[CANCER RESEARCH 58. 2036-2041.
May 1. 1998]
Absence of a Radiation-induced First-Cycle Gj-S Arrest in p53+ Human Tumor
Cells Synchronized by Mitotic Selection1
Ilalsumi Nagasawa, Peter Keng, Carl Maki, Yongjia Yu, and John B. Little2
Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115 [H. N., Y. Y., J. B. L.¡;Department of Pathology, Harvard Medical School,
Boston, Massachusetts ¡C.M.¡;and Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 [P. K.]
ABSTRACT
G0-G, arrest; these strains included moderately radiosensitive human
skin fibroblasts from patients with retinoblastoma (7, 8) and AT3
It is well known that normal human diploid fibrobtasts undergo a
significant, p53-dependent arrest in the G, phase of the cell cycle after
exposure to ionizing radiation. The presence and magnitude of a G, arrest
in human tumor cell lines, however, has been controversial, particularly in
cells derived from solid tumors and irradiated during exponential growth.
To examine this question more precisely, we synchronized cells by mitotic
selection and irradiated them in very early G, prior to any of the de
scribed G, checkpoints. Progression of cells from G, into the S phase was
monitored by autoradiographic measurement of cumulative labeling in
dices and by flow cytometric analysis. Three different human tumor cell
lines confirmed as expressing normal p53 function were examined, i.e.,
lines derived from an adenocarcinoma of the colon (RKO), a breast cancer
(MCF-7), and a squamous cell carcinoma (SCC61). Following irradiation
with 4-8 Gy, there was a transient delay in progression from G, into S
phase, lasting approximately 2 h, and in two of the three cell lines (RKO
and MCF-7), a small fraction of cells (5-8%) never entered the first S
hétérozygotes
(9, 10), as well as others (11), all of which express
wild-type p53. Cells derived from AT homozygotes showed neither
the G,-S delay nor the G0-G, arrest (7-10). Similarly, no radiationinduced G | delay or G0-G, arrest was observed for human diploid
fibroblasts transfected with the human papilloma virus E6 gene (11-
phase. Although there was no evidence for a prolonged G, arrest, the
expected G, delay was observed in all three cell lines. When irradiated
RKO cells were resynchronized at the next mitosis, approximately 30% of
the cells did not enter the second S phase. This latter finding is consistent
with earlier reports on the kinetics of radiation-induced
reproductive
failure in mammalian cells. These results indicate that cells derived from
human solid tumors that express normal p53 may respond to irradiation
quite differently than do normal cells in terms of G] checkpoint control.
The occurrence and nature of the G, arrest in human tumor cells,
however, is much less clear. It is well known that tumor cells undergo
multiple genetic changes involving loss of normal growth controls
during the development of a fully malignant, invasive tumor (16).
However, several investigators have reported the occurrence of a
prolonged, radiation-induced first-cycle G, arrest that is p53 depend
ent in asynchronously growing cell lines derived from human lymphomas (17-21) and solid tumors (22-24). We, on the other hand,
have been unable to demonstrate the presence of such a prolonged
first cycle G, arrest in p53+ human tumor cell lines irradiated during
INTRODUCTION
Information on radiation-induced cell cycle delays at the G,-S and
G2-M checkpoints in mammalian cells has been accumulating for over
30 years (1). In some studies with transformed rodent cells and HeLa
cells exposed to moderate doses of ionizing radiation, a transient
reduction that lasted for 1-4 h occurred in the rate at which cells
progressed from G, into S phase (2-4), particularly when synchro
nized cells were irradiated in G, (3, 4), whereas in other studies, no
such reduction was observed (1,5). On the other hand, a prolonged G,
arrest occurred in slowly growing human diploid fibroblasts following
exposure to 3-10 Gy, an arrest that, in some cases, lasted for several
days (6).
In later studies with human diploid fibroblasts, the semisynchronous movement of cells from G0-G, into S phase was examined in
cells irradiated and then released from the confluent, density-inhibited
phase of growth (7-10). Two distinct phenomena emerged, (a) A
dose-dependent delay in the movement of cells from G, into S phase
occurred in irradiated as opposed to nonirradiated cultures; the delay
was on the order of 4 h with a radiation dose of 4 Gy. (b) A significant
fraction of the cells appeared to be irreversibly arrested in G0-G,. In
some cell strains, as many as 80% of the cells were subject to this
Received 10/20/97; accepted 3/3/98.
