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(CANCER RESEARCH 58, 2817-2824. July I. 1998]
Explaining Differences in Sensitivity to Killing by Ionizing
Radiation between
Human Lymphoid Cell LinesDavid 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
We surveyed five human hematopoietic cell lines (IISlt-2, MOI, I
-4.Reh, CEM, and III,-60) to determine whether any simple
correlates withsensitivity to killing by y-irradiation might be
revealed. The clonogenicsurvival y-ray dose-response curves for
these cell lines cover a wide range
of sensitivities. Consistent with previous results for murine
hematopoieticcell lines, there was a clear correlation between the
rapidity with whichirradiation induced apoptosis and clonogenic
radiosensitivity of a cell line,although the relationship between
timing of apoptosis and radiosensitivitydiffered between human and
murine cell lines. Flow cytometric determination of cell cycle
distribution after irradiation showed that differencesbetween human
hematopoietic cell lines, in the rate of induction of apoptosis,
were generally related to the functioning of cell cycle
checkpoints.Whereas the rapidly dying and radiosensitive
1-1SB-2cell line underwent
apoptosis from different points in the cell cycle, the more
slowly dying celllines showed a variety of cell cycle arrest
profiles and initiated apoptosisafter accumulation of cells in the
G2 phase. The lag-phase between arrestin ( ;, and induction of
apoptosis was comparable for MOI ,1-4, Reh, andCEM; however.
III,-6(1 cells showed a markedly longer G2 arrest that
correlated with their greater radioresistance. The results
suggest that thetotal length of time available for DNA damage
repair (irrespective ofwhether this time accrues as blockage in G,,
S, or G2), prior to potentialactivation of apoptosis, is a critical
determinant of radiosensitivity inhuman hematopoietic cell lines.
Comparison of the p53 status of these celllines 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-associatedI25I decays was determined and was found to correlate
with the relative
sensitivity of these lines to y-irradiation. The highly
localized deposition ofenergy by I25I decays argues that DNA damage
is a potent initiator of
apoptosis in these cell lines. The results presented suggest
that differencesin the radiosensitivity of the cell lines examined
reflect differences in therapidity 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
thecontrol of cell cycling and/or the regulation of apoptosis.
Suchchanges can also alter radioresponsiveness, and there is now
extensiveliterature on the involvement of genes such as p53, c-myc,
and bcl-2
in determining the radiosensitivity of tumor cells. The impact
onradiosensitivity of mutation or change in the level of expression
ofsuch genes can, however, be variable and cell type dependent
(reviewed in Ref. 1), presumably reflecting the complexity of the
pathways that lead to cell death and the multiplicity of the
regulatorybiochemical interactions that together determine the fate
of an irradiated cell. We have examined different aspects of the
radiation response of murine lymphoid and myeloid cell lines to
determinewhether there are any simple, general correlates with
relative radio-
Received 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 with18 U.S.C. Section 1734 solely to
indicate this fact.
1To whom requests for reprints should be addressed, at Research
Division, PeterMacCallum Cancer Institute, Locked Bag No. 1,
A'Beckett Street. Melbourne. Victoria
8006. Australia. Phone: 61-3-9656-1291; Fax: 61-3-9656-1411;
E-mail: i.radford@
pmci.unimclb.cdu.au.
sensitivity. These studies suggested that sensitivity to
clonogenickilling by y-rays or DNA-associated 12il decays
correlated closely
with how rapidly apoptosis was induced after irradiation (2, 3).
Suchdifferences in timing reflected whether induction occurred soon
afterirradiation ("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
timesdependent upon the radiation dose they had received. It was
speculated that DNA dsb2 directly initiates the destruction of cell
lines
susceptible to rapid interphase death and that chromosomal
aberrations can trigger delayed interphase or mitotic apoptosis
(4). Theoccurrence of rapid interphase apoptosis appeared to be
dependentupon the presence of wild-type p53 in these cells (5).
There have been relatively few comparative radiobiological
studiesof human hematopoietic cell lines. The studies performed
have soughtto correlate radiosensitivity with differences in
capacity for sublethaldamage repair (6, 7). differences in lymphoid
lineage maturation orvarious biochemical parameters (8), or
differences in the rate of DNAdsb repair (9). However, aside from
noting that the most radiosensitive lines were derived from
immature lymphoid cells [we came to asimilar conclusion from
studies with murine lymphoid lines (3)], thesestudies have been
unable to explain the differences in radiosensitivityamong the cell
lines examined.
