dna repair and cellular resistance to alkylating agents in ... · cytotoxic dna lesions induced by...

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.

Research. on May 26, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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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



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).



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


�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



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.


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




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.

REFERENCES

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1997;3:2055-2061. Clin Cancer Res M R Müller, C Buschfort, J Thomale, et al. chronic lymphocytic leukemia.DNA repair and cellular resistance to alkylating agents in

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