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Increased -H2AX and Rad51 DNA Repair Biomarker Expression in
Human Cell Lines Resistant to the Chemotherapeutic Agents Nitrogen
Mustard and Cisplatin.
Sheba Adam-Zahir1, Piers N. Plowman2, Emma C. Bourton1, Fariha Sharif1 and Christopher N.
Parris1.
1Brunel
Institute of Cancer Genetics and Pharmacogenomics, Division of Biosciences, School of
Health Sciences and Social Care, Brunel University, Uxbridge, Middlesex UB8 3PH, United
Kingdom.
2Department of Radiotherapy, St Bartholomew's Hospital, West Smithfield, London EC1A 7BE,
United Kingdom.
Short title: DNA repair biomarkers and chemotherapeutic drug resistance.
Keywords
DNA repair, chemotherapy, biomarker, Rad51, -H2AX
Corresponding Author
Christopher N. Parris BSc, PhD.
Brunel Institute of Cancer Genetics and Pharmacogenomics,
Division of Biosciences,
School of Health Sciences and Social Care,
Brunel University,
Uxbridge,
Middlesex
UB8 3PH,
United Kingdom.
Tel: +44(0)1895 266293
Fax: +44(0)1895 269854
Author Email Address
[email protected]
Word Count: 4879 (excluding references)
No of tables: 1
No of figures: 6
1
Abstract
Chemotherapeutic anticancer drugs mediate cytotoxicity by a number of mechanisms.
However, alkylating agents which induce DNA interstrand cross links (ICL) are amongst the most
effective anticancer agents and often form the mainstay of many anticancer therapies. The
effectiveness of these drugs can be limited by the development of drug resistance in cancer
cells and many studies have demonstrated that alterations in DNA repair kinetics are
responsible for drug resistance. In this study we developed two cell lines resistant to the
alkylating agents nitrogen mustard (HN2) and cisplatin (Pt). To determine if drug resistance was
associated with enhanced ICL DNA repair we used immunocytochemistry and imaging flow
cytometry to quantitate the number of -H2AX and Rad51 foci in the nuclei of cells post drug
exposure. -H2AX was used to evaluate DNA strand breaks caused by repair incision nucleases
and Rad51 was used to measure the activity of homologous recombination (HR) in the repair of
ICL. In the drug resistant derivative cell lines, overall there was a significant increase in the
number and persistence of both -H2AX and Rad51 foci in the nuclei of cells over a 72 hr period,
when compared to the non-resistant parental cell lines (ANOVA P < 0.0001). Our data suggest
that using DNA repair biomarkers to evaluate mechanisms of resistance in cancer cell lines and
human tumours may be of experimental and clinical benefit. We concede however, that
examination of a larger population of cell lines and tumours is required to fully evaluate the
validity of this approach.
2
Introduction
The role of γ-H2AX in response to cellular exposure to ionising radiation (IR) has been
well established whereby phosphorylation on serine139 of H2AX corresponding to the formation
of DNA double strand breaks (DSB) was first identified nearly 15 years ago by Rogakou and coworkers (1). The induction of DSB by exposure to IR leads to the predictable induction of H2AX foci in the nuclei of non-lethally irradiated surviving cells, but within a 24 hr period DSB
are repaired and -H2AX foci are removed. However, in cell lines derived from individuals with
defects in DNA DSB repair, such as cells from ataxia telangiectasia patients, a failure to
efficiently repair DSB is associated with a persistence of -H2AX foci beyond 24 hrs (2). As a
result biomarkers of DSB such as -H2AX potentially lend themselves to the diagnostic setting in
the prediction of cancer patient response to clinical radiotherapy (RT). A retrospective study by
Bourton et al, 2011 (3) which employed γ-H2AX as a marker of DNA DSB successfully identified
patients who were hyper-sensitive to RT and experienced severe normal tissue toxicity (NTT). γH2AX analysis by flow cytometry revealed a persistence of foci in lymphocytes from patients
with severe NTT. Patients that tolerated RT with little or no NTT efficiently repaired DNA DSB
with the corresponding reduction in the expression of -H2AX foci.
Correspondingly, the use of -H2AX and other DNA repair biomarkers might be
informative in identifying both patient and tumour response to cytotoxic chemotherapy. Such
an approach is challenging given that: 1) chemotherapeutic agents in clinical use have widely
different mechanisms of action and may elicit different DNA repair pathways that cannot be
monitored by a single DNA repair biomarker; 2) many chemotherapy regimens used for cancer
treatment employ a combinatorial approach whereby multiple drugs are used concurrently and
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3) the development of drug resistance in cancer cells may occur by a number of mechanisms
that do not involve alteration or modulation of DNA repair pathways, an example here being
the development of multiple drug resistance due to p-glycoprotein upregulation (4).
