The capacity to repair DNA damage is an important factor that affects the therapeutic outcome in cancer treatment. To clarify the cellular repair response, we investigated the kinetics of DNA excision repair initiated by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in human leukemia CCRF-CEM cells at an exponential growth phase in vitro. Using the alkaline single-cell gel electrophoresis (comet) assay, we quantitated the repair kinetics as the amount of DNA single-strand breaks that were generated from the incision and were diminished by the rejoining in the repair process. CEM cells could initiate DNA excision repair in response to BCNU by starting an incision reaction. However, the incision capacity came to a plateau at a concentration of 80 to 1003M or after an incubation time of 90 to 120 minutes. When the cells were pulsed with 403M BCNU, the maximal incision occurred at the end of the incubation period, and the repair process was completed within 4 hours.When cells were treated with 1003M BCNU, the incised DNA was not rejoined at 4 hours, suggesting that the repair was not completed. Higher concentrations might surpass the cellular capacity for repair and would be associated with increased cell death. Evaluation of the repair process may provide a clue for therapeutic strategies to improve clinical efficacy if accelerated DNA repair is responsible for the drug resistance

Kawai, Yasukazu

Ueda, Takanori

Yamauchi, Takahiro

Community Repository of Fukui

International Journal of 
HEMATOLOGY
Alkylator-Induced DNA Excision Repair in 
Human Leukemia CCRF-CEM Cells In Vitro, 
   Measured Using the Single-Cell Gel
     Electrophoresis (Comet) Assay
Takahiro Yamauchi, 
        Takanori
Yasukazu Kawai, 
Ueda
  First  Department of Internal  Medicine, Fukui Medical  University, 
                       Fukui, Japan 
Received May  30,2002; received in revised form July  15,2002; accepted July  22,2002
Abstract 
  The capacity to repair DNA damage is an important factor that affects the therapeutic outcome in cancer treatment. To 
clarify the cellular repair response, we investigated the kinetics of DNA excision repair initiated by  1,3-bis(2-chloroethyl)-1- 
nitrosourea  (BCNU) in human leukemia CCRF-CEM cells at an exponential growth phase in vitro. Using the alkaline single-
cell gel electrophoresis (comet) assay, we quantitated the repair kinetics as the amount of  DNA  single-strand breaks that were 
generated from the incision and were diminished by the rejoining in the repair process. CEM cells could initiate DNA exci-
sion repair in response to BCNU by starting an incision reaction. However, the incision capacity came to a plateau at a con-
centration of 80 to  100p.M or after an incubation time of 90 to 120 minutes. When the cells were pulsed with  40p,M BCNU, the 
maximal incision occurred at the end of the incubation period, and the repair process was completed within 4 hours. When cells 
were treated with  1  OORM BCNU, the incised DNA was not rejoined at 4 hours, suggesting that the repair  was not completed. 
Higher concentrations might surpass the cellular capacity for repair and would  he associated with increased cell death. Eval-
uation of the repair process may provide a clue for therapeutic strategies to improve clinical efficacy ifaccelerated DNA repair 
is responsible for the drug resistance.  Mt  J  Hetnawl. 2002;76:328-332. 
©2002 The Japanese Society of Hematology
Key words: Alkylating agent; DNA excision repair; Drug resistance; Comet assay
1. Introduction
  Alkylating agents constitute a major class of anticancer 
drugs with well-established activity against hematologic 
malignancies  [I]. These compounds form various cytotoxic 
 alkylations, uch as mono-adducts on bases and di-adducts 
including  intrastrand and interstrand cross-links of DNA. 
The effectiveness of alkylating agents is limited by a number 
of factors, including drug resistance  [2,31. The capacity to
  Correspondence and reprint  requests:  Takahiro  Yamauchi, MD, 
 PhD, First Department of Internal Medicine, Fukui Medical 
 University, 23,  Shimoaizuki,  Matsuoka, Fukui,  910-1193,  Japan;  81-
776-61-3111; fax: 81-776-61-8109 (e-mail: tyamauch@fmsrsa. 
fukui-med.ac.jp).
repair alkylator-induced DNA lesions is an important mech-
anism underlying drug resistance  [2,3]. Such repair mecha-
nisms are classified into 4 general categories: direct repair, 
base excision repair, nucleotide xcision repair, and mis-
match repair  [3]. 
  Although direct repair with DNA alkyltransferase is
important  [4], this is not the only enzyme involved in repair 
because it is incapable of repairing interstrand i-adducts 
that are crucial to the cytotoxicity  [3]. DNA excision repairs 
including cross-link repairs that can eliminate interstrand 
cross-links may be more responsible for developing  resist-
ance. Therefore, the kinetic evaluation of DNA excision 
repairs will  be useful for clarifying the cellular response to 
alkylating agents, especially in the context of resistant 
diseases. 
