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MYELOID NEOPLASIA
Mechanisms of resistance to high and low linear energy transfer radiation in
myeloid leukemia cells
Kurtis J. Haro,1,2 Andrew C. Scott,1 and David A. Scheinberg1,2
1Molecular
Pharmacology and Chemistry Program and Leukemia Service, Memorial Sloan-Kettering Cancer Center, New York, NY; and 2Department of
Pharmacology, Weill Cornell Graduate School of Medical Sciences, New York, NY
Low linear energy transfer (LET) ionizing
radiation (IR) is an important form of
therapy for acute leukemias administered
externally or as radioimmunotherapy. IR
is also a potential source of DNA damage.
High LET IR produces structurally different forms of DNA damage and has
emerged as potential treatment of metastatic and hematopoietic malignancies.
Therefore, understanding mechanisms of
resistance is valuable. We created stable
myeloid leukemia HL60 cell clones radioresistant to either ␥-rays or ␣-particles to
understand possible mechanisms in radioresistance. Cross-resistance to each
type of IR was observed, but resistance to
clustered, complex ␣-particle damage was
substantially lower than to equivalent
doses of ␥-rays. The resistant phenotype
was driven by changes in: apoptosis; late
G2/M checkpoint accumulation that was
indicative of increased genomic instabil-
ity; stronger dependence on homologydirected repair; and more robust repair of
DNA double-strand breaks and sublethaltype damage induced by ␥-rays, but not
by ␣-particles. The more potent cytotoxicity of ␣-particles warrants their continued
investigation as therapies for leukemia
and other cancers. (Blood. 2012;120(10):
2087-2097)
Introduction
Nearly one-half of all cancer patients will undergo some form of
ionizing radiation (IR).1 In acute myeloid leukemia, total body
irradiation combined with chemotherapy before stem cell transplantation is an effective treatment for acute myeloid leukemia,2
although residual, radioresistant leukemic cell clones remain and
lead to relapse. Therefore, understanding the cellular and biochemical mechanisms of IR resistance is important for devising better
therapies and reducing adverse effects in normal tissues exposed to
IR during therapy, or inadvertently because of environmental
exposures or nuclear devices.
In contrast to the low linear energy transfer (LET) IR used in the
treatment of acute myeloid leukemia before stem cell transplantation or as radioimmunotherapy for leukemia or lymphoma, high
LET IR, including ␣-particles, deposit their energy in micron scale
distances in vivo. Although the damage that ␣-particles induce in
DNA and nearby biomolecules is chemically similar to that of
␥-rays, the relative impact of direct ionizations on biomolecules
from ␣-particles is much greater than that of ␥-rays as ␣-particles
typically induce highly clustered DNA damage, leading to complex
DNA double-strand breaks (DSBs) and chromosomal aberrations.3-5
These sites of highly clustered damage are thought to explain the
increased relative biologic effectiveness of ␣-particles.6 For example, DNA repair-deficient cell mutants become less radiosensitive compared with their wild-type counterparts when challenged
with ␣-particles versus low LET x-rays.7 The differential ability of
cells to cope with high and low LET IR is further underscored by
work demonstrating that chemo- and ␥-IR–resistance was circumvented with an ␣-emitting 213Bi-labeled anti-CD45 antibody in
leukemia cells.8 Thus, ␣-particle emitting nuclides are a promising
therapy of readily accessible tumors of the hematopoietic system,
sparing healthy tissues.9 Multiple clinical trials are currently
underway testing the ability of targeted ␣-particle emitters to kill
malignant cells in the hematopoietic compartments. They include
223Ra (Alpharadin), in phase 3 clinical trials for the treatment of
bone metastases in prostate and breast cancer,10 213Bi-labeled
anti-CD33 antibody and a 4 ␣-particle generator, 225Ac, also
conjugated to anti-CD33 antibody for treatment of myeloid
leukemia.11
We sought to address whether ␣-particle–induced radioresistance is possible in hematopoietic cancer cells and, if so, whether
observed mechanisms of high LET radioresistance could be
quantitatively and qualitatively similar to low LET radioresistance.
Hence, we created stable radioresistant clones derived from
myeloid leukemia HL60 cells irradiated with high or low LET IR.
Resistant cell clones demonstrated reduced IR-induced apoptosis,
desensitization of the late G2/M checkpoint, and improved repair of
specific forms of chromosomal DNA damage thought to result
from 2 DSB sites not in proximity to one another. Resistance to
␣-particle emitters was minimal, detected only at low ␣-particle doses.
Methods
IR selection and cloning of individual cell colonies
HL60 human myelocytic leukemia cells (ATCC) were maintained at
105-106 cells/mL. HL60 cells were irradiated 15 times over the course of
approximately 150 days with equitoxic, escalating doses with either a 137Cs
source or an 241Am source12 for low and high LET resistant cells, respectively
(Table 1). After each dose, when cells reached ⬎ 95% viability, cells were
immediately re-irradiated. The initial doses were determined from the doses
Submitted January 23, 2012; accepted July 3, 2012. Prepublished online as
Blood First Edition paper, July 24, 2012; DOI 10.1182/blood-2012-01-404509.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2012 by The American Society of Hematology
BLOOD, 6 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 10
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BLOOD, 6 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 10
HARO et al
Table 1. Dose selection scheme for creation of ␣ (RA) and ␥ (RG) resistant HL60 clones
Iteration
HL60
1
—
2
—
3
—
4
—
5
—
6
—
7
—
8
—
9
—
10
—
11
—
12
—
13
—
14
—
15
—
RA, Gy
0.9
0.9
1.3
1.3
1.3
1.3
1.7
1.7
1.9
2.0
2.0
2.2
2.2
2.2
2.4
RG, Gy
2.5
2.5
3.3
3.3
3.3
3.3
3.8
3.8
4.0
4.5
4.5
5
5.5
6
6.5
— indicates not applicable.
needed to kill 90% of naive HL60 cells (D10) in clonogenic survival assays and
increased, as indicated in “IR-induced apoptosis is reduced in all RA and RG
clones relative to HL60.” Unirradiated HL60 control cells were kept 150 days as
a control.
