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Vol. 3, 2055-2061. November 1997 Clinical Cancer Research 2055
DNA Repair and Cellular Resistance to Alkylating Agents in
Chronic Lymphocytic Leukemia’
Mark R. Muller,2 Claudia Buschfort,
J#{252}rgenThomale, Carmen Lensing,
Manfred F. Rajewsky, and Siegfried SeeberDepartment of Internal Medicine (Cancer Research) [M. R. M.. C. L.,
S. 5.1 and Institute of Cell Biology (Cancer Research) [J. T.. C. B.,M. F. R.], West German Cancer Center Essen, University of EssenMedical School. D-45122 Essen, Germany
ABSTRACT
The time course of the formation and persistence of
repair-induced DNA lesions such as single-strand breaks
(SSBs) were determined in isolated lymphocytes derived
from 32 patients with chronic lymphocytic leukemia (CLL)
using the single-cell gel electrophoresis (SCGE, “comet”)
assay. After pulse-exposure to N-ethyl-N-nitrosourea
(EtNU), the initial amount of SSBs (t0 SCGE values) and the
time periods required to reduce DNA damage by 50% (t50%
SCGE values) were determined in nuclear DNA of individual
cells. The 10 SCGE and t50% SCGE values varied interindividu-
ally between CLL specimens by factors of 16.6 and 8.2,
respectively. Regarding cell-to-cell variation, no major sub-
populations with significantly different DNA repair capaci-
ties were observed in cell specimens from a given patient. In
addition, a monoclonal antibody-based immunocytological
assay was used to determine the elimination kinetics for the
cytotoxic alkylation product 06-ethylguanine from nuclear
DNA. A strong correlation was observed between the rela-
tive times for SSB repair and the elimination of 06-ethyb-
guanine from nuclear DNA. Because SCGE and immunocy-
tologicab assay measure different steps of DNA repair, this
observation suggests coordinated regulation of the respec-
tive repair pathways. With regard to chemosensitivity pro-
files, a “fast” repair phenotype corresponded to enhanced
in vitro resistance to EtNU, 1,3-bis(2-chboroethyl).1-
nitrosourea, or chlorambucil. Accelerated SSB repair and
pronounced in vitro resistance to chborambucil, 1,3-bis(2-
chboroethyl)-1-nitrosourea, and EtNU were found in lym-
phocytes from CLL patients nonresponsive to chemotherapy
with alkylating agents. Distinct DNA repair processes thus
mediate resistance to alkylating agents in CLL lymphocytes.
Received 3/28/97; revised 7/I 1/97; accepted 7/I 1/97.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
I This work was supported by Grant W769/94/MU2 from Dr. Mildred
Scheel Stiftung.2 To whom requests for reprints should be addressed, at Innere Klinikund Poliklinik (Tumorforschung), Westdeutsches Tumorzentrum Essen.Universithtsklinikum Essen, Hufelandstrasse 55, D-45122 Essen,
Germany. Phone: 49-201-7233155: Fax: 49-201-7235924.
INTRODUCTION
Drug resistance to chemotherapy represents one of the key
problems in cancer chemotherapy and in the treatment of lym-
phomas including CLL3 in particular. The capacity to repair
cytotoxic DNA lesions induced by alkylating agents has been
identified as an important mechanism underlying drug resist-
ance in cell lines derived from primary tumors ( 1 , 2). The repair
of DNA monoadducts or cross-links is a complex process in-
volving the coordinated, successive action of many proteins in
parallel pathways ( 1 , 2). AT (EC 2. 1 . 1 .63) repairs DNA by
transferring alkyl groups from the O1�-atom of guanine (O6�
AlkGua) to one of its own cysteine residues (3-6). Recent
reports suggest that DNA repair pathways such as base or
nucleotide excision can also contribute to the removal of O�-
AlkGua in DNA (7-9). Furthermore, mismatch repair mutants
are tolerant to 06-AlkGua, which would otherwise be lethal (8).
