As
shown in Fig. la, DNA polymerase a activity in tsFT20 cells
decreased immediately after temperature upshift in contrast to
the activity in wild-type cells. Fig. \b shows the colony-forming
ability of tsFT20 cells after the temperature upshift. Exponen
tially growing tsFT20 cells lost colony-forming ability rapidly
after incubation at 39°C.When tsFT20 cells were synchronized
with FdUrd and shifted to the restrictive temperature after the
release from the FdUrd block, the decrease in colony-forming
ability of the synchronized cells became much more prominent
than that of exponentially growing cells. Only 8% of colonyforming ability was obtained with the synchronized cells after
incubation at 39°Cfor 8 h. Little decrease in colony-forming
ability was observed with synchronized wild-type cells.
Chromosome Aberrations Induced in tsFT20 Cells at the Re
strictive Temperature. We next studied the effect of the exposure
of tsFT20 cells to the restrictive temperature from the chro
mosome-morphological point of view, especially chromosome
aberrations. Fig. 2 shows typical metaphase figures containing
chromosome aberrations obtained from the cells which have
been incubated at 39°Cfor 4 h and then incubated at 33°Cfor
24 h in the presence of Colcemid. Various types of chromosome
aberrations were observed: simple chromatid-type chromosome
aberrations such as chromatid gaps and breaks (Fig. la; Arrows
E-H and J) and chromatid interchanges (Arrows A-D and /);
"complex-type" chromosome aberrations characterized by
many chromatid gaps, breaks, and exchanges (Fig. 2, b-d); and
~ loo
so
the cells were fixed on a glass slide and processed to autoradiography
as described previously (16). The existence of silver grains and chro
mosome aberrations was examined with about 300 metaphases at x
400.
Assay for SCEs. Exponentially growing cells (2.5 x IO5)were inoc
ulated into a culture flask (25 cm2) containing 5 ml of the growth
medium supplemented with 10% dialyzed calf serum and the antibiot
ics. 5-Bromo-2'-deoxyuridine (Sigma Chemical Co., St. Louis, MO)
012«
8
0124
e
Incubation
period al 39 *C (h)
Incubation
period at 39'C (h)
was added to the culture at a concentration of 5 Mg/ml, and then the
cells were incubated at 33°Cfor 18 h (approximately 1.2 cell cycles
Fig. 1. (a) Changes in the level of intracellular ONA polymerase a activity.
under these conditions) in the dark. The cells were incubated at 39°C Exponentially growing tsFT20 cells (•)and wild-type cells (O) were shifted up to
39'C
and cultured for the indicated periods. The preparation of crude cell extracts
for 4 h and further incubated at 33°Cfor 20 h. Colcemid was added at
a concentration of 0.08 Mg/ml to the culture, and the cells were
incubated at 33°Cfor 6 h. The cells were collected and fixed on a glass
slide as described under "Assays for Chromosome Aberrations and
Mitotic Index." Sister chromatids were differentially stained by a
modified method of Perry and Wolff (18) as follows. The glass was
dipped in 30 /¿g/mlHoechst 33258 solution at room temperature for
and assay for DNA polymerase a were performed as described previously (12).
(b) Changes in colony-forming ability of exponentially growing tsFT20 cells,
synchronized tsFT20 cells, and synchronized wild-type cells. Cells were synchro
nized at the d-S boundary with FdUrd as described under "Materials and
Methods." After the release from the FdUrd block, the cells were incubated at
39"C for the indicated periods and assayed colony-forming ability as described
under "Materials and Methods." Bars, SE. Synchronized tsFT20 cells (•),ex
ponentially growing tsFT20 cells (•),wild-type cells (O).
5163
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research.
ANALYSIS OF CHROMOSOME
INSTABILITY
IN tsFT20 CELLS
F
a
Fig. 2. Chromosome aberrations induced in tsFT20 cells by incubation at the restrictive temperature. Exponentially growing tsFT20 cells were incubated at 39'C
for 4 h and then incubated at 33'C for 24 h in the presence of 0.1 <¿g/mlColcemid. (a) Slightly damaged metaphase figure containing chromatid gaps or breaks
(Arrows ill, and J) and chromatid interchanges (Arrows A-D, and /); (¿>)
metaphase figure exhibiting slight complex-type chromosome aberrations, in which several
chromatid interchanges and breaks are observed; (c) and (</) metaphase figures exhibiting typical complex-type chromosome aberrations; (e) metaphase figure
exhibiting pulverization-type chromosome aberrations. Bars, 10 t/m.