The relationship between p53 status, the kinetics of induction
ofapoptosis, and radiosensitivity has been studied in human
lymphoidlines, although to a more limited extent than for mouse
cell lines.Human studies have compared the TK6 (wild-type p53) and
WI-L2-NS (mutant p53) cell lines, which were both derived from
theBurkitt's lymphoma WI-L2 cell line. WTK1 (a clonal derivative
of
WI-L2-NS, which is also a p53 mutant) showed delayed induction
ofapoptosis and was correspondingly more resistant to killing by
X-
irradiation than was TK6 (10, 11). Olive et al. (12) also showed
acorrelation between slower induction of apoptosis and increased
radioresistance in WI-L2-NS as compared with TK6. although,
this
correlation did not hold when TK6 cells in different phases of
the cellcycle were compared. More recent work has cast doubt on
whether thedifferences seen in the WI-L2 system are due solely to
the expression
of mutant p53 protein. Yu et al. (13) disrupted p53 function in
theTK6 cell line by transfection with the human papillomavirus E6
geneand showed that this had a relatively small effect on the
kinetics ofinduction of apoptosis and no effect on the clonogenic
survivaldose-response for y-irradiation. The rate of induction of
apoptosis inirradiated 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
moresensitive to radiation-induced growth inhibition.
There are various lines of evidence that support the assumption
thatdifferences in hematopoietic cell line radiosensitivity are
related insome way to the cell's response to DNA damage. For
example.
Warters (15) demonstrated that bromodeoxyuridine labeling
enhancedthe sensitivity to X-ray-induced killing of a murine T-cell
hybridoma.
2 The abbreviations used are: dsb. double strand break; IL,
interleukin; BrdUrd,
bromodeoxyuridine; ECL. enhanced chemiluminescence.
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RADIOSENSmVITY OF HUMAN LYMPHOID CELLS
and we have shown that murine lymphoid cell lines can be
exquisitelysensitive to killing by DNA-associated 125Idecays and
that, in gen
eral, their sensitivity to such radioactive decays correlates
closely withtheir sensitivity to external beam irradiation (2, 3).
The DNA-centric
view has, however, been challenged by investigators who assert
acrucial role for ceramide production, following activation of
sphin-
gomyelinase at cell membranes, in the induction of apoptosis
byirradiation (16). Consistent with the latter hypothesis, it was
shownthat acid sphingomyelinase-deficient human or murine lymphoid
cellsshowed reduced levels of radiation-induced apoptosis (17).
Recently,
a correlation between the level of postirradiation ceramide
productionand the relative radiosensitivity of a panel of human
lymphoid celllines has been adduced as further support for the
importance ofmembrane 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
radiosensitivityand the rate of induction of apoptosis and between
sensitivity tokilling by external beam radiation and DNA-associated
125I decays
are also observed in human hematopoietic cell lines that show a
broadrange of radioresponsiveness. For these purposes, we have used
apanel of four human lymphoid (CEM, HSB-2, MOLT-4, and Reh) andone
myeloid (HL-60) cell line.
MATERIALS AND METHODS
Cell Lines. All cell lines were obtained from the American Type
CultureCollection. 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
wereperformed using asynchronous, log-phase cultures. The cell
lines were rou
tinely checked for Mycoplasma contamination using a PCR method
(19). Theorigin and some of the characteristic features of each of
the cell lines used arepresented in Table 1.
y-Irradiation, 125IIncorporation, Clonogenic Survival Assay, and
Sta
tistical Treatment of Survival Data. Cell cultures were
irradiated at roomtemperature 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 ¿IMthymidine. Cells
were then
washed, and unincorporated label was chased by incubation in
growth mediumcontaining 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
werecorrected to decays per minute using the method of Eldridge
(20). Cell pelletswere then resuspended in a known volume of growth
medium, aliquots weretaken, and cell number was determined by
Coulter counting. The mean numberof I25I decays occurring per cell,
per day was then calculated. After slow
addition of an equal volume of growth medium plus 20% DMSO, the
cellsuspension 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 ofI25I decays per cell had occurred, and assayed for
clonogenic survival. Accu-
Table 1 General properties of human hematopoielic cell lines
CelllineHSB-2
MOLT-4RehCEMHL-60OriginPre-T
Pre-TEarly BTPre-myelocyleKaryotype"Pseudodiploid
PseudotetraploidPseudodiploidPseudodiploidPseudodiploidDoubling
time(h)29
23212233Cloning
efficiency(% ±SE)15
±363 ±741 ±348 ±850 ±5TP53
status"Wild
typeWild type/mutant^Mutant
Deleted" Data from American Type Culture Collection catalog.*
Data from other studies (see "Results" for references).