Enhanced DNA cross-link repair has been attributed to resist-
ance to melphalan in human cell lines ( I 0, 1 1 ). Overexpression
or loss of various DNA repair proteins, such as 3-methybadenine
DNA glycosylase, the ERCCI protein, DNA topoisomerase ha,
or the damage recognition protein XP-A, modulate cellular
sensitivity to DNA-damaging agents (12-15). On the other
hand, diminished, rather than increased, resistance to alkylating
agents has been observed in hamster cells overexpressing 3-
methyladenine DNA glycosylase, which catalyzes an early step
in DNA base excision repair ( 16). It is thus probable that
deregulation of DNA repair and/or the accumulation of repair
intermediates such as SSBs contribute significantly to the cyto-
toxicity of alkylators in addition to primary drug-DNA reaction
products.
Substantial interindividual differences in the expresssion or
activity of DNA repair proteins have been described ( I 7-20). It
is not yet known whether this wide interindividual range in
DNA repair phenotypes translates into clinical responsiveness to
treatment with alkylating anticancer drugs.
CLL represents a model particularly suited to study the
development of drug resistance. At first presentation. CLL pa-
tients are usually either untreated or on oral CLB chemotherapy
and become resistant to this regimen later on. In extracts of
lymphocytes from nonresponsive CLL patients, increased ex-
pression of the DNA excision repair protein ERCCI and en-
hanced repair of DNA cross-links were observed (21 , 22). Other
studies, however, did not find a relation between repair of
3 The abbreviations used are: CLL, chronic lymphocytic leukemia; AT.06-alkylguanine-DNA alkyltransferase; 06-AlkGua, 06-alkylguanine:
SCGE. single-cell gel electrophoresis: ICA, immunocytological analy-sis; CLB, chlorambucil: MiT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide; BCNU, 1.3-bis(2-chloroethyl)-l-nitrosourea:DMSO. dernethyl sulfoxide; EtNU, N-ethyl-N-nitrosourea: � drugconcentration required to reduce A)4)) to 50% of control; O6-EtGua.
06-ethylguanine; SSB, single-strand break.
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2056 DNA Repair in CLL Lymphocytes
cross-links or the expression of DNA repair proteins and treat-
ment outcome in leukemia patients (23-25). The clinical signif-
icance of DNA repair as a major determinant of drug resistance
thus remains controversial.
Methods for the measurement of DNA repair in individ-
ual cells have been developed recently (19, 20, 26-30). One
of these techniques, the SCGE “comet” assay, has been
applied for the measurement of DNA damage and repair at
the single-cell level (26-30). Following neutral or alkaline
lysis of cells, this assay uses the electrophoretic mobility of
DNA fragments in agarose gels that, after staining with a
fluorescent DNA dye, appear as “comets” emerging from the
compact nuclear DNA. The formation and gradual disappear-
ance of “comets” correspond, respectively, to the number of
DNA double-strand breaks (neutral lysis) or alkali-labile
lesions and SSBs (26-30). Several different SCGE protocols
exist, using either irradiation or various DNA-reactive agents
for the induction of DNA damage and, furthermore, various
lysis conditions, incubation periods, and end points (27, 30).
The SCGE assay has been used for the single time assessment
of (unprocessed) DNA damage [corresponding to the t0 SCGE
values (SSB formation after 20 mm EtNU exposure) in the
present protocol] in human lymphocytes (29) but has not yet
been applied to the study of repair kinetics in a larger number
of clinical specimens. Besides the measurement of alkali-
labile DNA lesions, this assay also allows us to determine the
kinetics of incision and rejoining of DNA strands during
excision repair. In the present protocol, CLL lymphocytes
were pulse-exposed to EtNU and analyzed at different time
points for comet formation. One advantage of using EtNU as
a DNA-damaging agent, although it is not used clinically as
a chemotherapeutic drug, is that all of its major DNA reaction
products have been structurally well characterized, and their
relative rates of formation in DNA are known (31). A further
advantage of EtNU is that the electrophoretic mobility of
DNA fragments in the comet is directly related to the number
of SSBs and not superimposed by DNA cross-links as in-
duced by bifunctional alkylating agents.