5164
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research.
ANALYSIS OF CHROMOSOME
INSTABILITY
IN tsFT20 CELLS
fsfÃ-ÕL.*
^«^
AM--^
'%^>^
?.'£)& "*¿W^
t * ¿e.A -
:^ -
•
^»
\
** ^i.
'W
»
x. V
Fig. 3. Relationship between the induction of chromosome aberrations and DNA replication. Exponentially growing tsFT20 cells were incubated at 39'C for 4 h
and pulse-labeled with ['Hjthymidine for 10 min. After being washed, the cells were cultured at 33*C for 24 h in the presence of Colcemid and fixed. Metaphases
containing normal chromosomes and pulverization-type chromosomes and that exhibiting complex-type chromosome aberrations, which were stained with Giemsa,
are shown in (<•)
and (¡I).The samples were destained with isopropyl alcohol and processed for autoradiography. (a) and (h) are autoradiograms of the same
metaphases of (c) and (</), respectively. Hun, 10 »mi.
chromosome pulverization forming pulverized chromosome
fragments (Fig. 2e). The complex-type chromosome aberration
and chromosome pulverization characterized the chromosome
aberration induced in tsFT20 cells by exposing the cells to the
restrictive temperature, and such aberrations were induced at
very low frequency in wild-type cells and the revenants derived
from tsFT20 cells (data not shown).
Analysis of Relationship between Induction of Chromosome
Aberrations and Position of Cells in the Cell Cycle. In order to
determine the relationship between the induction of chromo
some aberrations and DNA replication, tsFT20 cells were pulselabeled for 10 min with [3H]thymidine after incubation at the
restrictive temperature for 4 h and then incubated at the per
missive temperature for 32 h in the presence of Colcemid.
Fig. 3 shows metaphase figures exhibiting chromosome ab
errations (c, pulverization type; d, exchange type) and autora
diograms of the same metaphases containing silver grains (a,
lì).
From the results summarized in Table 1, it is revealed that
all metaphases exhibiting chromosome aberrations contain sil
ver grains, suggesting that chromosome aberrations are induced
only in the cells which were in the S phase when they were
incubated at the restrictive temperature. On the other hand, the
Table 1 Relationship between DNA synthesis and the induction of chromosome
aberrations in lsFT20 cells by incubation at the restrictive temperature
Exponentially growing tsFT20 cells were pulse-labeled with [3H]thymidine for
10 min after incubation at 39'C for 4 h. After washing, mitotic cells were
accumulated at 33*C for 32 h in the presence of Colcemid. They were processed
as described under "Materials and Methods."
no.Sample
Chromosome aberration
Grain (+)
Chromosome aberration
Grain (—
)
Chromosome aberration
Grain (+)
Chromosome aberration
Grain (-)Sample
1103
2118
3103
(+)
(+)
0
0
0
(—
)
92
116
91
(—
)
55Cell
95Sample
67
metaphases containing silver grains do not always show chro
mosome aberrations.
To analyze in detail susceptible points to induce chromosome
aberrations in the cell cycle, we have performed experiments
the scheme of which is shown in Fig. 4. At the indicated times
after release from synchronization, cells were shifted to 39°C
for 2 h and then incubated at 33°Cfor 24 or 32 h in the presence
5165
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research.
ANALYSIS OF CHROMOSOME
INSTABILITY
IN tsFT20 CELLS
of Colcemid. As shown in Fig. 5, the cells which were shifted
to 39°Cduring S phase exhibited various types of chromosome
20
aberrations at high frequencies (Fig. 5; a, gap or break type; b,
exchange type; c, pulverization type; d, complex type). The
classification of chromosome aberrations is described under
"Materials and Methods." The exchange type consisted of
10
0
20
2h
39 «C
10
0
20
10
33 «C
Xh
K h
0
colcemid
40
-»32
h
release
FdUrd
sample fixation
20
Fig. 4. Scheme for analysis of the relationship between the induction of
chromosome aberrations and the position of cells in the cell cycle.