Despite these caveats, a limited approach to monitoring chemotherapy responses by
assessing DNA repair capacity might be both possible and of clinical and experimental benefit.
The mainstay of many chemotherapeutic regimens is the use of alkylating agents such as HN2,
cyclophosphamide and Pt which are amongst the most effective of chemotherapeutic drugs (5).
Here cytotoxicity is mediated by the introduction of DNA ICL and the degree of cytotoxicity is
directly related to their ability to introduce ICL (6). ICL cause strand distortion and prevent
strand dissociation thus inhibiting DNA synthesis and replication, leading to cell death. Cellular
repair of ICL poses a significant challenge to the DNA repair machinery and involves the coordinated interaction of distinct DNA repair pathways. In brief, the strand distortion caused by
an ICL is recognised by proteins of the Fanconi Anaemia (FA) pathway whereby Fanconiassociated nuclease 1 (FAN1) with a 5’-3’ exonuclease activity and a 5’-FLAP endonuclease
function cleaves the ICL in a process known as “unhooking”. This converts a stalled replication
fork into a one-ended DSB. Other endonucleases including MUS81-EME1 and XPF-ERCC1 cleave
the DNA on the 3’ and 5’ ends of the ICL respectively. Subsequently, the strand break caused by
the action of the endonucleases creates a substrate which is repaired by HR via a Holliday
junction pathway mediated by the Rad51 protein (7). Therefore in order to monitor this activity
in vitro, measuring the level of biomarkers such as -H2AX and Rad51 might be valuable. For IR
exposure, the appearance of γ-H2AX foci post-irradiation is indicative of DNA DSB formation.
On the other hand, γ-H2AX foci appearing post treatment with chemotherapeutic agents
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causing ICL, may be reflective of both direct chemotherapy induced DNA damage or repair
processes taking place since -H2AX will be activated by the action of nucleases excising the
damage (8). This is further supported by Clingen et al,2008 (9) who demonstrated that repair
nuclease-induced DSB were initiated in both Chinese hamster and human ovarian cancer cells
in response to the formation of ICL with a concomitant increase in γ-H2AX foci. Furthermore
the appearance and quantitation of Rad51 foci following exposure to ICL inducing
chemotherapeutic drugs might indicate the extent of DNA repair occurring by HR at the site of
DNA damage and the extent of tumour cells resistance or sensitivity to the chemotherapeutic
drug.
Development of resistance to chemotherapeutic drugs poses a serious limitation to the
effectiveness of treatment (10 – 11). For example it has been shown that acquired resistance
to Pt accounted for treatment failure and deaths in up to 90% of patients with ovarian cancer
(12). Moreover, it has been demonstrated that increased Rad51 expression, evident of HR, is
associated with poor treatment outcomes in breast cancer patients (13).
To evaluate the role of both the -H2AX and Rad51 DNA repair biomarkers we employed
immunocytochemical methods combined with multispectral imaging flow cytometry to
evaluate DNA repair in human cells resistant and sensitive to the cross-linking agents HN2 and
Pt. We demonstrated that in cell lines resistant to these drugs, there was in general elevated
and persistent expression of -H2AX and Rad51 foci in the nuclei of cells. These data indicate
that evaluation of these biomarkers in both normal and tumour cells may predict patient
response to therapy and determine mechanisms of patient resistance to treatment.
5
Materials and Methods
Cell Culture
Cells were routinely cultured in Dulbecco’s Modified Eagle Medium (DMEM) (PAA
Laboratories Ltd., Yeovil, Somerset, UK) which was supplemented with 10% foetal calf serum,
2 mM L-glutamine and 100 units/mL penicillin and streptomycin (PAA). Cells were grown in
100mm Petri dishes (Sarstedt Ltd., Leicester, UK) as monolayers at 37oC in a humidified
atmosphere of 5% CO2 in air. All cell culture was carried out in a temperature controlled
laboratory within a Heraeus Class II Laminar Flow hood.
Cell Lines
Immortalised human fibroblast cell lines derived from normal and DNA repair defective
individuals as well as two ovarian cancer cell lines from an untreated cancer patient were
selected for this study. Details of these cell lines are shown in Table 1. The A278 ovarian cancer
cell line derived from an untreated cancer patient. Also the A2780 cisplatin resistant variant
was used for this study. These cell lies were obtained rom the Porton Down
Development of Cell Lines Resistant to Nitrogen Mustard and Cisplatin
Two DNA repair normal cell lines, MRC5-SV1 and NB1-HTERT were selected to develop
cell lines resistant to HN2. IC50 values, defined as the concentration of drug that kills
approximately 50% of the cell population post 1 hr exposure to each chemotherapeutic agent,
were derived for the two cell lines using clonogenic assays. These concentrations provided a
starting point for drug treatment and development of resistance. The cell lines were
continuously exposed to 0.50 μg/mL HN2 (Sigma Aldrich Ltd., Dorset, UK) in culture medium
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until they reached confluency. Cells were then sub-cultured and exposed to a higher
concentration of HN2. This concentration was increased by a geometric ratio of 1.5-fold of the
previous concentration (i.e. 0.50 μg/mL was increased to 0.75 μg/mL). Cells were continuously
exposed to HN2 until they reached a concentration of drug that was 10-fold of their respective
IC50 values (3.50 μg/mL for NB1-HTERTR and 5.30 μg/mL for MRC5-SV1R).