After the final round of irradiation, individual cell clones from the
irradiated progeny and unirradiated HL60 cells were randomly selected
after 2 weeks of growth in semisolid methycellulose medium. These
colonies were expanded then aliquoted for long-term liquid nitrogen
storage. All cells were mycoplasma free at all times.
␥-H2A.X foci quantitation
Cells were fixed in 2% paraformaldehyde (Sigma-Aldrich) for 15 minutes
in the dark, blocked with 2% BSA in PBS, then incubated for 1 hour with a
monoclonal antibody anti–phospho-H2A.X (clone JBW301; Millipore) at a
1:1000 dilution, then probed with secondary antibody goat anti–mouse IgG
conjugated to AlexaFluor-488 in PBS ⫹ 2% BSA at 1:1000 dilution for
30 minutes in the dark. After further washing, cells were mounted in
ProLong antifade mounting solution (Invitrogen) containing 4,6-diamidino2-phenylindole and imaged by wide-field microscopy (Mirax). Foci size
and threshold were adjusted to fit the images, and all samples were
quantified by automated analysis using Volocity with identical exposure
times and threshold settings.
Neutral comet assay
Cells were irradiated on ice at 105 cells/mL in PBS with various doses of
␥-IR and immediately processed for DSB formation using the comet assay
kit (Trevigen) and analyzed using CometScore Version 1.5 (TriTek
Corporation) software. The tail moments were reported for at least 100 cells
per sample.
Annexin V and propidium iodide apoptotic assay
The annexin V apoptosis kit (BD Biosciences PharMingen) was used to
quantitate early cell death. A total of 2 ⫻ 104 events were acquired on a
C6 Accuri flow cytometer (Accuri Cytometers) for each sample.
Executioner caspase assay
Cell lysates were prepared in nondenaturing conditions, normalized by
protein content, and incubated with fluorogenic substrate Ac-DEVD-AMC
(Calbiochem) at 37°C for 90-120 minutes and analyzed for fluorescence at
485 excitation/535 emission in a 384-well black plate.
Measurement of cell nuclei and geometry for ␣-particle
exposure
Cells at 5 ⫻ 105 cells/mL were fixed in regular medium containing
2% paraformaldehyde for 15 minutes at room temperature, stained with
4,6-diamidino-2-phenylindole, and added to aluminum dishes containing
Mylar seals. Cells were settled on a Leica TCS AOBS SP2 inverted
microscope (Leica Microsystems) for 20 minutes before z-stacked images
were taken in 1-␮m increments. Reconstructed images taken parallel to the
culture surface were used to make planar projections for cross-sectional
measurements of cell nuclear area parallel to the culture surface and thus
perpendicular to the path length of ␣-particle traversal using Volocity
Version 5.4.1 software (PerkinElmer Life and Analytical Sciences).
0.25-second exposure. For a nuclear radius of 5.15 ␮m (eg, “parental”
HL60 cells), this requires an exposure of 16 seconds to achieve an average
of 1 nuclear traversal. The mean ␣-particle energy is estimated to be
2.9 MeV incident to the surface with a full-width half-maximum value
of ⬃ 0.9 MeV, corresponding to a mean LET of 132 keV/␮m.13 For
spherical cells, this corresponds to a conservative estimated nuclear dose of
1 Gy for every 4 nuclear ␣-particle traversals.14 Any differences in dose to
specific cell clones were accounted for by adjusting the number of
␣-particle traversals for a given exposure time according to measured
nuclear area (supplemental Figure 4, available on the Blood Web site; see
the Supplemental Materials link at the top of the online article). All cell
samples were settled on the benchtop irradiator at ambient temperature for
20 minutes before exposure to ensure a uniform cell layer on the mylar cell
culture surface.
Cells were exposed to low LET IR by a Shepherd Mark I 137Cs source
irradiator (JL Shepherd) at ⬃ 2 Gy/min.
Cell cycle analysis
Approximately 106 cells were fixed with 1 mL ice-cold 70% ethanol
for ⬎ 2 hours at ⫺20°C, then washed and stained with PBS, 20 ␮g/mL
propidium iodide (Invitrogen), 0.1% Triton-X 100, and 100 ␮g/mL RNAse
A (Sigma-Aldrich), and incubated 30 minutes at 37°C in the dark. Cells
were collected on a flow cytometer (Accuri Cytometers) and gated for cells
containing 2n and 4n DNA content.
Mitotic index assay
At indicated times, cells were fixed and assayed for mitotic index as
described.15 Samples were normalized to unirradiated controls (including
each time point in time-course studies).