¿n^FinriH
nrppn-,Pn„
r-, n
20
n n
I
3
4
S
6
32
Time after
n_ n 1
1
•
Time otter
release
from
Z
3
i
5
S
E
B
10
cells.
2 h or 4 h
sample fixation
it inn it
33 *C
Kh
t I l t t t t
colcemid
( 2h or Ah interval
FäUrd
synchronization
40
( h )
( h )
Fig. S. Histograms indicating the frequencies of various types of chromosome
aberrations induced at various phases in the cell cycle. Experiments were per
formed as described in Fig. 4. The histograms indicate the frequencies of gap- or
break-type (a), exchange-type (b), pulverization-type (c), and complex-type (d)
chromosome aberrations observed in mitotic cells which had been incubated at
33°Cfor 24 h (solid column) or for 32 h (broken column) after temperature
upshift for 2 h at the indicated times. The phases of cell cycle in which the
majority of the synchronized cell population exist are shown under these columns
by arrows. The classification of chromosome aberration types is described under
"Materials and Methods." Lower columns, tsFT20 cells; upper columns, wild-type
39'C
32
type. The classification of the chromosome aberration types is described under
"Materials and Methods."
12
..-02-M-».—G1-»
synchronization
from
24
Fig. 7. Histograms indicating the temporal order of the appearance of various
types of chromosome aberrations in tsFT20 cells. Experiments were performed
as described in Fig. 6. Shadowed regions, period of temperature upshift (2 and 4
10-(a)(</)
andand
(g),( ;'),
Mitotic
index;
(b)(c)
andand
(A),(A),
gappulverization
or break type;type;
(c) and
type;
complex
type;
(/) (i),
andexchange
(/), ring
H
O
release
16
40
î
)
release
Fig. 6. Scheme for analysis of the temporal order of the appearance of various
types of chromosome aberrations.
mainly interchange-type chromosome aberrations, especially
chromatid interchanges and a small number of chromosome/
chromatid interchanges.
The cells shifted to the restrictive temperature during non-S
phase and wild-type cells showed few chromosome aberrations.
It was observed that the metaphases exhibiting heavy chromo
some aberrations, complex type and pulverization type, in
creased as the cells were incubated for longer period at 33°C
after temperature upshift.
Temporal Order of the Appearance of Various Types of Chro
mosome Aberrations. In order to analyze the temporal order of
the appearance of various types of chromosome aberrations,
the experiment designed as shown in Fig. 6 was performed.
The cells synchronized by the treatment of FdUrd were shifted
to 39°Cfor 2 or 4 h after the release from the FdUrd block and
then harvested at 2- or 4-h intervals to measure mitotic index
and the frequency of each type of chromosome aberration. The
results obtained with tsFT20 cells are shown in Fig. 7.
Chromosome aberrations appeared in the cells exposed to
the restrictive temperature for 2 h as the following order: gap
or break type (mode time, 8-10 h); exchange type (14-16 h);
pulverization type (18-20 h); complex type (20-22 h); and ring
type (38-40 h). Double minute-like chromosomes were often
5166
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research.
ANALYSIS OF CHROMOSOME
INSTABILITY
IN tsFT20 CELLS
X
30
a/
TJ
c
20
u
o
¡I
IO
(b)
O
20
IO
u
C
3
CT
O
20
.c
o
16
O
C
O»
D
a>
ov>
o
ok_
Time after release
IO
er
32
from synchronization
( h )
cu
Fig. 9. Schematic illustration of the fluctuation pattern of mitotic index and
various types of chromosome aberrations. The curves were drawn according to
the results shown in Fig. 7 (a-f). (a) Mitotic index; (b) chromosome aberrations;
Curve A, gap or break type; Curve B, exchange type; Curve C, complex type;
Curve D, pulverization type; Curve E, ring type.
40
20
40
Time after
release
from
synchronization
( h )
Fig. 8. Histograms indicating the temporal order of the appearance of various
types of chromosome aberrations in wild-type cells. Experiments were performed
as described in Fig. 6. The shadowed regions indicate the period of temperature
upshift (2 h). (a) Mitotic index; (A) gap or break type; (c) exchange type; (d)
complex type. The classification of the chromosome aberration types is described
under "Materials and Methods."
and exchange type (Fig. 8c), were detected at a considerable
frequency but few complex-type aberrations (Fig. Sd) and no
pulverization-type aberrations were observed in these cells. The
aberrations observed in the wild-type cells may be mainly due
to thymidylate stress by the treatment of FdUrd as described
previously (6).
