(CANCER RESEARCH 58, 2817-2824.
July I. 1998]
Explaining Differences in Sensitivity to Killing by Ionizing Radiation between
Human Lymphoid Cell Lines
David R. Aldridge and Ian R. Radford1
Sir Donald and Lady Trescowihick Research Laboratories, Peter MacCallum Cancer Institute, St. Andrews Place, East Melbourne, Victoria 3002, Australia
ABSTRACT
sensitivity. These studies suggested that sensitivity to clonogenic
killing by y-rays or DNA-associated 12il decays correlated closely
We surveyed five human hematopoietic cell lines (IISlt-2, MOI, I -4.
Reh, CEM, and III,-60) to determine whether any simple correlates with
sensitivity to killing by y-irradiation might be revealed. The clonogenic
survival y-ray dose-response curves for these cell lines cover a wide range
of sensitivities. Consistent with previous results for murine hematopoietic
cell lines, there was a clear correlation between the rapidity with which
irradiation induced apoptosis and clonogenic radiosensitivity of a cell line,
although the relationship between timing of apoptosis and radiosensitivity
differed between human and murine cell lines. Flow cytometric determi
nation of cell cycle distribution after irradiation showed that differences
between human hematopoietic cell lines, in the rate of induction of apop
tosis, were generally related to the functioning of cell cycle checkpoints.
Whereas the rapidly dying and radiosensitive 1-1SB-2 cell line underwent
apoptosis from different points in the cell cycle, the more slowly dying cell
lines showed a variety of cell cycle arrest profiles and initiated apoptosis
after accumulation of cells in the G2 phase. The lag-phase between arrest
in ( ;, and induction of apoptosis was comparable for MOI ,1-4, Reh, and
CEM; however. III,-6(1 cells showed a markedly longer G2 arrest that
correlated with their greater radioresistance. The results suggest that the
total length of time available for DNA damage repair (irrespective of
whether this time accrues as blockage in G,, S, or G2), prior to potential
activation of apoptosis, is a critical determinant of radiosensitivity in
human hematopoietic cell lines. Comparison of the p53 status of these cell
lines suggested that mutations in the 77*53 gene are contributing to the
delay of induction of apoptosis seen in the more radioresistant cell lines.
The sensitivity of MOLT-4 and HL-60 cells to killing by DNA-associated
I25I decays was determined and was found to correlate with the relative
sensitivity of these lines to y-irradiation. The highly localized deposition of
energy by I25I decays argues that DNA damage is a potent initiator of
apoptosis in these cell lines. The results presented suggest that differences
in the radiosensitivity of the cell lines examined reflect differences in the
rapidity of induction of apoptosis and that radiation-induced cell death in
hematopoietic
cells can be explained as a response to DNA damage.
INTRODUCTION
Tumorigenesis frequently involves genetic changes that impair the
control of cell cycling and/or the regulation of apoptosis. Such
changes can also alter radioresponsiveness, and there is now extensive
literature on the involvement of genes such as p53, c-myc, and bcl-2
in determining the radiosensitivity of tumor cells. The impact on
radiosensitivity of mutation or change in the level of expression of
such genes can, however, be variable and cell type dependent (re
viewed in Ref. 1), presumably reflecting the complexity of the path
ways that lead to cell death and the multiplicity of the regulatory
biochemical interactions that together determine the fate of an irradi
ated cell. We have examined different aspects of the radiation re
sponse of murine lymphoid and myeloid cell lines to determine
whether there are any simple, general correlates with relative radioReceived 12/9/97; accepted 4/23/98.
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.
1To whom requests for reprints should be addressed, at Research Division, Peter
MacCallum Cancer Institute, Locked Bag No. 1, A'Beckett Street. Melbourne. Victoria
8006. Australia. Phone: 61-3-9656-1291;
pmci.unimclb.cdu.au.
Fax: 61-3-9656-1411;
E-mail:
with how rapidly apoptosis was induced after irradiation (2, 3). Such
differences in timing reflected whether induction occurred soon after
irradiation ("rapid interphase" death), following arrest in the G2 phase
("delayed interphase" death), or after cell division ("mitotic" death).
Cell lines could show different proportions of death at these times
dependent upon the radiation dose they had received. It was specu
lated that DNA dsb2 directly initiates the destruction of cell lines
susceptible to rapid interphase death and that chromosomal aberra
tions can trigger delayed interphase or mitotic apoptosis (4). The
occurrence of rapid interphase apoptosis appeared to be dependent
upon the presence of wild-type p53 in these cells (5).
There have been relatively few comparative radiobiological studies
of human hematopoietic cell lines. The studies performed have sought
to correlate radiosensitivity with differences in capacity for sublethal
damage repair (6, 7). differences in lymphoid lineage maturation or
various biochemical parameters (8), or differences in the rate of DNA
dsb repair (9). However, aside from noting that the most radiosensi
tive lines were derived from immature lymphoid cells [we came to a
similar conclusion from studies with murine lymphoid lines (3)], these
studies have been unable to explain the differences in radiosensitivity
among the cell lines examined.
The relationship between p53 status, the kinetics of induction of
apoptosis, and radiosensitivity has been studied in human lymphoid
lines, although to a more limited extent than for mouse cell lines.
Human studies have compared the TK6 (wild-type p53) and WIL2-NS (mutant p53) cell lines, which were both derived from the
Burkitt's lymphoma WI-L2 cell line. WTK1 (a clonal derivative of
WI-L2-NS, which is also a p53 mutant) showed delayed induction of
apoptosis and was correspondingly more resistant to killing by Xirradiation than was TK6 (10, 11). Olive et al. (12) also showed a
correlation between slower induction of apoptosis and increased ra
dioresistance in WI-L2-NS as compared with TK6. although, this
correlation did not hold when TK6 cells in different phases of the cell
cycle were compared. More recent work has cast doubt on whether the
differences seen in the WI-L2 system are due solely to the expression
of mutant p53 protein. Yu et al. (13) disrupted p53 function in the
TK6 cell line by transfection with the human papillomavirus E6 gene
and showed that this had a relatively small effect on the kinetics of
induction of apoptosis and no effect on the clonogenic survival
dose-response for y-irradiation. The rate of induction of apoptosis in
irradiated WI-L2-derived cell lines is, however, significantly slower
than is seen for many of the murine cell lines that we have examined.
O'Connor et al. (14) examined a panel of Burkitt's lymphoma lines
and found that cells containing wild-type p53 were generally more
sensitive to radiation-induced growth inhibition.
There are various lines of evidence that support the assumption that
differences in hematopoietic cell line radiosensitivity are related in
some way to the cell's response to DNA damage. For example.
Warters (15) demonstrated that bromodeoxyuridine labeling enhanced
the sensitivity to X-ray-induced killing of a murine T-cell hybridoma.
