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NEOPLASIA
Reduction in drug-induced DNA double-strand breaks associated with
␤1 integrin–mediated adhesion correlates with drug resistance in U937 cells
Lori A. Hazlehurst, Nikola Valkov, Lee Wisner, Jonathan A. Storey, David Boulware, Daniel M. Sullivan, and William S. Dalton
We previously showed that adhesion of
myeloma cells to fibronectin (FN) by
means of ␤1 integrins causes resistance
to certain cytotoxic drugs. The study described here found that adhesion of U937
human histiocytic lymphoma cells to FN
provides a survival advantage with respect to damage induced by the topoisomerase (topo) II inhibitors mitoxantrone,
doxorubicin, and etoposide. Apoptosis
induced by a topo II inhibitor is thought to
be initiated by DNA damage. The neutral
comet assay was used to determine
whether initial drug-induced DNA damage correlated with cellular-adhesion–
mediated drug resistance. Cellular adhe-
sion by means of ␤1 integrins resulted in
a 40% to 60% reduction in mitoxantroneand etoposide-induced DNA doublestrand breaks. When the mechanisms
regulating the initial drug-induced DNA
damage were examined, a ␤1 integrin–
mediated reduction in drug-induced DNA
double-strand breaks was found to correlate with reduced topo II activity and
decreased salt-extractable nuclear topo
II␤ protein levels. Confocal studies
showed changes in the nuclear localization of topo II␤; however, alterations in
the nuclear-to-cytoplasmic ratio of topo
II␤ in FN-adhered cells were not significantly different. Furthermore, after a high
level of salt extraction of nuclear proteins, higher levels of topo II␤–associated
DNA binding were observed in FN-adhered cells than in cells in suspension.
Together, these data suggest that topo II␤
is more tightly bound to the nucleus of
FN-adhered cells. Thus, FN adhesion by
means of ␤1 integrins appears to protect
U937 cells from initial drug-induced DNA
damage by reducing topo II activity secondarily to alterations in the nuclear distribution of topo II␤. (Blood. 2001;98:
1897-1903)
© 2001 by The American Society of Hematology
Introduction
Studies have found that cellular adhesion by means of ␤1 integrins
inhibits cell death induced by DNA cross-linking agents and
topoisomerase (topo) II inhibitors.1,2 The mechanisms of resistance
associated with ␤1-mediated adhesion are unknown. It is thought
that DNA cross-linking agents and topo II inhibitors initiate
cellular death by inducing DNA damage. Thus, cellular adhesion
by means of ␤1 integrins could confer resistance by either
decreasing drug-induced DNA damage or increasing cellular
tolerance to such damage. To address this issue, we examined the
effects of ␤1-mediated cellular adhesion to fibronectin (FN) on
DNA damage induced by pharmacologic inhibitors of topo II. If ␤1
integrin–mediated adhesion reduces DNA damage induced by this
class of drugs, then alterations in the putative target (topo II) may
represent one mechanism of cellular-adhesion–mediated drug
resistance (CAM-DR).
Topo II is an adenosine triphosphate (ATP)–dependent enzyme
that reversibly cuts double-stranded DNA and is transiently linked
to the 5⬘ end of the break site by phosphotyrosyl bonds. Mammalian cells contain 2 isoforms of topo II (topo II␣ and topo II␤,
which are 170 kd and 180 kd, respectively). These 2 isoforms are
encoded by separate human genes and differ with respect to
molecular mass, sequence specificity for DNA cleavage, regulation
of expression, and tissue distribution.3,4 Many topo II inhibitors
stabilize this normally transiently bound DNA-protein complex
and form what is referred to as the cleavable complex.5 Stabiliza-
From the Department of Interdisciplinary Oncology and Clinical Investigations
Program and the Biostatistical Department, H. Lee Moffitt Cancer Center and
Research Institute, University of South Florida, Tampa, FL.
Submitted December 18, 2000; accepted May 10, 2001.
Supported in part by National Cancer Institute grants CA77859 (W.S.D.) and
CA82533 (W.S.D.).
BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
tion of this complex is thought to initiate apoptotic cell-death
pathways. Qualitative and quantitative changes in topo II␣ and
topo II␤ are associated with drug resistance and decreased druginduced DNA damage.6-8 The amount of drug-induced cleavable
complex can be detected indirectly by measuring DNA doublestrand breaks.9 In this study, we found that ␤1-mediated adhesion
of U937 cells to FN reduced the number of mitoxantrone- and
etoposide-induced DNA double-strand breaks as assessed by the
neutral single-cell gel-electrophoresis (comet) assay. Furthermore,
the ␤1-adhesion–mediated reduction in DNA double-strand breaks
correlated with decreased salt-extractable topo II␤ protein levels
and alterations in the nuclear localization of topo II␤.