mulated decays at day "t" (At) were determined using the
equationA, = 86.56/l0(l - i'-0-01155»),where Aa represents the
number of decays per
cell per day at day 0.Survival assays were performed using
single-cell suspensions that were
obtained by gently pipetting the cultures approximately 10 times
through a5-ml glass pipette. Dilutions of this cell suspension were
then counted using aCoulter counter. For each of the cell lines
except HSB-2, clonogenic survival
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 theaddition of 0.01% crystal violet in 1.5%
acetic acid. Colonies containing >50cells 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 ingrowth medium in 96-well, U-bottomed microtiter
plates (Greiner) and check
ing for growth after 14 days. Microtiter plate wells were scored
as positive ifthey contained greater than 50 cells. Cell numbers
added per well wereadjusted 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 and5% conditioned medium from the IL-2-secreting gibbon
leukaemic cell line
MLA 144 (data not shown). Although Guizani et al. (21)
demonstrated thatHSB-2 does not constitutively express the IL-2
receptor, growth factors present
in either the human serum or the conditioned media may be
inducing expression of IL-2 receptor. Alternatively, other factors
secreted by MLA-144 cellsmay be contributing to the improved
growth. Results for HSB-2 cells werecalculated 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
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 least-
squares fit. The fit was minimized according to the criterion of
£¡(0â̂€”¿�£¡)2/V¡,
where O is observed value, E is calculated value, and V is
variance. Thestatistical errors associated with the survival data
were calculated, after themethod proposed by Boag (22), by assuming
a simple Poisson variance on thetotal number of control and test
colonies counted.
Determination of Apoptotic Morphology. Cell samples, taken at
intervalsafter irradiation, were pelleted by centrifugation at 1000
rpm, gently resuspended in a small volume of residual growth
medium, and then mixed with anequal volume of growth medium
containing 10 fig/ml of ethidium bromide and3 p.g/ml of acridine
orange. At least 500 cells were then scored for apoptoticnuclear
morphology under a fluorescence microscope.
Cell Cycle Distribution. Cell cultures were labeled with 10
JXMBrdUrd for30 min and then analyzed using the anti-BrdUrd
antibody technique (23).
Cellular DNA content was determined by staining samples with
propidiumiodide. Samples were processed using a Becton Dickinson
FACStar Plus flowcytometer, 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 sortingand microscopic examination. Levels of apoptosis
determined by flow cytom-
etry were generally lower than values obtained by scoring
morphology (presumably due to overlap between the DNA content of
apoptotic and G, or G2cells); however, the two methods gave
qualitatively similar results (compareFigs. 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
sampleviscosity, 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 at10,000 rpm in a microcentrifuge to remove insoluble
material. Supematantswere assayed for protein concentration using
bicinchoninic acid and thenstored at —¿�70°C.Prior to
electrophoresis, buffer containing bromphenol blue
was added to give a final concentration of 10% glycerol, 10
HIMDTT, and 50mM Tris (pH 7.5). Samples were boiled for 3 min and
allowed to coolimmediately prior to loading.
Equal amounts of total protein (100 /j,g unless stated
otherwise) wereseparated 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 containing25 mM Tris base, 192 mM glycine, and 20% methanol
at pH 8.3 (24). Themembrane was then allowed to dry overnight at
room temperature, stained for
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RADIOSENSmVITY OF HUMAN LYMPHOID CELLS
(0>3CO
.1
I .010)§>o .001o
.0001
Dose (Gy)
Fig. I. Clonogenic survival of different human hemalopoietic
cell lines as a functionof -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 atroom 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 withperoxidase-labeled secondary antibody for l h at
room temperature. Afterfour 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, 0.1mg/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 notquantitative, information about protein
levels.
tion are presented in Fig. 1. The a and ßvalues derived from
fittingthe data to a linear-quadratic function of dose are given in
the figurelegend. The results show clearly that HSB-2 and HL-60
are. respec
tively, the most radiosensitive and the most radioresistant of
the celllines surveyed, with approximately a 200-fold difference
between thelines in the level of survival after a dose of 4 Gy.