2 The abbreviations
i.radford@
bromodeoxyuridine;
used are: dsb. double strand break; IL, interleukin;
ECL. enhanced chemiluminescence.
2817
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1998 American Association for Cancer Research.
BrdUrd,
RADIOSENSmVITY
OF HUMAN LYMPHOID
CELLS
and we have shown that murine lymphoid cell lines can be exquisitely
sensitive to killing by DNA-associated 125I decays and that, in gen
mulated decays at day "t" (At) were determined using the equation
A, = 86.56/l0(l - i'-0-01155»),where Aa represents the number of decays per
eral, their sensitivity to such radioactive decays correlates closely with
their sensitivity to external beam irradiation (2, 3). The DNA-centric
view has, however, been challenged by investigators who assert a
crucial role for ceramide production, following activation of sphingomyelinase at cell membranes, in the induction of apoptosis by
irradiation (16). Consistent with the latter hypothesis, it was shown
that acid sphingomyelinase-deficient human or murine lymphoid cells
showed reduced levels of radiation-induced apoptosis (17). Recently,
a correlation between the level of postirradiation ceramide production
and the relative radiosensitivity of a panel of human lymphoid cell
lines has been adduced as further support for the importance of
membrane damage and for the inferred irrelevancy of DNA damage
(18).
Accordingly, we wished to determine whether the correlations,
found using murine cell lines, between external beam radiosensitivity
and the rate of induction of apoptosis and between sensitivity to
killing by external beam radiation and DNA-associated 125I decays
cell per day at day 0.
Survival assays were performed
are also observed in human hematopoietic cell lines that show a broad
range of radioresponsiveness. For these purposes, we have used a
panel of four human lymphoid (CEM, HSB-2, MOLT-4, and Reh) and
one myeloid (HL-60) cell line.
MATERIALS
Cell Lines. All cell lines were obtained from the American Type Culture
Collection. Cells were grown in the a modification of Eagle's medium (ICN)
plus 10% PCS (CSL, Melbourne, Australia) at 37°C,using sealed flasks that
had been flushed with 5% CO2, 5% O2, and balance N2. All studies were
performed using asynchronous, log-phase cultures. The cell lines were rou
tinely checked for Mycoplasma contamination using a PCR method (19). The
origin and some of the characteristic features of each of the cell lines used are
presented in Table 1.
y-Irradiation, 125I Incorporation, Clonogenic Survival Assay, and Sta
tistical Treatment of Survival Data. Cell cultures were irradiated at room
temperature in a '37Cs -y-ray source at a dose rate of approximately 0.9 Gy/min.
I25I labeling was performed by incubating cells for greater than 1.5 popu
lation doubling times in growth medium containing 50-100 Bq/ml of
[125I]iododeoxyuridine (NEN/DuPont) and 2.5 ¿IM
thymidine. Cells were then
washed, and unincorporated label was chased by incubation in growth medium
containing 20 /UMthymidine and 20 /J.Mdeoxycytidine for 3 h. After chasing,
incorporated '~5I was measured by pelleting the cells and counting the level of
radioactivity in a Compugamma CS (LKB) gamma counter. Counts were
corrected to decays per minute using the method of Eldridge (20). Cell pellets
were then resuspended in a known volume of growth medium, aliquots were
taken, and cell number was determined by Coulter counting. The mean number
of I25I decays occurring per cell, per day was then calculated. After slow
addition of an equal volume of growth medium plus 20% DMSO, the cell
suspension was aliquoted, frozen at -PC/min
in a controlled-rate freezer
[Cryologic (Melbourne) model CL3000 with Cryogenesis V3 software] to
—¿
60°Cand then transferred to liquid nitrogen for storage. Cell aliquots were
removed from liquid nitrogen storage at various times, after known numbers of
I25I decays per cell had occurred, and assayed for clonogenic survival. Accu-
lineHSB-2
Cell
suspensions
that were
was determined by plating cells in growth medium plus 0.3% Noble agar
(Difco) and 0.4% (packed cell volume) sheep RBCs (Amadeus International,
Melbourne, Australia). After 14 days, plates were fixed and stained by the
addition of 0.01% crystal violet in 1.5% acetic acid. Colonies containing >50
cells were then scored. The HSB-2 cell line could not be cloned in soft agar.
Consequently, the survival of this cell line was assayed by plating cells in
growth medium in 96-well, U-bottomed microtiter plates (Greiner) and check
ing for growth after 14 days. Microtiter plate wells were scored as positive if
they contained greater than 50 cells. Cell numbers added per well were
adjusted to give —¿50%
positive wells. In control platings, this corresponded to
around three cells per well. The cloning efficiency of HSB-2 cells was
significantly improved by the addition of 10% pooled human AB serum and
5% conditioned medium from the IL-2-secreting gibbon leukaemic cell line
MLA 144 (data not shown). Although Guizani et al. (21) demonstrated that
HSB-2 does not constitutively express the IL-2 receptor, growth factors present
in either the human serum or the conditioned media may be inducing expres
sion of IL-2 receptor. Alternatively, other factors secreted by MLA-144 cells
may be contributing to the improved growth. Results for HSB-2 cells were
calculated using the equation Ce = [-ln(AO]/Cw, where cloning efficiency (Ce)
is equal to the negative natural log of the fraction of wells without cell growth
(N) divided by the number of cells plated per well (Cw). Clonogenic -y-ray
AND METHODS
Table 1 General properties
using single-cell
obtained by gently pipetting the cultures approximately 10 times through a
5-ml glass pipette. Dilutions of this cell suspension were then counted using a
Coulter counter. For each of the cell lines except HSB-2, clonogenic survival
of human hematopoielic
cell lines
efficiency
status"Wild
±
time
(h)29 (%SE)15
±3
type
MOLT-4 Pre-T
Pseudotetraploid
63 ±7 Wild type/mutant
23
Early B
Pseudodiploid
41 ±3 ^Mutant
Reh
21
48 ±8
CEM
T
Pseudodiploid
2233Cloning
HL-60OriginPre-T
Pre-myelocyleKaryotype"Pseudodiploid
PseudodiploidDoubling
50 ±5TP53
Deleted
" Data from American Type Culture Collection catalog.
* Data from other studies (see "Results" for references).
dose-response
survival curves for CEM cells plated in either soft agar or
microtiter wells showed no significant difference (data not shown).
Survival data were fitted to equations using a computer-generated
leastsquares fit. The fit was minimized according to the criterion of £¡(0^
—¿
£¡)2/V¡,
where O is observed value, E is calculated value, and V is variance. The
statistical errors associated with the survival data were calculated, after the
method proposed by Boag (22), by assuming a simple Poisson variance on the
total number of control and test colonies counted.