In conjunction with SCGE, a sensitive ICA has been used
as a functional test for the accumulation and repair of a specific
alkylation product, 06-EtGua, in the nuclear DNA of individual
leukemic cells (19, 20). Using this assay, increased DNA repair
rates were observed in lymphocytes from CLL patients nonre-
sponsive to treatment with alkylating agents (19). Furthermore,
the chemosensitivity profiles of isolated CLL lymphocytes were
assessed by the MIT assay. This test has been successfully
applied to predict drug resistance in leukemia patients (32).
Because different alkylating agents vary with respect to the
relative formation of different DNA reaction products, the cy-
totoxic effects of the monofunctional alkylator EtNU and the
cross-linking agents BCNU and CLB were evaluated in the
present analysis.
The aim of this study was to determine the kinetics of
repair-induced SSBs in individual CLL lymphocytes and to
correlate these data with the elimination kinetics for the primary
DNA adduct 06-EtGua and with the chemosensitivity to alky-
lating agents in vitro and with treatment outcome.
MATERIALS AND METHODS
Preparation of Lymphocytes and Clinical Data. Blood
samples obtained from CLL patients were taken before treat-
ment and analyzed on the same day. Heparmnized blood (10 ml)
was layered onto 10 ml of Ficoll-Hypaque and centrifuged for
25 mm at 200 x g at room temperature, and mononuclear cells
at the interface were removed. The resulting cell suspension
contained more than 90% lymphocytes, as confirmed by light
microscopy. With regard to clinical status, specimens were
derived either from untreated, treated sensitive, or treated resist-
ant CLL patients. In case of treated sensitive patients, the
peripheral lymphocytes count returned to < 15,000/jil after in-
termittent treatment with CLB. Therapy with CLB or intermit-
tent high dose cyclophosphamide, vincristine, and prednisone
failed to reduce peripheral lymphocyte counts by 25-30% in
treated resistant patients. Specimens were derived from B-CLL
cases, and no transformation into Richter’s syndrome was noted.
SCGE (“Comet Assay”). The comet assay was used as
essentially described earlier (28). Lymphocytes in PBS supple-
mented with 900 jiM Ca2� , 490 jiM Mg2� , and 25 jiM HEPES
(pH 7.25) were pulse-exposed for 20 mm to EtNU at 37#{176}C.Cells
were washed twice in PBS and resuspended in RPM! 1640 kept
at 37#{176}Cin a humidified atmosphere. Samples were taken im-
mediately or at several time points up to 9 h after EtNU
treatment. After sampling, cells were washed with PBS and
suspended in low-melting-point agarose (0.6%, Biozym). The
cell suspension (5 x 10�/10 p.1) was spread evenly onto a fully
frosted microscope slide covered with a thin layer of 0.6% LE
agarose. A top layer of 0.5% low melting point agarose was
used to protect the cells. Cells were then lysed by 2.5 M NaC1,
100 m�i EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100, and
1% sodium sarcosinate (pH 10) at 4#{176}Covernight. Afterward,
slides were incubated in an alkaline electrophoresis solution
(300 ms� NaOH, 1 mM EDTA, pH 12) for 20 mm with two
buffer changes to unwind the DNA. The slides were then sub-
jected to electrophoresis in the same solution for 20 mm at 4#{176}C
and 4 V/cm, neutralized (3 X 10 mm in 0.4 M TrisHCL, pH
7.5), and stained with ethidium bromide (2 jig/ml).
Measurement of 06-EtGua in the Nuclear DNA of In-
dividuab Cells: ICA. Lymphocytes were exposed to EtNU
essentially as described for the SCGE assay. After 20 mm of
incubation, cells were washed twice in PBS and resuspended in
fresh, prewarmed RPMI 1640. Samples were taken immediately
or 1.5, 3, 6, 9, and 24 h after EtNU treatment and transferred to
microscopic slides. The immunofluorescence staining of
06-EtGua in nuclear DNA was performed as described previ-
ously (19, 20). Briefly, cells on slides were fixed with methanol,
and DNA was partly denatured by alkali treatment. Cells were
then incubated with the (anti-06-alkylguanine) monoclonal an-
tibody ER-17 at a concentration of 0.2 jig/ml in PBS/l% BSA
for 16 h at 4#{176}C.Cells were washed and stained with goat anti-rat
IgG F(ab)2 fragment conjugated with rhodamine isothiocyanate
(Dianova; 2 jig/mI PBS/1% BSA) for 45 mm at 37#{176}C.Nuclear
DNA was counterstained with the fluorescent dye 4,6-dia-
midino-2-phenylindole (3 X l0� M in PBS for 10 mm).