For easy understanding of the temporal order of the appear
ance of various types of chromosome aberrations, Fig. 9 shows
the fluctuation pattern of mitotic index and various types of
chromosome aberrations, which was made on the basis of the
results shown in Fig. 7.
Effect of Cycloheximide on the Induction of Chromosome
Aberrations in tsFT20 Cells. It has been observed that addition
of cycloheximide prevents cells from thymineless death and
decreases the frequency of chromosome aberrations induced by
thymidylate stress (10, 19). Therefore, we examined the effect
of cycloheximide on the induction of chromosome aberrations
in tsFT20 cells exposed to the restrictive temperature.
tsFT20 cells were incubated at 39°Cfor 4 h in the presence
or absence of cycloheximide, and mitotic cells were accumulated
by an incubation at 33°Cfor 32 h in the presence of Colcemid.
observed in the cells containing ring-type chromosome aberra
tions (data not shown).
In the cells incubated for 4 h at the restrictive temperature,
the temporal order of the appearance of various types of chro
mosome aberrations was the same as that of 2-h-exposed cells
but it was observed that appearance of heavy aberrations such
as complex type and pulverization type was retarded as com
pared to that of 2-h-exposed cells. The time courses of the
appearance of chromosome aberrations in wild-type cells are
shown in Fig. 8. Slight aberrations, gap or break type (Fig. 8A)
As shown in Table 2, the induction of heavy chromosome
aberrations (complex-type) was repressed dramatically by the
addition of cycloheximide. It was found that the addition of
cycloheximide alone had little effect on the induction of chro
mosome aberrations as reported previously (10) (data not
shown).
Induction of Sister Chromaticl Exchanges in tsFT20 Cells
Exposed to the Restrictive Temperature. From many studies on
SCE, it has been suggested that SCE is a specific phenomenon
related to DNA replication (20-23). Therefore, the induction
5167
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research.
ANALYSIS OF CHROMOSOME
INSTABILITY
IN tsFT20 CELLS
Fig. 10. Concurrent occurrence of SCEs
and chromosome aberrations. Samples were
prepared as described under "Materials and
Methods." Bars, 10ptn.(a) Metaphases exhib
iting slight complex-type aberration; (/>) metaphase exhibiting heavy complex-type aber
ration; IM metaphase containing two sites of
exchange-type aberration; (¡I)control meta
phase (cultured at 33*C).
Table 2 Effect of cycloheximide on the induction of chromosome aberrations in
tsFT20 cells
Exponentially growing cells were incubated at 39'C for 4 h in the presence or
Fig. 5 clearly indicate that chromosome aberrations were in
duced only in the cells synthesizing DNA at the time when they
were exposed to the restrictive temperature. In addition, it is
absence of 3 jig/ml cycloheximide. After the cycloheximide was washed, mitotic
cells were accumulated by incubation at 33'C for 32 h in the presence of Colcemid.
apparent from the results shown in Fig. 5 that the cells in early
and middle S phase are more sensitive to the chromosomeFrequency of chromosome aberrations
damaging effect than the cells in the late S phase. It seems
of
possible that the cells in the late S phase had already proceeded
exchange2.53.03.11.83.7Gap,
break1.51.55.20.82.3
ConditiontsFT20
into G2 before the influence of temperature upshift became
cellsNon+
effective. The relatively high frequency of the induction of
chromosome aberrations in the next d phase may be due to
Cycloheximide+
Cycloheximide"Wild-type
the S-phase cells contaminated in the population because of the
decay of synchronization.
cellsNon+
The analysis of the temporal order of the appearance of
CycloheximideComplex60.50.90.400Types
various types of chromosome aberrations revealed that slightly
* Cycloheximide was added l h before the temperature upshift.
damaged aberrations such as gap or break type appear earlier
than heavily damaged aberrations such as complex and pulver
of SCEs in tsFT20 cells was examined in relation to chromo
ization type (Figs. 7 and 9). The observation that the peaks of
complex-type and pulverization-type chromosome aberrations
some aberrations.