Materials and methods
Cell culture
The U937 human histiocytic lymphoma cell line was obtained from the
American Type Culture Collection (Rockville, MD). The cells were grown
in suspension in RPMI 1640 medium (Cellgro; Fischer Scientific, Pittsburgh, PA) supplemented with 10% fetal-calf serum (FCS; Omega Scientific, Tarzana, CA), penicillin (100 ␮g/mL), streptomycin (100 ␮g/mL), and
2 mM L-glutamine (Gibco, Grand Island, NY). Cells were maintained at
37°C in an atmosphere of 5% carbon dioxide and 95% air and underwent
passage twice weekly.
Reprints: William S. Dalton, H. Lee Moffitt Cancer Center, 12902 Magnolia
Drive, Tampa, FL 33612; e-mail: [email protected].
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 U.S.C. section 1734.
© 2001 by The American Society of Hematology
1897
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1898
BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
HAZLEHURST et al
Drugs and antibodies
Mitoxantrone and doxorubicin were obtained from Sigma (St Louis, MO)
and dissolved in sterile double-distilled water. Etoposide was obtained from
Sigma and dissolved in dimethyl sulfoxide (DMSO). The integrin-blocking
antibodies included ␣4 blocking antibody (P4G9; Dako, Carpinteria, CA),
␣5 blocking antibody (P1D6; Dako), and ␤1 blocking antibody (P4C10;
Gibco). MAR4 antibody obtained from Pharmingen (San Diego, CA) was
used to detect expression of ␤1 integrin on the cell surface. Topo II␤ and
topoII␣ polyclonal antibodies were generated in the laboratory of Dr D.
Sullivan (Moffitt Cancer Center, Tampa, FL),10 and topo I monoclonal
antibody was a generous gift from Dr Y.-C. Cheng (Yale University Medical
School, New Haven, CT).11
Cell-surface expression and functional adhesion assay
Cell-surface expression of integrins was measured by incubating 1 million
cells with primary or isotype control antibody for 30 minutes on ice.1 After
2 washes with phosphate-buffered saline (PBS), cells were incubated with a
secondary fluorescein isothiocyanate (FITC)–conjugated goat antimouse
antibody (Dako). After incubation with the secondary antibody, the samples
were washed twice with PBS. Fluorescence was analyzed by flow
cytometry using a fluorescence-activated cell-sorter scanner (Becton Dickinson, Mountain View, CA) to record 10 000 events. Mean fluorescence
values for the isotype control were subtracted from the mean fluorescence
values for integrin staining. Mean and SD values were calculated from the
results of 3 independent experiments.
The adhesion assay was done as described previously.1 Briefly, 96-well
Immunosorp (Nunc, Denmark) plates were coated with either 50 ␮L (40
␮g/mL) of soluble FN (Gibco) or bovine serum albumin (BSA) and allowed
to evaporate overnight at room temperature. Cells were washed once in
serum-free RPMI medium and resuspended at a density of 1 ⫻ 106
cells/mL. Before cell adhesion, cells were incubated for 30 minutes with a
1:100 dilution of either isotype control, ␣4 blocking antibody (P4G9), ␣5
blocking antibody (P1D6), or ␤1 blocking antibody (P4C10). After
incubation with antibodies, 1 ⫻ 105 cells were added to each well. After 2
hours of adhesion, unattached cells were removed by 3 washes with RPMI
medium, and adherent cells were fixed with 70% methanol for 10 minutes,
dried, and subsequently stained with a solution of 0.02% crystal violet and
0.2% ethanol. The stained cells were solubilized in 100 ␮L Sorenson
solution, and absorbance was read at 540 nm with an automated 96-well
plate reader (Dynex, Chantilly, VA). Mean and SE values were calculated
from the results in 4 independent wells. Experiments were repeated at least
twice, and results of representative experiments are shown (Figure 1).
Inhibition of cell growth
Inhibition of cell growth was determined by using a modified monotetrazolium (MTT) dye assay with the following modifications.1 A 96-well
Immunosorp plate was coated with FN as described previously.1 Briefly,
cells were washed once in serum-free RPMI medium, and FN samples were
plated at a density of 150 000 cells/mL. Cells in suspension were incubated
in serum-free RPMI medium in a conical tube at the same density as cells
attached to FN. After 2 hours of cellular adhesion, wells were aspirated and
180 ␮L RPMI medium containing 10% FCS was added to each well. Cells
maintained in suspension were centrifuged and resuspended in RPMI
medium containing 10% FCS at a concentration of 100 000 cells/mL. Plates
were treated with various concentrations of drug for 1 hour. After 1 hour of
drug exposure, plates were washed 3 times with RPMI containing 10%
FCS. After a 72-hour incubation at 37°C, 50 ␮L MTT dye (2 mg/mL) was
added to each well, and the cells were incubated for an additional 4 hours.