Micronucleus assay
After IR treatment, cells were treated with 10 ␮g/mL cytocholasin B for
20 hours and assayed for micronucleus formation as described previously.16
At least 250 nuclei were counted for each sample from at least 5 different
fields. For micronuclei quantification, at least 50 binucleated cells were
counted for each sample and were scored as positive if the size of the
micronucleus was between approximately one-tenth and one-third the size
of a normal nucleus. Cells containing 4 nuclei were not scored.
Clonogenic survival assay
Cells were adjusted to 10 times plating concentration in 1:10 methylcellulose (StemCell Technologies) containing 40% methylcellulose, 10% FBS,
2mM L-glutamine, and 50% RPMI with a blunt-ended 16-gauge needle
(StemCell Technologies) before plating 1.1 mL per 35-mm2 dish in
duplicate for each sample. After ⬃ 14 days in a 37°C humidified incubator,
colonies containing ⬎ 50 cells were enumerated blindly.
ATP Lite viability and thymidine proliferation assays
The ATP Lite assay kit was used (PerkinElmer Life and Analytical
Sciences) on 104 cells in 100 ␮L in quadruplicate. Proliferation was
performed with [6-3H]-thymidine (PerkinElmer Life and Analytical Sciences), added 12 hours before harvest, as originally described, with a
Tomtec harvester (Tomtec).17
Alpha particle and ␥-ray exposure
The design of the 259-MBq 241Am source has been described in detail
elsewhere.12 The flux of ␣-particles per unit area using the CR-39 plastic
etching technique was found to be 198 ⫾ 14 particles/mm⫺2 from a
Low oxygen clonogenic cell culture
Cells were equilibrated for a minimum of 6 hours in Nunc 25-cm2
vent/close cap tissue culture flasks (Nalgene Nunc International) in an In
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BLOOD, 6 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 10
Vivo2 400 Hypoxic workstation (Ruskinn), containing a gas mixture of
5% CO2, 95% N2 at 37°C and 90% humidity, which achieved a maximum
oxygen concentration of 1000 ppm measured in air with the In Vivo2
oxygen probe. Cells were manipulated in the workstation, sealed, and
immediately taken to the ␥-ray source for irradiation. Cells were maintained
under aerobic conditions after IR exposure.
Knockdown of Rad51 using shRNA
MISSION-targeted shRNA constructs were obtained from Sigma-Aldrich
for the Rad51 protein using gene accession number NM_002875. The
sequence used was 5⬘-CCGGCGGTCAGAGATCATACAGATTCTCGAGAATCTGTATGATCTCTGACCGTTTTT-3⬘. shRNA-containing plasmid
constructs were packaged with lentiviral plasmids per the manufacturer’s
protocol in HEK293T (ATCC). Viral soups were titered using the HIV-1
p24 antigen ELISA assay kit (Zeptometrix). Target cells were incubated
overnight with virus at a multiplicity of infection of 10, placed in normal
medium for another 24 hours, and selected on 5 ␮g/mL puromycin for
10 days. Cells were assayed for Rad51 knockdown via Western blot using a
rabbit polyclonal antibody specific to Rad51 (Novus Biologicals).
Cell cycle synchronization
Cell synchronization was achieved using the double thymidine block
technique18 (12 hours “on,” 10 hours “off,” 14 hours “on”). Greater than
80% of cells were accumulated at the G1/S border as assessed by cell cycle
flow cytometry in all experiments.
Results
IR-induced apoptosis is reduced in all RA and RG clones
relative to HL60
The dose scheme used for selection of radioresistant cell clones is
displayed in Table 1. Each increase in dose was equivalent in terms
of expected increases in cell death in HL60 cells. We initially
screened clones derived after 15 irradiations of either ␥-rays (RG)
or ␣-particles (RA) by assaying for apoptosis at 48 hours after IR
(where apoptosis is maximal; supplemental Figure 1) and observed
substantial reductions in apoptosis in response to IR (supplemental
Figure 2A-B). RA clones were more uniformly resistant to
IR-induced apoptosis than RG clones (supplemental Figure 2A-B).
We validated these initial results with 3 clones from RA and RG
cells and found the phenotype to be stable over several days in
culture (Figure 1A-B). Notably, RA and RG clones displayed
cross-resistance to ␥-ray and ␣-particle exposures, respectively
(Figure 1C-D). We performed detailed analyses of specific clones
RA5 and RG2, chosen to identify any potential mechanistic
differences between the cell clones in response to different chronic
radiation treatments. In these resistant clones, reduced apoptosis
correlated with lack of executioner caspase activation 24 hours
after ␥-ray exposure (Figure 1E). The lack of IR-induced apoptosis
in these cells was stable over a total culture time of at least 2 months.
To ensure that the RA and RG clones’ resistance to IR-induced
apoptosis was not the result of variability in parental HL60 cells,
we also analyzed the apoptotic response to a single dose of 4 Gy of
␥-rays in 15 naive HL60 clones and found 1 with significantly
reduced apoptosis (supplemental Figure 2). However, clonogenic
survival assays on the naive HL60 clones revealed that no
radiation-naive clone had significantly reduced mitotic death
(supplemental Figure 3A-B). Collectively, the IR-induced apoptotic response of RA and RG progeny were significantly different
from those derived from naive HL60 clones, indicating that
repeated exposure to radiation created radioresistant clones that
MECHANISMS OF RADIORESISTANCE IN MYELOID LEUKEMIA
2089
were not resistant because of random variability (supplemental
Figure 2D).