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.
1 This work was supported by NIH Grants CA^t7542 and CA73725 and Center Grant
ES-00002; Massachusetts Department of Public Health Grant 3408799 DO35; and a
British Cancer Research Award (to C. M.).
2 To whom requests for reprints should be addressed, at Harvard School of Public
Health, 665 Huntington Avenue, Boston, MA 02115.
13) or in fibroblasts that had lost p53 function derived from a patient
with the Li-Fraumeni syndrome (11). When cells from normal indi
viduals were released from density-inhibited growth and then irradi
ated near the G,-S border, no radiation-induced G, delay or G[ arrest
occurred (14), presumably because the cells had progressed beyond
the critical checkpoints in G,. On the basis of these findings, it has
been proposed that the expression of functional p53 plays an impor
tant role in G, checkpoint control and the induction of an irreversible
arrest in irradiated human diploid fibroblasts (11-13, 15).
exponential growth and examined by flow cytometry (25, 26) or
following irradiation of density-inhibited G, phase cells subsequently
released and examined by continuous labeling autoradiographic tech
niques (27).
To gain definitive information on the effects of radiation on the
progression of cells from G, into S phase during the first postirradiation cell cycle, we studied highly synchronized tumor cell popula
tions obtained by mitotic selection and irradiated in very early G,
prior to any of the described G, checkpoints (28). Cell lines derived
from three different types of human solid tumors were used, all of
which were confirmed to express wild-type p53. Although there was
a transient delay in progression from G, into S phase, lasting approx
imately 2 h, there was no evidence for a prolonged radiation-induced
G, arrest in any of these tumor cell lines.
MATERIALS
AND METHODS
Cell Culture and Synchrony. Three human tumor cell lines with normal
p53 function (MCF7, RKO, and SCC61) were used in these studies. They were
cultured in Eagle's MEM supplemented with 10% heat-inactivated FBS (56°
for 30 min), penicillin (100 unit/ml), and streptomycin (100 /*g/ml). Diploid
G, phase cells were separated from the original tumor cell populations by
centrifugal elutriation prior to use for experiments, to eliminate nondiploid
cells that could confound flow cytometric analyses. These actively growing
diploid cells were subcultured into T-150 plastic tissue culture flasks (Costar
3150) 2 days before synchronization
by selection of mitotic cells.
3 The abbreviations used are: AT, ataxia telangiectasia;
propidium iodide; BrdUrd, 5-bromo-2'-deoxyuridine.
FBS, fetal bovine serum; PI,
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NO G,-S ARREST IN p53 HUMAN TUMOR CELLS
Synchronous cells were obtained by shaking loose the mitotic cells from the
actively growing asynchronous cultures (29, 30). The shaking procedure (2-4
shakes/s on a reciprocating platform shaker for a total of 10-20 s, depending
upon the cell line) was carried out at 37°C in a walk-in incubator. The
synchronous cells were immediately cooled to 0-4°C by pouring the medium
aP
* fis*
.'i,
**fc
**"*
S*** ,J»•
containing the mitotic cells into a glass Erlenmyer flask placed in an ice bath.
Ten ml of conditioned medium were then added to the T-150 culture flasks in
preparation for the next shake. Conditioned medium was obtained from near
confluent cultures 24 h after incubation in fresh medium. The shaking proce
dure was repeated every 10 min until sufficient cells were collected (3-5 X IO6
cells for autoradiographic and IO7 cells for flow cytometric analysis). At each
shake, the cell suspension was added to the Erlenmyer flask in the ice bath. An
aliquot of the cell suspension was removed after each shake and monitored for
the cell count and percentage mitotic cells. If the mitotic index was less than
80%, the cell suspension from that shake was discarded. The pooled suspen
sion of mitotic cells was concentrated immediately after the last shake by
centrifugation at 1000 rpm (300 X g) for 10 min. The mitotic indices were
80-99.5%, as shown in Table 1. A representative photomicrograph of such a
IfcjA
" ^
'.**
(30). To control for any effects of the procedure whereby mitotic cells were
accumulated and pooled, the cells in some experiments were studied immedi
ately after a single mitotic shake. The results of these latter experiments did not
differ from those with the pooled suspension derived from multiple shakes.