MOLT-4 cells show
a comparable, although somewhat more radioresistant, response
tothat of HSB-2. Reh and CEM are considerably more
radioresistantthan HSB-2 and MOLT-4. Both show similar clonogenic
dose re
sponses, although CEM is significantly more radioresistant than
Rehat 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
givenequitoxic radiation doses sufficient to reduce clonogenic
survival to0.5% of the control value and were then incubated at
37°C.The timing
of occurrence of cell death was then determined in culture
samplestaken at various times after irradiation. All five cell
lines showedinduction of apoptotic nuclear morphology after
irradiation. HL-60
cultures showed some necrotic death, although apoptosis was
thepredominant form of death over the time interval examined (data
notshown). As shown in Fig. 2, the rate of induction of apoptosis
afterequitoxic doses of y-irradiation varied greatly between the
five cell
lines examined, and the relative ordering of these responses
reflectedthe clonogenic survival dose responses. For example, HSB-2
is the
most radiosensitive of the cell lines examined and also shows
the mostrapid induction of apoptosis. The more radioresistant
MOLT-4 cellline shows a pronounced delay of —¿�4-6h before
commencement ofinduction of apoptosis at a similar rate to that of
HSB-2. Reh culturesare more radioresistant than MOLT-4 and show a
somewhat longer
delay before initiating apoptosis. The subsequent entry of Reh
cellsinto apoptosis occurs at a much slower rate than is seen for
MOLT-4or 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 ofaccumulation of apoptotic cells appears to be similar to
that of Reh.
RESULTS
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 highlevel of inter-experimental variability. This result was of
concern
given claims that, at least for some cell lines, there is a
correlationbetween cloning efficiency and radiosensitivity (25).
The controlcloning efficiencies of CEM, HL-60, MOLT-4, and Reh
were, how
ever, found to be greatly increased by the addition of sheep
RBCs tothe medium. For example, the cloning efficiency of HL-60
increasedfrom 1% to ~50% in the presence of sheep RBCs. The ability
of
sheep RBCs to increase the cloning efficiency of hematopoietic
cellswas first demonstrated by Bradley et al. (26). Even with the
additionof 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.
Controlcloning efficiencies, obtained for each of the cell lines
using the abovemethods, 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-responses
for the five human hematopoietic cell lines under investiga-
oOu
oo.
o"'S
SSIL
1.0
0.8
0.6
0.2
10 20
Time (h)
30 40
Fig. 2. Rate of induction of apoptosis. as determined by nuclear
morphology, in•¿�y-irradiatedhuman 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 incubatedat
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 fromat least two separate experiments.
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RADIOSENSmVITY OF HUMAN LYMPHOID CELLS
.1
.01
HL-60(31.211.9)
MOLT-4(22.110.6)
20 40
125
60 80
I decays/cell
100
Fig. 3. Clonogenic survival responses of MOLT-4 and HL-60 cells
exposed toDNA-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 associatederror are shown on the figure. Each
data set is from two separate experiments.
The apparent decline in the numbers of apoptotic CEM cells at
—¿�20
h after irradiation is an artifact caused by the fragility of
apoptoticCEM cells (i.e., the true kinetics of appearance of
apoptotic CEM cellsare probably more comparable with those of Reh
than to HL-60).HL-60 is clearly the most radioresistant cell line
examined and also
shows the most pronounced delay prior to the induction of
apoptosis.Accumulation of apoptotic cells was not observed in HL-60
cultures
until at least 24 h after irradiation. It is of interest to note
that even themost rapidly dying of the human cell lines (HSB-2)
shows markedlyslower kinetics of induction of apoptosis than does a
murine lym-
phoma such as ST4 (Fig. 2).Sensitivity to Killing by
DNA-associated 125I Decays. Because
of the continuing debate over the importance of DNA damage
ininitiating radiation-induced apoptotic cell death and because the
levelof DNA damage induced per unit dose of y-irradiation can
vary
between cell lines (28), we were interested in comparing the
sensitivity of our panel of cell lines to killing by DNA-associated
I25I
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
interference from DNA repair activities. The cells must then show
reasonablecloning efficiencies after thawing. Our attempts to
determine thesensitivity of these cell lines to DNA-associated 125I
decays were,
however, generally unsuccessful due to low and variable
cloningefficiencies after freezing and thawing. MOLT-4 and HL-60
were the
only lines to show reasonable cloning efficiencies after
freezing andthawing (30 ±9% for MOLT-4 and 6.7 ±0.4% for HL-60).