Plates were centrifuged once at 500g, medium was aspirated, the waterinsoluble product was dissolved in DMSO, and absorbance was read at 490
nm on an automatic plate reader. The concentration of drug that produced
50% inhibition of growth (IC50) was calculated by using linear regression analysis.
Apoptosis
A flow cytometric assay assessing annexin V staining was used to count
apoptotic cells after drug exposure, as described previously.1 After 2 hours
of adhesion to FN, cells were exposed to drug for 1 hour and extracellular
drug was removed by 3 washes with RPMI medium containing 10% FCS.
For experiments examining etoposide-induced apoptosis, cells were exposed continuously to various doses of etoposide. Apoptotic cells were
detected 20 hours after initial drug exposure by using annexin V staining
and flow cytometric analysis. Ten thousand events were analyzed by flow
cytometry (Becton Dickinson, San Jose, CA). Mean and SD values were
calculated from the results of at least 3 independent experiments done in
duplicate. Statistical comparisons used the Student t test.
Comet assay
Cells were placed in serum-free RPMI medium (750 000 cells/mL) and
either adhered to FN-coated, 35-mm plates (Nunc) or placed in suspension
for 2 hours. After 2 hours of adhesion, cells were exposed to various
concentrations of drug for 1 hour. Subsequently, 5000 cells were placed in a
microcentrifuge tube containing 1 mL cold PBS, and the neutral comet
assay was done as described by Kent et al.12 Briefly, cells were centrifuged
and resuspended in 500 ␮L cold PBS, and 1.5 mL 1% agarose was added to
each sample. The agarose-cell suspension was gently layered on a
frosted-glass microscope slide, allowed to solidify for 5 minutes, and then
placed immediately in ice-cold lysis buffer containing 30 mM disodium
ethylenediamine tetraacetic acid (EDTA, pH 8.0), 0.5% sodium dodecyl
sulfate (SDS), and 0.25 mg/mL proteinase K (Fisher Scientific, Norcross,
GA). The samples were lysed for 1 hour at 4°C and then kept at 37°C for 12
to 16 hours. After cell lysis and digestion of protein-DNA complexes with
proteinase K, the agar slides were re-equilibrated in TBE (90 mM
Tris-hydrochloric acid, 90 mM boric acid, and 2 mM EDTA [pH 8.0]) for 2
hours, with a change of buffer every 15 minutes. The samples were
electrophoresed with TBE buffer for 20 minutes at 25 V. The DNA was then
stained with a 1:10 000 dilution of Sybr Green (Molecular Probes, Eugene,
OR) for 20 minutes and slides were washed twice for 5 minutes in TBE. To
ensure random sampling, 50 images/slide were captured and, in some
experiments, the observer was blinded to the conditions. The images were
captured on a fluorescent microscope (Vysis, Downers Grove, IL) and
quantified by using Imagequant software (Molecular Dynamics, Sunnyvale,
Ca). The comet moment was calculated by using the following equation
described by Kent et al12: comet moment ⫽ ⌺0⫺n ((intensity of DNA at
distance X) ⫻ (distance))/intensity of total DNA.
The mean comet-moment value obtained from vehicle-control samples was
subtracted from the mean comet-moment value for each drug dosage. Data
shown are the mean and SD values from 3 independent experiments (50 images
for each dose of each independent experiment). An analysis of variance
(ANOVA) model was used to quantify the relation between the response variable
and the 2 independent variables. The response variable in the analysis was the
difference between the mean comet-moment values for the control and drugtreated samples. Independent variables were the dosage of mitoxantrone (0.1
␮M, 1 ␮M, and 10 ␮M) and the treatment type (FN versus suspension). The
variance estimate for the test statistic was calculated by pooling the variances
from each of the 2 groups (control and treated).
Accumulation of carbon 14 (14C)–mitoxantrone
Cellular accumulation of 14C-mitoxantrone in FN-adhered cells and cells in
suspension (1 ⫻ 106) was compared after a 1-hour exposure to 2.5 ␮M
14C-mitoxantrone (specific activity, 0.3 GBq/mM). After exposure to drug
at 37°C for 1 hour, FN-adhered and suspension cells were washed 3 times in
cold PBS. The cells were counted before cell lysis with 10% SDS, and the
data were normalized to counts per minute of 14C-mitoxantrone/1 million cells.
