Quantification of DNA interstrand crosslinks induced by ACNU in NIH/3T3 and L1210 cells using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Article abstract of DOI:10.1002/rcm.6800

RATIONALE Chloroethylnitrosoureas (CENUs) are important alkylating agents employed for the clinical treatment of cancer. The cellular toxicity of CENUs is primarily due to induction of DNA interstrand crosslinks (ICLs), which has been characterized as l-(3-deoxycytidyl), 2-(l-deoxyguanosinyl)ethane (dG-dC). However, the formation of dG-dC crosslinks can be prevented by O ⁶-alkylguanine-DNA alkyltransferase (AGT), which removes the O ⁶-chloroethyl group from O⁶-chloroethylguanine (O ⁶-ClEt-Gua), and ultimately its increased expression can result in drug resistance. Differing levels of AGT expression can lead to varying amounts of dG-dC crosslinking, which influences the sensitivity of cells to CENUs. METHODS In this work, a sensitive method for the quantitation of dG-dC crosslinks in cellular DNA has been established using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). RESULTS The limit of detection (LOD) and limit of quantitation (LOQ) of the method were determined to be 2 fmol and 8 fmol on-column, respectively, and the recovery ranged from 96% to 105% with the relative standard deviation (RSD) below 5%. Using this method, the levels of dG-dC crosslink induced by 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)- 3-nitrosourea hydrochloride (ACNU) were determined in NIH/3T3 fibroblasts cells (high level of expression of AGT) and L1210 leukemia cells (low level of expression of AGT). The time-course profile indicated that the levels of dG-dC crosslink uniformly increased in the early incubation period and reached the maximum at 12 h. Subsequently, the amount of dG-dC crosslinking decreased to very low levels presumably owing to the repair of O⁶-ClEt-Gua by AGT. The crosslinking levels in L1210 cells were significantly higher than those in NIH/3T3 cells at each time point. This provides strong evidence that high express of AGT in CENU-resistant cells inhibits the formation of dG-dC crosslinks. CONCLUSIONS This work will contribute to the further understanding of the drug resistance of CENUs, and will provide a means to evaluate the anticancer activity of new bifunctional anticancer agents. Copyright

Full text of DOI:10.1002/rcm.6800

Research Article
Received: 2 September 2013
Revised: 28 October 2013
Accepted: 3 December 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 439–447
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6800
Quantification of DNA interstrand crosslinks induced by ACNU in
NIH/3T3 and L1210 cells using high-performance liquid
chromatography/electrospray ionization tandem mass spectrometry
*
Lili Li, Lijiao Zhao and Rugang Zhong
Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Sciences and Bioengineering, Beijing University of
Technology, Beijing 100124, P.R. China
RATIONALE: Chloroethylnitrosoureas (CENUs) are important alkylating agents employed for the clinical treatment of
cancer. The cellular toxicity of CENUs is primarily due to induction of DNA interstrand crosslinks (ICLs), which has been
characterized as l-(3-deoxycytidyl), 2-(l-deoxyguanosinyl)ethane (dG-dC). However, the formation of dG-dC crosslinks
can be prevented by O6-alkylguanine-DNA alkyltransferase (AGT), which removes the O6-chloroethyl group from
O6-chloroethylguanine (O6-ClEt-Gua), and ultimately its increased expression can result in drug resistance. Differing
levels of AGT expression can lead to varying amounts of dG-dC crosslinking, which influences the sensitivity of cells
to CENUs.
METHODS: In this work, a sensitive method for the quantitation of dG-dC crosslinks in cellular DNA has been
established using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry
(HPLC/ESI-MS/MS).
RESULTS: The limit of detection (LOD) and limit of quantitation (LOQ) of the method were determined to be 2 fmol and
8 fmol on-column, respectively, and the recovery ranged from 96% to 105% with the relative standard deviation (RSD)
below 5%. Using this method, the levels of dG-dC crosslink induced by 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-
(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) were determined in NIH/3T3 fibroblasts cells (high level of
expression of AGT) and L1210 leukemia cells (low level of expression of AGT). The time-course profile indicated that
the levels of dG-dC crosslink uniformly increased in the early incubation period and reached the maximum at 12 h.