The
clonogenic survival curves for these cell lines, after
accumulation of125I-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 tobe 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
cycleblockage or mitosis) and how differences in radiosensitivity
might berelated to these events (4). Cell cultures were pulse
labeled with
BrdUrd at intervals after exposure to equitoxic y-ray doses
(sufficient
to reduce clonogenic survival to 0.5% of the control value) and
werethen analyzed by flow cytometry. Cell cycle distributions, as a
function of time after irradiation, for each of the five cell lines
are shownin 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 anincrease in the levels of apoptosis. Irradiation has
rapid and profoundeffects on these cells as evidenced by an almost
total suppression ofmitotic activity after 3 Gy of y-irradiation
and a marked decrease inBrdUrd incorporation into S-phase cells
within 2 h after irradiation
(data not shown). Flow cytometric profiles of cultures that had
beenincubated for 4 or more hours after irradiation, showed a
significantincrease in the level of cells with sub-G, or
intermediate to G, and G2
(but lacking BrdUrd incorporation) DNA content (Fig. 5),
suggestingthat 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
contrastto the results obtained for HSB-2, the other more rapidly
dying cellline, MOLT-4, shows an accumulation of cells in G2 that
parallels andprecedes the induction of apoptosis. Consistent with
MOLT-4 cells
undergoing apoptotic death after blockage in G2, flow
cytometricprofiles showed a progressive increase, with time after
irradiation, inthe proportion of cells showing a DNA content
intermediate to G, andG2 (but lacking BrdUrd incorporation; Fig.
5). Similar to MOLT-4,
the increase in the fraction of apoptotic Reh cells follows and
occursin parallel with the accumulation of cells in the G2 phase.
Thisaccumulation occurs more slowly than is seen for MOLT-4,
appar
ently due to a more prolonged blockage of Reh cells in Gl and/or
S.Consistent with induction of apoptosis in G2, flow cytometric
profilesfrom irradiated Reh cultures undergoing apoptosis showed
increasedlevels of cells with a DNA content intermediate to G, and
G2 (butlacking BrdUrd incorporation; see Fig. 5). Although Reh and
CEMhave comparable sensitivities to killing by y-irradiation, their
cell
cycle responses are significantly different (Fig. 4). Unlike
Reh, CEMcells show no postirradiation blockage in the G, phase, but
they doshow a pronounced blockage in mid- to late-S phase that is
followed
by a rapid exit from S and accumulation in the G2 phase. As
withMOLT-4 and Reh, the latter event precedes the appearance of
apop
totic cells in culture. Consistent with the death of CEM cells
in G2phase, flow cytometric profiles of apoptosing cultures showed
increased levels of cells with a DNA content intermediate to G( and
G2(but lacking BrdUrd incorporation; data not shown). HL-60, the
most
radioresistant and the slowest dying of the cell lines in our
panel, lacksa marked G, arrest but shows prolonged blockage of
cells in G2 priorto the appearance of apoptosis in the culture
(Fig. 4). The timing ofoccurrence 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 thesecells 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
lymphoidcells. Accordingly, we sought to correlate the status and
expression ofthe TP53 gene with the radiosensitivity and cell cycle
response of thecell 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
Rehcells could not be found in the literature. We tested for the
presenceof p53 protein in each of the cell lines by Western
blotting. The blotswere probed with the PAb 421 monoclonal
antibody, which is specificfor an epitope in the COOH-terminal
region of the protein (29), and
reactive proteins were detected using ECL. Expression of p53
proteinwas found in all of the cell lines, except for HL-60 (Fig.