The A2780Cis cell line was developed through continual exposure of the A2780 parental
cell line to Pt. This cell line was obtained from the ECACC, Porton Down.
Induction of ICL in Cell Lines by Drug Exposure
To monitor the induction of ICL by drug exposure, cells were first exposed to an IC50
drug concentration. This was followed by immunological detection of -H2AX and Rad51 foci.
The IC50 used for both sensitive and resistant derivatives was derived from clonogenic assays of
the parental cells to allow for meaningful comparisons. IC50 values for HN2 was 0.30 µg/mL for
NB1-HTERT cells (parent and resistant) and 0.50 µg/mL for MRC5-SV1 cells (parent and
resistant). For Pt, the IC50 concentration was 12.00 µg/mL for the MRC5-SV1 cell line and 6.00
µg/mL for NB1-HTERT cells. All cell lines as proliferating monolayers and at approximately 80%
confluency were treated for 1 hr with the IC50 drug concentration.
Immunocytochemistry to Detect γ-H2AX and Rad51 Foci
Immunocytochemistry was carried out as detailed in Bourton et al, 2013 (16). Untreated
cells and those exposed to HN2 were fixed in 50:50 methanol:acetone (V:V) at 3, 5, 24, 30 and
48 hrs post treatment with HN2. For Pt exposures, the fixation time points were 6, 12, 24, 30,
48 and 72 hrs post treatment. Cells were blocked using 10% rabbit serum (PAA) in phosphate
7
buffered saline pH 7.4 (PBS) (Severn Biotech, Gloucestershire, UK) and stained with an mouse
monoclonal anti-serine139 γ-H2AX antibody (Clone JBW 301, Millipore UK Ltd., Hampshire, UK)
at 1:10 000 dilution in block buffer. Cells were then counterstained with Alexa Fluor488 (AF488)
rabbit anti-mouse IgG (Life Technologies, Paisley, UK) at 1:1000 dilution in block buffer and
5 µM Draq5 for nuclear staining (Biostatus Ltd., Leicestershire, UK).
For Rad51 antibody staining, cells were fixed in 100% methanol at 6, 24, 30 and 48 hrs post
treatment with HN2 and at 6, 12, 24, 30, 48 and 72 hrs post treatment with Pt. They were
blocked using 20% rabbit serum in PBS and stained with a mouse monoclonal anti-Rad51
antibody (Clone 14B4, Abcam, 330 Cambridge Science Park, Cambridge, CB4 0FL) diluted 1:200
in block buffer. Cells were then counterstained with AF488 rabbit anti-mouse IgG and 5 µM
Draq5 for nuclear staining.
Imaging Flow Cytometry
Imaging flow cytometry was conducted using the ImagestreamX (Amnis Inc., Seattle,
Washington, USA) which can capture images on up to six optical channels. Following excitation
with a 488 nm laser, images of each individual cell were captured using a 40X objective on
Channel 1 for brightfield (BF), Channel 2 for AF488 which represents the green staining of γH2AX and Rad51 foci, and on Channel 5 for Draq5 staining which represents the nuclear region
of each cell. Images were acquired at a rate of approximately 100 images per second and 10
000 images were captured for each sample at each time point.
Image Compensation
8
Compensation was performed on populations of cells that had been fixed 24 hrs post
treatment with either HN2 or Pt due to the intensity of γ-H2AX and Rad51 likely being the
highest in these samples.
Cells were stained with either AF488 or Draq5 and images were captured using the 488
nm laser as the sole source of illumination. The IDEAS® analysis software compensation wizard
generates a table of coefficients whereby detected light displayed by each image is placed into
the proper channel (Channel 2 for AF488 and Channel 5 for Draq5) on a pixel-by-pixel basis. The
coefficients were normalised to 1 and each coefficient represents the leakage of fluorescent
signal into juxtaposed channels. This compensation matrix was then applied to all subsequent
analyses.
Analysis of Cell Images and -H2Ax or Rad51 Foci Number Calculation
γ-H2AX foci were quantified using the IDEAS® analysis software. Foci were quantified in
a similar manner as previously described in Bourton et al, 2013 (16). In brief, a series of
predefined “building blocks” provided within the software distinguished the population of
single cells that were in the correct focal plane. Two truth populations with a minimum of 40
cells were then identified by the operator; one to represent low numbers of foci (less than 2)
and the other representing cells with high numbers of foci (greater than 5-6 foci). The
populations were selected to encompass the range of staining achieved (i.e. weakly stained
cells to bleached cells) which permitted the software to select the most sensitive mask that
accurately enumerated the foci.