Clonogenic survival data show that clones are resistant to low
LET IR at all doses tested, but resistant only to low doses of
high LET ␣-particles
To confirm that these apoptotic results were indicative of improved
survival at the single-cell level, 3 representative sublines from RA
and RG cells were subjected to both types of IR and analyzed for
their capacity for single cells to form colonies. Resistant cell clones
had increased survival after low LET ␥-rays compared with HL60
at all doses tested (Figure 2A and Table 2).
In contrast to low LET IR, clonogenic survival after ␣-particle
exposure was qualitatively different for the resistant cells. Only at
very low doses of ␣-particles were differences between resistant
clones and HL60 cells observed (Figure 2B,D). In the low-dose
portion of the ␣-particle curve, the induction of a small shoulder
was observed of ⬃ 1 or 2 additional ␣-particle traversals in the
RA5 and RG2 clones (Figure 2C). Resistance to a low dose
of ⬃ 0.23 Gy of ␣-particles was confirmed in 5 of 6 clones (Figure
2D). Following Poisson statistics, these data imply radioresistance
to high LET ␣-particles in most RA and RG clones can only be
achieved following doses that result in most cells receiving either
0 (41%), 1 (36.5%), or 2 (16%) nuclear ␣-particle traversals. Thus,
resistant cells show differences to HL60 cells after individual cell
doses of less than ⬃ 0.5 Gy. Because of the ␣-particle probability
distribution, at higher doses, the assay is no longer capable of
resolving differences between sensitive and resistant cells. These
results were not because of differences in cell morphology and
associated ␣-particle dosimetry between cell sublines because
mean nuclear areas were similar, as demonstrated by confocal
microscopy (supplemental Figure 4).
Apoptosis is reduced in RA5 and RG2 cells with internal high
LET IR-emitters of the radioimmunotherapy construct
225Ac–anti-CD33 (lintuzumab) and 125I-dUdr
We wanted to confirm our studies of survival with the external
application of ␣-particle flux with the clinically relevant construct
225Ac-labeled lintuzumab, which targets ␣-particles to CD33⫹
cells. We treated cells in culture with a dose escalation of
225Ac-conjugated to lintuzumab (HuM195). There was an approximately 1-log shift in survival in the ATP Lite viability assay of RA5
and RG2 cells compared with HL60 at 48 hours after treatment
(Figure 3A; supplemental Figure 5). A 100-fold excess unlabeled
antibody blocked the effect (Figure 3B), demonstrating its
specificity.
Similar results were obtained from 125I-dUdr treatment of cells,
where decay primarily through electron capture produces Auger
electrons that act as high LET particles, creating complex DSBs.19,20
Despite nearly identical uptake of the molecule in the 3 cell lines
(Figure 3D), apoptosis was reduced in RA5 and RG2 cells relative
to HL60 at 48 hours after treatment (Figure 3C). It was difficult to
control the dose of internalized high LET emitters to levels low
enough to ascertain differences between RA5 and RG2 compared
with HL60. These data are consistent with that obtained with the
external ␣ irradiator.
Late G2/M arrest is reduced in RA5 and RG2 cells and depends
significantly on the LET of the IR
Radiosensitivity is dependent on cell cycle stage.21 Cell cycle
analysis showed that the doubling time and cell cycle distribution
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Figure 1. RA and RG clones have significantly reduced IR-induced apoptosis compared with HL60
cells after both ␥-ray and ␣-particle exposure. Selected RA and RG cell clones and HL60 cells were
irradiated with indicated doses of ␥-IR (A,C) or ␣-IR
(B,D), incubated 48 hours under standard cell culture
conditions, and then assayed for apoptosis by annexin V
and propidium iodide staining. Data are mean ⫾ SEM
from at least 3 independent experiments. Two-way,
repeated-measures ANOVA with a Dunnett test showed
all clones to be statistically different from HL60 at
P ⫽ .05 level or better. (E) Data from a representative
executioner caspase activity assay repeated 3 times at
24 hours after ␥-IR. *P ⬍ .05, **P ⬍ .01, and ***P ⬍ .001,
least significant of the 3 resistant clones at each dose.
kinetics of the 3 cell lines were similar (Table 3). However, RA5
and RG2 cells had an ⬃ 2-fold increase in the dose of ␥-rays
needed to achieve 50% inhibition of thymidine incorporation at
36 hours after ␥-IR compared with HL60 (supplemental Figure 6A).
HL60 lacks a functional G1/S checkpoint,22 so we restricted our
analysis to the 2 molecularly distinct G2/M checkpoints23 to
determine whether changes in checkpoint functionality could
explain the survival data in resistant clones. We performed
bivariate flow cytometric analysis to quantitate the number of cells
attempting division after IR. We found that the early, ATMdependent G2/M checkpoint for cells in G2 during IR exposure to
Table 2. Parameters of ␥-ray survival curves for HL60, RA5, and
RG2 fit with the linear-quadratic model
Cell line
D0, Gy
D1, Gy
HL60
0.76
1.48
4
RA5
1.02
2.41
20
RG2
1.17
1.96
9.5
n
be similar in all 3 cell lines 1 hour and 2 hours after IR
(supplemental Figure 6B-C). These data suggested that ATM
functionality is largely intact and that early DNA damage-sensing
machinery is similar in sensitive and resistant cell lines.