Quantitation of Fluorescence Signals and Data Analy-
sis. A fluorescence photomicroscope (Zeiss Axioplan)
equipped with a HBO lOO-W mercury arc lamp and Zeiss
Research. on May 26, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
A SCGE
B
4Lua4
LA.
0
LUU)4LUaC.)z
LU>
4-jLua
LuC.)
LUU)Qi-Cn�
0�LU
I-.
tUtU..ia0’S-
z
I I I
0 20 40 60
TIME AFTER ETNU PULSE (mm)
300
200
1 00
0#{149}
1 50
1 00
50
0�
ICA
0 5 10 15 20 25
standard filter combinations 02 (for 4,6-diamidino-2-phe-
nylindole), 14 (for tetramethylrhodamine isothiocyanate),
and 15 (for ethidium bromide) were used. Fluorescence sig-
nals were amplified and recorded by a dual-mode charge-
coupled device camera (Phototonics, Hamamatsu City, Ja-
pan) and fed into a multiparameter image analysis program
(ACAS Cytometry Analysis System; Ahrens Electronics,
Bargteheide, Germany). In case of the SCGE, the amount of
DNA damage in single cells was determined by measuring
the total area of stained nuclear DNA and the fluorescence
intensity by image analysis. Comet sizes were defined as the
relative increase of fluorescent area of EtNU-treated lympho-
cytes compared to the area of nuclei of untreated control cells
from the same donor in the same experiment. For ICA, signal
intensities of 06-EtGua immunostaining and DNA staining
were expressed as the integrated values (average signal X
number of selected pixels) for each nucleus. 06-EtGua sig-
nals were corrected for nuclear DNA content.
MTT Assay. For the tetrazolium dye (MTT) assay, cells
were seeded into 96-well microtiter plates using 200 p.1/well of
a cell suspension containing 106 lymphocytes/ml of RPMI 1640
supplemented with 10% FCS. Dissolved drugs were added in 20
p.1 of PBS. On day 4, 20 p.1 of a solution of S mg MTT/ml of
PBS were added to each well, and the plates were further
incubated for 5 h. Thereafter, the plates were centrifuged for 10
mm at 100 X g. The medium was removed from each well, 200
Clinical Cancer Research 2057
Fig. 1 “Comet” formation in a CLL lymphocyte after exposure toEtNU for 20 mm (A) compared with nuclear fluorescence from an
untreated CLL lymphocyte as a control (B). The labile sites in DNA dueto repair processes appear as a comet attached to the nucleus. Theamount of DNA damage in single cells was determined by measuring
the total area of stained nuclear DNA and the fluorescence intensity byimage analysis. Comet sizes were defined as the relative increase of
fluorescence area of EtNU-treated lymphocytes compared to the area of
nuclei of untreated control cells.TIME AFTER ETNU PULSE (h)
Fig. 2 Kinetics of comet formation and disappearance (top) and of the
elimination of 06-EtGua from nuclear DNA (bottom) in two specimens
of CLL lymphocytes after in vitro pulse exposure to EtNU. Curves were
obtained from two CLL patients who were either sensitive (#{149})orresistant (0) to treatment with alkylating agents. In case of SCGE, the
area of stained nuclear DNA (mean of 150 cells; coefficient of variation,15%) is given as the increase relative to untreated control cells. Regard-
ing ICA, mean values of relative nuclear fluorescence signals of 100cells/time point are plotted (coefficient of variation. 25%). The t3�.3
values were determined graphically in all plots. lnterindividual variation
was not only observed for t5� values but also for the form of the curves
that do not follow a first-order kinetic (20, 33).
Research. on May 26, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
500
400
300
200
1 00
S
I..
S
I
I U
60
50
40-
30-
20-
1 0�
0�
S.
S
SII
II
I
I
I
Fig. 3 Interindividual varia-
tion of the capacity of CLLlymphocytes for repair of SSBs
in nuclear DNA. t0 SCGE’
“initial” comet size after 20
mm of EtNU pulse exposure.