Statistical Analysis
9
Statistical analysis was carried out using the data analysis feature in Microsoft Excel.
Two-way analysis of variance was used to compare distribution of foci in drug-resistant and
parental cell lines post exposure to the chemotherapeutic agents, HN2 and Pt. This was carried
out across the whole time course of the experiment with a P-value of <0.001 being considered
as significant.
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Results
Cellular Sensitivity to HN2 and Pt
Clonogenic assays were carried out on all cell lines after exposure to increasing
concentrations of HN2 (Fig. 1A) and Pt (Fig. 1B) to determine cellular sensitivity to these drugs.
It was observed that the MRC5-SV1R cell line had an IC50 value of 1.40 µg/mL in response
to treatment with HN2. This was a 3-fold increase in resistance when compared to the MRC5SV1 cell line whose IC50 value was 0.50 µg/mL. The NB1-HTERTR cell line showed a 2.7-fold
increase in resistance to HN2 in comparison to the NB1-HTERT cell line with IC50 values of 0.82
and 0.30 µg/mL respectively. Both HN2 resistant cell lines also displayed cross-resistance to Pt.
The MRC5-SV1R cell line had an IC50 value of 23.00 μg/mL in comparison to 12.00 μg/mL seen in
the parental cell line thus exhibiting an approximate 2-fold resistance to Pt. The NB1-HTERT cell
line had an IC50 of 6.00 μg/mL whilst the NB1-HTERTR cell line had an IC50 of 19.00 μg/mL Pt
demonstrating a 3-fold resistance. Therefore resistance to HN2 and cross-resistance to Pt was
induced in the MRC5-SV1R and NB1-HTERTR cell lines. As expected, the GM08437B cell line
showed an increased sensitivity to HN2 in comparison to all other cell lines observed with an
IC50 value of 0.20 μg/mL for HN2. However it was seen to have a similar sensitivity to Pt as the
NB1-HTERT cell line with an IC50 value of 7.00µg/mL.
DNA Repair Assays
To determine if drug resistance was associated with elevated γ-H2AX or Rad51 foci
expression, foci numbers were quantified at different time points over a maximum of 72 hrs
post 1 hr exposure to either HN2 or Pt (Fig. 2 – 5).
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-H2AX Foci Induction in MRC5-SV1, MRC5-SV1R , A2780 and A2780Cis post HN2 treatment
Average γ-H2AX foci induction was determined in the MRC5-SV1 and MRC5-SV1R cell
lines post 1 hr treatment with 0.50 μg/mL HN2 over a 48 hr period (Fig. 2A). Foci images were
analysed and quantified using the ImagestreamX and enumerated by applying a Morphology
and Peak mask as previously described in Bourton et al, 2013 (16).
The MRC5-SV1 parental cell line exhibited fewer γ-H2AX foci compared to MRC5-SV1R
cell line in the majority of the time points sampled. In MRC5-SV1 untreated cells, an average of
2.28 foci per cell was observed while the MRC5-SV1 cell line showed an average of 10.98 foci
per cell. At 24 hrs, there was a clear increase in foci induction in the MRC5-SV1 cell line with an
average of 13.51 foci per cell. By 48 hrs, the level of γ-H2AX foci decreased dramatically to 3.67
foci per cell. However the MRC5-SV1R cell line did not exhibit any dramatic fluctuations in foci
number over the same time period with an average of 12.04 and 13.38 foci seen at 24 and 48
hrs respectively.
Average γ-H2AX foci induction was also determined in the A2780 and A2780Cis cell lines
post 1 hr treatment with 0.50 μg/mL HN2 over a 48 hr period (Fig. 2A). The A2780 parental cell
line also exhibited peak γ-H2AX foci formation at 24 hours, averaging 5.67 foci per cell. This was
a 2.25 fold increase from the untreated controls which showed an average of 2.56 foci per cell.
The A2780Cis cell line had similar levels of foci in the earlier time points to the A2780 cell line
with an average of 3.515 foci per cell but at 24 and 30 hrs, foci formation had nearly tripled in
comparison to the untreated control with an average of 10.39 foci and 9.67 foci seen
respectively.