6). The latterresult is consistent with the finding that HL-60 has
a large deletion
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RADIOSENSmVITY OF HUMAN LYMPHOID CELLS
1.0
Fig. 4. Effect of -y-irradiation on cell cycle progression
of human lymphoid lines. The distribution of cells between the
different phases of the cycle (G,, S. and G2)and the level of
apoptosis (Api were determined by flowcytometry using cultures
given equitoxic doses and thenincubated at 37°C.Each data set was
pooled from two
separate experiments.
o -
0 2 4 602468 10 0
Time (h) Time (h)
8 12 16 20 24
Time (h)
1.0
.2 .8
CL
.4
8.2
CEM (5 Gy) HL-60 (6.5 Gy)
4 8 12 16 20 240
Time (h)
10 15 20 25 30 35
Time (h)
within the TP53 gene that prevents protein expression (30). The
twomost rapidly dying and radiosensitive cell lines, HSB-2 and
MOLT-4,have both been shown to express wild-type p53 (31, 32). In
additionto a wild-type gene, the MOLT-4 cell line also contains a
TP53
mutation that leads to aberrant splicing of the mRNA, causing a
frameshift and truncating the protein before translation of exons
10 and 11(32). Of the more slowly dying cell lines, none are known
to expresswild-type p53. Both copies of the TP53 gene in CEM cells
contain
single base substitutions that produce missense mutations (31),
and asmentioned above, the HL-60 cell line does not express p53
protein due
to gene deletions. Reh does express p53 protein (Fig. 6):
however, wedo 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
reactiveprotein of Mr -26,000 (Fig. 6). Reh and HL-60 differed from
the
other lines in showing an additional reactive band of slightly
highermolecular weight. The additional band might represent
expression ofa 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 ofHL-60 has been shown to express Bcl-xL (35).
DISCUSSION
We have examined whether the correlation between the rate
ofinduction of apoptosis and the clonogenic survival y-ray
dose-
response, observed for murine hematopoietic cell lines, is also
seen inhuman hematopoietic cell lines. The results presented in
this reportclearly demonstrate, for the cell lines examined, a
correlation betweenthe rapidity with which apoptosis is induced
after irradiation and theclonogenic 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
equitoxicradiation dose, are correspondingly more resistant to
killing byy-irradiation.
Although a general correlation between the relative rate of
induction of apoptosis and the clonogenic survival y-ray
dose-response is
seen for both human and murine hematopoietic cell lines, one
notabledifference is that although the survival dose-response
curves for the
more radiosensitive human and murine cell lines are similar,
equitoxicradiation 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
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RADIOSENSÕTMTY OF HUMAN LYMPHOID CELLS
HSB-2 (Control) HSB-2 (3 Gy + 5 hrs)
uO
O3E'
2mO
MOLT-4 (Control) 2
Reh (Control)
O
mO
MOLT-4 (3.5 Gy + 10 hrs)
O
Reh (5 Gy + 24 hrs)
DNA ContentFig. 5. Dot-pio! distributions for BrdUrd
incorporation (ordinale) versus DNA content
(abscissa) of cells from control and irradiated MOLT-4 and Reh
cultures. Regionscorresponding 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 thisstudy (HSB-2) does not reach this level of
apoptosis until at least 24 hafter receiving an equitoxic y-ray
dose, despite having a similarclonogenic survival y-ray
dose-response to ST4. Unfortunately, because we were unable to
determine the sensitivity of HSB-2 cells tokilling by
DNA-associated 125Idecays, we cannot rigorously compare
the response to DNA damage of ST4 and HSB-2. If we assume
thatthere are similar y-ray dose-responses for DNA lesion induction
in the
two cell lines, then perhaps the simplest explanation for the
differencein timing is that, after the detection of lethal cellular
injury, thebiochemical events that lead to the triggering of
apoptosis occur muchmore 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 isa marked difference in the population doubling times of
cell lines suchas 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
doubling time (around 12 h) and yet show induction of apoptosis
afterirradiation 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
relapsedafter intensive radiotherapy and/or chemotherapy of the
primary tumor, whereas the derivation of murine lines like ST4 did
not involveexposure to cytotoxic agents. Such treatment may well
have selectedfor cells with down-regulated apoptotic responses, and
it is perhaps
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) andp53-independent blocks in S (38) and G2 (39). These blocks
areultimately caused by inhibition of cyclin-dependent kinases that
are
necessary for cycle progression. In the cell lines that we have
examined, 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
undergoinduction of apoptosis in different phases of the cell
cycle. Consistentwith previous observations that the more rapidly
dying and radiosensitive murine lymphoid cell lines contain
wild-type p53 (5), HSB-2encodes wild-type TP53 (31), and we have
shown that it expressesp53 protein. It may also be relevant to the
radiosensitivity of HSB-2
that, due to homozygous deletion of the Rb gene (40), this cell
linedoes not express pRb. Expression of pRb was associated with
p53-independent protection against radiation-induced apoptosis in
both a
human osteosarcoma and a lymphocytic cell line (41, 42).