Cell Cycle Analysis. The mitotic cells were seeded onto 35-mm plastic
Petri dishes in complete medium containing 1 /j.Ci/ml [3H]thymidine (specific
activity, 20 Ci/mmol) at 37°Cto score the cumulative labeling index as a
^.'
Then, cells were treated with 1 ml of RNase (1 mg/ml) prior to being
counterstained with PI (10 /Ag/ml) for flow cytometric analysis (31).
Fluorescence distributions of at least 10,000 cells/sample were collected by
a EPICS Elite ESP flow cytometer. The distribution of green fluorescence from
FITC expressed on a logarithmic scale was collected as a measure of BrdUrd
content, and the distribution of red fluorescence from PI on a linear scale was
collected as a measure of DNA content. Filters used were 550-nm dichroic,
525-nm band pass for green fluorescence and 610-nm band pass for red
measure of the progression of G, cells into S. The cells were irradiated 1-4 h
after seeding when cytokinesis was complete with a ^Co y ray source (United
States Nuclear model GR-9) at a dose rate of 0.12 Gy/sec. At regular intervals
after seeding, one dish was removed, and the cells were fixed directly on the
dish (7). Kodak nuclear emulsion NTB2 (Eastman Kodak, Rochester, NY) was
applied to the fixed dishes for autoradiography by standard techniques, as
described previously (7). After development, the cells were stained through the
emulsion with 0.1% crystal violet; labeled cells were clearly recognized, and
200 total cells were scored on each dish to determine the labeling index.
Flow Cytometric Analysis. For analysis of cell cycle distributions of
control and irradiated samples by PI staining, cells were removed by
trypsinization at various times after irradiation, washed twice with PBS, and
then fixed in 75% ethanol for 24 h before DNA analysis. After the removal of
ethanol by centrifugation, cells were treated with RNase (1 mg/ml) for 30 min
and then stained with PI (10 ¿¿g/ml)
for 15 min. DNA fluorescence of
Pi-stained cells was measured with an EPICS Elite ESP flow cytometer/cell
fluorescence.
Two-parameter
distributions
of BrdUrd content and DNA content were
generated using the Elite program (Coulter Electronics) on a PC workstation.
The actual percentage of BrdUrd-positive cells, as defined by the box shown
in Fig. 5, was determined by subtracting the percentage of cells from the
negative control. The distribution of G,, S, and G2-M cells in each sample was
analyzed by the Multicycle AV program (31).
Western Blot Analysis. Exponentially growing cells were irradiated with
6 Gy. Protein extracts were prepared 5.5 h after irradiation treatment (32).
Cells were washed twice with PBS, scraped into 0.5 ml of lysis buffer [50 mM
Tris (pH 8.0), 5 iriM EDTA, 150 mM NaCl, 0.5% NP40, and 1 mM phenylmethylsulfonyl fluoride] and incubated on ice for 15 min with occasional mild
vortexing. Cells were then sonicated for 10 pulses at setting 5, 50% output,
with a Branson 450 sonifier. Lysates were spun at 15,000 X g for 15 min to
remove cellular debris. One hundred fig of protein extract from the resulting
supernatants were resolved by SDS-PAGE and transferred to Immobilon-P
membranes (Millipore) for detection with the p53 monoclonal antibody Ab-6
sorter (Coulter Electronics, Hialeah, FL). An argon ion laser operating at a
488-nm wavelength and 15 mW was used for excitation. DNA fluorescence
was monitored through a 610-nm band pass filter. The percentages of G,, S,
and G2-M cells were determined from the DNA histograms using the Multiple
AV program of the Phoenix Row System (San Diego, CA).
For two-parameter analysis of cell cycle progression, cells were continu
ously cultured with IO"6 M BrdUrd in complete medium. At various times after
(Oncogene Science) or the p21 polyclonal antibody 15431E (PharMingen; Ref.
32).