Topo II activity and expression
Cells in log-phase growth were washed once in serum-free RPMI medium,
resuspended at density of 1 ⫻ 106 cells/mL in serum-free RPMI medium,
and adhered to FN or placed in suspension as described previously.1
Nuclear extracts were prepared as described by Sullivan et al.7 All the
following procedures were done at 4°C, and 1 mM phenylmethylsulfonyl
fluoride, 5 ␮g/mL leupeptin, and 5 ␮g/mL aprotinin were added to buffers B
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BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
REDUCTION IN DRUG-INDUCED DNA DOUBLE-STRAND BREAKS
to F. For FN samples, cells remained on FN-coated plates until placed in a dounce
homogenizer. Approximately 25 million cells were washed once in PBS and then
washed once with 25 mL buffer A (0.15 M sodium chloride [NaCl] and 10 mM
potassium phosphate, monobasic [KH2PO4]). Samples were incubated for 30
minutes on ice with 4 mL buffer B (5 mM KH2PO4, 2 mM magnesium chloride
[MgCl2], 4 mM dithiothreitol [DTT], and 0.1 mM sodium (NA2) EDTA). Cells
were then dounce homogenized for 15 strokes; the release of nuclei was followed
microscopically before proceeding to the next step. Nuclei were collected at
2500g for 15 minutes, further purified by resuspension in 2 mL buffer C (buffer B
and 0.25 M sucrose), and layered over 1 mL buffer D (buffer B and 0.6 M
sucrose). The sucrose gradient was centrifuged in a swinging-bucket rotor for 20
minutes at 2000g. The nuclear pellet was resuspended in 100 ␮L buffer E (5 mM
KH2PO4, 4 mM DTT, and 1 mM Na2 EDTA), and the total volume was
measured. An equal volume of buffer F (40 mM Tris [pH 7.5], 2 M NaCl, and 4
mM DTT) was added to the solution, which was incubated for an additional 30
minutes on ice. The lysate was centrifuged at 100 000g for 1 hour, and the
supernatant was adjusted to 10% glycerol (vol/vol). To decrease the chance of
topo II degradation, all topo activity assays were done on the same day the
nuclear extracts were obtained.
For immunoblotting, 30 ␮g fresh nuclear extract from suspension and
FN-adhered samples were separated on a 7.5% SDS-polyacrylamide gel
and transferred to a polyvinylidene difluoride membrane. The blot was
probed with either a topo II␣ or topo II␤ polyclonal antibody or a topo I
monoclonal antibody. The band of interest was detected by chemiluminescence (NEN, Boston, MA) and quantified by using Imagequant (Molecular Dynamics).
The catalytic activity of topo II was measured as the decatenation of
networks of kinetoplast DNA (kDNA). The kDNA was labeled with
tritium-thymidine and isolated from Crithidia fasciculata as described
previously.7 One microgram nuclear protein extract and 0.40 ␮g kDNA was
incubated in a total volume of 40 ␮L at 30°C for selected times. The
reaction buffer consisted of the following: 50 mM Tris (pH 7.5), 100 mM
NaCl, 10 mM MgCl2, 1.0 mM ATP, 0.5 mM DTT, and 30 ␮g/mL BSA. The
reaction was terminated by the addition of 5 ␮L 2.5% SDS. The samples
were then microcentrifuged for 10 minutes at 12 000 rpm at room
temperature. After centrifugation, 30 ␮L of the supernatant, which contained the released kDNA minicircles, was removed, liquid scintillation
fluid was added, and radioactivity was measured with a scintillation counter
(Beckman, Palo Alto, CA).
adhesion with blocking antibodies were measured in U937 cells.
Surface expression of ␣4, ␣5, and ␤1 integrin subunits is shown in
Figure 1A. The U937 cells expressed more ␣4 (mean fluorescence,
91.32 ⫾ 31.74) than ␣5 (mean fluorescence, 36.11 ⫾ 15.97);
however, the use of blocking antibodies showed that adhesion of
U937 cells is mediated primarily by ␣5␤1 integrin (Figure 1B).
Immunohistochemical analysis
Confocal microscopy was used to determine whether cellular adhesion
changed the intracellular localization of topo II. U937 cells were adhered to
FN for 2 hours. After 2 hours of cellular adhesion, cells maintained in
suspension or cells adhered to FN were fixed with 4% paraformaldehyde for
10 minutes. The cells were then cytospinned and subsequently permeabilized with 0.5% Triton-X, 1% glycine, and PBS for 1 hour as described
previously.10 Briefly, after permeabilization, slides were incubated with
either a 1:100 dilution of topo II␣ or topo II␤ polyclonal antibody for 1 hour
(0.1% NP-40 and 1% BSA in PBS). After several washes in PBS for 2 hours,
slides were incubated with a goat anti–rabbit immunoglobulin G (IgG)–
tetrarhodamine isothiocyanate–labeled antibody (Sigma) at a 1:80 dilution in
0.1% NP-40 and 1% BSA in PBS for 35 minutes in the dark. After incubation
with the secondary antibody, slides were washed several times in PBS for 2
hours. Immunofluorescence was observed with a scanning confocal microscope
(LSM 510; Zeiss, Göttingen, Germany). To obtain nuclear-to-cytoplasmic ratios
of FN-adhered cells and cells in suspension, 100 individual cells were analyzed
for pixel density of the nucleus and cytosol. The mean pixel density of the
background was subtracted from all values before calculation of the nuclear-tocytoplasmic ratio.