Rad51 Foci Induction in MRC5-SV1 and MRC5-SV1R post HN2 treatment
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Average Rad51 foci induction was determined in the MRC5-SV1, MRC5-SV1R, A2780 and
A2780Cis cell lines post 1 hr treatment with 0.50 μg/mL HN2 (Fig. 2B). The MRC5-SV1 cell line
showed fewer Rad51 foci than the MRC5-SV1R cell line at all time points examined. There was a
3.7-fold difference seen between the untreated controls of the two cell lines with an average of
11.57 foci seen in the MRC5-SV1R cell line and 3.16 foci seen in the parental cell line. This
difference in RAD51 foci induction was furthermore maintained at all time points tested up to
48hrs with significantly higher foci numbers observed in the MRC5-SV1R cell line (ANOVA p <
0.0001).
In contrast, the A2780 cell line showed increased foci formation at every time point in
comparison to the A2780Cis cell line with both showing a more modest induction of Rad51 foci
in comparison to the γ-H2AX foci induction. Peak foci formation was seen at 30 hrs with an
average of 23.41 foci in the A2780 cell line and 18.26 foci in the A2780Cis cell line.
-H2AX Foci Induction in NB1-HTERT, NB1-HTERTR and GM08437B post HN2 treatment
The NB1-HTERT, NB1-HTERTR and GM08437B cell lines were exposed to 0.30 μg/mL HN2
for 1 hr and γ-H2AX foci induction was observed in these cells lines over a 48 hr period (Fig. 3A).
The NB1-HTERT cell line showed a lower number of γ-H2AX foci across the whole time period in
comparison to the NB1-HTERTR cell line. In the NB1-HTERT cell line, the untreated control
displayed an average of 5.00 foci per cell. A modest induction of γ-H2AX foci was shown at 24
hrs with an average of 7.50 foci per cell which then decreased to 3.70 foci per cell at 48 hrs. In
contrast, the NB1-HTERTR cell line had an average of 6.90 foci in the untreated control which
13
increased to 8.08 foci per cell at 24 hrs. Retention of foci was seen at 48 hrs with an average of
8.10 foci per cell. Surprisingly the GM08347B (XPF deficient) cell line showed the highest
induction of γ-H2AX foci at 24 hrs with an average of 13.60 foci per cell in comparison to an
average of 5.70 foci per cell seen in the untreated control. Foci retention was observed at 48
hrs in this cell line with an average of 8.70 foci per cell.
Rad51 Foci Induction in NB1-HTERT, NB1-HTERTR and GM08437B post HN2 treatment
Average Rad51 foci numbers were calculated in the same cell lines post treatment with
HN2 (Fig. 3B). The untreated control of the NB1-HTERT cell line exhibited 4.18 foci per cell
which then moderately increased to 5.89 foci per cell at 24 hrs. This was then seen to decrease
to 5.37 foci per cell at 48 hrs. In the NB1-HTERTR cell line, foci numbers were slightly lower and
untreated control cells exhibited 1.56 foci per cell. This increased to 2.37 and 3.36 foci per cell
at 24 hrs and 48 hrs respectively. The GM08347B cell line showed higher numbers of Rad51 foci
in untreated control and drug treated cells at all time points when compared to the NB1-HTERT
and NB1-HTERTR cell lines (ANOVA p<0.0001). This observation largely mimics that displayed
with the -H2AX biomarker using the same cell lines.
-H2AX Foci Induction in MRC5-SV1 and MRC5-SV1R post Pt treatment
Average γ-H2AX foci were enumerated in the MRC5-SV1 and MRC5-SV1R cell lines post 1
hr treatment with 12 μg/mL Pt (Fig. 4A). Similar results were yielded for γ-H2AX expression
levels post treatment with Pt compared to those obtained using HN2 treatment in these cell
lines. It was observed that the MRC5-SV1R cell line showed an increased level of γ-H2AX foci at
every time point sampled. For example, in untreated controls there were 4.96 foci per cell in
the MRC5-SV1 cell line but 17.08 foci in the MRC5-SV1R cell line. The MRC5-SV1 cell line
14
exhibited a peak of γ-H2AX foci formation at 24 hrs with an average of 7.78 foci per cell which
was then seen to decline at 72 hrs to 6.06 foci per cell. The MRC5-SV1R cell line showed very
little increase in foci induction with peak foci induction seen at 30 hrs averaging 19.92 foci
which decreased to 18.47 foci per cell at 72 hrs.
Rad51 Foci Induction in MRC5-SV1 and MRC5-SV1R post Pt treatment
Post treatment with 12 μg/mL Pt, an increase in Rad51 expression was seen in the
MRC5-SV1R cell line at all time points in comparison to the MRC5-SV1 cell line (Fig. 4B).