ISO’) SC(,I’ time required to reducecomet formation by 50%. Notethe wide interindividual rangefor both values.
to SCGE t50% SCGE
7.5.
2.5-
0 10 20 30 40
2058 DNA Repair in CLL Lymphocytes
�1<0
aI-
LA.#{176}
U.
4LUAU
LUU��)
‘-LU<a.-a.-,
LUa
0
0Ar)
t50% SCGE (mm)
AU
010
LUa>-0
0�.�
�
�U.
OW
UiC.)a.
50
Fig. 4 Relationship between DNA repair time determined simulta-
neously by SCGE � SCGE) and ICA (t�� iCA) in individual samplesof CLL lymphocytes. A strong correlation was observed for the relative
times required for the repair of 50% of SSB and 06-EtGua (r = 0.98.
P < 0.015).
p.1 of DMSO were added to dissolve the crystals, and the plates
were shaken for I 0 mm. Absorbances at 540 and 690 nm were
read using a dual wavelength Dynatech MR 7000 plate-reader.
In all experiments, four replicate wells were used for each drug
concentration. The drug concentration required to reduce the
absorbance at 540 nm to 50% of the control was taken to be the
ID50 of the sample.
Statistics. The Spearman rank correlation coefficient was
applied to ID50 and t50% values. The Student’s t test was used to
calculate statistical significance of any differences between var-
ious groups of patients.
RESULTSFormation and Repair of SSB and 06-EtGua in the
Nuclear DNA of CLL Lymphocytes. CLL lymphocytes
were exposed to EtNU for 20 mm and analyzed for the initial
increase, and subsequent decrease, in electrophoretic mobility of
nuclear DNA (t0 scGE) The initial comet size, representing the
number of DNA SSBs and alkali-labile sites after 20 mm of
EtNU exposure, was determined by relating the increased
stained DNA area of treated cells to the area of untreated control
cells from the same donor (Fig. 1 ). The time periods required to
reduce the “initial” comet area by 50% (t50% scGE)’ were deter-
mined as a measure for the cellular capacity to process second-
ary DNA lesions. Typical examples of the DNA repair kinetics
for induced DNA strand breaks are shown in Fig. 2. Initial
comet areas varied interindividuably between 26 and 435% (Fig.
3). Similarly, the kinetics ofcomet formation and disappearance
showed considerable interindividual differences, with the t50%
SCGL values varying by factors up to 8.2 between CLL speci-
mens (Fig. 3). No correlation was found between “initial” DNA
damage (t0 scGE) and the t5�f SCGE repair values (P > 0.05).
The pattern of comet formation was generally uniform among
CLL lymphocytes within a given sample (coefficients of vari-
ation, 7.2-19.6). Subpopulations displaying degrees of DNA
damage deviating significantly from the mean value of a spec-
imen were not observed.
In parallel, the repair kinetics of the alkylation product
06-EtGua from DNA were measured after 20 mm of expo-
sure to EtNU in selected specimens using the monoclonal
antibody-based ICA (Fig. 2). For each time point, antibody-
mediated fluorescence signals were corrected for nuclear
DNA content. The time periods required to eliminate 50%
(t ) of 06-EtGua residues from DNA were determined as
a measure of the efficiency of early DNA repair steps con-
tributing to adduct removal. In specimens from four CLL
patients, cellular DNA repair kinetics were determined by
Research. on May 26, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
EtNU BCNU CLB
EDi
,�,I)
0
(5
>
00
a
12.5
10’
7.5
5’
2.5
S.
#{163}5
SS � S
S
s:-s 0
S
S
55 5
t50% values (mm)
6
5.
4�.
3 I,
S � �
2 55
S S�S
1 #{149}$
0 I 1 I I I
0 10 20 30 40 50 60 70
t50% values (mm)
0 10 20 30 40 50 60 70
E
Lu
0Cl)
0Ar)
70’
60’
50
40
30
20-
1 0-
0-
I
S
S
SS
S S S
SS I
T
U TS TR
Fig. 6 Repair time for SSB in the nuclear DNA of CLL lymphocytesin relation to treatment outcome. The t50� SCGE repair values were
determined in lymphocytes obtained from CLL patients who were eitheruntreated, sensitive, or resistant to treatment with alkylating agents.Horizontal lines, means of the distributions.