  In the present study, we  investigated the kinetics of  DNA 
excision repairs initiated by a popular bifunctional lkylator,
328
Alkylator-Induced Excision Repair in CEM Cells 329
1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), in human 
leukemia CCRF-CEM cells in vitro [5]. The processes of 
excision repairs initiated by BCNU include incision  for 
removal of the damaged DNA, gap filling by DNA  resynthe-
sis, and rejoining by ligation  [6]. Therefore, the repair kinet-
ics were quantitated with the alkaline single-cell gel elec-
trophoresis (comet) assay [7] as the amount of DNA 
single-strand breaks.
2. Materials and Methods
2.1. Chemicals and Reagents
A B
  BCNU was purchased from Sigma Chemical Company 
(St. Louis, MO, USA).The compound was dissolved in 100% 
ethanol immediately before use.
Figure 1. The comet formation.  CEM cells were incubated with 
 10011M  1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) for 30 minutes, 
followed by washing the cells into fresh media. The cells were immedi-
ately examined by the comet assay. Control (A); the comet generated by 
treatment with BCNU (B).
2.2. Cell Culture
  Human leukemia CCRF-CEM cells were cultured in 
RPMI 1640 supplemented with 10% heat-inactivated fetal 
calf serum and 2mM L-glutamine. The cells were maintained 
at 37°C in a humidified atmosphere with 5% carbon dioxide. 
When the cells were cultured for 8  days, the growth curve 
was the most log-linear between 24 and 96 hours, suggesting 
that the cells were in an exponential growth phase during this 
period. Therefore, subsequent studies were performed with 
cells in this phase.
2.3. Drug Treatment
  CEM cells at 5  X  105 cells/mL were incubated with vari-
ous concentrations of BCNU for the indicated time periods, 
followed by washing the cells into fresh media and a subse-
quent incubation for the indicated time periods.
2.4. The Alkaline Single-Cell Gel Electrophoresis 
(Comet) Assay
  To evaluate the DNA repair kinetics, we performed the 
alkaline comet assay according to the method previously 
described but with a slight modification [7]. Approximately 
3000 CEM cells after the treatment were mixed with 20  pi 
of 0.5% low-melting-point agarose in phosphate buffered 
saline (PBS) at 37°C. The mixture was layered onto a frosted 
microscope slide previously coated with 70  pt of 0.65% nor-
mal agarose in PBS, followed by a top layer of 80  ILL of low-
melting-point agarose. After solidification, the slide was left 
in the lysis solution (2.5M sodium chloride, 10mM Tris, 
100mM ethylenediamine tetraacetic acid, 10% dimethylsul-
foxide,  1% Triton X-100, pH 10) at 4°C for 1 hour. The slide 
was then placed in the electrophoresis buffer (1mM ethyl-
enediamine tetraacetic acid, 300mM sodium hydroxide, pH 
13) for 40 minutes at  4°C to allow the unwinding of DNA. 
Electrophoresis was conducted for the next 15 minutes at 
90 V and 450 mA. After electrophoresis the slide was washed 
in neutralization buffer (0.4M Tris, pH 7.5) and stained with 
25  pi of 20  p,g/mL of ethidium bromide. One hundred cells 
per treatment were analyzed by means of the computer-
based image-analysis ystem (Kinetic Imaging Komet Sys-
tern, Version 4.0, Kinetic Imaging, Ltd., Liverpool, UK). The 
amount of DNA single-strand breaks was  expressed as the 
"tail moment," which combines measurement of the length 
of DNA migration and the relative DNA content herein [7].
2.5. Quantitation of Apoptotic Cell Death
  To evaluate cytotoxicity, apoptotic ell death was deter-
mined by Hoechst staining at 24 hours after the treatment 
[8]. Cells that had been treated and washed into fresh media 
were incubated with 2  p,g/mL Hoechst No. 33342 for 30 min-
utes at 37°C. Nuclei, 200 per treatment condition, were 
counted under UV illumination. Apoptotic cell death was 
determined from the nuclear morphology of nuclear  con-
densation and fragmentation.
3. Results
3.1. Comet Formation
  A typical comet image is shown in Figure 1. An untreated 
cell did not exhibit a comet ail, which indicated there were 
 no DNA strand breaks (Figure  1A), whereas a cell treated 
with BCNU showed comet formation, suggesting that DNA 
strand breaks had migrated in the electric field (Figure 1B).