In contrast, using the same mitotic assay, we found differences
in the dose-dependent late G2 accumulation and subsequent release
into mitosis as both a function of time (Figure 4A-C,E-G) and dose
(Figure 4D,H). These differences depended on the type of LET of
the IR. RA5 and RG2 cells more rapidly entered mitosis compared
with HL60, particularly at higher doses of ␥-IR (Figure 4A-C).
Similar, less robust results were seen with ␣-particles (Figure
4E-G). These data indicated that more substantial cell cycle arrest
(ie, reduced replicative capacity) in resistant cells after ␣-particle
exposure may have prevented increased colony formation, thus
explaining the survival data observed after ␣-particle exposure
(Figure 2). Similarly, when we compared mitotic indices at 24 hours
after IR over a range of doses, the resistant lines showed significant
increases in mitotic indices compared with HL60 cells, but these
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BLOOD, 6 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 10
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Figure 2. Clonogenic survival is increased for various RA and RG clones after IR exposure. Log-linear
clonogenic survival curves for cells treated with ␥-rays
(A) or ␣-particles (B). (C) The low ␣-particle doseresponse is displayed for RA5 and RG2. (D) Normalized
survival to a single dose of ⬃ 0.23 Gy ␣-particles is
shown for select clones. *P ⬍ .05, relative to HL60. Data
are mean ⫾ SEM of at least 3 independent experiments
differences were substantially less after high LET IR (Figure
4D,H). Thus, although resistant cells were much less likely to
undergo apoptosis after IR exposure, the relatively large differences in checkpoint desensitization for resistant cells treated with
Figure 3. RA5 and RG2 have reduced apoptosis to high LET-emitting constructs 225Ac-lintuzumab and 125I-dUdr. Cells were treated in log-phase growth with indicated
concentrations of radioimmunotherapy construct for 48 hours in a 96-well plate and then assayed by the ATP Lite viability assay. (A) Treatment with 225Ac-lintuzumab.
(B) Treatment in the presence of 100-fold excess unlabeled antibody. Data are mean ⫾ SD of a representative experiment repeated 3 times. The curves were fitted using
sigmoidal dose-response nonlinear regression with variable slope. The corresponding IC50 values for HL60, RA5, and RG2 were 0.59, 5.25, and 8.7 kBq/mL, respectively.
(C) Cells were treated with indicated doses of 125I-Udr, and cell viability was measured by scoring of nuclear morphology, normalized to controls. (D) Percent uptake of 125I-Udr
at 24 and 48 hours after treatment. 125I-Udr experiments are mean ⫾ SEM from 2 independent experiments. A 2-way, repeated-measures ANOVA was performed to determine
significance at each dose. *P ⬍ .05. **P ⬍ .01. ***P ⬍ .001.
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Table 3. Baseline cell cycle distributions and doubling times of
HL60, RA5, and RG2 cells
Cell line
% G1
%S
% G2
Td , h
HL60
50.8
30.8
18.9
18.6 ⫾ 0.6
RA5
48.8
29.5
22.7
17.3 ⫾ 2.4
RG2
51.4
29.8
19.8
17.9 ⫾ 1.1
iso-effective ␥-ray and ␣-particle exposures suggested that the
quality and type of the DNA damage cells sustain after IR exposure
still played a large role in G2/M checkpoint arrest and subsequent
colony formation.
To gain a better understanding of the checkpoint behavior of
RA5 and RG2 cells, we used the micronucleus assay to measure the
extent of attempted cell division after IR and residual chromosomal
DNA damage in the form of micronuclei.24 At both high and low
doses of ␥-IR, RA5 and RG2 cells had higher proportions of
mitotic cells, but this effect was accompanied by a higher
percentages of cells with micronuclei (Figure 5A-D), indicative of
increased chromosomal instability.25 After a mean dose of ⬃ 1.5 Gy
of ␣-particles, the radioresistant cells still had significantly higher
levels of micronuclei, but not significant increases in the percentage of mitotic cells after IR (Figure 5E-F), consistent with the data
from the mitotic index assay (Figure 4). Thus, at iso-effective doses
(Figure 5C,E), ␣-particles were more able than ␥-rays to prevent
premature entry into mitosis in radioresistant cells versus naive cells.
We tested whether inhibition of late G2 arrest alone by pretreatment
of cells with caffeine22 1 hour before IR was sufficient to improve the
naive HL60 cell’s ability to survive ␥-rays. We measured micronuclei formation at 20 hours after IR, the accumulation of G2 arrest at
18 hours after IR, and clonogenic survival. Although caffeine was
capable of almost completely abolishing G2 arrest (supplemental
Figure 7A-B), there was no improvement in clonogenic survival
after 4 Gy ␥-IR (supplemental Figure 7C). Moreover, inhibition of
Figure 4. Late G2 checkpoint arrest is attenuated in
RA5 and RG2 cells after IR. Cells were irradiated with
indicated doses of ␥-IR (A-D) or ␣-IR (E-H) and assayed
for mitotic index as described in “Mitotic index assay.”
Data are normalized to unirradiated controls and are
mean ⫾ SEM from 3 independent experiments. Cells were
analyzed for both time responses following specific doses
(A-C,E-G) and dose-response at 24 hours after IR.