Results
Adhesion to FN is mediated by ␣5␤1 integrin for U937 cells
Because surface expression of integrin subunits may not reflect
functional adhesion, both cell-surface expression and functional
1899
Adhesion of U937 cells to FN for 2 hours causes resistance to
topo II inhibitors
The MTT assay was used to determine whether the adhesion of
U937 cells to FN protects cells from drug-induced cytotoxicity. As
shown in Figure 2, adhesion of U937 cells to FN for 2 hours before
drug exposure increased the IC50 value of mitoxantrone approximately 10 fold (range, 5-17 fold). The IC50 value of doxorubicin
was increased approximately 3 fold (range, 2-5 fold). The degree of
resistance for the nonintercalating topo II inhibitor etoposide, as
measured by MTT assay, was less than that for mitoxantrone and
doxorubicin, being approximately 2 fold (range, 1.3-4 fold).
In addition to measuring cytotoxicity with the MTT assay, we
used an apoptosis assay to assess the effects of FN adhesion on
drug-induced apoptosis. FITC-conjugated annexin V, which binds
to inverted phosphatidylserine on the surface of the plasma
membrane, was used to identify apoptotic cells. As shown in Figure
3A, cells adhered to FN before a 1-hour exposure to various doses
of mitoxantrone had reduced apoptosis on annexin V staining. Cells
treated while adhered to FN were also protected from etoposideinduced apoptosis (Figure 3B). These data indicate that adhesion of
U937 cells to FN protects against mitoxantrone-induced apoptosis
and, to a lesser extent, etoposide-induced apoptosis. Studies using
doxorubicin were not done because the drug interfered with this
fluorescence assay.
Adhesion of U937 cells to FN reduces mitoxantrone- and
etoposide-induced DNA double-strand breaks as measured by
neutral comet assay
Mitoxantrone and etoposide are known to stabilize topo II–DNA
complexes, resulting in DNA double-strand breaks.13-15 We used
the neutral comet assay to compare the amount of mitoxantrone- or
etoposide-induced DNA double-strand breaks in U937 cells that
were either exposed to drug in suspension or adhered to FN. The
comet moment is a function of both the distance and the amount
(measured in pixel density) of DNA that migrates from the center
of the head of the comet. As shown in Figure 4, the tail length, tail
intensity, and tail shape differed according to whether the cells
were treated with drug in suspension or while adhered to FN. After
Figure 1. U937 cells expressed both very late antigen (VLA) 4 (␣4␤1 heterodimer) and VLA-5 (␣5␤1 heterodimer) integrin but adhered to FN primarily
by means of VLA-5 integrin. (A) Cell-surface expression of integrin subunits was
measured using flow cytometry. The mean fluorescence of 10 000 events for each
subunit (3 independent experiments) is shown. (B) Blocking antibodies were used to
determine the specificity of binding of U937 cells to FN. The presence of the ␣5 or the
␤1 blocking antibody reduced adhesion of U937 cells to levels comparable to those
with BSA. In contrast, the ␣4 blocking antibody did not reduce cellular adhesion
to FN.
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1900
HAZLEHURST et al
BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
Figure 3. U937 cells adhered to FN for 2 hours were resistant to apoptosis
induced by mitoxantrone and etoposide. (A) Adhesion to FN significantly reduced
mitoxantrone-induced apoptosis (P ⬍ .05 on ANOVA). Shown are the mean results
and 95% confidence intervals (CIs) from 3 independent experiments done in
duplicate (the percentage of apoptosis is equal to the percentage of apoptosis in
drug-treated samples minus the percentage of apoptosis in the vehicle control).
(B) Adhesion to FN significantly decreased etopside-induced apoptosis (P ⬍ .05 by
ANOVA). Shown are the mean results and 95% CIs from 4 independent experiments
done in duplicate (the percentage of apoptosis is equal to the percentage of apoptosis
in drug-treated samples minus the percentage of apoptosis in the vehicle control).
no significant difference at the P ⬍ .05 level). These data indicate
that the reduction in cytotoxicity and DNA damage that occurs
when cells are adhered to FN is not due to a decrease in
intracellular concentration of the drug. These findings are consistent with our previous study showing that adhesion of the multiple
myeloma 8226 cell line to FN did not alter the intracellular
concentration of doxorubicin.1
Adhesion of U937 cells to FN reduces salt-extractable topo II
activity and topo II␤ protein levels and alters the nuclear
localization of topo II␤ but does not change the total levels or
nuclear-to-cytoplasmic ratio of topo II␤
Mitoxantrone and etoposide were previously reported to stabilize
topo II–DNA complexes.8,13,14 Furthermore, cell lines selected in
vitro for resistance to mitoxantrone and etoposide often have
qualitative or quantitative changes in topo II.8,16,17 To determine
Figure 2. U937 cells adhered to FN had increased survival (MTT analysis) after
exposure to mitoxantrone (A), doxorubicin (B), or etoposide (C). Shown are the
mean results from a representative experiment done in quadruplicate wells. Three to
six independent experiments were done, and similar results were obtained.