Subsequently, the amount of dG-dC crosslinking decreased to very low levels presumably owing to the repair of
O6-ClEt-Gua by AGT. The crosslinking levels in L1210 cells were significantly higher than those in NIH/3T3 cells at each
time point. This provides strong evidence that high express of AGT in CENU-resistant cells inhibits the formation of
dG-dC crosslinks.
CONCLUSIONS: This work will contribute to the further understanding of the drug resistance of CENUs, and will
provide a means to evaluate the anticancer activity of new bifunctional anticancer agents. Copyright © 2014 John Wiley
& Sons, Ltd.
Chloroethylnitrosoureas (CENUs) are an important family of
alkylating agents widely used in the clinical treatment
of various cancers, including lymphomas, melanomas and
brain tumors.[1–4] As shown in Fig. 1, carmustine (BCNU),
lomustine (CCNU), semustine (MeCCNU), nimustine
(ACNU) and fotemustine (FTMS) represent the typical CENU
chemotherapeutics. CENUs are unstable under physiological
conditions and spontaneously undergo decomposition to
chloroethyldiazohydroxides followed by further degradation
to diazonium ions.[5–7] Diazonium ions are very active
electrophilic agents, which can directly alkylate DNA and
induce various types of DNA damage, including double/
single strand breaks and interstrand/intrastrand crosslinks.
The formation of DNA interstrand crosslinks (ICLs) is a
crucial damage mechanism involved in the anticancer effect
of CENUs.[1,3,8–10] Previous investigations indicated that the
ICLs induced by CENUs mainly formed between guanine
and the complimentary cytosine.[11] As shown in Fig. 2,
the mechanism for the formation of dG-dC crosslinks was
proposed whereby the chloroethyl diazonium ion originating
from CENUs reacted with guanine at the O6 site to form
O6-chloroethylguanine (O6-ClEt-Gua) followed by further
alkylation of the complimentary cytosine on the N3 site
via a cationic intermediate, N1,O6-ethanoguanine.[10,12] As
a result, this covalent crosslink interferes with normal
DNA replication by preventing the separation of the
double strands, which finally leads to the cytotoxicity
of CENUs.
* Correspondence to: L. J. Zhao, Beijing Key Laboratory of
Environmental & Viral Oncology, College of Life Sciences
and Bioengineering, Beijing University of Technology,
Beijing 100124, P.R. China.
E-mail: zhaolijiao@bjut.edu.cn
Rapid Commun. Mass Spectrom. 2014, 28, 439–447
Copyright © 2014 John Wiley & Sons, Ltd.

L. Li, L. Zhao and R. Zhong
CENUs, was designed and synthesized by Sartorelli’s
group.[15] They treated leukemia cells with cloretazine and
found that its therapeutic action was largely due to the
production of dG-dC crosslinks. Due to the high sensitivity
and specificity, mass spectrometry has been frequently
employed for the determination of DNA adducts in biological
samples. Wang’s group[16–18] reported the application of liquid
chromatography/tandem mass spectrometry (LC/MS/MS) in
assessing the levels of ICLs and monoadducts in human cells
treated by psoralen and various derivatives. Malayappan
et al.[19] investigated the formation and repair of the N,N-bis
[2-(N7-guanyl)ethyl]amine crosslink in human blood induced
by cyclophosphamide using LC/MS/MS. Paz et al.[20] used
LC/MS/MS to map the DNA adducts, including interstrand
and intrastrand crosslinks, in different cell lines exposed to
mitomycin C and derivatives. In our previous work,[21,22]
dG-dC crosslinks induced by MeCCNU in calf thymus DNA
and in synthetic oligonucleotides were determined by high-
performance liquid chromatography/electrospray ionization
tandem mass spectrometry (HPLC/ESI-MS/MS). The results
indicated that the dG-dC crosslink level stayed relatively low
during the first 2 h of treatment and then underwent an
obvious increase, which suggested that the reaction was
initiated by the formation of the monoadduct followed by the
second alkylation on the complementary strand of DNA to
form the crosslink. This provided convincing evidence for
the crosslinking mechanism proposed in our theoretical
studies.[23,24]
As an important family of alkylating agents, CENUs have
been widely employed as chemotherapeutics against gliomas,
lymphoma and solid tumors since N-methyl-N-nitrosourea
was found to have anticancer activity in 1960s. However,
the clinical efficacy of CENUs is greatly limited by drug
resistance. Previous studies revealed that O6-alkylguanine-
DNA alkyltransferase (AGT), which acts as a single agent to
directly remove the alkyl groups located on the O6-position
of guanine from DNA, provides a powerful resistance
mechanism to cancer chemotherapy.[25–30] AGT prevents the
formation of CENUs-induced DNA ICL by transferring the
chloroethyl groups from O6-ClEt-Gua to an active-site
cysteine residue.[25,31,32] In addition, AGT reacts with the
cyclic intermediate N1,O6-ethanoguanine[33,34] to prevent
crosslink formation. For understanding the physiological role
Figure 1. Typical CENU chemotherapeutics used in the
clinical treatments of cancer.