Determination of Apoptotic Morphology. Cell samples, taken at intervals
after irradiation, were pelleted by centrifugation at 1000 rpm, gently resus
pended in a small volume of residual growth medium, and then mixed with an
equal volume of growth medium containing 10 fig/ml of ethidium bromide and
3 p.g/ml of acridine orange. At least 500 cells were then scored for apoptotic
nuclear morphology under a fluorescence microscope.
Cell Cycle Distribution. Cell cultures were labeled with 10 JXMBrdUrd for
30 min and then analyzed using the anti-BrdUrd antibody technique (23).
Cellular DNA content was determined by staining samples with propidium
iodide. Samples were processed using a Becton Dickinson FACStar Plus flow
cytometer, and the data were analyzed using the WinMDI version 2.5 program.
Cells were counted as apoptotic if they had a sub-G, or sub-G2 DNA content
and lacked BrdUrd incorporation. This assignment was checked by cell sorting
and microscopic examination. Levels of apoptosis determined by flow cytometry were generally lower than values obtained by scoring morphology (pre
sumably due to overlap between the DNA content of apoptotic and G, or G2
cells); however, the two methods gave qualitatively similar results (compare
Figs. 2 and 4).
Western Blotting. Protein was extracted from each cell line by lysing IO7
cells in 0.5 ml of 50 mm Tris (pH 7.5) plus 2% SDS. To reduce sample
viscosity, DNA was sheared by 20 passages through an 18-gauge needle.
Samples were then boiled for 3 min, cooled on ice, and spun for 5 min at
10,000 rpm in a microcentrifuge to remove insoluble material. Supematants
were assayed for protein concentration using bicinchoninic acid and then
stored at —¿
70°C.Prior to electrophoresis, buffer containing bromphenol blue
was added to give a final concentration of 10% glycerol, 10 HIMDTT, and 50
mM Tris (pH 7.5). Samples were boiled for 3 min and allowed to cool
immediately prior to loading.
Equal amounts of total protein (100 /j,g unless stated otherwise) were
separated on a 12.5% SDS-PAGE gel and then transferred to a polyvinylidene
difluoride membrane (Millipore) for 1 h at 50 V in transfer buffer containing
25 mM Tris base, 192 mM glycine, and 20% methanol at pH 8.3 (24). The
membrane was then allowed to dry overnight at room temperature, stained for
2818
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1998 American Association for Cancer Research.
RADIOSENSmVITY
OF HUMAN LYMPHOID
CELLS
tion are presented in Fig. 1. The a and ßvalues derived from fitting
the data to a linear-quadratic function of dose are given in the figure
legend. The results show clearly that HSB-2 and HL-60 are. respec
(0
>
tively, the most radiosensitive and the most radioresistant of the cell
lines surveyed, with approximately a 200-fold difference between the
lines in the level of survival after a dose of 4 Gy. MOLT-4 cells show
a comparable, although somewhat more radioresistant, response to
that of HSB-2. Reh and CEM are considerably more radioresistant
than HSB-2 and MOLT-4. Both show similar clonogenic dose re
sponses, although CEM is significantly more radioresistant than Reh
at lower radiation doses. Our dose responses for MOLT-4 and HL-60
are comparable with those of previous studies (7, 27).
Rate of Induction of Cell Death after y-Irradiation Correlates
with Radiosensitivity. Based on the y-ray survival dose-responses
presented in Fig. 1. cultures of the different cell lines were given
equitoxic radiation doses sufficient to reduce clonogenic survival to
0.5% of the control value and were then incubated at 37°C.The timing
.1
3
CO
I
.01
§>
0)
o .001
o
.0001
Dose (Gy)
Fig. I. Clonogenic survival of different human hemalopoietic cell lines as a function
of -y-ray dose. Data were fitted to the equation S = exp - [aD + ßD2].where S is
survival, D is dose, and a and ßare constants. Calculated values of a and ß:HSB-2
(1.75 ±0.06 and 0.087 ±0.029); MOLT-4 (1.49 ±0.05 and 0.064 ±0.018); Reh
(1.06 ±0.01 and 0.005 ±0.005); CEM (0.61 ±0.03 and 0.081 ±0.006); and HL-60
(0.62 ±0.02 and 0.029 ±0.004). Each data set is from at least two separate experiments.
total protein with Ponceau-S (Sigma), and blocked with 3% skim milk powder,
2% gelatin, and 0.1% Tween 20 in PBS (pH 7.4). After incubation for 2 h at
room temperature, fresh blocking solution containing the primary antibody
(AC 21 (Santa Cruz Biotechnology) for Bcl-2 or PAb 421 for p53) was added,
and the membrane was incubated overnight at 4°C.The membrane was then
washed three times in PBS plus 0.2% Tween 20, followed by incubation with
peroxidase-labeled secondary antibody for l h at room temperature. After
four X 15-min washes in PBS plus 0.2% Tween 20 at room temperature, the
membrane was incubated for 1 min in ECL buffer [0.4 mg/ml luminol, 0.1
mg/ml 4-iodophenol, and 1 /¿I/mlH2O2 (36%) in 60 mM Tris buffer, pH 7.5],
and proteins were then visualized on Fuji Rx medical X-ray film using
exposure times varying from 30 s to 5 min. ECL provides qualitative, but not
quantitative, information about protein levels.
of occurrence of cell death was then determined in culture samples
taken at various times after irradiation. All five cell lines showed
induction of apoptotic nuclear morphology after irradiation. HL-60
cultures showed some necrotic death, although apoptosis was the
predominant form of death over the time interval examined (data not
shown). As shown in Fig. 2, the rate of induction of apoptosis after
equitoxic doses of y-irradiation varied greatly between the five cell
lines examined, and the relative ordering of these responses reflected
the clonogenic survival dose responses. For example, HSB-2 is the
most radiosensitive of the cell lines examined and also shows the most
rapid induction of apoptosis. The more radioresistant MOLT-4 cell
line shows a pronounced delay of —¿4-6h before commencement of
induction of apoptosis at a similar rate to that of HSB-2. Reh cultures
are more radioresistant than MOLT-4 and show a somewhat longer
delay before initiating apoptosis. The subsequent entry of Reh cells
into apoptosis occurs at a much slower rate than is seen for MOLT-4
or HSB-2. CEM cells are more resistant to y-irradiation than Reh and
show a significantly longer delay prior to the induction of apoptosis.
Once apoptosis has begun in irradiated CEM cultures, the rate of
accumulation of apoptotic cells appears to be similar to that of Reh.