3.2. Incision Response to BCNU
  To confirm that the comet was generated via the cellular 
response to BCNU, CEM cells were incubated with various 
concentrations of BCNU for 30 minutes or with  40p,M 
BCNU for various  times.  The tail moment values increased in 
both concentration- and time-dependent manners (Figure 2). 
suggesting an enhanced incision reaction that corresponded 
to  the increased BCNU-induced DNA damage. However, 
the tail moment value reached aplateau at a concentration 
of 80 to  100pLM or after an incubation period of 90 to 120 
minutes. Whereas the former esult suggests hat the incision 
capacity was saturated, the latter suggests a saturated  inci-
330 Yamauchi  et  al / International Journal of  Hematology 76 (2002) 328-332
 C 
a) 
E O
12
8
4
0
A
0 20 40 60 80 100
O
12
8
4
0
B
0 30 60 90 120
BCNU  (11M) Minutes
Figure 2. CEM cells were incubated for 30 minutes with various concentrations (0,  20,  40, 60, 80, or  'ALM) of BCNU (A), or with  40RM BCNU 
for various times  (0,15,  30.60, 90, and  120 minutes) (B). After cells were washed into fresh  media, they were immediately examined by the comet 
assay.The tail moment value of the untreated cells was set as a control  (hairline).  The values are means  ± SD of triplicate determinations. BCNU indi-
cates  1,3-bis(2-chloroethy0-1-nitrosourea.
 sion  capacity or the initiation of repair that enabled rejoining 
of the incised DNA.
3.3. DNA-Repair Process Initiated by  BCNU
  To determine the kinetics of DNA repair initiated by 
BCNU, CEM cells were examined by the  cornet assay at the 
indicated time points after the cells had been pulsed with 
BCNU and washed into fresh media. When the cells were 
treated with  40ILM BCNU, the tail moment was greatest at 
the end of the incubation period,  suggesting the maximal 
DNA strand breaks resulting from the incision of the repair 
process (Figure 3A). The tail moment decreased promptly 
thereafter, suggesting a rapid repair process enabling the 
rejoining of incised  DNA.  The tail moment value returned to 
the control evel at 4 hours, representing a successful com-
pletion of the process (Figure 3A).The figure also shows that 
the repair process was most linear between 0 and 2 hours, 
whereas the slope became lower thereafter. This result sug-
gests that the value of the 2-hour tail moment may be more 
indicative of completion of the repair process, compared with 
the value at 4  hours. 
  When the cells were treated with  100g.M  BCNU, the tail 
moment value was also greatest at the end of the incubation 
period (Figure 3B). However, in contrast o the result with 
 40KM BCNU (Figure 3A), the tail moment did not return to 
the control level within 4 hours, a result suggesting that 
repair had not been completed.
3.4. Completion  of  the Repair Process and the 
Subsequent  Cywtoxicity
  To identify the ability to complete the repair process, we 
incubated CEM cells with various concentrations of BCNU. 
The tail moment values at 2 hours appeared to increase with 
concentrations of BCNU beyond  40p,M  (60-100p.M), 
whereas the tail moment generated by lesser concentrations 
 (0-40pM) of BCNU remained low (Figure 4A). The  100p,M
4
 (1)  3 
E O 
2   2 
  1
 0
 ININIBCNU  40uM
A
0 1 2 3 
 Hours
4
E O 
2 
F-
12
8
4
0
 IIIIIIIBCNU  100uM
B
0 1 2 
Hours
3 4
Figure 3. CEM cells were pulsed for 30 minutes with  401.LM (A) or  100µM (B) of  1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), followed by wash-
ing the cells into fresh media. The cells were then examined by the comet assay at 0, 1, 2, and 4 hours after washing.  The tail moment value of the 
untreated cells was set as a control (hairline). The values are means  ± SD of triplicate determinations.
Alkylator-Induced Excision Repair in CEM Cells 331
a) 
E 0 
co
8
6
4
2
A
(/) 
 15 
 0 
0 0 
a
80
60
40
20
0
0 20 40 60 80 100
BCNU  (tM)
0
0 20 40 60 80 100 
  BCNU  (pM)
Figure 4.  A, CEM cells were incubated for 30 minutes with  1,3-bis(2-chloroethyl)-1-nitrosourea  (BCNU) at  0,  20,  40,  60, 80, or  100KM, followed by 
washing the cells into fresh  media.  The tail moment values were measured 2hours after  washing.  The tail moment value of the untreated cells was set 
as a control  (hairline).  The values are means ± SD of triplicate determinations.  I3,  The subsequent cell viabilities were assessed as apoptotic ell death 
via staining with Hoechst 33342 staining at 24 hours. The values are means ±  SD of triplicate determinations.
concentration was highly toxic to cells, whereas  40RM 
BCNU was minimally toxic (Figure 4B). These results ug-
gest that the higher concentrations surpass the cellular 
capacity for repair and thereby induce cell death.