PCR/Single-Strand Conformation Polymorphism Analysis. Five exons
(exons 5-9) of the p53 gene were amplified by PCR using oligonucleotide
primers as follows: exon 5, 5'-TTATCTGTTCACTTGTGCCC-3'
(forward)
and 5'-TCATGTGCTGTGA
CTGCTTG-3' (reverse); exon 6, 5'-ACGACAGGG CTGGTTGCCCA-3'
(forward) and 5'-CTCCCAGAGACCC
CAGTTGC-3' (reverse); exon 7, 5'-GGCCTCATCTTG
GGCCTGTG-3' (forward)
and 5'-CAGTGTGCAGGGTGG
CAAGT-3' (reverse); exon 8, 5'-CTGCCTCTTGCTTCTCTTTT-3'
(forward) and 5-TCTCCTCCACCGCTTCTTGT-3' (reverse); and exon 9, 5'-GCAGTTATGCCTCAGATTCA-3'
(for-
labeling, cells were removed from flasks by trypsin and fixed in 75% ice-cold
ethanol. The DNA needed to be denatured to allow the anti-BrdUrd antibody
to access. Therefore, cell pellets were resuspended and incubated in 3 ml of
pepsin-HCl solution (0.2 mg/ml pepsin in 2 N HC1) at 37°Cfor 20 min. After
PBS with 0.5% FBS and
in 1 ml of PBS with 2%
cells were repelleted and
Mannheim) for 45 min.
Table 1 Characteristics
I * 4.
Fig. 1. Photomicrograph of the SCC61 cell population immediately after mitotic
selection. Cells were fixed with 50% acetic acid, stained with 2% aceto-orcein, and then
squashed. Nearly 100% of the cells are in mitosis.
synchronized population is shown in Fig. 1. We verified that most of these
mitotic cells completed cytokinesis within l h at 37°C,as previously reported
denaturation, cells were permeabilized by 1 ml of
0.5% Tween 20, spun down, and then resuspended
FBS for 20 min to block nonspecific binding. The
stained with 10 /ml anti-BrdUrd/FITC (Boehringer
*
of human tumor cell lines
p53 status
Cell line
Origin
RKOMCF7SCC61Colon
adenocarcinomaBreast
SSCP°-'
Function'
MPDT (h)
Radiosensitivity,
D0 (Gy)
Mitotic index after selection (%)
+WT
+WT
adenocarcinomaSquamous
+1520201.050.951.0785-9380-8595-99.5
cell carcinomaWT
a Five exons (exons 5-9) of the p53 gene were amplified by PCR using oligonucleotide primers, as described previously (20).
SSCP, single-strand conformation polymorphism; MPDT, mean population doubling time; WT. wild type.
'' Induction with 6 Gy of 7 irradiation (Fig. 2).
2037
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NO G,-S ARREST IN p53 HUMAN TUMOR CELLS
SCC-61
RKO
MCF-7
Fig. 2. Western analysis of p53 and p2l protein levels 5.5 h after irradiation with 0
(controls) or 6 Gy. Significant induction of p53 and p2l protein expression by radiation
was verified by densitometry in all cases (ratios of >2), with the exception of p53 in the
SCC61 cell line (ratio = 1.0).
ward) and 5'-GGCATTTTGAGTGTTAGACT-3'
(reverse). Twenty ml of
PCR mixture contained 200 ng of sample DNA; 2.5 mCi of [35S]dATP (1069
Ci/mmol; DuPont New England Nuclear, Boston, MA); 20 inM dATP; 200 mM
dCTP. dGTP, and dTTP; 10 pmol of each primer; 10 mM Tris-HCl (pH 8.3);
50 mM KC1; 1.5 mM MgCl,; and 1 unit of AmpliTaq DNA polymerase
(Perkin-Elmer/Cetus,
Norwalk, CT). After 30 cycles of reaction (1 min at
94°C,1 min at 58°C,and 1 min at 72°C),the PCR product was analyzed on
a 6% polyacrylamide gel with 10% glycerol. The gel was run for 16 h at 6 W
and was exposed for 15 h for autoradiographic detection of bands (33).