RESULTS
1.0
Optimizing Clonogenic Assays. Our initial studies suggested that,
when plated in growth medium plus semi-solid agar, the human cell
lines chosen generally had low cloning efficiencies that showed a high
level of inter-experimental variability. This result was of concern
given claims that, at least for some cell lines, there is a correlation
between cloning efficiency and radiosensitivity (25). The control
cloning efficiencies of CEM, HL-60, MOLT-4, and Reh were, how
o
O
u
o
o.
ever, found to be greatly increased by the addition of sheep RBCs to
the medium. For example, the cloning efficiency of HL-60 increased
from 1% to ~50% in the presence of sheep RBCs. The ability of
0.6
o
sheep RBCs to increase the cloning efficiency of hematopoietic cells
was first demonstrated by Bradley et al. (26). Even with the addition
of conditioned media, sheep RBCs, or lethally irradiated feeder cells,
we were unable to clone HSB-2 cells in soft agar. This cell line was,
however, found to be clonable in microtiter plates as described in
"Materials and Methods." Our inability to clone HSB-2 cells in soft
agar may indicate a requirement for cell-to-cell contact or that some
aspect of the cloning methodology is lethal to this cell line. Control
cloning efficiencies, obtained for each of the cell lines using the above
methods, are shown in the Table. The values obtained are, in general,
significantly greater than those reported in previous studies.
Clonogenic Survival y-Irradiation Dose Responses. Dose-re
sponses for the five human hematopoietic cell lines under investiga-
0.8
"'S
0.2
SS
IL
10
20
30
40
Time (h)
Fig. 2. Rate of induction of apoptosis. as determined by nuclear morphology, in
•¿y-irradiated
human lymphoid cell lines. Cell cultures were given equitoxic doses (HSB-2.
3 Gy; MOLT-4. 3.5 Gy; Reh, 5 Gy; CEM. 5 Gy; and HL-60. 6.5 Gy) and then incubated
at 37°C.The response of the murine ST4 lymphoid cell line after 3 Gy of -y-irradialion is
included for comparison [data from Radford et al. (36)|. Each data set was pooled from
at least two separate experiments.
2819
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1998 American Association for Cancer Research.
RADIOSENSmVITY
OF HUMAN LYMPHOID
CELLS
BrdUrd at intervals after exposure to equitoxic y-ray doses (sufficient
HL-60
(31.211.9)
MOLT-4
(22.110.6)
.1
.01
20
40
125
60
80
100
I decays/cell
Fig. 3. Clonogenic survival responses of MOLT-4 and HL-60 cells exposed to
DNA-associated 125Idecays. Data were fitted to the equation S = exp - [DID„\,where
S is survival, D is dose, and ö„
is a constant. Calculated values for D0 and its associated
error are shown on the figure. Each data set is from two separate experiments.
to reduce clonogenic survival to 0.5% of the control value) and were
then analyzed by flow cytometry. Cell cycle distributions, as a func
tion of time after irradiation, for each of the five cell lines are shown
in Fig. 4.
Flow cytometric analysis of irradiated HSB-2 cultures showed no
marked accumulation of cells in any phase of the cell cycle prior to an
increase in the levels of apoptosis. Irradiation has rapid and profound
effects on these cells as evidenced by an almost total suppression of
mitotic activity after 3 Gy of y-irradiation and a marked decrease in
BrdUrd incorporation into S-phase cells within 2 h after irradiation
(data not shown). Flow cytometric profiles of cultures that had been
incubated for 4 or more hours after irradiation, showed a significant
increase in the level of cells with sub-G, or intermediate to G, and G2
(but lacking BrdUrd incorporation) DNA content (Fig. 5), suggesting
that the induction of apoptosis in HSB-2 cells can occur at different
points in the cell cycle and is not linked to one checkpoint. In contrast
to the results obtained for HSB-2, the other more rapidly dying cell
line, MOLT-4, shows an accumulation of cells in G2 that parallels and
precedes the induction of apoptosis. Consistent with MOLT-4 cells
undergoing apoptotic death after blockage in G2, flow cytometric
profiles showed a progressive increase, with time after irradiation, in
the proportion of cells showing a DNA content intermediate to G, and
G2 (but lacking BrdUrd incorporation; Fig. 5). Similar to MOLT-4,
the increase in the fraction of apoptotic Reh cells follows and occurs
The apparent decline in the numbers of apoptotic CEM cells at —¿20 in parallel with the accumulation of cells in the G2 phase. This
accumulation occurs more slowly than is seen for MOLT-4, appar
h after irradiation is an artifact caused by the fragility of apoptotic
ently due to a more prolonged blockage of Reh cells in Gl and/or S.
CEM cells (i.e., the true kinetics of appearance of apoptotic CEM cells
are probably more comparable with those of Reh than to HL-60).
Consistent with induction of apoptosis in G2, flow cytometric profiles
HL-60 is clearly the most radioresistant cell line examined and also
from irradiated Reh cultures undergoing apoptosis showed increased
levels of cells with a DNA content intermediate to G, and G2 (but
shows the most pronounced delay prior to the induction of apoptosis.
Accumulation of apoptotic cells was not observed in HL-60 cultures
lacking BrdUrd incorporation; see Fig. 5). Although Reh and CEM
have comparable sensitivities to killing by y-irradiation, their cell
until at least 24 h after irradiation. It is of interest to note that even the
most rapidly dying of the human cell lines (HSB-2) shows markedly
cycle responses are significantly different (Fig. 4). Unlike Reh, CEM
slower kinetics of induction of apoptosis than does a murine lymcells show no postirradiation blockage in the G, phase, but they do
show a pronounced blockage in mid- to late-S phase that is followed
phoma such as ST4 (Fig. 2).
Sensitivity to Killing by DNA-associated 125I Decays. Because
by a rapid exit from S and accumulation in the G2 phase. As with
MOLT-4 and Reh, the latter event precedes the appearance of apop
of the continuing debate over the importance of DNA damage in
initiating radiation-induced apoptotic cell death and because the level
totic cells in culture. Consistent with the death of CEM cells in G2
of DNA damage induced per unit dose of y-irradiation can vary
phase, flow cytometric profiles of apoptosing cultures showed in
creased levels of cells with a DNA content intermediate to G( and G2
between cell lines (28), we were interested in comparing the sensi
tivity of our panel of cell lines to killing by DNA-associated I25I (but lacking BrdUrd incorporation; data not shown). HL-60, the most
decays.
The technique used for I25I decay experiments involves freezing
cells and then storing them over liquid nitrogen for periods up to 2-3
months to allow the accumulation of DNA damage without interfer
ence from DNA repair activities. The cells must then show reasonable
cloning efficiencies after thawing. Our attempts to determine the
sensitivity of these cell lines to DNA-associated 125I decays were,
however, generally unsuccessful due to low and variable cloning
efficiencies after freezing and thawing. MOLT-4 and HL-60 were the
only lines to show reasonable cloning efficiencies after freezing and
thawing (30 ±9% for MOLT-4 and 6.7 ±0.4% for HL-60). The
clonogenic survival curves for these cell lines, after accumulation of
125I-induced DNA damage, are shown in Fig. 3. As for the y-ray
survival results, HL-60 cells (D0 = 38.2 ±1.9 decays) were found to
be far more resistant to 125I decay-induced DNA damage than are
MOLT-4 cells (D0 = 22.1 ±0.6 decays).