(D,H) Two-way, repeated measures ANOVA was used to test
for differences at each dose. *P ⬍ .05. **P ⬍ .01.***P ⬍ .001.
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Figure 5. Mitotic indices and proportion of binucleated cells with micronuclei are increased in RA5 and
RG2 cells compared with HL60 after IR. Cells were
irradiated with 2 Gy ␥-IR (A-B), 6 Gy ␥-IR (C-D), or ⬃ 1.5 Gy
␣-particles (E-F) and assayed for mitotic index (A,C,E) and
percent of binucleated cells with micronuclei (B,D,F). Data
are mean ⫾ SEM from 3 independent experiments.
Samples were scored as indicated in “Micronucleus
assay.” Two-way, repeated-measures ANOVA with Bonferroni posttests were performed to analyze significance
of each treatment between HL60 and resistant cells.
*P ⬍ .05. **P ⬍ .01.***P ⬍ .001.
late G2 arrest led to increased IR-induced micronuclei compared
with cells irradiated in the absence of caffeine (supplemental
Figure 7D). These results demonstrated that perturbing late G2
checkpoint activity alone is not sufficient to produce radioresistance in HL60 cells.
Lesion-specific DNA damage is better repaired in RA5 and RG2
cells compared with HL60 cells
To assess IR-induced DNA damage and repair of DSBs, we assayed
for phosphorylated serine-139 on the histone H2A variant H2A.X
(also known as ␥H2A.X), a biochemical marker of DNA DSBs.26,27
Because the majority (⬎ 95%) of untreated cells in the 3 cell lines
under investigation had 10 or fewer foci, we scored cells as positive
for having IR-induced foci if the nucleus contained 11 or more foci.
These experiments revealed that at 30 minutes after IR, ␥H2A.X
foci were similar in HL60 and RG2 cells but substantially lower in
RA5 cells (Figure 6A). At 4 hours after IR, however, greater than
50% of foci had been resolved in the resistant cell lines, whereas
HL60 cells had only resolved ⬃ 15% of their foci-positive cells
(Figure 6A). These data indicated that the repair of DNA DSBs was
more rapid in the radioresistant cells. Analysis of the data by the
reduction in mean ␥H2A.X foci/cell yielded similar conclusions
(supplemental Figure 9). In contrast to the data after ␥-IR, there
were similar levels of foci-positive cells in HL60, RA5, and RG2
cells after an equitoxic dose of ⬃ 0.5 Gy of ␣-particles at 4 hours
(Figure 6B, see supplemental Discussion for further analysis).
Therefore, the DNA damage after ␣-particles is more difficult to
resolve, limiting radioresistance to ␣-particles.
Comet assays confirmed that the lower initial formation of
␥H2A.X foci in RA5 cells after ␥-IR was not a reflection of lower
induction of DNA damage (eg, through up-regulated intracellular
radical-scavenging molecules), as all 3 cell lines had similar
increases in comet tail moments (measured by both mean and
median) immediately after 2 Gy of ␥-IR (Figure 6C).
The level and persistence of DSBs (particularly at early time
points) induced by IR do not always correlate with the ability of the
cell to survive IR-induced DNA damage.28,29 The quality and
proximity of DSBs can have a large impact on whether a lethal
lesion is formed.3 When we split doses of ␥-IR in time, allowing for
sublethal damage repair, we found that this type of repair was
enhanced in RA5 and RG2 cells compared with HL60 (Figure
6D-F). The quasi-threshold dose, Dq, for HL60 cells improved to
0.5 Gy after splitting the dose by 1 hour and increased to 1 Gy
when the dose was split by 4 hours. In contrast, the Dq for both RA5
and RG2 cells was 1 Gy with 1 hour of incubation at 37°C between
irradiations and increased to ⬃ 2 Gy after 4 hours of split-dose
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2094
HARO et al
BLOOD, 6 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 10
Figure 6. Nonclustered DNA damage and DSB repair are improved in RA5 and RG2 cells compared with HL60. Cells were irradiated with 2 Gy ␥-IR (A) or an
approximate dose of 0.5 Gy ␣-particles per nucleus (B) and assayed for percent foci-positive cells (n ⫽ 500) relative to controls at 30 minutes and 4 hours after IR. (C) Cells
were irradiated with 2 Gy ␥-IR on ice in PBS and immediately processed for DSB formation by the neutral comet assay. Data are box and whisker plots: whiskers are 1st and
99th percentiles; and ⫹, means (calculated from at least 100 cells per sample). (D-F) Cells were irradiated with a test dose of 3 Gy of ␥-rays and allowed to incubate under
standard cell culture conditions for the indicated times before additional irradiation with indicated doses of ␥-rays, and then assayed for clonogenic survival. Cells were
irradiated under hypoxic conditions (⬍ 0.1% O2) with indicated doses of ␥-rays before assaying for clonogenic survival (G). (H-I) Cells were treated with indicated doses of
bleomycin (H) or neocarzinostatin (I) and assayed for apoptosis 48 hours after treatment. They were are fitted by nonlinear regression. IC50 values exceed the 95% CI for both
resistant clones. Data are mean ⫾ SEM from at least 2 independent experiments. *P ⬍ .05. **P ⬍ .01. ***P ⬍ .001.
incubation time. Sublethal damage damage repair would be
expected to have little effect on survival after ␣-particles because
the complex DNA lesions would be difficult to restore to nonlethal
lesions. Indeed, splitting a single dose of 3 mean ␣-particles per
nucleus into 2 exposures separated by 2 hours showed no increase
in survival than the same dose in 1 exposure in all 3 cell lines
(supplemental Figure 10).