RESULTS
The three p53+ tumor cell lines studied were derived from a
squamous cell carcinoma (SCC61), an adenocarcinoma of the colon
(RKO), and an adenocarcinoma of the breast (MCF-7). These lines are
all relatively radiosensitive as compared with those from many human
tumors (34), with the D0 (inverse of the slope of the survival curve)
ranging from 0.95 to 1.07 Gy (Table 1). Their wild-type p53 status
was confirmed by single-strand conformation polymorphism analysis
of exons 5-9, the DNA-binding region (Table 1). Each of these cell
lines was irradiated, and p53 and p21 were levels examined by
Western blot analysis. As can be seen in Fig. 2, p53 and p21 protein
levels were significantly increased in RKO and MCF-7 cells 5.5 h
after irradiation, the time of maximal induction. Although SCC61
cells showed little or no enhancement in p53 protein levels after
irradiation, there was a significant enhancement in p21 expression
postirradiation. To examine the effect of irradiation on the progression
of cells from G, into S phase (G, phase checkpoint), synchronized cell
populations obtained by mitotic selection were incubated with
[3H]thymidine and irradiated 1, 2, or 4 h later when cytokinesis was
complete, and their movement into S phase monitored by measuring
the cumulative labeling index by autoradiography. As can be seen in
Table 2, where the maximum cumulative labeling indices are shown,
5 and 8% of RKO and MCF7 cells, respectively, did not enter the first
S phase after irradiation with 4 Gy l h after mitosis. Although there
was a small difference in the fraction of MCF-7 cells arrested in G,
after irradiation at 4 h as compared with l h after mitosis, the
maximum cumulative labeling indices appeared otherwise to be in
dependent the time of irradiation up to 4 h after mitosis.
The effect of increasing radiation doses on the entry of cells into S
was examined in RKO cells. As can be seen in Table 3, there was no
significant increase in the fraction of cells arrested in the first G,
phase with increasing doses of radiation; the slight increase seen
following 8 Gy is not statistically significant. When synchronized
RKO cells with and without irradiation reached the second mitosis,
the mitotic cells were harvested by a second mitotic selection, and the
movement of cells through the second postirradiation cell cycle was
monitored. Of the cells initially irradiated with 6 Gy, 28% never
entered the S phase during the second cell cycle after irradiation
(Table 3).
The kinetics of progression of cells from G, into S during the first
cycle were monitored by measuring the cumulative labeling index at
multiple time points up to 80 h following mitotic selection and
irradiation 1 h later with 6 Gy. Results for the RKO and SCC61 cell
lines are shown in Fig. 3. As can be seen, there was a transient delay
of approximately 2 h in the movement of cells into the S phase in both
cell lines. In addition, as shown in Table 2, a small fraction of RKO
cells never entered S phase, as evidenced by the maximum continuous
labeling index.
The progression of cells through the entire life cycle was examined
up to 27 h postirradiation by flow cytometric techniques. As can be
seen in Fig. 4, synchronous RKO and SCC61 cells exhibited a single
peak of G, DNA content at 2 or 3 h after mitosis (Fig. 4, A, E, I, and
M). These G, cell populations progressed into the S phase over the
next 6-9 h (Fig. 4, B, F, J, and A/), the irradiated cells showing
evidence of a slight transient delay in progression from G, into S
phase, consistent with the results in Fig. 3. Nonirradiated cells had
begun entering the second cell cycle G, phase by 18 h after mitosis
(Fig. 4, C and K, and Fig. 5A) and subsequently progressed into the
second-cycle S phase (Fig. 4, D and L, and Fig. 5ß).Cells irradiated
with 6 Gy not only showed a slight transient delay in their movement
from G | into S phase but also displayed a characteristic G,-phase
arrest 18 h after mitosis (Fig. 4, G and O, and Fig. 5Q. These
G2-arrested cells moved slowing into the second cell cycle G, phase
by 24-27 h after original mitotic selection (Fig. 4, H and P, and Fig.
5D). Two-parameter analysis with continuous labeling with BrdUrd
(Fig. 5) indicated that a small fraction of cells did not incorporate
BrdUrd in the irradiated cell populations, as compared with controls;
however, the percentage of noncycling cells was less than 10% in each
Table 3 Effects of different radiation doses on maximum cumulative labeling index
after release of RKO cells from mitotic synchronization"
Dose(cGy)0
cycle95.0cell
cycle98.3 cell
±4.6(100)
±1.0(100)
95.1 ±0.9(96
±0.7)94.0
4
±1.9(95 ±1.7)
70.2 ±1.2(72± 1.2)
6gFirst
88.5 ±5.3 (89 ±5.4)Second
" Values given are mean percentage ±SE of results from three or more independent
experiments. Figures in parentheses are values normalized to 100% for 0 dose controls.