Clinical Cancer Research 2059
25U�
200
1 50
100� #{149}#{149}#{149}.#{149}I�
�, ,#{149} S50 #{149}#{149} S
0#{149} I I I I I I
0 10 20 30 40 50 60 70
t50% values (mm)
Fig. 5 Correlation between chemosensitivity in vitro and DNA repair time for SSBs in CLL lymphocytes. The Y-axes display ID��s for EtNU.
BCNU, and CLB, respectively, and the X-axes the time interval required for the reduction of initial comet formation by 50% (t5�% �4-� values). The
Spearman rank correlation coefficients (rfl) for DNA repair time and cytotoxicity were r� = -0.71 for EtNU, r� = -0.54 for BCNU, and r� = -0.69
for CLB, respectively (P < 0.001).
SCGE and ICA simultaneously (Fig. 4). A positive correla-
tion was observed between the t50% values for both the
elimination of 06-EtGua from nuclear DNA and the repair of
SSBs (r = 0.98, P < 0.015).
Table I Chemosensitivity of isolated lymphocytes derived from
untreated (U), treated sensitive (TS), and treated resistant (TR)
CLL patients
The ID50s were determined after a 4-day exposure to alkylatingagents using the MiT assay.
U TS TR
(‘a = 12) (ii = 10) (‘a = 8)
EtNU (jig/ml) 78.4 ± 32.0 89.4 ± 21.7 144.0 ± 39.0”
BCNU (jig/mi) 5.6 ± 2.7 6.6 ± 1.4 9.2 ± 2.6”
CLB (jig/ml) 2.1 ± 0.7 2.5 ± 0.9 3.7 ± 1.1”
“ P < 0.001.
Relationship of DNA Repair Capacity to Chemosensi-
tivity to Alkybating Agents in Vitro. DNA repair capacities,
as determined by SCGE, were related to the chemosensitivity of
CLL lymphocytes to the alkylating agents EtNU, BCNU, and
CLB. A significant ranking correlation was found between the
t5#{216}% SCGE and ID50 values for these DNA-reactive drugs (Fig.
5). No relationship was found between t#{216}%SCGE values and in
vitro drug resistance (P > 0.05). The ID50s of EtNU, BCNU,
and CLB were positively correlated (EtNUIBCNU, r� = 0.51;
EtNU/CLB, r� 0.70; BCNU/CLB, r� 0.52; P < 0.001; n
26) and suggested cross-resistance of CLL lymphocytes to al-
kylating agents forming different DNA adducts.
Relationship of DNA Repair Capacity to Clinical Sta-
tus. Fig. 6 displays the DNA repair rates of CLL lymphocytes
in relation to the patients’ clinical status. The mean t50� S(�GE
values for secondary DNA lesions were not significantly differ-
ent between specimens derived from untreated (n 1 2) or
treated sensitive (n - 10) CLL patients. In contrast, high rates
of DNA strand break processing were observed in specimens
from 10 CLL patients resistant to treatment with alkylating
agents. The respective mean t5�% SCGE values were significantly
lower in comparison to untreated or treated sensitive CLL
patients. The degree of initial DNA damage (t0 SCIE) was
Research. on May 26, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
2060 DNA Repair in CLL Lymphocytes
unrelated to clinical status (P > 0.05). Table 1 shows the ID50s
for alkylating agents determined in CLL lymphocytes in relation
to clinical status. The mean ID50s for EtNU, BCNU, and CLB
were significantly elevated in lymphocytes from CLL patients
resistant to chemotherapy with CLB in comparison to untreated
or treated sensitive patients.
DISCUSSION
The SCGE comet assay was applied to evaluate in vitro the
efficiency of DNA excision repair in individual CLL lympho-
cytes in relation to DNA monoadduct elimination, chemosensi-
tivity to alkylating agents, and to clinical status. EtNU-induced
DNA monoadducts, such as 06-EtGua, are partly removed from
DNA by the repair protein AT in a one-step process (3). Fur-
thermore, 06-AlkGua is a substrate for mismatch repair pro-
cesses, and excision repair also contributes to the elimination of
06-AlkGua from nuclear DNA (7-9). During these processes,
SSB in DNA result from the incision by endonucleases as repair
intermediates. Following pulse-exposure to EtNU, the initial
numbers of SSBs varied considerably between CLL specimens.