2 hours of adhesion to FN, both mitoxantrone- and etoposideinduced comet-moment values were decreased by approximately
40% to 60% compared with results in cells treated in suspension
(Figure 4E-F). The ANOVA showed a significant difference
between cells treated with drug in suspension and adhered to FN
(P ⬍ .01 for all doses tested).
A drug-accumulation assay was done to determine whether the
reduction in drug-induced DNA double-strand breaks correlated
with reduced intracellular drug accumulation. Total intracellular
14C-mitoxantrone was measured after a drug exposure of 1 hour, a
time consistent with the measurement of drug-induced DNA
damage. The adhesion-dependent decrease in DNA double-strand
breaks could not be attributed to decreased intracellular drug
accumulation (counts per minute for 14C-mitoxantrone, 8853 ⫾
1329 in cells in suspension and 10 266 ⫾ 1052 in FN-adhered cells;
Figure 4. The comet tail shape, intensity, and length differed according to
whether the cells were treated in suspension or adhered to FN. The comet
moment is a function of the distance and intensity of DNA from the center of the comet
head. (A) Results with 0.1% DMSO. (B) Results with 0.5 ␮M etoposide. (C) Results
with 5.0 ␮M etoposide. (D) Results with 50 ␮M etoposide. (D-E) Cells adhered to FN
for 2 hours showed a significant (P ⬍ .01) reduction in mitoxantrone- and etoposideinduced DNA double-strand breaks. The neutral comet assay was used to compare
drug-induced DNA double-strand breaks in cells adhered to FN and those cultured in
suspension. Cells were exposed to various concentrations of either mitoxantrone
(E) or etoposide (F) for 1 hour. The comet moment was then calculated. Fifty images
were captured for each dose, and 3 independent experiments were done. The graph
represents the mean values and 95% CIs from 3 independent experiments.
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BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
Figure 5. Adhesion of U937 cells to FN decreased topo II activity and nuclear
topo II␤ protein levels. (A) Nuclear extracts were obtained, and 1 ␮g nuclear extract
from cells in suspension and FN-adhered cells were incubated with 0.4 ␮g
tritium-kDNA for the times indicated. The amount of cleaved kDNA was reduced by
approximately 20% to 30% in FN-adhered cells compared with cells grown in
suspension (results were identical for 2 independent experiments, and the figure
represents results from one experiment). (B) Western blot analysis of 1.0-M NaCl
nuclear extracts showed a 66% reduction (mean value from 3 independent experiments) in nuclear topo II␤ protein levels when cells were adhered to FN. (C) Topo II␣
protein levels were unchanged when cells were adhered to FN. (D) Topo I levels were
unchanged when cells were adhered to FN.
whether the decrease in drug-induced DNA double-strand breaks
correlated with alterations in topo II, we examined nuclear topo II
activity by measuring the decatenation activity of the enzyme.
After 2 hours of cellular adhesion to FN, the amount of cleaved
kDNA was approximately 20% to 30% lower (Figure 5A) than that
in cells grown in suspension (2 independent nuclear extracts).
Western blotting was done to determine whether the adhesiondependent decrease in topo II catalytic activity correlated with
decreased nuclear protein levels. As shown in Figure 5B, Western
blot analysis of nuclear extracts found that topo II␤ levels were
decreased by 66% (mean value from 3 independent experiments) in
cells adhered to FN compared with cells grown in suspension. In
contrast, adhesion of cells to FN did not alter the nuclear levels of
topo II␣ (Figure 5C). Furthermore, topo I levels remained constant
in nuclear extracts prepared from either cells in suspension or
FN-adhered cells (Figure 5D).
Confocal microscopy was used to determine whether cellular
adhesion to FN altered the cellular distribution of topo II␣ and
topoII␤. We found that adhesion of U937 cells to FN resulted in
distinct punctate clusters of topo II␤ in the nuclei of adhered cells
(Figure 6), suggesting that adhesion alters the nuclear distribution of topo II␤. After cellular adhesion to FN, most topo II remained nuclear (Figure 6; nuclear-to-cytoplasmic ratio for topo
II␣, 7.56 ⫾ 0.51 in cells in suspension and 5.39 ⫾ 0.37 in
FN-adhered cells; ratio for topo II␤, 6.01 ⫾ 1.58 in cells in
suspension and 4.87 ⫾ 0.33 in FN-adhered cells). Furthermore, the
mean total fluorescence/cell was not altered in FN-adhered cells
(pixel density for topo II␣/cell, 174 537 ⫾ 34 112 in cells in
suspension and 134 275 ⫾ 24 173 in FN-adhered cells; density for
REDUCTION IN DRUG-INDUCED DNA DOUBLE-STRAND BREAKS
1901
Figure 6. Cellular adhesion to FN altered the nuclear distribution of topo II␤.