Table 2 Maximum cumulative labeling index in control cells and after irradiation (4 Gy) al various times after release from mitotic synchronization"
Time of irradiation after release from mitosis
Cell line
Control
Ih
2h
4h
92.4 ±2.4(100)
87.3 ±3.6 (95 ±2.0)
87.5 ±3.6 (95 ±2.0)
SCC61
95.8 ±0.8(100)
92.4 ±0.2(98 ±1.0)
95.4 ±1.2 (100 ±1.0)
95.3 ±1.0(98 ±1.0)
95.9 ±1.16(100)
MCF7
87.5 ±1.7(92± 1.5)
90.1 ±1.55 (94 ±1.2)
93.1 ±0.186(97 ±1.3)
" Values given are mean percentage ±SE of results from three or more independent experiments. Figures in parentheses are values normalized to 100% for controls for each
individual experiment ±SE.
RKO
2038
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NO G,-S ARREST IN p53 HUMAN TUMOR CELLS
RKO
SCC61
100-eo-eo-4020-O-ln"»U"11glIJE«•Q'i.
.......i
10
20
30
40
50
Time after Mitosis ( HRS )
10
20
30
40
50
II
60
70
80
Time after Mitosis ( HRS }
Fig. 3. Cumulative labeling indices after release of RKO and SCC61 cells, synchro
nized by mitotic selection and irradiated l h later. D, controls; •6-Gy y rays.
of the tumor cell lines, in agreement with the autoradiographic results
in Tables 2 and 3 and Fig. 3.
DISCUSSION
In early investigations, it was shown that the primary mode of death
after irradiation of most proliferating mammalian cells was reproduc
tive failure; that is, loss of the capacity of cells to continue dividing
and form macroscopic colonies in vitro (35). Interestingly, irradiated
cells undergoing reproductive failure could progress through one or
more rounds of cell division before mitosis ceased (35-37). The
number of division cycles nonsurviving cells could complete was dose
dependent (36, 37). Many cells did not die during mitosis, however,
but eventually became arrested in G, (38), where they could persist as
viable cells for periods of many days (36).
In these early experiments, a reduction in the rate at which cells
progressed from G, into S phase was reduced in some cell lines (2),
whereas in others, no effect on entry into S phase was demonstrated
(39, 40). However, all of these studies were carried out with either a
human tumor cell line (HeLa) or various transformed rodent cell lines.
When the progression of irradiated cells from G, into S phase was
examined in human diploid fibroblasts, a prominent G, arrest was
observed; following doses as low as 1-4 Gy, up to 60% of the
population became irreversibly arrested in the first postirradiation G,
phase (6-8). Recently, this irreversible G, arrest has been shown to
be p53 dependent (11-13). Moreover, Linke et al. (15) showed that a
significant fraction of the irradiated cells that escaped arrest in the first
cell cycle become irreversibly arrested in the second G, phase; this
effect continued for at least three rounds of cell division. The pro
gressive loss of damaged cells from the population over multiple
generations accounts for the enhanced radiosensitivity of human dip
loid fibroblasts expressing wild-type p53. The loss of cells in subse
quent cycles occurred but to a much lesser extent in p53~ cells (15).
The response to irradiation of cell lines derived from human solid
tumors, however, appears to differ significantly. First, unlike the case
with normal cells, clear evidence is lacking for a systematic effect of
p53 expression on radiosensitivity. Although Mcllwrath et al. (41)
reported that six tumor lines showing normal p53 function were
significantly more radiosensitive than were cell lines with p53 muta
tions, the tumor types in the two groups differed completely. When
Brachman et al. (42) examined the radiosensitivity of 24 tumor cell
lines derived from squamous cell carcinomas of the head and neck,
they found no difference in the range of radiosensitivities for the nine
lines expressing wild-type p53, compared with those with mutant p53.
Furthermore, abrogation of p53 function in several human tumor cell
lines expressing wild-type p53 by transfection with the human papilloma virus E6 gene led to no significant change in their radiosensi
tivity (19, 43, 44).
The function of the G, checkpoint in irradiated cell lines derived
from human solid tumors, as opposed to lymphomas or cells of
hematopoietic origins, has also been controversial. Kuerbitz et al. (22)
initially reported that irradiation of several tumor cell lines during
exponential growth induced a prolonged, transient first-cycle G, arrest
lasting 24 h or longer. On the other hand, we have been unable to find
evidence for such an arrest either in exponentially growing tumor cells
RKO
SCC61
Control
6Gy
Control
6 Gy
A
2Hr
E
2Hr
I
3Hr
M
3hr
B
6Hr
F
6Hr
J
9Hr
N
9Hr
fi
Fig. 4. Flow cytometric profiles of Pi-stained single cell populations.