We have shown previously that this variation is not due to
different levels of primary adduct formation but rather is related
to different efficiencies of early steps in DNA repair (33). For
example, EtNU failed to induce SSBs in vitro prior to the
addition of DNA repair proteins. Widely varying time intervals
were required for the disappearance of repair-induced SSBs, as
observed among specimens from different CLL patients. This
may reflect large interindividuab differences in the concentra-
tions/activities of DNA repair proteins in human cells, as re-
ported previously (17-20). However, no obvious relationship
was found between the initial comet size after EtNU (t0 scGE)’
used as an end point for DNA damage in most studies (27),
and #{231} S(’(iI values or any other parameter tested. One explana-
tion for this lack of correlation is that the number of SSBs
measured at a given time point after drug exposure is dependent
on several factors, such as the activity of early steps in DNA
repair (glycosybases and endonucleases) and the efficiency of
the downstream processing of DNA lesions by, e.g. , DNA
polymerases/-bigases during the incubation period.
DNA repair phenotypes are heterogeneous not only be-
tween individuals but also among different cell types (34, 35).
To monitor the DNA repair capacity of tumor cells from cancer
patients. reliable, quantitative assays are required that allow the
selective analysis of individual cells in heterogeneous biopsy
material. The present approach does not permit the measurement
of DNA repair efficiency in a given cell as a function of time;
however, it enabled us to evaluate cell-to-cell variation among a
small number of cells by SCGE and ICA. Nevertheless, detect-
able subpopulations of CLL lymphocytes with DNA repair
phenotypes significantly different from the average value for the
total cell population were not observed among the lymphocyte
specimens examined in this study.
ICA measures the efficiency of early steps of 06-EtGua
repair including those effected by AT or excision repair pro-
teins. Later stages of DNA damage processing, such as gap
filling and rejoining of SSB during excision repair, are moni-
tored by SCGE. It is, therefore, interesting to note that the
relative repair rates determined by these functional assays coy-
ering different areas of the DNA repair network were correlated.
This correlation suggests at beast partial coordination of differ-
ent rate-limiting repair components. It remains to be determined
whether this reflects the predominant action of a certain repair
pathway measured by both assays or coregulation of key com-
ponents of distinct repair pathways. The latter possibility is
supported by the observed cross-resistance of CLL lymphocytes
to structurally unrelated alkylating agents inducing different
patterns of DNA adducts.
In cell lines, an inverse relationship has been reported
previously between the repair capacity of tumor cell lines for
SSB or AT activity and the chemosensitivity to alkylating
agents (36, 37). In the present study, increased in vitro chemore-
sistance to alkylating agents was observed in CLL lymphocytes
exhibiting a fast DNA repair phenotype. This suggests the
clinical importance of DNA repair in mediating cellular resist-
ance to alkylating agents in human leukemic cells. It is not yet
established whether the observed broad range of interindividual
DNA repair capacities translates into clinical responsiveness to
alkylating drugs. We have shown previously that CLL lympho-
cytes of chemotherapy-resistant patients displayed higher rates
of adduct elimination in comparison to CLL lymphocytes of
responsive patients (19). The present study has complemented
this observation by the finding that CLL lymphocytes from
nonresponsive patients exhibited faster processing of secondary,
repair-induced DNA lesions, such as SSBs, compared to control
cells. Both observations underline the clinical significance of
DNA repair as an important mechanism of resistance to alky-
lating agents in leukemic cells. Thus far, the number of speci-
mens studied is relatively small. Future studies should encom-
pass a larger number of patients to exclude biased selection. In
any event, use of functional assays such as SCGE and ICA will
provide a means to monitor DNA repair in cancer patients to
facilitate the rational design of chemotherapeutic regimens.
ACKNOWLEDGMENTS
We thank Bettina Baack for excellent technical assistance.
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