We also observed that the relative radioresistance of RA5 and
RG2 cells was nearly abolished by hypoxia (0.1%; Figure 6G).
Although these data suggest that RA5 and RG2 cells have
mechanisms that protect them against reactive oxygen species
during IR, efforts to test this hypothesis by probing for changes in
cellular gluthathione levels, catalase activity, and resistance to
hydrogen peroxide and doxorubicin (reactive oxygen speciesdependent death) did not support a reactive oxygen speciesmediated mechanism (supplemental Figure 11).
Alternatively, RA5 and RG2 may be less sensitive to damage
under hypoxia because hypoxic conditions reduce a substantial
portion of the indirect, radical-mediated damage to DNA.30 This
could, therefore, cause a shift in the balance from 2-track ionization
cell killing to single-track ionization cell killing because 2-track
cell killing, more dependent on free radical formation, would be
reduced under such circumstances.31,32 The striking resemblance of
the survival curve in Figure 6G and the ␣-particle survival curve in
Figure 2C, where differences in survival exist at only low doses,
illustrate this point. It is also possible that the lack of oxygen may
reduce the chemical complexity of DNA damages, reducing the
relative radioresistance of RA5 and RG2 cells compared with
HL60, which under hypoxia may have similar DNA repair
capacity. In either case, the data demonstrated the qualitative
dependence on the type of DNA damage that can be withstood in
radioresistant clones.
The apoptotic response to the radiomimetic drugs bleomycin
and neocarzinostatin, which create a high frequency of DNA
damage clusters,33,34 was only mildly reduced in resistant clones
relative to HL60, with IC50 increases of approximately 2-fold for
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BLOOD, 6 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 10
MECHANISMS OF RADIORESISTANCE IN MYELOID LEUKEMIA
2095
Figure 7. RA5 and RG2 depend on homology directed repair for radioresistance. Cells were synchronized to the early G1/S border by double thymidine block and were
irradiated 1 hour (A) or 5 hours (B) after release into regular medium and assayed for clonogenic survival. (C) Percent knockdown of Rad51 relative to cells infected with control
virus after normalization to ␤-actin. (D-F) Cells were irradiated with indicated doses of ␥-IR and assayed for clonogenic survival. Data are mean ⫾ SEM from at least
2 independent experiments. *P ⬍ .05, **P ⬍ .01, and ***P ⬍ .001, for indicated dose relative to HL60 (A-B) or in Rad51-depleted cells relative to “Empty” control cells (D-F).
each drug (Figure 6H-I). Therefore, the survival and DNA damage
data from Figure 6 suggest that clustered versus nonclustered DNA
damage has a substantial effect on the radioresistance that can be
achieved in radioresistant cells, consistent with the survival data
after ␣-particle exposure (Figure 2C).
RA5 and RG2 cells depend on homology directed repair for
significant radioresistance to ␥-IR
DSB repair can occur via 2 distinct mechanisms that largely depend
on cell cycle stage, nonhomologous end-joining, and homology
directed repair (HDR).35 HDR is primarily used in the late S or
G2 phases of the cell cycle, when the sister chromatid is available
for template-mediate repair. We tested whether HDR could be
leading to radioresistance in RA5 and RG2. Cells were enriched at
the G1/S border by double thymidine block and irradiated 1 hour or
5 hours after release from the G1/S border (supplemental Figure
12). When irradiated in early S phase, RA5 and RG2 cells were
almost as sensitive to ␥-rays as HL60 (Figure 7A). RA5 and RG2
cells irradiated at 5 hours after G1/S release, however, demonstrated radioresistance closer to their baseline levels, indicating that
HDR might be playing a key role in radioresistance for RA5 and
RG2 cells (Figure 7B). To confirm these data, we created viral
particles expressing shRNA targeted to Rad51, which is essential
for HDR. Knockdown of the Rad51 protein (Figure 7C; supplemental Figure 13) decreased clonogenic survival in RA5 and RG2 cells
to a much greater extent than in HL60 cells (Figure 7D-F). These
data demonstrate that radioresistance in RA5 and RG2 is exquisitely dependent on proper DNA repair, perhaps because of shorter
G2 checkpoint arrest after IR.
Discussion
We have examined radioresistance of myeloid leukemia cells to IR
of different LET, using ␥-rays and ␣-particles, with incident LETs
of ⬃ 1 and 132 keV/␮m13, respectively. We are not aware of other
␣-particle–resistant clones of leukemia cells. The data revealed a
number of possible mechanisms that could account for resistance.