Untreated control samples of the MRC5-SV1 cell line exhibited 3.59 foci per cell whilst the
MRC5-SV1R cell line showed 10.23 foci per cell. The peak of foci formation in the MRC5-SV1R
cell line was seen at 30 hrs with an average of 16.54 foci which decreased to 12.29 foci at 72
hrs. In the MRC5-SV1 cell line, foci formation peaked at 24 hrs with an average of 7.78 foci and
this decreased to 6.06 foci per cell at 72 hrs. Comparison of foci numbers for both -H2AX and
Rad51 revealed a significant difference between the cell lines at all time points (ANOVA: P <
0.0001)
-H2AX Foci Induction in NB1-HTERT, NB1-HTERTR and GM08437B post Pt treatment
Post treatment with 6 μg/mL Pt, the untreated control of the NB1-HTERT cell line
exhibited 3.93 γ-H2AX foci whilst the NB1-HTERTR cell line showed 2.06 foci per cell (Fig. 5A).
The peak of foci formation was seen in the NB1-HTERT cell line at 24 hrs with an average of 6.92
foci. Foci formation peaked in the NB1-HTERTR cell line at 30 hrs with 5.94 foci per cell. Both cell
lines showed γ-H2AX foci levels to have decreased at 72 hrs with an average of 3.39 foci in the
NB1-HTERT cell line and 2.52 foci in the NB1-HTERTR cell line. The GM08437B cell line showed
the highest induction of γ-H2AX foci with an increase from 7.46 foci per cell in the untreated
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control to 15.81 foci per cell seen at 30 hrs. There is retention of foci seen with this cell line at
72 hrs with an average of 12.83 foci per cell.
Rad51 Foci Induction in NB1-HTERT, NB1-HTERTR and GM08437B post Pt treatment
Average Rad51 foci numbers were determined in the same cell lines post treatment
with Pt (Fig. 5B). The untreated control of the NB1-HTERT cell line exhibits an average of 6.14
foci per cell. Rad51 foci in the NB1-HTERTR cell line was slightly lower with an average of 4.97
foci per cell. The GM08437B cell line shows an average of 6.74 foci in the untreated control.
Peak of Rad51 foci formation was seen at 24 hrs in the NB1-HTERT cell line, showing an average
of 8.06 foci per cell and at 30 hrs for NB1-HTERTR cell line, averaging 7.81 foci per cell. Rad51
foci formation also peaked at 30 hrs in the GM08347B cell line with an average of 12.51 foci per
cell. At 48 hrs, Rad51 levels in the NB1-HTERT and NB1-HTERTR decreased to similar levels as
exhibited in their respective untreated controls but foci levels remained high in the GM08437B
cell line. Statistical analysis using ANOVA revealed significant differences in foci induction
between the cell lines at all time points tested (P < 0.0001).
Figure 6 show representative examples of increasing foci number for -H2AX (Fig. 6A)
and Rad51 (Fig. 6B). Here images of cells are shown in BF on Channel one while the foci stained
with AF488 are shown in Channel 2. The third column depicts the spot mask (overlaid in cyan)
created to enumerate foci and finally a Draq 5 stained image of the cell nuclei is shown on
Channel 5.
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Discussion
This study examined at the DNA damage response of different cell lines to the crosslinking chemotherapeutic agents; HN2 and Pt by analysing γ-H2AX and Rad51 foci induction
over a maximum time period of 72 hrs. Three of the cell lines, MRC5-SV1, A2780 and NB1HTERT were repair normal cell lines with no known DNA repair defects. Two cell lines resistant
to the crosslinking agents HN2 and Pt were created in our lab; MRC5-SV1R and NB1-HTERTR. A
third cell line A2780Cis was created in a similar manner independently of our lab.
In response to treatment with HN2 and Pt which are effective at inducing ICL, γ-H2AX
foci formation indicate repair nuclease incisions being made during the resolution of DNA
damage. In broad terms, we demonstrated that in cell lines exhibiting resistance to HN2 and Pt
there was an elevated induction of γ-H2AX foci at most time points sampled. This increased foci
induction is consistent with DNA strand breaks created by the action of DNA repair
endonucleases removing the ICL from the DNA. This reasoning is supported by in vitro studies
from, for example Niedernhofer et al, 2004 (16) and Clingen et al, 2007 (17) who have shown
the accumulation of γ-H2AX foci in the nuclei of cells following exposure to Pt and Mitomycin C
which mediate cytotoxicity via ICL induction in the DNA.
Treatment with both HN2 and Pt revealed increased Rad51 foci levels in one of the
resistant cell lines, MRC5-SV1R in comparison with its parental cell line (MRC5-SV1). Since HR is
known to have a critical role in completing repair of ICL, the increased Rad51 foci levels is
indicative of an upregulation of the HR pathway in this cell line. Interestingly, the other two cell
17
lines NB1-HTERTR and A2780Cis did not consistently show increased Rad51 foci levels post
treatment with HN2 and Pt. This may indicate that these cell lines might have acquired drug
resistance through other mechanisms. For example increased tolerance to the formation of
DNA breaks caused by incision nucleases may account for the low Rad51 foci levels but high γH2AX foci levels observed in the NB1-HTERTR cell line.