Effect of y-Irradiation on Cell Cycling. Studying the effect of
y-irradiation on subsequent cell cycling can indicate whether induc
tion of apoptosis is related to cell cycle events (e.g., cell cycle
blockage or mitosis) and how differences in radiosensitivity might be
related to these events (4). Cell cultures were pulse labeled with
radioresistant and the slowest dying of the cell lines in our panel, lacks
a marked G, arrest but shows prolonged blockage of cells in G2 prior
to the appearance of apoptosis in the culture (Fig. 4). The timing of
occurrence of apoptosis in irradiated HL-60 cells also coincides with
the resumption of mitotic activity in these cultures (data not shown).
This result and the flow cytometric data suggest that death of these
cells occurs both in G2 and during or around the time of mitosis.
p53 and Bcl-2 Status. The p53 protein is an important modulator
of the cell cycle and a determinant of radiosensitivity in lymphoid
cells. Accordingly, we sought to correlate the status and expression of
the TP53 gene with the radiosensitivity and cell cycle response of the
cell lines examined. The TP53 gene of the HSB-2, MOLT-4, CEM,
and HL-60 cell lines has been sequenced by other investigators, and
the results are summarized in Table 1. Similar sequence data for Reh
cells could not be found in the literature. We tested for the presence
of p53 protein in each of the cell lines by Western blotting. The blots
were probed with the PAb 421 monoclonal antibody, which is specific
for an epitope in the COOH-terminal region of the protein (29), and
reactive proteins were detected using ECL. Expression of p53 protein
was found in all of the cell lines, except for HL-60 (Fig. 6). The latter
result is consistent with the finding that HL-60 has a large deletion
820
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1998 American Association for Cancer Research.
RADIOSENSmVITY
OF HUMAN LYMPHOID
CELLS
1.0
o 0
Fig. 4. Effect of -y-irradiation on cell cycle progression
of human lymphoid lines. The distribution of cells be
tween the different phases of the cycle (G,, S. and G2)
and the level of apoptosis (Api were determined by flow
cytometry using cultures given equitoxic doses and then
incubated at 37°C.Each data set was pooled from two
2
4
602468
10 0
Time (h)
8
Time (h)
1.0
12
20
24
Time (h)
HL-60 (6.5 Gy)
CEM (5 Gy)
separate experiments.
16
.2 .8
CL
.4
8.2
4
8
12
16
20
Time (h)
within the TP53 gene that prevents protein expression (30). The two
most rapidly dying and radiosensitive cell lines, HSB-2 and MOLT-4,
have both been shown to express wild-type p53 (31, 32). In addition
to a wild-type gene, the MOLT-4 cell line also contains a TP53
mutation that leads to aberrant splicing of the mRNA, causing a frame
shift and truncating the protein before translation of exons 10 and 11
(32). Of the more slowly dying cell lines, none are known to express
wild-type p53. Both copies of the TP53 gene in CEM cells contain
single base substitutions that produce missense mutations (31), and as
mentioned above, the HL-60 cell line does not express p53 protein due
to gene deletions. Reh does express p53 protein (Fig. 6): however, we
do not know whether this protein is mutant or wild type.
Members of the Bcl-2 family of proteins are important modulators
of apoptosis and have been shown to influence radiosensitivity (33).
Western blotting, using a polyclonal antibody to Bcl-2 protein,
showed that all of the cell lines used in this study expressed a reactive
protein of Mr -26,000 (Fig. 6). Reh and HL-60 differed from the
other lines in showing an additional reactive band of slightly higher
molecular weight. The additional band might represent expression of
a Bcl-2-related protein, such as Bcl-x,. which has a molecular weight
of 29.000 (34), or a phosphorylated form of the protein. A variant of
HL-60 has been shown to express Bcl-xL (35).
240
10
15
20
25
30
35
Time (h)
DISCUSSION
We have examined whether the correlation between the rate of
induction of apoptosis and the clonogenic survival y-ray doseresponse, observed for murine hematopoietic cell lines, is also seen in
human hematopoietic cell lines. The results presented in this report
clearly demonstrate, for the cell lines examined, a correlation between
the rapidity with which apoptosis is induced after irradiation and the
clonogenic survival dose-response. For example, the HSB-2 cell line,
which shows the most rapid induction of apoptosis after y-irradiation,
was also found to be the cell line most sensitive to clonogenic killing,
whereas MOLT-4, Reh, CEM, and HL-60, which show progressively
longer delays prior to the induction of apoptosis after an equitoxic
radiation dose, are correspondingly more resistant to killing by
y-irradiation.
Although a general correlation between the relative rate of induc
tion of apoptosis and the clonogenic survival y-ray dose-response is
seen for both human and murine hematopoietic cell lines, one notable
difference is that although the survival dose-response curves for the
more radiosensitive human and murine cell lines are similar, equitoxic
radiation doses induce apoptosis far more rapidly in the murine lines.
For example, we found that at least 90% of ST4 cells (a murine pre-T
2821
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1998 American Association for Cancer Research.
RADIOSENSÕTMTY OF HUMAN LYMPHOID CELLS
HSB-2 (Control)
HSB-2 (3 Gy + 5 hrs)
MOLT-4 (Control) 2
MOLT-4 (3.5 Gy + 10 hrs)
relevant that Bcl-2 protein is readily detectable in the human cell lines
that we have used.
Irradiation of cells can induce blockage at checkpoints in different
phases of the cell cycle, including p53-dependent arrest in G, (37) and
p53-independent blocks in S (38) and G2 (39). These blocks are
ultimately caused by inhibition of cyclin-dependent kinases that are
necessary for cycle progression. In the cell lines that we have exam
ined, the rate of induction of apoptosis, after y-irradiation, generally
appears to be related to the functioning of these cell cycle checkpoints.