However, the limited number of clones examined do not exclude
other pathways that may also be involved. Compared with their
unirradiated parental HL60 cells, the defined cell lines showed
significantly increased radioresistance. However, radioresistance,
as measured by assays of apoptosis and clonogenic survival, was
qualitatively and quantitatively different after different types of test
radiation. Resistance to ␥-rays was far more pronounced. Increases
in clonogenic survival after ␣-IR were seen only at low doses,
where the majority of cells received 2 or fewer ␣-particle traversals
through the nucleus. In the RA5 and RG2 clones studied in detail,
early DNA damage sensing appeared to be similar in sensitive and
resistant cells. Late G2 arrest was reduced in the radioresistant cell
lines, apoptosis was markedly reduced, and improved DNA repair
was necessary to impart radioresistance in RA5 and RG2 cells,
particularly through HDR. Meanwhile, DNA damage by high LET
IR was more difficult to resolve, and repair of ␣-particle–induced
DSBs was not improved in the radioresistant cells. These results are
consistent with the increased tendency to divide (and thus avoid
apoptosis) with extant DNA damage that imparted resistance to
RA5 and RG2 cells when exposed to low numbers of ␣-particles,
but not at higher ␣-particle doses, where this mechanism probably
became overwhelmed by genomic damage.
The 15 rounds of irradiation used to create the radioresistant cell
clones began and terminated at equitoxic doses in naive cells.
Therefore, it is not unexpected that the increase in radioresistance
was similar for RA and RG clones after exposure to either type of
IR. However, whereas resistance to ␥-rays was observed at all
doses tested, resistance to ␣-particles quickly saturated after mean
doses that exceeded 1 ␣-particle traversal, leading to an increase in
D50 of ⬃ 0.25 Gy in resistant clones, when calculated according to
the methods of Charlton and Sephton.14 The data suggest that the
mechanisms we have attributed to radioresistance in these specific
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2096
BLOOD, 6 SEPTEMBER 2012 䡠 VOLUME 120, NUMBER 10
HARO et al
clones, derived from both chronic ␥-ray and ␣-particle exposures,
were quantitatively and qualitatively similar. Not surprisingly, the
limited ␣-particle resistance appeared to correlate with the inability
of resistant cells to cope with highly complex DNA damages, as
previously described.36 Indeed, low LET irradiation under hypoxia
almost eliminated radioresistance in RA5 and RG2 cells, whereas
resistance to the clustered damage-inducing radiomimetics bleomycin and neocarzinostatin was modest in RA5 and RG2 cells, further
supporting the hypothesis that DNA lesion type largely determines
cell fate after IR exposure, even in cells technically defined as
“radioresistant.”37
From a mechanistic standpoint, the changes in the cell survival
curve after IR exposure in RA5 and RG2 cells could simply be
attributed to reductions in IR-induced apoptosis, often observed in
␥-IR–resistant cancer cell variants, including HL60.38-40 Similarly,
the increased propensity to undergo mitosis in the radioresistant
cells after IR exposure could account for radioresistance, despite
residual chromosomal damage, as reported by other studies41 (and
J. Seideman, N. Veomett, D.A.S., observations under review,
September 2009). However, naive HL60 cells treated with caffeine
before IR demonstrated that inhibition of late G2 arrest after IR did
not increase clonogenic survival. In addition, the stronger dependence of RA5 and RG2 cells on Rad51-mediated HDR for cell
survival after ␥-rays compared with HL60, suggested that lack of
IR-induced apoptosis is necessary, but not sufficient, for radioresistance in these cells. Therefore, it is likely a combination of
perturbations in apoptosis, G2 checkpoint maintenance, and DNA
repair that led to radioresistance in RA5 and RG2 cells. It is likely,
however, that additional mechanisms, perhaps in different combinations, would also be uncovered in other radioresistant HL60 clones.
The application of ␣-particle therapies currently in clinical
trials, including lintuzumab conjugated to 213Bi or 225Ac for
myeloid leukemia, 223Ra to treat bone metastases in prostate and
breast cancer patients, and 211At for glioblastoma, make a better
understanding of ␣-particle–induced radioresistance and sensitivity
critical. This information may help to understand how to protect
normal hematopoietic progenitors from damage or mutagenesis
after ␣-particle therapy. The results presented here shed light on
potential mechanisms that myeloid leukemia cells acquired to
become radioresistant. Although resistance mechanisms may differ
between individual RA and RG clones, our survival data from other
clones suggest that induced radioresistance is at least quantitatively
similar for all clones in the population. Importantly, the data
demonstrate the potent cytotoxicity of ␣-particles and limited
resistance cancer cells can achieve to high LET IR, warranting
further efforts to include them as part of cancer therapies.
Acknowledgments
The authors thank Dr Jonathan Seideman, Dr John Humm, Dr John
Petrini, and Dr Richard Kolesnick for helpful discussions and
feedback; Dr Michael McDevitt for providing 225Ac-labeled lintuzumab; and Oakley Olson for assistance with some experiments.
This work was supported by the National Institutes of Health
(R01-CA55349), the Weill Cornell Department of Pharmacology
(training grant T32 GM073546), the Lymphoma Foundation, the
Reuven Merker Foundation, the Tudor Foundation, and the Glades
Foundation.
Authorship
Contribution: K.J.H. designed and performed research, collected,
analyzed, and interpreted data, and wrote the manuscript; A.C.S.
performed research and collected data; and D.A.S. designed
research, analyzed and interpreted data, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: David A. Scheinberg, Memorial Sloan Kettering Cancer Center, 1275 York Ave, New York, NY 10065; e-mail:
[email protected].
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2012 120: 2087-2097
doi:10.1182/blood-2012-01-404509 originally published
online July 24, 2012
Mechanisms of resistance to high and low linear energy transfer
radiation in myeloid leukemia cells
Kurtis J. Haro, Andrew C. Scott and David A. Scheinberg
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