The observations obtained with the GM08437B (XPF defective cell line) is prima facie,
inconsistent with the hypothesis that increased γ-H2AX and Rad51 expression is associated with
elevated DNA repair. We demonstrated that this cell line was hypersensitive to the lethal
effects of both drugs in a clonogenic assay. Moreover, we have asserted that high foci levels are
associated with both increased repair incision activity and elevated HR in the drug resistant and
normal cell lines. Other studies too have shown that cells deficient in either ERCC1 or XPF
render them highly sensitive to ICL agents (9, 18-21). The XPF-ERCC1 site specific endonuclease
is an important heterodimer in the repair of ICL where the heterodimer “unhooks” ICL by
making dual cuts on one strand of the cross-linked DNA thus initiating the repair process of ICL
(9, 19). However it has also been demonstrated that ERCC1-deficient cells can induce γ-H2AX
foci formation where ICL-induced DSB formation was independent of the XPF -ERCC1
endonuclease (18, 22). This correlates with findings in this study which showed the GM08437B
cell line, deficient in XPF, also induced γ-H2AX formation at very high levels. We propose that
retention of both γ-H2AX and Rad51 foci seen in the GM08437B cell line is due to stalled DNA
repair due to the defective XPF-ERCC1 excision nuclease.
Recent studies have revealed that 15 genes belonging to the FA pathway play a vital role
in the maintenance of genomic stability (23) and have a central role in ICL repair. They are
18
involved in the coordination of multiple repair processes; particularly nucleases that are vital
for incising the ICL. Bhagwat et al, 2009 (22) demonstrated that the MUS81-EME1 heterodimer
endonuclease, recruited by various FA proteins, makes the initial incision at the site of the
cross-link; thereby creating a DSB. XPF -ERCC1 then makes the second incision, resulting in the
“un-hooking” of the cross-link. This may provide an explanation for the induction of γ-H2AX foci
in the XPF-deficient cell line where the defective XPF-ERCC1 endonuclease cannot complete the
repair thus leaving residual DSB and high -H2AX signal.
To summarise, we have developed drug resistance to HN2 and Pt in two human
immortalised cell lines and shown that, in general, alterations in the dynamics and level of DNA
repair of ICL may partly account for the elevated resistance to these drugs. We employed two
DNA repair biomarkers, -H2AX to monitor repair incision nuclease activity and Rad51 to
monitor HR activation. We conclude that these biomarkers may prove useful in providing a
mechanistic understanding of induced drug resistance in those cases where alterations in DNA
repair dynamics underlies the response. The application of such biomarkers may be particularly
appropriate in cancers such as ovarian where treatment failure due to drug resistance is a
major issue in the clinical management of these cancers (12). However, we concede that to
exclusively focus on DNA repair as mechanism of drug resistance in cancer would be naïve given
that drug resistance can occur by a number of mutually exclusive cellular mechanisms.
Notwithstanding, a comprehensive analysis of DNA repair kinetcs in drug resistance human cell
lines and tumours using -H2AX and Rad51 (and other) DNA repair biomarkers may prove useful
in identifying cancers where inhibition of specific DNA repair pathways may be of significant
clinical benefit.
19
Acknowledgements
S. Adam-Zahir & E. C. Bourton are supported by two grants from the Barts Charity, East London,
UK
20
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Pharmacol 2010;65(5);989-994.
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22
Tables
Table 1: Catalogue of Cell Lines Used in Study
Cell line
DNA repair
status
Details of cell lines
MRC5-SV1 (14)
DNA repair
normal
Derived from fibroblast foetal lung cells and
immortalised by SV 40 Large T antigen.
NB1-HTERT (15)
DNA repair
normal
Derived from normal individual and
immortalised by the HTERT gene.
MRC5-SV1R
Resistant to
HN2, Pt
NB1-HTERTR
Resistant to
HN2, Pt
GM08437B (Coriell
Institute, New Jersey,
USA)
Defective
nucleotide
excision repair
A2780 (European
Collection of Cell
Cultures, Porton Down)
DNA repair
normal
Developed through continual exposure of
parental cell line MRC5-SV1 to gradually
incremented doses of HN2.
Developed through continual exposure of
parental cell line NB1-HTERT to gradually
incremented doses of HN2.
Derived from xeroderma pigmentosum
complementation group F patient (XPF) which
forms part of the endonuclease complex with
ERCC1. Exhibits sensitivity to cross-linking
agents such as Pt and HN2.
Derived from an untreated cancer patient
A2780Cis (European
Collection of Cell
Cultures, Porton Down)
Resistant to Pt,
Mephalan,
Adriamycin
Developed through continual exposure of
parental cell A2780 to increasing doses of Pt.
The above table provides details of the origins of all cell lines used in this study.