To understand the anticancer mechanism of CENUs and
develop more efficient chemotherapeutics, in vitro and
in vivo studies have been performed to investigate the
CENUs-induced DNA ICLs. Fischhaber et al.[12] identified
dG-dC crosslinking induced by N,N’-bis(2-chloroethyl)
nitrosourea (BCNU) using synthetic oligonucleotides and
provided the first direct evidence that BCNU had no
strong sequence preference for interstrand crosslinking.
Subsequently, Bodell and colleagues[13] demonstrated that
there was a significant correlation between LD10 of CENUs
and the levels of dG-dC crosslinks, which suggested that the
level of the dG-dC crosslink could be used as a molecular
dosimeter for the therapeutic response after treatment with
CENUs. Ueda-Kawamitsu et al.[14] measured the time course
of DNA ICLs in L1210 cells treated with BCNU. The results
showed that the percentage of crosslinks reached the
maximum after 6 h exposure and subsequently decreased
presumably because of DNA repair. Cloretazine, which is a
relatively new prodrug with a similar molecular structure to
Figure 2. Supposed mechanism for the formation of dG-dC crosslinks induced by CENUs.
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Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 439–447

Quantitation of DNA interstrand crosslinks induced by ACNU in cells
1260 mg (4.8 mmol) of triphenylphosphine, 750 μL (4.8 mmol)
of AGT in drug resistance, a number of cellular experiments
were performed by treating various cell lines with CENUs.
Melanoma-transfected cells with high AGT level were
found to be significantly less sensitive to carmustine and
fotemustine.[35] Erickson et al.[36] measured the levels of
DNA ICL and DNA-protein crosslinks in CENU-treated
human cell strains from malignant tumors using alkaline
elution assays. They found that the strains deficient in AGT
(Mer– phenotype) produced consistently higher levels of
DNA ICL than did the Mer+ strains. Also using alkaline
elution, Bodell et al.[37] determined the number of DNA ICLs
formed in BCNU-treated cells. The number of DNA ICLs
formed in BCNU-resistant cells 9L-2, HU-188, and HU-252-2
are approximately 50% that of the corresponding sensitive
cell lines 9L and HU-126. All these studies demonstrated that
the formation of DNA ICL induced by CENUs was closely
related to the AGT levels in different cell lines. In this work,
the levels of dG-dC crosslinks were determined in L1210
leukemia cells (Mer–) and NIH/3T3 fibroblasts cells (Mer+)
treated with ACNU using HPLC/ESI-MS/MS. The purpose
of this study is to establish a highly sensitive quantitation
method for DNA ICLs in cells, which can be used to evaluate
the anticancer activity and drug resistance of CENUs. We
expect this work will contribute to the development of novel
bifunctional alkylating anticancer agents with high efficiency
and low toxicity.
of diethylazodicarboxylate, and 312 μL (4.8 mmol) of
2-fluoroethanol dioxane at room temperature for 1 h. Then
30 mL of 5% NaHCO3 solution were added. The mixture
was stirred at room temperature for 15 min. The product
was extracted three times with 50 mL of methylene chloride.
The oily residue was dissolved in 10 mL of methanol after
evaporation of CH2Cl2. After the addition of 60 mL of
concentrated ammonium hydroxide, the mixture was
kept at 60°C for 3 h. After evaporation of methanolic
NH4OH, the product, O6-(2-fluoroethyl)-2’-deoxyguanosine
(O6-FEt-dGuo), was dissolved in methanol and purified by
silica gel column chromatography (200–300 mesh) using
ethyl acetate and petroleum ether as the solvent. The
obtained O6-FEt-dGuo was used as the material for the
following synthesis of dG-dC crosslinks. O6-FEt-dGuo
(20 mg, 64 μmol) was mixed with 2’-deoxycytidine (5 mg,
22 μmol). The mixture was suspended in DMSO (100 μL)
at 55°C for 20 days. HPLC purification with fraction
collection of dG-dC was performed using a 4.6ꢀ 250 mm
ZORBAX SB-C18 column (5 μm particle size) and a 10 mM
ammonium acetate buffer (0.1% acetic acid, pH 6.8) (A)
and acetonitrile (B) at a flow rate of 1 mL/min. A gradient
of 5 to 10% B in 20 min was employed with a linear
gradient to 30% B over 10 min with a 3 min isocratic wash
at 30% B. The fraction during 20 to 22 min was collected.