HSB-2 may be an exception in this regard, because it shows no clear
evidence for accumulation at checkpoints and appears to undergo
induction of apoptosis in different phases of the cell cycle. Consistent
with previous observations that the more rapidly dying and radiosen
sitive murine lymphoid cell lines contain wild-type p53 (5), HSB-2
encodes wild-type TP53 (31), and we have shown that it expresses
p53 protein. It may also be relevant to the radiosensitivity of HSB-2
u
O
O
3
E
'
O
2
m
O
Reh (Control)
m
O
Reh (5 Gy + 24 hrs)
O
DNA Content
Fig. 5. Dot-pio! distributions for BrdUrd incorporation (ordinale) versus DNA content
(abscissa) of cells from control and irradiated MOLT-4 and Reh cultures. Regions
corresponding to cells in G, and G2 phases of the cell cycle are indicated.
cell line), exposed to a y-ray dose sufficient to reduce clonogenic
survival to 0.5%, showed apoptotic morphology 3 h after irradiation
(36). In contrast, the most rapidly dying human cell line used in this
study (HSB-2) does not reach this level of apoptosis until at least 24 h
after receiving an equitoxic y-ray dose, despite having a similar
clonogenic survival y-ray dose-response to ST4. Unfortunately, be
cause we were unable to determine the sensitivity of HSB-2 cells to
killing by DNA-associated 125Idecays, we cannot rigorously compare
the response to DNA damage of ST4 and HSB-2. If we assume that
there are similar y-ray dose-responses for DNA lesion induction in the
two cell lines, then perhaps the simplest explanation for the difference
in timing is that, after the detection of lethal cellular injury, the
biochemical events that lead to the triggering of apoptosis occur much
more slowly in HSB-2 than in a murine line such as ST4. Two factors
that may be relevant to this human/murine difference are: (a) there is
a marked difference in the population doubling times of cell lines such
as ST4 and HSB-2 (9 h versus 29 h). The data of Olive et al. (12),
however, suggest that human TK6 cells have a relatively rapid dou
bling time (around 12 h) and yet show induction of apoptosis after
irradiation at a comparable rate to HSB-2 cells; and (b) the human cell
lines that were used in this study all derived from tumors that relapsed
after intensive radiotherapy and/or chemotherapy of the primary tu
mor, whereas the derivation of murine lines like ST4 did not involve
exposure to cytotoxic agents. Such treatment may well have selected
for cells with down-regulated apoptotic responses, and it is perhaps
that, due to homozygous deletion of the Rb gene (40), this cell line
does not express pRb. Expression of pRb was associated with p53independent protection against radiation-induced apoptosis in both a
human osteosarcoma and a lymphocytic cell line (41, 42). When
MOLT-4 or Reh cells were exposed to a y-ray dose that reduces
survival to 0.5% (unlike HSB-2), both showed induction of apoptosis
after accumulation of cells in G-,. Due apparently to blockage earlier
in the cycle, Reh cells accumulated in G2 much more slowly than did
MOLT-4 cells and showed a corresponding increase in the time
between irradiation and the appearance of apoptotic cells that corre
lated with their greater radioresistance. The molecular basis for this
difference is unclear. Both MOLT-4 and Reh express pRb (40, 43)
and, as shown in this study, p53. Although MOLT-4 cells express both
wild-type and a truncated p53 (32), the status of the p53 expressed by
Reh cells is not known to us. The relative slowness of the induction
of apoptosis in Reh cells suggests that they contain mutant p53. Like
MOLT-4 and Reh, irradiated CEM cells appeared to die after block
age in G2. CEM cells differed, however, in showing a prominent
blockage in mid- to late-S phase that preceded accumulation in G2.
Consistent with their slow kinetics of induction of apoptosis and their
radioresistance, CEM cells express pRb (40) and mutant p53 (31).
Finally, the response of HL-60 to a y-ray dose that reduces survival to
0.5% is characterized by transient arrest in S and a marked and
prolonged blockage in G2. Other authors have also described radia
tion-induced G-, arrest and apoptosis of HL-60 cells (39, 44). Al
though MOLT-4, Reh, and CEM cultures showed an increase in the
level of apoptosis that lagged about 4 h behind the accumulation of
cells in G2, our results suggest that HL-60 cells can remain arrested in
G2 for much longer times before undergoing apoptosis. The basis for
this difference has not been examined, although in other cell types
expression of a ras oncogene was associated with a decrease in cyclin
Bl mRNA levels and a lengthening of G2 arrest (45).
HSB-2
MOLT-4
Reh
CEM
HL-60
p53
bcl-2
Fig. 6. Western blotting analysis of p53 and Bcl-2 protein expression
hematopoietic cell lines.
2822
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1998 American Association for Cancer Research.
in human
RADIOSENSITIVE
The importance of the duration
and G2 checkpoints in determining
various nonlymphoid cell types
evidence to suggest that although
OF HUMAN LYMPHOID
of postirradiation arrest at the G,
radiosensitivity has been tested in
that undergo apoptosis. There is
the p53-dependent G, checkpoint
may not be critical in this regard (46), the length of the G2 delay may
be directly related to radiosensitivity (45). Consistent with our previ
ous results for murine hematopoietic cell lines (4), the data presented
in this paper suggest that the total length of time available for DNA
damage repair (irrespective of whether this time accrues as blockage
in G|, S, or G-,), prior to potential activation of apoptosis, is a critical
determinant of radiosensitivity in human hematopoietic cell lines.
We have assumed that the explanation for the differences in radiosensitivity between hematopoietic cell lines lies in understanding the
response of the cell to DNA damage. Consistent with this view, we
have shown in this report a correlation between the sensitivity of
MOLT-4 and HL-60 to y-irradiation and to DNA-associated 125I
decays. It is interesting to note that the D0 values for I25I decayinduced
cell killing of MOLT-4
and HL-60 (22.1
± 0.6 and
38.2 ±1.9 decays, respectively) are very similar to the values for the
murine lines WEHI-7 and 18-81 (22.4 ±1.3 and 40.8 ±2.8 decays,
respectively; Ref. 3). Both MOLT-4 and WEHI-7 cells show postirradiation accumulation in and subsequent apoptosis in G2 phase,
whereas HL-60 and 18-81 both exhibit a very delayed induction of
apoptosis (4). I25I decays have been shown to deposit the majority of
their energy in close proximity to the site of decay (47) and, when
occurring in DNA, induce dsb with close to 100% efficiency (48).
Accordingly, the 125Iresults (presented in this and previous studies)
suggest that radiation-induced DNA dsb is a potent initiator of apop
tosis, and they are a strong indicator that these lesions are also
responsible for y-ray-induced apoptosis. An opposing view, that ra
diation-induced apoptosis is triggered by membrane damage-initiated
production of ceramide, has been supported recently by data showing
lower postirradiation production of ceramide in HL-60 than in a more
radiosensitive Burkitt's lymphoma line (18). The latter study did not,
however, compare the ceramide responses of the cell lines at equitoxic
radiation doses; in addition, it has been suggested that the ceramide
assay method used by Michael et al. (18) gives artifactual results (49).
We have shown that differences in the radiosensitivity of a panel of
human hematopoietic cell lines can be related to differences in the
rapidity of induction of apoptosis and that radiation-induced apoptosis
can be explained as a response to DNA damage.