Fig. 10 shows metaphase figures exhibiting typical complexappeared later than the peak of mitosis, which was derived from
the cells synchronized at d-S boundary, suggests that the cells
type aberrations derived from the cells which have been exposed
at 39°Cfor 4 h and then incubated at 33°Cfor 26 h. Apparently
exhibiting heavy chromosome aberrations are late in traversing
S phase. The gap- or break-type chromosome aberrations that
a large number of SCEs are observed in the aberrant chromo
somes. As for metaphases exhibiting complex-type and pulver
appeared soon after the release from the FdUrd block may be
ization-type chromosome aberrations, hyperinduction of SCE
derived from cells that were in late S phase during treatment of
was observed without exception.
FdUrd.
From the studies on chromosome aberrations induced by Xray, which is known to cause double-stranded DNA lesions, it
DISCUSSION
has been suggested that DNA lesions on chromosomes before
In this study, we have shown that tsFT20 cells, which have replication produce isochromatid-type aberrations, and DNA
lesions after replication produce chromatid-type aberrations
ts defects in the DNA replication enzyme, are rapidly commit
(24, 25). The majority of aberrations induced in tsFT20 cells
ted to cell death and exhibit extensive chromosome aberrations
were chromatid-type; therefore, it seems likely that the DNA
when the cells are exposed to the restrictive temperature. Re
markable aberrations induced in these cells were complex type, lesions that cause the aberrations were induced on newly repli
which contains a large number of chromai id gaps, breaks, and cated enromadas. However, the other possibility that singleexchanges, and pulverization type, containing a large number
stranded lesions were induced on nonreplicated DNA may also
of fragmented chromosomes. The results shown in Table 1 and be probable.
5168
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research.
ANALYSIS OF CHROMOSOME
INSTABILITY
Ring-type chromosome aberrations were often observed in
the metaphases containing double minute chromosomes which
belong to isochromatid-type aberrations (not shown). The
mechanism of formation of the pulverized chromosomes is not
clear at present; however, it must be noted that the morphology
of the pulverized chromosomes is very similar to that of pre
mature condensed chromosomes of late S-phase cells (26).
The precise mechanism by which DNA lesions are induced
in tsFT20 cells is unknown. The observations that a large
number of SCEs were induced in the cells with chromosome
aberrations (Fig. 10) and that cycloheximide inhibited the in
duction of chromosome aberrations (Table 2) may provide a
clue to consider the mechanism. It is known that SCEs are
formed via DNA double-strand breaks. One can speculate that
the mechanism which induces DNA strand breaks in tsFT20
cells is as follows. Upon temperature upshift, although DNA
replication once started can proceed normally within a replicón,
the initiation of DNA replication at replicónorigins is inhibited
due to the inactivation of DNA polymerase a as reported
previously (16). Under these conditions, replication forks may
be arrested near or at the termination points of active replicons
or replicónclusters adjacent to inactive nonreplicated replicons,
and subsequent persistence of single-stranded gaps is suscepti
ble to endonuclease attack. Furthermore, replicónorigins which
are activated to initiate replication but cannot initiate due to
limiting supply of functional DNA polymerase a may become
the target of endonuclease attack. The inhibiting effect of
cycloheximide on the induction of chromosome aberrations can
be explained by the decrease in activated replicons because the
initiation of DNA replication at replicón origins is suggested
to be dependent on protein(s) which turn over very rapidly (27,
28). However, it also may be possible that the abnormal cessa
tion of DNA replication induces an enzyme or a protein in
volved in DNA strand breaks and that cycloheximide blocks
the synthesis of such an inducible protein.
Several studies on chromosomal instability have been per
formed with mammalian cell mutants. Besides tsF'l 20 cells,
only a small number of mutants with defects in DNA poly
merase a activity have been reported (13, 29-31), and one of
the mutants, aph'-4 strain isolated from Chinese hamster V79
cells, which contains aphidicolin-resistant DNA polymerase a,
has been shown to exhibit chromosome instability as well as
hypermutability and UV sensitivity. Hori et al. (11) have re
ported that extensive chromosome aberrations were induced by
thymidylate stress in thy" mutants isolated from FM3A cells.
Under slight thymidylate stress, the induction of somatic mu
tations at two genetic loci (the 6-thioguanine-resistant
locus
and the ouabain-resistant locus) was observed in the thy" mu
tant (32). In addition, it is well known that the interruption of
DNA synthesis by the treatment of inhibitors for DNA precur
sor synthesis induces chromosome instability and sometimes
results in gene amplification to produce drug-resistant mutants
(33). However, in our tests, the induction of mutation in tsFT20
cells by incubation at 39°Cwas not observed at least at the
ouabain locus.