UV detection was performed at 258 nm. The synthesis of
the internal standard, 15N3-dG-dC, was carried out using
the same procedure as the unlabeled dG-dC except 15N3-
2’-deoxycytidine was used in the final step. The final
product was characterized by NMR, MS, IR and UV
spectroscopy. The data were consistent with the results
obtained previously.[11]
EXPERIMENTAL
Chemicals and materials
ACNU, acetonitrile (HPLC grade), 2’-deoxyguanosine,
2’-deoxycytidine and phosphodiesterase I were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Nuclease S1,
alkaline phosphatase (CIAP) and deoxyribonuclease I were
obtained from TaKaRa Biotechnology (Tokyo, Japan). 15N3-
2’-Deoxycytidine was purchased from Cambridge Isotope
Laboratories (Andover, MA, USA). All other chemicals,
reagents and solvents were purchased from Sigma-Aldrich.
Microcon YM-10 centrifugal columns were purchased from
Millipore (Billerica, MA, USA). Deionized water was purified
by a PALL deionizer.
Mouse lymphoid leukemia L1210 cells were purchased
from Cell Center of Peking Union Medical College. The
NIH/3T3 fibroblasts cells were a gift from the Chinese
National Institute for Viral Disease Control and Prevention.
All cell culture media and reagents were obtained from
Peking Union Medical College.
Cell culture
Murine lymphoid leukemia L1210 cells were grown, as
suspension cultures, in Dulbecco’s modified Eagle’s medium
(DMEM) high glucose medium supplemented with 10% (v/v)
horse serum (HS) and antibiotics (100 U/mL penicillin and
100 μg/mL streptomycin) at 37°C under a 5% CO2 atmosphere.
The mouse embryonic fibroblast cell line NIH/3T3 was
routinely sub-cloned and then cultivated in DMEM high
glucose medium supplemented with 10% fetal calf serum
(FCS), penicillin (100 U/mL) and streptomycin (100 μg/mL)
at 37°C in an air atmosphere containing 5% CO2. Cells were
passaged as required every 2–3 days. The culture medium
was discarded when cells achieved a saturation density of
1–2ꢀ 106 cells/mL. Afterwards, the HS-free and FCS-free
medium containing ACNU was supplemented.
Synthesis of the standards
Treatment of NIH/3T3 and L1210 cells with ACNU
The synthesis of [l-(3-deoxycytidyl),2-(l-deoxyguanosinyl)
ethane (dG-dC) was carried out according to a previously
published method, with some modifications.[11] Briefly,
N2,3’,5’-triacetyl-2’-deoxyguanosine was produced from the
reaction of 650 mg (2.3 mmol) of 2’-deoxyguanosine with
2.4 mL (26 mmol) of acetic anhydride, 30 mg (0.25 mmol)
of 4-dimethylaminopyridine, and 3.8 mL (27 mmol)
triethylamine in 100 mL of dry pyridine at 50°C for 20 h. After
crystallization and filtration, the obtained residue was
dissolved in 6 mL of dry dioxane followed by incubation with
ACNU was dissolved in deionized water immediately prior
to use and directly added to fresh culture medium to achieve
final concentrations of 0.2, 0.6 and 1 mM, respectively. Cells
were incubated in freshly prepared culture solutions
containing ACNU at 37°C for various times of treatment
(3 h, 6 h, 9 h, 12 h, 15 h, 18 h, 21 h and 24 h). NIH/3T3 cells
were trypsinized after the treatment. Cells were collected by
centrifugation followed by washing with phosphate-buffered
saline (PBS). The obtained cell pellets were kept at –20°C for
Rapid Commun. Mass Spectrom. 2014, 28, 439–447
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L. Li, L. Zhao and R. Zhong
DNA extraction. For each time point, the control cells were
cultured under the same conditions as the treated cells
without addition of ACNU to the culture medium.