REFERENCES
1. Olive. P. L.. and Durand, R.E. Apoptosis: an indicator of radiosensitivity in vitro? Int.
J. Radial. Biol.. 71: 695-707, 1997.
2. Radford. I. R. Mouse lymphoma cells that undergo interphase death show markedly
increased sensitivity to radiation-induced DNA double-strand breakage as compared
with cells that undergo mitotic death. Int. J. Radiât.Biol., 59: 1353-1369, 1991.
3. Radford. I. R. Radiation response of mouse lymphoid and myeloid cell lines. Part I.
Sensitivity to killing by ionizing radiation, rate of loss of viability, and cell type of
origin. Int. J. Radial. Biol.. 65: 203-215. 1994.
4. Radford, I. R., and Murphy. T. K. Radiation response of mouse lymphoid and
myeloid cell lines. Part III. Different signals can lead to apoptosis and may influence
sensitivity to killing by DNA double-strand breakage. Int. J. Radiât. Biol.. 65:
229-239, 1994.
5. Radford. I. R. p53 status, DNA double-strand break repair proficiency, and radiation
response of mouse lymphoid and myeloid cell lines. Ini. J. Radial. Biol.. 66: 557-560.
1994.
6. FitzGerald. T. J., McKenna. M., Kase. K.. Daugherty, C.. Rothstein. L.. and
Greenberger. J. S. Effect of X-irradiation dose rate on the clonagenic survival of
human and experimental animal hematopoietic tumor cell lines: evidence for heter
ogeneity. Int. J. Radial. Oncol. Biol. Phys., 12: 69-73, 1986.
7. Lehnen. S.. Rybka. W. B.. Suissa. S., and Giambaltisto. D. Radialion response of
haematopoietic cell lines of human origin. Ini. J. Radiât.Biol.. 49: 423-431. 1986.
8. Uckun. F. M.. Mitchell. J. B., Obuz. V.. Park. C. H., Waddick, K.. Friedman, N..
Oubaha. L.. Min, W. S., and Song. C. W. Radiation sensitivity of human B-lineage
lymphoid precursor cells. Int. J. Radial. Oncol. Biol. Phys., 21: 1553-1560, 1991.
CELLS
Evans. H. H.. Ricanati. M.. Horng. M. F.. Jiang. Q.. Menci. J.. and Olive. P. DNA
double-strand break rejoining deficiency in TK6 and other human B-lymphoblast cell
lines. Radiât.Res.. 134: 307-315. 1993.
10. Amundson, S. A., Xia. F.. Wolfson. K.. and Liber. H. L. Different cytotoxic and
tnutagenic responses induced by X-rays in two human lymphoblastoid cell lines
derived from a single donor. Mutât.Res.. 286: 233-241. 1993.
Xia. F., Wang, X.. Wang. Y. H.. Tsang. N. M.. Yandell. D. W., Kelsey. K. T.. and
Liber, H. L. Altered p53 status correlates with differences in sensitivity to radiationinduced mutation and apoptosis in two closely related human lymphohlasl lines.
Cancer Res., 55: 12-15, 1995.
Olive, P. L., Banath. J. P., and Durand. R. E. Development of apoptosis and
polyploidy in human lymphohlast cells as a function of position in the cell cycle at the
time of irradiation. Radial. Res.. 146: 595-602. 1996.
Yu. Y.. Li. C. Y.. and Little. J. B. Abrogation of p53 function by HPV16 £6gene
delays apoptosis and enhances mutagenesis hut does not alter radiosensitivity in TK6
human lymphoblast cells. Oncogene. 14: 1661-1667. 1997.
O'Connor, P. M., Jackman. J.. Jondle. D.. Bhatia. K.. Magralh. !.. and Kohn. K. W.
Role of the p53 tumor suppressor gene in cell cycle arrest and radiosensitivity
Burkitt's lymphoma cell lines. Cancer Res.. 5.?: 4776-4780. 1993.
of
Warters. R. L. Radiation-induced apoptosis in a murine T-cell hybridoma. Cancer
Res., 52: 883-890, 1992.
Haimovitz-Friedman. A.. Kan. C. C.. Ehleiter. D.. Persaud. R. S., McLoughlin, M .
Fuks. / and Kolesnick. R. N. loni/ing radiation acts on cellular membranes to
generate ceramide and initiate apoptosis. J. Exp. Med.. 1X0: 525-535, 1994.
Santana. P.. Pena. L. A.. Haimovitz-Fricdman. A.. Martin, S.. Green. D., McLoughlin,
M.. Cordon-Cardo. C.. Schuchman, E. H.. Fuks, Z., and Kolesnick. R. Acid sphingotnyelinase-deficient
human lymphoblasts and mice are defective in radiationinduced apoptosis. Cell. 86: 189-199. 19%.
18. Michael. J. M., Lavin, M. F.. and Watters. D. J. Resistance to radiation-induced
apoptosis in Burkitt's lymphoma cells is associated with defective ceramide signaling.
Cancer Res.. 57: 3600-3605, 1997.
19. Pruckler, J. M.. Pruckler. J. M.. and Ades. E. W. Detection by polymerase chain
reaction of all common Mvcoplasma in a cell culture facility. Pathobiology, 63: 9-11.
1995.
20. Eldridge. J. S.. and Crowther, P. Absolute determination of 1125 in clinical applica
tions. Nucleonics. 22: 56-59. 1964.
21. Guizani, L.. Lang. P., Stancou, R.. and Bertoglio. J. Regulation of interleukin-2 and
interleukin-4 receptor expression in human and ape lymphoid cell lines. Lymphokine
Cytokine Res.. 12: 25-32. 1993.
22. Boag. J. W. The statistical treatment of cell survival dala. In: Cell survival after low
doses of radiation, pp. 40-53. London: John Wiley and Sons. 1975.
23. Dean. P. N., Dolbeare. F.. Gratzner. H.. Rice. G. C., and Gray, J. W. Cell-cycle
analysis using a monoclonal amibody lo BrdUrd. Cell Tissue Kinel.. 17: 427-436.
1984.
24. Towbin. H.. Staehelin, T., and Gordon. J. Electrophorelic transfer of proteins from
polyacrylamide gels lo nitrocellulose sheets: procedure and some applications. Proc.
Nati. Acad. Sci. USA. 76: 4350-4354. 1979.
25. Brenner. D. J., Zaider. M.. Geard, C. R., and Georgsson. M. A. Cell survival and
plating efficiency. Radial. Res.. ///: 572-576. 1987.
26. Bradley. T. R., Telfer, P. A., and Fry, P. The effect of erythrocyles on mouse bone
marrow colony developmenl in vitro. Blood. 38: 353-359, 1971.