RKO (¡eft)
and SCC61 (right) cells were synchronized by mitotic selec
tion, released, and irradiated l h later, then examined at the times shown
(A-P).
m
3
G
18 Hr
C
18 Hr
HI
U
O
18 Hr
H
24 hr
100
Gì G2/M
200
0
100
Gt
DMA CONTENT
GilM
200
100
Gì
200
G!/M
0
100
Gì
200
Gi/M
DMA CONTENT
2039
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NO O,-S ARREST IN p53 HUMAN TUMOR CELLS
Control
6Gy
18 Hr
18 Hr
showed a prominent radiation-induced G, block; however, we found
no evidence for a significant G, arrest in a culture of this cell line
obtained from the American Type Culture Collection. Interestingly,
when cells from the RKO line were resynchronized at the first mitosis
after irradiation, approximately 30% of the cells appeared to be
irreversibly blocked in the second postirradiation G, phase. This
finding is consistent with earlier observations reported by Hurwitz and
Tolmach (36, 48), who found with HeLa cells that a similar fraction
of the cells irradiated with 4 Gy completed only one round of cell
division, becoming reproductively inactive in the second cycle.
It is now generally accepted that tumor cells have undergone a
multiplicity of genetic changes that permit them to escape normal
growth controls. It is not unexpected, therefore, that normal G,
checkpoint function would be reduced or absent in tumor cell lines. It
is of interest in this context, however, that the function of the G2
checkpoint did not appear to be reduced or abrogated in these human
tumor cells.
1000
•too
O
1°:
S
O
B
•O
m
"•C
100
24 Hr
24 Hr
10
ACKNOWLEDGMENTS
We thank K. Moss and R. Harley for technical assistance.
0.1
20
GìS
40
G2/M
60
20
Gì S
40
G2/M
60
REFERENCES
DNA CONTENT (PI)
Fig. 5. Flow cytometric profiles of two-parameter distributions of continuous BrdUrdlabeled RKO cells from the same experiment shown in Fig. 4. Control and 6-Gy-irradiated
cells were synchronized by mitotic selection and irradiated l h later. BrdUrd-positive cells
are shown inside the gating box. A and C, 18 h; ßand D, 24 h postirradiation.
studied under similar conditions (25) or following irradiation of
density-inhibited cells, subsequently released by subculture to low
density and monitored for progression to S phase by autoradiography
(27). In cells studied during exponential growth, it may be argued that
most cells in the population will be beyond the Gt checkpoint, thus
masking the effect in the small population in early G,. Furthermore,
it is conceivable that the colcemid and [3H]thymidine added to the
cultures to arrest them in the first mitosis (and thus prevent contam
ination of the flow cytometric profiles by second-cycle G, cells) could
influence the results. In the second type of experiment, the incubation
of the cells in low serum, as is required for tumor cells to attain
density inhibition of growth, could result in cells being distributed
throughout G, rather than in early G,, thus bypassing a critical G,
checkpoint. Thus, we carried out the present experiments in which the
entire cell population was synchronized in very early G, by mitotic
selection. Clearly, two of the cell lines examined showed normal p53
function (Fig. 2). The lack of apparent induction of p53 by radiation
in the SCC61 line may indicate a p53-independent induction of p21
(45). MDM-2 expression was also low in this cell line.
As is evident in Table 1, the mitotic selection technique we have
used provides nearly pure mitotic cell populations and thus permits
the study of a highly synchronized cell population in very early G,
without the use of any chemical inhibitors or growth manipulations.
This high degree of synchrony is visually represented in Fig. 1. When
cells were irradiated immediately after cytokinesis was complete, the
cells moved synchronously into S phase 7-10 h later. As can be seen
in Fig. 3, a transient delay of progression from Gj into S phase of
approximately 2 h occurred in irradiated cells, consistent with the
results reported by Pellegata et al. (46). There was clearly no pro
longed G, arrest, although in two of the three tumor cell lines
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Absence of a Radiation-induced First-Cycle G1-S Arrest in p53+
Human Tumor Cells Synchronized by Mitotic Selection
Hatsumi Nagasawa, Peter Keng, Carl Maki, et al.
Cancer Res 1998;58:2036-2041.
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