(solvent B). The initial elution step was 2% B for the first
5 min. The gradient was linearly increased to 80% B in
25 min and held at that composition for 3 min. Then the
solvent composition was brought back to the initial
composition of 2% B in the next 2 min and equilibrated for
30 min. The instrumental parameters of the mass
spectrometer were set as follows: spray voltage 4000 V;
sheath gas (nitrogen) pressure 50 psi; auxillary gas (nitrogen)
pressure 15 psi; capillary temperature 300°C; and tube lens
offset 89 V. Collision energy was 20 eV with an argon
pressure of 1.0 mTorr. The source collision-induced
dissociation (CID) was set to 8 V. The fragmentation patterns
of dG-dC and 15N3-dG-dC are shown in Fig. 3. The amounts
of dG-dC crosslink were quantified by selecting reaction
monitoring (SRM) with the transition of m/z 521 → 289 for
dG-dC and 524 → 292 for 15N3-dG-dC.
DNA extraction from cell samples
DNA extraction from cell pellets was performed following
the previously reported procedures with modifications.[38–40]
Typically, L1210 and NIH/3T3 cells were homogenized in
10 mL of lysis buffer (10 mM Tris-HCl, 150 mM NaCl, and
10 mM EDTA, pH 8.0) with addition of 60 μL of proteinase
K solution (20 mg/mL), shaking at 37°C for 12 h. The mixture
was extracted with phenol/chloroform/isoamyl alcohol
solution (25:24:1, v/v/v) twice. Then the supernatant
containing DNA was collected and the DNA was precipitated
by addition of 20 mL of ice-cold ethanol followed by
centrifugation at 12000 rpm for 5 min. The DNA pellets were
washed with 70% ethanol and then 100% ethanol. All the
DNA samples were dried with a stream of nitrogen and
stored at –20°C until enzymatic hydrolysis.
According to previous work,[23,24] the level of dG-dC
crosslink in DNA from cells were reported as the number of
crosslinked dG and dC in every 109 base pairs calculated by
Eqn. (1). In Eqn. (1), C refers to the determined molar
concentrations of the dG-dC crosslink; V refers to the volume
of the enzymatic digestion solution, which is 300 μL; C0 is the
concentration of the DNA duplexes; V0 is the volume of the
DNA sample before enzymatic digestion, which is 200 μL;
and M0 represents the average molecular weight of the four
deoxynucleotides (325 g/mol):
Enzymatic hydrolysis of DNA
The purity and concentration of the DNA samples were
determined before enzymatic hydrolysis. The obtained DNA
pellets were redissolved in 1 mL of Tris-HCl buffer (10 mM,
pH 7.0). The purity of DNA was confirmed by measuring
UV absorption at 230, 260 and 280 with the ratio of 260/230
and 260/280 at 2.4 and 1.8, respectively. The concentration
of the DNA solution was determined by UV absorbance at
260 nm. One OD260nm corresponded to approximately
50 μg/mL for double-stranded DNA.
dG-dC crosslinks=109 base pairs
À
Á
¼ C ꢀ V ꢀ 109 =ðC0 ꢀ V0=2M0Þ
(1)
Following the previously reported protocols,[21,22] DNA
samples were digested by four enzymes, including DNase I,
nuclease S1, alkaline phosphatase and snake venom
phosphodiesterase. Briefly, the solutions were heated at 98°
C for 5 min and promptly chilled in an ice-bath for 10 min.
Each solution (200 μL) was hydrolyzed using 150 units of
DNase I (30 μL, buffered in CH3COONa 20 mM, NaCl
150 mM, pH 5.0) and 500 units of nuclease S1 (20 μL, buffered
in CH3COONa 10 mM, NaCl 150 mM, ZnSO4 0.05 mM,
pH 4.6). After incubation at 37°C for 6 h, the mixture was
further incubated overnight at 37°C with the addition of
40 units (40 μL) of alkaline phosphatase and 1 milliunit of
phosphodiesterase I (10 μL) buffered in Tris-HCl 500 mM,
MgCl2 10 mM (pH 9.0). Finally, the DNA samples were
filtered with molecular weight centrifugal filters (Microcon
YM-10) for HPLC/ESI-MS/MS analysis. A buffer control
without DNA was prepared for each set of samples and
processed as negative controls following the same procedure.