After 2 hours of cellular adhesion to FN, cells were fixed and stained for either topo II␣
or topo II␤. One hundred cells were analyzed for pixel density of the cytoplasm and
nucleus. The nuclear-to-cytoplasmic ratio for topo II␣ for cells grown in suspension
was 7.56 ⫾ 0.51, whereas that for FN-adhered cells was 5.39 ⫾ 0.37. For topo II␤,
the nuclear-to-cytoplasmic ratio was 6.01 ⫾ 1.58 for cells grown in suspension and
4.87 ⫾ 0.33 for cells adhered to FN.
topo II␤/cell, 77 534 ⫾ 10 263 in cells in suspension and 83 732 ⫾
7845 in FN-adhered cells).
To assess whether cellular adhesion to FN altered the nuclear
binding properties of topo II␤, we measured the amount of topo II␤
remaining in the nuclear fraction after 2 sequential salt extracts of
0.5 M and 1.0 M NaCl. After the last salt extract, the remaining
pellet was digested with DNase, and proteins were separated by
SDS–polyacrylamide gel electrophoresis (PAGE). We found that
salt extracts from FN-adhered samples contained less topo II␤ than
cells grown in suspension (Figure 7). In contrast, the remaining
DNase-digested pellet contained more topo II␤ than cells grown in
suspension. These results are consistent with increased nuclear
binding of topo II␤ in cells adhered to FN.
Figure 7. Adhesion of cells to FN increased the amount of topo II␤ bound to the
nucleus after a 1.0-M NaCl extract. Nuclei isolated from FN-adhered cells or cells in
suspension were sequentially extracted in 0.5 M and 1 M NaCl. After the 1.0-M salt
extract, the remaining pellet was sonicated and digested with DNase. Ten micrograms of the 0.5-M and 1.0-M NaCl extracts and 100 ␮g of the DNase-digested
extracts were separated by SDS-PAGE and immunoblotted for topo II␤.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1902
HAZLEHURST et al
Discussion
Durand and Sutherland18 were among the first investigators to
show that changes in the microenvironment can alter the response
to radiation. In their model, cells grown as a spheroid were more
resistant to drugs and radiation than cells grown as a monolayer.
The increase in drug resistance associated with spheroid cultures
compared with monolayer cultures was shown to correlate with
decreased drug-induced DNA damage.19 These results suggest that
cell-cell contact or cell-matrix contact modulates the cellular
response to drug-induced DNA damage. Moreover, the decrease in
drug-induced DNA damage was found to be correlated with the
redistribution of topo II␣ from the nucleus to the cytosol.20
However, studies using a cell-spheroid model have not identified a
cell-surface receptor responsible for drug resistance. We chose to
focus on the role of ␤1 integrin–mediated adhesion and the
contribution of this specific receptor-ligand interaction to mediating drug resistance and drug-induced DNA damage.
We previously showed that ␤1 integrin–mediated adhesion
inhibits drug-induced apoptosis in myeloma cell lines.1 Similarly,
Sethi et al2 reported that ␤1–mediated adhesion confers resistance
to chemotherapeutic drugs in small-cell lung cancer cell lines.
Similar to the results in the spheroid model was the finding by Hoyt
et al21 that adhesion of murine tumor–derived endothelial cells by
means of ␤1 integrins resulted in diminished etoposide-induced
DNA damage, indicating that ␤1 integrins modulate drug-induced
DNA damage in nontransformed cells. We previously determined
that adhesion of myeloma cells to FN resulted in inhibition of
cell-cycle progression and increased p27kip1 protein levels. Furthermore, the increase in p27kip1 protein was casually related to the
CAM-DR phenotype.22 However, it is not known how the FNinduced increase in p27kip1 confers drug resistance.
In this study, we found that adhesion of U937 cells to FN by
means of ␤1 integrin for 2 hours is sufficient to inhibit initial DNA
double-strand breaks induced by the topo II inhibitors mitoxantrone and etoposide. After a 1-hour exposure to mitoxantrone,
the decrease in drug-induced DNA double-strand breaks did not
correlate with decreased total intracellular drug concentration.
However, these results do not exclude the possibility that changes
in drug efflux over time or alterations in the intracellular localization of drug contribute to a decrease in initial DNA damage and
drug resistance.