WhenMOLT-4 or Reh cells were exposed to a y-ray dose that
reducessurvival to 0.5% (unlike HSB-2), both showed induction of
apoptosis
after accumulation of cells in G-,. Due apparently to blockage
earlierin the cycle, Reh cells accumulated in G2 much more slowly
than didMOLT-4 cells and showed a corresponding increase in the
time
between irradiation and the appearance of apoptotic cells that
correlated with their greater radioresistance. The molecular basis
for thisdifference is unclear. Both MOLT-4 and Reh express pRb (40,
43)and, as shown in this study, p53. Although MOLT-4 cells express
bothwild-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
inductionof apoptosis in Reh cells suggests that they contain
mutant p53. LikeMOLT-4 and Reh, irradiated CEM cells appeared to
die after block
age in G2. CEM cells differed, however, in showing a
prominentblockage in mid- to late-S phase that preceded
accumulation in G2.
Consistent with their slow kinetics of induction of apoptosis
and theirradioresistance, 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
andprolonged blockage in G2. Other authors have also described
radiation-induced G-, arrest and apoptosis of HL-60 cells (39, 44).
Although MOLT-4, Reh, and CEM cultures showed an increase in
the
level of apoptosis that lagged about 4 h behind the accumulation
ofcells in G2, our results suggest that HL-60 cells can remain
arrested in
G2 for much longer times before undergoing apoptosis. The basis
forthis difference has not been examined, although in other cell
typesexpression of a ras oncogene was associated with a decrease in
cyclinBl 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 in humanhematopoietic cell lines.
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RADIOSENSITIVE OF HUMAN LYMPHOID CELLS
The importance of the duration of postirradiation arrest at the
G,and G2 checkpoints in determining radiosensitivity has been
tested invarious nonlymphoid cell types that undergo apoptosis.
There isevidence to suggest that although the p53-dependent G,
checkpoint
may not be critical in this regard (46), the length of the G2
delay maybe directly related to radiosensitivity (45). Consistent
with our previous results for murine hematopoietic cell lines (4),
the data presentedin this paper suggest that the total length of
time available for DNAdamage repair (irrespective of whether this
time accrues as blockagein 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
radio-
sensitivity between hematopoietic cell lines lies in
understanding theresponse of the cell to DNA damage. Consistent
with this view, wehave shown in this report a correlation between
the sensitivity ofMOLT-4 and HL-60 to y-irradiation and to
DNA-associated 125Idecays. It is interesting to note that the D0
values for I25I decay-
induced 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 themurine 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
postir-
radiation accumulation in and subsequent apoptosis in G2
phase,whereas HL-60 and 18-81 both exhibit a very delayed induction
ofapoptosis (4). I25I decays have been shown to deposit the
majority of
their energy in close proximity to the site of decay (47) and,
whenoccurring 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
alsoresponsible for y-ray-induced apoptosis. An opposing view, that
radiation-induced apoptosis is triggered by membrane
damage-initiated
production of ceramide, has been supported recently by data
showinglower postirradiation production of ceramide in HL-60 than
in a moreradiosensitive Burkitt's lymphoma line (18). The latter
study did not,
however, compare the ceramide responses of the cell lines at
equitoxicradiation doses; in addition, it has been suggested that
the ceramideassay method used by Michael et al. (18) gives
artifactual results (49).
We have shown that differences in the radiosensitivity of a
panel ofhuman hematopoietic cell lines can be related to
differences in therapidity of induction of apoptosis and that
radiation-induced apoptosis
can be explained as a response to DNA damage.
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1998;58:2817-2824. Cancer Res David R. Aldridge and Ian R.
Radford Radiation between Human Lymphoid Cell LinesExplaining
Differences in Sensitivity to Killing by Ionizing
Updated version
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