RESULTS AND DISCUSSION
Method validation
The synthesized dG-dC and isotope-labeled 15N3-dG-dC were
purified by HPLC and identified by MS employing ESI in
positive mode. As shown in Fig. 3, the [M+H]+ ion of dG-dC
(m/z 521) yields two fragment ions at m/z 405 and 289 after
losing one and two deoxyribose moieties, respectively. A
transition of m/z 521 → 289 was selected for SRM quantitative
analysis of dG-dC crosslinks because m/z 289 is the dominant
fragment ion. Similarly, neutral loss of deoxyribose leads to
two fragment ions of m/z 408 and 292 from the [M+H]+ ion of
15N3-dG-dC (m/z 524), and the transition m/z 524 → 292 was
monitored for the internal standard 15N3-dG-dC. Under the
HPLC conditions used here, dG-dC and 15N3-dG-dC coeluted
at 22.6 min (see Fig. S1, Supporting Information).
The standards were prepared by mixing the synthesized
dG-dC (0.32, 1.6, 8, 16, 32 and 60 nM) with a fixed amount
of 15N3-dG-dC (9.6 nM) in deionized water. They were
subjected to HPLC/ESI-MS/MS analysis as described
above for quantitative analysis. The calibration curve was
constructed by plotting the corresponding SRM peak area
ratios of dG-dC/15N3-dG-dC versus the corresponding
concentration ratios. As shown in Fig. 4, the calibration curve
was linear over the range from 0.32 to 60 nM with a
correlation coefficient (R2) of 0.9999.
Quantitation of dG-dC crosslinks by HPLC/ESI-MS/MS
HPLC/ESI-MS/MS analysis was carried out on a Thermo
TSQ Quantum Discovery MAX triple quadrupole tandem
mass spectrometer interfaced with a HPLC system (Thermo
Finnigan, San Jose, CA, USA). The electrospray ionization
(ESI) was performed in positive mode. Chromatography
was performed with a 2.1ꢀ150 mm (5 μm particle size)
Zorbax SB-C18 column (Agilent Technologies, Palo Alto,
CA, USA) and a flow rate of 0.1 mL/min. The injection
volume was 25 μL. The mobile phase consisted of deionized
water with 0.01% acetic acid (solvent A) and acetonitrile
The sensitivity, accuracy and specificity of the method were
validated by determining the limit of detection (LOD), limit
of quantification (LOQ), recovery and analysis of control
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Quantitation of DNA interstrand crosslinks induced by ACNU in cells
Figure 3. Positive ESI product ion spectra of the [M+H]+ ions of dG-dC (A) and
15N3-dG-dC (B).
Table 1. Recovery of the dG-dC quantitation method
Added
concentration
(nmol/L)
Determined
concentration
(nmol/L) (n = 3)
Recovery
(%)
RSD
(%)
2
1.9
4.0
96
100
105
99
3.7
4.3
2.9
3.9
4
8
16
8.4
15.9
with dG-dC and 15N3-dG-dC. Appropriate amounts of
dG-dC were prepared in the calf thymus DNA solution at the
concentration of 2, 4, 8 and 16 nM with a fixed 15N3-dG-dC
concentration of 9.6 nM to obtain the quality control (QC)
samples. Enzyme hydrolysis was conducted for the QC
samples. The resulting mixtures were subjected to HPLC/
ESI-MS/MS analysis. As summarized in Table 1, the mean
recovery was demonstrated to fall within the range of 96 to
105%. The precision of the assay was evaluated by determining
the relative standard deviation (RSD). Each QC sample was
analyzed in triplicate. The measured RSD levels were below
5% for all samples. The measured recovery and precision
indicated that the influence of sample preparation and matrix
effects is negligible on the obtained results of the dG-dC
concentration. Figure 5 shows the SRM chromatograms of
dG-dC and 15N3-dG-dC in the DNA enzymatic hydrolysates
Figure 4. Calibration curve of dG-dC crosslinks by plotting
SRM peak area ratios versus the concentration ratio between
dG-dC and 15N3-dG-dC.