4. Discussion
  The kinetics of DNA excision repair have been widely 
investigated inthe various contexts of using normal or malig-
nant lymphocytes, normal  fibroblasts, or malignant cell lines 
[9-13]. Lymphocytes can be a paradigm for the investigation 
of excision repair function, because the cells are quiescent 
and are not affected by the cell cycle; however, they may not 
be representative of cycling tumor cells. In addition, the 
incorporation of  thymidine into DNA that has commonly 
been used to quantitate DNA-repair kinetics may be masked 
by DNA replication in cycling  cells.  Thus, it has been difficult 
to evaluate repair kinetics in the clinically more relevant con-
ditions of actively dividing cells. 
  In the present study, the  cornet assay (Figure 1) was suc-
cessfully used to evaluate the kinetics of alkylator-induced 
DNA excision repair (Figure 3) in cycling CEM cells in a log-
arithmic growth phase. Several excision repair processes ini-
tiated by DNA alkylation involve the incision and excision of 
the oligonucleotides that include the damaged nucleotide. 
Subsequently, there is gap  filling by resynthesis ofDNA, and 
ligation rejoins the strands. The comet assay was able to 
quantitate the first step of the incision and the final step 
of the rejoining. Previous tudies have demonstrated that 
alkylator-induced DNA lesions do not produce DNA single-
strand breaks in the comet assay  [12,13]. Therefore, the 
0-hour tail moment would appear to reflect he incision step 
of the DNA-repair process, whereas the decrease in the tail 
moment hereafter suggests the completion of the repair 
process by rejoining. 
  As demonstrated in Figure 3A, malignant CEM cells were 
able to initiate the repair of DNA damage after alkylation by 
 4011M BCNU. The significant increase in the tail moment 
observed at the end of the 30-minute incubation period sug-
gested that repair was initiated immediately. The  steep
decrease in the tail moment at subsequent times suggested 
that ligation had occurred and that the repair process was 
completed quickly. 
 Conversely, the tail moment value generated by  10011M 
BCNU did not return to the control evel at 4 hours (Fig-
ure  3B), suggesting that the cells failed to repair the damage 
induced by this high concentration. At 6 to  8 hours after the 
end of the incubation period, cells formed a variety of 
 comets; some had almost  no comets, and others exhibited 
larger comet ails (data not shown). Because 50% of the cells 
treated with  100RM BCNU were dead after 24 hours  (Fig-
ure 4B),  the cells that had  the  larger comet ails at 2 hours 
 might have been induced to die without completing the 
repair process. In addition, the tail moment at 2 hours gener-
ated by 0 to  40RM BCNU was minimal, whereas itappeared 
to increase with concentrations beyond  40p,M  (60-100p.M; 
Figure 4A). These results uggest that BCNU at higher con-
centrations might induce DNA damage that surpasses the 
cell's capacity to repair it and thereby induce cell death. 
  In conclusion, with the comet assay we have precisely 
demonstrated the kinetics of DNA excision repair initiated 
by BCNU in human leukemia CEM cells in vitro. Although 
BCNU is not used in the treatment of  hematologic  malig-
nancies, it induces the most common bifunctional lkylations 
with 90% of mono-adducts and 3% to 5% of interstrand 
cross-links, effects that are similar to those induced by other 
alkylators [5]. The agent is commercially available, and it 
does not need to  be metabolized for activation, in contrast to 
cyclophosphamide. Thus,  the repair process initiated by 
BCNU can be a model for evaluating alkylator-induced 
DNA excision repair in leukemic ells. Accelerated DNA 
repair is important to consider in view of chemotherapy. In 
this regard, the evaluation of the repair process may provide 
a clue for therapeutic strategies to overcome resistance and 
improve clinical efficacy.
 Acknowledgments
  This work was supported in part by a grant-in-aid from 
the Ministry of Education, Science, and Culture, Japan, by a
332 Yamauchi et al /  International Journal  of Hematology 76(2002)328-332
grant-in-aid from the Ministry of Health and Welfare,  Japan, 
and by a grant from the Japan Research Foundation for Clin-
ical Pharmacology.
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Alkylator-Induced DNA Excision Repair in Human Leukemia CCRF-CEM Cells In Vitro, Measured Using the Single-Cell Gel Electrophoresis (Comet) Assay

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