27. Szekely. J. G.. and Lobreau. A. U. High radiosensitivity of the MOLT-4 leukaemic
cell line. Int. J. Radial. Biol., 48: 277-284. 1985.
28. Radford. I. R. Evidence for a general relationship between the induced level of DNA
double-strand breakage and cell-killing after X-irradiation of mammalian cells. Int. J.
Radial. Biol.. 49: 611-620. 1986.
29. Gannon. J. V., Greaves, R., Iggo, R., and Lane, D. P. Aclivating mutalions in p53
produce a common conformational effect. A monoclonal antibody specific for the
mutant form. EMBO J.. 9: 1595-1602. 1990.
30. Wolf, D.. and Roller. V. Major dételionsin Ihe gene encoding Ihe p53 lumor antigen
cause lack of p53 expression in HL-60 cells. Proc. Nati. Acad. Sci. USA, 82:
790-794. 1985.
3 j Cheng. J.. and Haas. M. Frequenl mutations in the p5J tumor suppressor gene in
human leukemia T-cell lines. Mol. Cell. Biol.. 10: 5502-5509, 1990.
32 Chow. V. T.. Quek, H. H.. and Tock. E. P. Alternative splicing of ihe p5J tumor
suppressor gene in the Moll-4 T-lymphohlaslic leukemia cell line. Cancer Lett., 73:
141-148, 1993.
33 Strasser. A., Harris, A. W.. Jacks. T.. and Cory, S. DNA damage can induce apoptosis
in proliferating lymphoid cells via p53-independenl mechanisms inhibitahle by Bcl-2.
Cell. 79: 329-339. 1994.
34 Boise, L. H.. Gonzalez-Garcia. M.. Poslema, C. E., Ding, L.. Lindslen. T.. Turka,
L. A.. Mao. X., Nunez. G., and Thompson. C. B. bcl-x, a bcl-2-relaled gene that
functions as a dominant regulator of apoptotic cell death. Cell, 74: 597-608. 1993.
35. Han, Z., Chatterjee. D.. Early. J.. Pantazis. P.. Hendrickson. E. A., and Wyche. J. H.
Isolalion and characlerizalion of an apoptosis-resistanl varianl of human leukemia
HL-60 cells lhat has switched expression from Bcl-2 to Bcl-xL. Cancer Res., 56:
1621-1628. 1996.
36. Radford. I. R., Murphy. T. K.. Radley. J. M.. and Ellis. S. L. Radiation response of
mouse lymphoid and myeloid cell lines. Part II. Apoptolic dcalh is shown by all lines
examined. Int. J. Radial. Biol., 65: 217-227. 1994.
37. Kuerbilz. S. J.. Plunkell. B. S., Walsh. W. V.. and Kaslan. M. B. Wild-type p53 is a
cell cycle checkpoint determinant following irradiation. Proc. Nati. Acad. Sci. USA,
«9..7491-7495. 1992.
D'Anna. J. A.. Valdez. J. G.. Habbersell. R. C.. and Crissman, H. A. Associalion of
38.
G,-S-phase and lale S-phase checkpoints wilh regulation of cyclin-dependent kinases
in Chinese hamsler ovary cells. Radial. Res.. 148: 260-271. 1997.
2823
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1998 American Association for Cancer Research.
RADIOSHNSmVITY
OF HUMAN LYMPHOID
39. Han. Z.. Chatterjee. D.. He, D. M.. Early, J.. Pantazis, P.. Wyche, J. H., and
Hendrickson, E. A. Evidence for a G2 checkpoint in p53-independent apoptosis
induction by X-irradiation. Mol. Cell. Biol., 15: 5849-5857, 1995.
40. Cheng. J.. Scully, P.. Shew. J. Y.. Lee. W. H.. Vila, V., and Haas, M. Homozygous
deletion of the retinoblastoma gene in an acute lymphoblaslic leukemia (T) cell line.
Blood, 75: 730-735, 1990.
41. Haas-Kogan, D. A.. Kogan. S. C.. Levi. D.. Dazin, P.. A. T. A.. Fung, Y. K.. and
Israel, M. A. Inhibition of apoplosis by the retinoblastoma gene produci. EMBO J.,
14: 461-472. 1995.
42. Ishii. H., Igarashi, T., Saito, T., Nakano, T., Mori. M., Ohyama, H., Miyamoto, T.,
Saito. Y., and Oh. H. Retinoblastoma protein expressed in human non-Hodgkin's
lymphoma cells generates resistance against radiation-induced apoptosis. Am. J.
Hematol., 55: 46-48, 1997.
43. Christoffersen. J.. Smeland, E. B.. Stokke. T.. Tasken. K.. Andersson. K. B.. and
Blomhoff. H. K. Retinoblastoma protein is rapidly dephosphorylated by elevated
cyclic adenosine monophosphate levels in human B-lymphoid cells. Cancer Res., 54:
2245-2250, 1994.
CELLS
44. Abend. M., Gilbert/, K. P.. Rhein. A., and van Beuningen. D. Early and late G2 arrest
of cells undergoing radiation-induced apoptosis or micronucleation. Cell Prolifera
tion, 29: 101-113, 1996.
45. McKenna, W. G.. Bernhard. E. J.. Markiewicz. D. A., Rudoltz. M. S.. Maity. A., and
Muschel, R. J. Regulation of radiation-induced apoptosis in oncogene-transfected
fibroblasts: influence of H-ras on the G2 delay. Oncogene, 12: 237-245, 1996.
46. Gupta, N., Vij, R.. Haas-Kogan. D. A.. Israel. M. A.. Deen, D. F.. and Morgan, W. F.
Cytogenetic damage and the radiation-induced G1-phase checkpoint. Radial. Res..
¡45:289-298, 1996.
47. Charlton. D. E. The range of high LET effects from I25I decays. Radiât.Res., 107:
163-171. 1986.
48. Kandaiya, I. S.. Lobachevsky. P. N., D'Cunha. G.. and Martin. R. F. DNA strand
breakage by I2il-decay in a synthetic oligodeoxynucleotide I. Fragment distribution
and evaluation of DMSO protection effect. Acta Oncol., 35: 803-808. 1996.
49. Walts, J. D.. Gu. M.. Polverino. A. J.. Patterson. S. D., and Aebersold, R. Fas-induced
apoptosis of T cells occurs independently of ceramide generation. Proc. Nati. Acad.
Sci. USA. 94: 7292-7296, 1997.
2824
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1998 American Association for Cancer Research.
Explaining Differences in Sensitivity to Killing by Ionizing
Radiation between Human Lymphoid Cell Lines
David R. Aldridge and Ian R. Radford
Cancer Res 1998;58:2817-2824.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/58/13/2817
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1998 American Association for Cancer Research.