Topo II is the putative target of mitoxantrone and etoposide that
causes DNA double-strand breaks. Thus, qualitative or quantitative
changes in topo II can decrease such drug-induced breaks. We
observed that adhesion of U937 cells to FN resulted in diminished
topo II catalytic activity as measured by the release of minicircles
from kDNA. The decrease in enzymatic activity correlated with a
decrease in salt-extractable nuclear topo II␤ protein levels. However, the nuclear-to-cytoplasmic ratio of topo II␤ was only
marginally decreased (by approximately 20%), and no significant
changes in total pixel density were observed. The discrepancy
between the confocal data and Western blot results regarding
salt-extractable topo II␤ suggests that cellular adhesion to FN
results in changes in the affinity of topo II␤ for the nucleus. The
confocal studies showed a redistribution of topo II␤ in the nucleus,
suggesting the presence of qualitative differences in topo II␤ in
cells in suspension and FN-adhered cells. Furthermore, the decrease in salt-extractable nuclear protein was confirmed by demonstration of an increase in topo II␤ associated with DNA after a 1M
NaCl extraction. Additional studies are needed to determine
BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
whether these changes in the nuclear localization of Topo II␤
produce the reduction in drug-induced DNA damage.
Topo II has been shown to be posttranscriptionally modified by
phosphorylation and ribosylation,23,24 and perhaps posttranscriptional modifications alter the affinity of topo II␤ for DNA. An
alternative explanation for our results is that topo II␤ is sequestered
by DNA-binding proteins in the nucleus. Topo II␤ was shown to
bind specifically to several nuclear binding proteins, including
histone deacetylase (HDAC) 1 and HDAC2, and this protein
complex was found to contain HDAC activity.25 The investigators
also showed that topo II␤ coprecipitated with metastasis-associated
protein 2, a component of the nucleosome remodeling and deacetylating (NuRD) complex. The NuRD complex contains both chromatin remodeling activity and HDAC activity.26,27 It is possible that
recruitment of topo II␤ into the NuRD complex decreases the
ability to extract topo II␤ from the nucleus with 1.0 M NaCl.
Additional studies are needed to determine whether FN adhesion
results in alterations in the binding or activity of protein complexes
associated with topo II␤.
There is evidence that etoposide and mitoxantrone inhibit both
topo II␣ and topo II␤. However, cells derived from a murine topo
II␤ knockout were more resistant to mitoxantrone and amsacrine
than to etoposide.28 We found that reduced extractable topo II␤
protein levels correlated with decreased mitoxantrone- and etoposide-induced DNA double-strand breaks; however, the protection
of mitoxantrone was greater than that of etoposide. A possible
explanation for the greater protection observed for mitoxantrone is
that mitoxantrone-induced topo II␤ complexes are more lethal than
etoposide-stabilized topo II␤–DNA complexes. Alternatively, the
increased resistance observed with mitoxantrone compared with
etoposide may have resulted from activation of separate signal
transduction pathways initiated by binding to FN that are unrelated
to DNA double-strand breaks.
Little is known about extracellular signals that regulate expression and activity of topo II␤. Activation of ␤1 integrin signaling is
known to activate several signal transduction pathways, including
phosphatidylinositol 3 kinase,29 protein kinase B,30,31 mitogenactivated protein kinase,32 and focal adhesion kinase.33 We are
currently investigating whether activation of these known ␤1–
mediated signaling pathways contributes to changes in the nuclear
distribution of topo II␤ or diminished drug-induced DNA doublestrand breaks.
In summary, we found that adhesion of U937 cells to FN
attenuates DNA double-strand breaks induced by mitoxantrone and
etoposide. The decrease in these breaks correlated with a decrease
in topo II catalytic activity and salt-extractable topo II␤ protein
levels. Collectively, these data suggest that cell-adhesion–mediated
protection from drug-induced apoptosis mediated by topo II
inhibitors is the result of decreased drug-induced DNA damage.
Furthermore, the decrease in drug-induced DNA damage may be
related to the changes in the nuclear localization or binding
properties of the nuclear pool of topo II␤ protein.
Acknowledgments
We thank the H. Lee Moffitt Analytical Microscopy Core Facility,
the H. Lee Moffitt Flow Cytometry Core Facility, the H. Lee
Moffitt Biostatistics Core Facility, all of which are supported by
National Institutes of Health grant P30-CA76292-04-08; and
Peggy Farrell for careful editing of the manuscript.
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BLOOD, 15 SEPTEMBER 2001 䡠 VOLUME 98, NUMBER 6
REDUCTION IN DRUG-INDUCED DNA DOUBLE-STRAND BREAKS
1903
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2001 98: 1897-1903
doi:10.1182/blood.V98.6.1897
Reduction in drug-induced DNA double-strand breaks associated with β1
integrin−mediated adhesion correlates with drug resistance in U937 cells
Lori A. Hazlehurst, Nikola Valkov, Lee Wisner, Jonathan A. Storey, David Boulware, Daniel M. Sullivan and
William S. Dalton
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