samples to measure any contamination or artifactual
formation of dG-dC. The LOD and LOQ were estimated at
the levels giving signal-to-noise ratios (S/N) of 5 and 15,
respectively. The LOD and LOQ for a dG-dC crosslink
standard spiked in calf thymus DNA were determined to be
2 and 8 fmol on-column, respectively. In order to evaluate
the influence of sample preparation and matrix effects on
the measured results, the recovery of dG-dC in the
hydrolysates was studied using calf thymus DNA spiked
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L. Li, L. Zhao and R. Zhong
Figure 5. SRM chromatograms of dG-dC crosslinks in the DNA digestion
mixtures from cells treated with 0.6 mM ACNU for 12 h (A) and from control
cells (B).
from the cells treated with 0.6 mM ACNU for 12 h (Fig. 5(A))
crosslinks/109 base pairs in NIH/3T3 cells at the ACNU
concentrations of 0.2, 0.6 and 1 mM, respectively. In L1210
cells, the crosslinking levels also reached the maximum at
12 h with 986 and 1306 dG-dC crosslinks/109 base pairs at
the ACNU concentrations of 0.6 and 1 mM, while at 0.2 mM
ACNU the maximum was reached at 9 h with 490 dG-dC
crosslinks/109 base pairs. Subsequently, the crosslinking
levels decreased to very low levels in both the cell lines. The
crosslinking levels at 24 h fell back to levels lower than those
at 3 h. Most notably, in NIH/3T3 cells the crosslinking level
was below the detection limit at 24 h and an ACNU
concentration of 0.2 mM. The decrease in the dG-dC
crosslinking levels reflected the repair of alkylated guanine
by AGT. The decrease in the crosslinking level is also possibly
due to death of the cells containing high levels of DNA damage.
Moreover, besides the AGT-mediated repair mechanism, there
are other pathways repairing the crosslinked base pair, such
as the base excision repair (BER) processing, in which the BER
proteins are capable of recognizing and removing the
crosslinked base pairs induced by chemotherapeutic agents.[41]
and from the control cells (Fig. 5(B)). In Fig. 5(A), the retention
time for dG-dC in the digestion mixture is 22.9 min, and its
corresponding isotope-labeled standard, 15N3-dG-dC, has the
same retention time. Figure 5(B) indicates that there is no signal
detected with the SRM transition for dG-dC in the DNA
hydrolysates from the control cells. This indicates that there is
no significant matrix interference or contamination in the
analyte channels from internal standards or hydrolytic
enzymes, so the specificity of the method was acceptable.
Quantitation of dG-dC crosslinks in the hydrolysates of
ACNU-treated NIH/3T3 and L1210 cells
To provide insight into the relationship between crosslinking
level and cellular sensitivity to CENUs, the established
quantitation method was employed to determine the dG-dC
crosslink levels in DNA extracted from mouse leukemia
L1210 cells and fibroblast NIH/3T3 cells treated with ACNU.
The determined time courses for the levels of dG-dC crosslink
are indicated in Fig. 6, and the corresponding values of the
crosslinking level at each time point were summarized in
Supplementary Table S1 (Supporting Information). In both
L1210 and NIH/3T3 cells, the levels of dG-dC crosslink
displayed dose-dependence with increasing ACNU
concentrations from 0.2 to 1 mM. For the treatments at three
different ACNU concentrations, the crosslinking levels
showed a common increasing trend from 0 to 12 h, and
reached a maximum at 12 h with 359, 594, and 895 dG-dC
Ueda-Kawamitsu et al.[14] performed
a quantitative
analysis of BCNU-induced dG-dC crosslinks in L1210
leukemia cells by fluorescence assay with BCNU exposure
for 24 h. The results indicated that the maximum level of
DNA ICL was observed at 6 h followed by a decline, which
was attributed to the repair mechanism. Both of the two
studies gave direct evidence for the AGT-mediated repair of
DNA ICLs by observing obvious decreases in the crosslinking
levels, although different peak times of the crosslinking levels
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Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 439–447

Quantitation of DNA interstrand crosslinks induced by ACNU in cells
Figure 6. Profiles of dG-dC crosslinks in NIH/3T3 cells (A) and in L1210 cells
(B) treated with ACNU for 0–24 h.
were observed. This difference may be due to a different
CENU being used in treatment of the cells. Further studies
of dG-dC crosslink levels in cells induced by ACNU, BCNU
and other CENUs, which have different crosslinking activities
and half-lives, are being performed in our laboratory.