As to production of SCEs, several models have been pro
posed. Painter (23) proposed a model based on the idea that
double-strand breaks are generated at junctions between a com
pletely replicated replicón cluster and a partially replicated
replicón cluster. This model seems to fit the case of tsFT20
cells. Therefore, tsFT20 cells may become a good tool for the
analysis of the mechanism of formation of SCEs as well as the
analysis of the molecular mechanism of eukaryotic DNA rep
lication.
IN tsFT20 CELLS
ACKNOWLEDGMENTS
We thank Y. Eguchi for his technical assistance.
REFERENCES
1. Cohen, S. S., and Barner, H. D. Studies on unbalanced growth in Escherichia
coli. Proc. Nati. Acad. Sci. USA, 40: 885-893, 1954.
2. Barclay, B. J., Kunz, B. A., Little, J. G., and Haynes, R. H. Genetic and
biochemical consequences of thymidylate stress. Can. J. Biochem., 60: 172194, 1982.
3. Borsa, J., and Whitmore, G. J. Cell killing studies on the mode of action of
methotrexate on L-cells in vitro. Cancer Res., 29: 737-744, 1969.
4. Hrynick, W. M., and Berlino, J. R. Growth rate and cell kill. Ann. NY Acad.
Sci., 186: 330-342, 1971.
5. Reuckert, R. R., and Mueller, G. C. Studies on unbalanced growth in tissue
culture. I. Induction and consequences of thymidine deficiency. Cancer Res.,
20:1584-1591,1960.
6. Taylor, J. H., Haut, W. F., and Tung, J. Effect of fluorodeoxyuridine on
DNA replication, chromosome breakage and reunion. Proc. Nati. Acad. Sci.
USA, 4«:190-198, 1962.
7. Ayusawa, D., Koyama, H., Iwata, K.. and Seno, T. Single-step selection of
mouse FM3A cell mutants defective in thymidylate synthase. Somatic Cell
Genet., 6: 261-270, 1980.
8. Ayusawa, D., Koyama, H., Iwata, K., and Seno, T. Selection of mammalian
thymidine auxotrophic cell mutants defective in thymidylate synthase by
their reduced sensitivity to methotrexate. Somatic Cell Genet., 7: 523-534,
1981.
9. Little, J. G., and Haynes, R. H. Isolation and characterization of yeast
mutants auxotrophic for 2-deoxythymidine-5'-monophosphate.
Mol. Gen.
Genet., J68: 141-151, 1979.
10. Ayusawa, D., Shimizu, K., Koyama, H., Takeishi, K., and Seno, T. Accu
mulation of DNA strand breaks during thymineless death in thymidylate
synthase-negative mutants of mouse FM3 A cells. J. Biol. Chem., 258:1244812454, 1983.
11. Hori, T., Ayusawa, D., Shimizu, K., Koyama, H., and Seno, T. Chromosome
breakage induced by thymidylate stress in thymidylate synthase-negative
mutants of mouse FM3A cells. Cancer Res., 44: 703-709, 1984.
12. Murakami, Y., Yasuda, H., Miyazawa, H., Hanaoka, F., and Yamada, M.
Characterization of a temperature-sensitive mutant of mouse FM3A cells
defective in DNA replication. Proc. Nati. Acad. Sci. USA, 82: 1761-1765,
1985.
13. Tsai. Y.-.I., Hanaoka, F., Nakano, M. M., and Yamada, M. A mammalian
DNA" mutant decreasing nuclear DNA polymerase a activity at nonpermis
sive temperature. Biochem. Biophys. Res. Commun., 91:1190-1195, 1979.
14. Yasuda, H., Matsumoto, Y., Mita, S., Marunouchi, T., and Yamada, M. A
mouse temperature-sensitive mutant defective in HI histone phosphorylation
is defective in deoxyribonucleic acid synthesis and chromosome condensa
tion. Biochemistry, 20:4414-4419, 1981.
15. Murakami, Y., Eki, T., Miyazawa, H., Enomoto, T., Hanaoka, F., and
Yamada, M. Further characterization of a murine temperature-sensitive
mutant, tsFT20 strain, containing heat-labile DNA polymerase a activity.