As shown in Fig. 6, there were significant differences
(P <0.05) between the dG-dC levels in the DNA from L1210
cells and NIH/3T3 cells. At each time point, the levels of
dG-dC crosslink were higher in L1210 cells than in NIH/
3T3 cells. The levels of dG-dC crosslink in L1210 cells were
1.02 to 2.19 times higher than those in NIH/3T3 cells
throughout the experiment. This result suggested that the
dG-dC crosslinks induced by ACNU were more efficiently
repaired in NIH/3T3 cells than in L1210 cells. NIH/3T3
the levels of dG-dC crosslinks in the three glioma lines,
BTRC-19, 9L-2 and 9L cells. They concluded that the
reduction in the levels of dG-dC crosslink in 9L-2 and
BRC-19 cells, which were 10-fold more resistant to CENU
than 9L cells, was associated with increased levels of AGT
activity. These results convincingly demonstrated that the
suppression of the formation of CENU-induced crosslinks
occurred as a result of chloroethyl transferase action of AGT.
CONCLUSIONS
In summary, a method for quantitation of dG-dC interstrand
crosslinks in cells was established using HPLC/ESI-MS/MS
providing high sensitivity, specificity and accuracy. Using this
method, the levels of dG-dC crosslinks induced by ACNU
were determined in NIH/3T3 fibroblasts cells (high level of
expression of AGT) and L1210 leukemia cells (low level of
express of AGT). The time-course profiles of dG-dC crosslinks
in the two cell lines both showed a time-dependent pattern
over a 24 h period. The crosslinking levels upon exposure to
ACNU at various concentrations uniformly increased in the
early incubation period reaching the maximum at 12 h.
mouse fibroblasts represent
a stable diploid line that
expresses stable levels of AGT activity.[42] Dunn et al.[43]
demonstrated that NIH/3T3 cells were more resistant to
ACNU than MGMT-deficient cells because of their relatively
high levels of AGT. Previous studies indicated that L1210
cells are deficient in expression of AGT,[44] although they
were not considered completely defective in crosslink repair
because low levels of AGT activity are present.[45] Bodell
et al.[46] obtained similar results as our study by comparing
Rapid Commun. Mass Spectrom. 2014, 28, 439–447
Copyright © 2014 John Wiley & Sons, Ltd.
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L. Li, L. Zhao and R. Zhong
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Subsequently, the crosslinking levels declined after 12 h,
presumably due to AGT-mediated DNA repair. The
crosslinking levels in L1210 cells were significantly higher
than those in NIH/3T3 cells at each time point, which
suggested that NIH/3T3 cells have higher repairing activity
than L1210. Moreover, the rate of disappearance of the dG-dC
crosslink from 12 to 24 h is more rapid in L1210 than in
NIH/3T3 cells, reflecting that the higher levels of the dG-dC
crosslink in L1210 cells might be due to the higher efficiency
of its formation by better uptake of the drug. The difference
in the crosslinking levels of L1210 and NIH/3T3 provides
direct evidence for AGT-repair of dG-dC crosslinks.
This work provided a robust method for quantitation of
DNA ICLs in cell lines. This method provides direct
quantitation of dG-dC crosslinks allowing for higher
specificity and accuracy than previous methods, such as
fluorescence and alkaline elution based assays, which
measure the crosslinked double strands. This work not only
contributes to a further understanding of the drug resistance
to CENU-based anticancer drugs induced by AGT, but also
will assist in the development of new bifunctional anticancer
agents with high specificity and efficiency.
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N’-bis(2-chloroethyl)nitrosourea
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Acknowledgements
This work was supported by grants from the National
Natural Science Foundation of China (No. 21277001), the
Beijing Nova Program (No. 2009B08), and the Beijing
Municipal Education Commission Science and Technology
Project (No. KZ201110005003).
[17] C. F. Lai, H. C. Cao, J. E. Hearst, L. Corash, H. Luo, Y. S. Wang.
Quantitative analysis of DNA interstrand cross-links and
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and UVA irradiation. Anal. Chem. 2008, 80, 8790.
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P. Jacobson, N. Tretyakova. Quantitative high-performance
liquid chromatography-electrospray ionization tandem
mass spectrometry analysis of bis-N7-guanine DNA-DNA
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[21] B. Q. Bai, L. J. Zhao, R. G. Zhong. Quantification of meCCNU-
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SUPPORTING INFORMATION
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