Exp. Cell Res., 163: 135-142, 1986.
16. Eki, T., Murakami, Y., Enomoto, T., Hanaoka, F., and Yamada, M. Char
acterization of DNA replication at a restrictive temperature in a mouse DNA
temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA
polymerase a activity. J. Biol. Chem., 261:8888-8893, 1986.
17. Nakano, N. Establishment of cell lines in vitro from a mammary ascites
tumor of mouse and biological properties of the established lines in a serumcontaining medium. Tohoku J. Exp. Med., 88: 69-84, 1966.
18. Perry, P., and Wolff, S. New Giemsa method for the differential staining of
sister chromatids. Nature (Lond.), 251: 156-158, 1974.
19. Hori, T., Ayusawa, D., and Seno, T. Thymidylate stress and sister chromâtid
exchanges. In: R. R. Tice and A. Hollaender (eds.), Sister Chromatid Ex
changes, pp. 149-159. New York: Plenum Publishing Corporation, 1984.
20. Cleaver, J. E. Correlation between sister chromatid exchange frequencies and
replicón sizes. A model for the mechanism of SCE production. Exp. Cell
Res., 136: 27-30, 1981.
21. Ishii, Y., and Bender, M. Effects of inhibitors of DNA synthesis on sponta
neous and ultraviolet light-induced sister-chromatid exchanges in Chinese
hamster cells. Mutât.Res., 79: 19-32, 1980.
22. Kato, H. Mechanisms for sister chromatid exchanges and their relation to
the production of chromosome aberrations. Chromosoma (Beri.), 59: 179191, 1977.
23. Painter, R. B. A replication model for sister-chromatid exchange. Mutât.
Res., 70:337-341, 1980.
24. Evans, H. J. Chromosome aberrations induced by ionizing radiations. Int.
Rev. Cytol., 13:221-321, 1962.
25. Savage, J. R. K. Classification and relationships of induced chromosomal
structural changes. J. Med. Genet., 13: 103-122, 1975.
26. Hirschberg, J„and Marcus, M. Isolation by a replica-plating technique of
Chinese hamster temperature-sensitive cell cycle mutants. J. Cell. Physiol.,
113:159-166,1982.
27. Fujiwara, Y. Effect of cycloheximide on regulatory protein for initiating
DNA replication at the nuclear membrane. Cancer Res., 32: 2089-2095,
1972.
5169
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research.
ANALYSIS OF CHROMOSOME
INSTABILITY
28. Hori, T., and Lark, K. G. Effect of puromycin on DNA replication in Chinese
hamster cells. J. Mol. Biol., 77: 391-404, 1973.
29. Liu, P. K., Chang, C.-C, Trosko, J. E., Dube, D. K., Martin, G. M., and
Loeb, L. A. Mammalian mutator mutant with an aphidicolin-resistant DNA
polymerase a. Proc. Nati. Acad. Sci. LISA, 80: 797-801, 1983.
30. Nishimura, M., Yasuda, H.. Ikegami, S., Ohashi, M., and Yamada, M.
Aphidicolin resistant mutant of which DNA polymerase a is induced by this
drug. Biochem. Biophys. Res. Commun., 91: 939-945. 1979.
IN tsFT20 CELLS
31. Sugino, A., and Nakayama, K. DNA polymerase a mutants from a Drosophila
melanogaster cell line. Proc. Nati. Acad. Sci. USA, 70: 245-248, 1980.
32. Koyama, H., Ayusawa. D., Tsuji, M., and Seno. T. Thymineless death and
mutation induction in cultured mouse FM3A cell mutants deficient in thymidylate synthase. Mutât.Res., 105:433-438, 1982.
33. Shimke, R. T. Gene amplification in cultured animal cells. Cell, 37: 705713, 1984.
5170
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1987 American Association for Cancer Research.
Characterization of Chromosome Aberrations Induced by
Incubation at a Restrictive Temperature in the Mouse
Temperature-sensitive Mutant tsFT20 Strain Containing
Heat-labile DNA Polymerase α
Toshihiko Eki, Takemi Enomoto, Yasufumi Murakami, et al.
Cancer Res 1987;47:5162-5170.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/47/19/5162
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 17, 2017. © 1987 American Association for Cancer Research.