23
Figure Legends
Figure 1
The above shows clonogenic survival rates of all cell lines post treatment with HN2 (A) and Pt (B). It can
be seen that the two resistant cell lines (indicated by the dotted lines) exhibited increased resistance to
all doses of both drugs. The GM08437B (XPF deficient) cell line shows an increased sensitivity to the two
drugs with a steep decline in survival seen as the dosage was increased. All data is representative of 3
independent experiments and error bars were calculated by dividing the standard deviation of the mean
by the square root of the number of repeats.
Figure 2
Fig. 2 represents average γ-H2AX (A) and Rad51 (B) foci numbers in the MRC5-SV1, MRC5-SV1R, A2780
and A2708CIS cell lines over a 48 hr period post exposure to 0.5 μg/mL HN2. In Fig. 2A, the MRC5-SV1
parental cell line indicates a peak of γ-H2AX foci formation at 24 hrs which has largely disappeared by
48 hrs. The MRC5-SV1R cell line indicates increased γ-H2AX foci formation in comparison to the MRC5SV1 cell line across the time period examined. The A2780 cell line exhibits a similar peak at 24 hours.
The A2780Cis cell line shows a similar repair profile to the parental cell line with peak foci formation also
seen at 24 hours. At 24 and 30 hours, foci formation in the A2780Cis cell line is doubled or more in
comparison to the A2780 cell line. Fig. 2B shows nuclear RAD51 foci counts for the MRC5-SV1 and
MRC5-SV1R cell lines. There is a 3.7 fold difference between the MRC5-SV1 and MRC5-SV1R cell lines in
the untreated controls. Although the foci counts are seen to increase in the MRC5-SV1 cell line, there is
little difference seen between the time points. In contrast, the RAD51 foci counts in the MRC5-SV1R cell
line are seen to continually increase over the 48 hr time period. With the A2780 and A2780 cell lines,
increased foci formation is seen in the A2780 cell line in comparison to the A2780Cis cell line at every
time point examined. Error bars represent the standard error of the mean derived from approximately
10000 cells analysed at each time point for each cell line.
Figure 3
Fig. 3 represents average γ-H2AX (A) and Rad51 (B) foci numbers in the NB1-HTERT, NB1-HTERTR and
GM08437B cell line over a 48 hr period post exposure to 0.3 μg/mL HN2. In Fig. 3A, a vast induction of γH2AX foci at 24 hrs was seen in the GM08437B cell line. Foci numbers for the NB1-HTERTR remained
relatively similar over the 48 hr period while the NB1-HTERT cell line showed a clear pattern of induction
and disappearance of γ-H2AX foci over the same time period. At 48 hrs, there is still a clear retention of
24
foci seen in the GM08437B cell line. In Fig. 3B, it can be seen that the NB1-HTERTR cell line showed little
change in RAD51 foci counts over the 48hr time period. The NB1-HTERT cell line showed an increased
expression of RAD51 foci in comparison with the NB1-HTERTR cell line at all time points examined. The
GM08437B cell line showed the highest expression of RAD51 foci of all 3 cell lines examined and this
was seen to continually increase over the 48 hr time period. Error bars represent the standard error of
the mean derived from approximately 10000 cells analysed at each time point for each cell line.
Figure 4
Fig. 4 represents average γ-H2AX (A) and Rad51 (B) foci numbers in the MRC5-SV1 parental and resistant
cell lines over a 72 hr period post 1hr treatment with 12 µg/mL Pt. Fig. 4A shows increased levels of γH2AX foci in the MRC5-SV1R cell line at all time points in comparison to the parental line with no clear
peak of foci formation seen. The MRC5-SV1 parental cell line shows a peak of foci formation at 24 hrs
which was seen to largely be resolved at 48 hrs. Fig. 4B shows the peak of Rad51 foci formation occurred
earlier in the resistant cell line with peak formation seen at 30hrs whilst this was seen at 48 hrs in the
parental cell line. Error bars represent the standard error of the mean derived from approximately
10000 cells analysed at each time point for each cell line.
Figure 5
Fig. 5 represents average γ-H2AX (A) and Rad51 (B) foci numbers in the NB1-HTERT, NB1-HTERTR and
GM08437B cell lines over a 72 hr period post 1hr treatment with 6 µg/mL Pt. Fig. 5A indicates increased
γ-H2AX foci in both the NB1-HTERTR and GM08437B cell lines in comparison to the NB1-HTERT parental
cell line. Peak of foci formation was seen at 24 hours in the parental cell line and at 30 hours in the other
two cell lines. Foci retention was observed in the GM08437B cell line at 72 hrs. Fig. 5B indicates the
peak of Rad51 foci formation was observed at 6hrs in the NB1-HTERTR cell line whilst the peak of foci
formation was seen in the NB1-HTERT parental cell line at 24 hrs. Peak of foci formation was seen at 30
hrs in the GM08437B cell line with foci retention observed at 72 hrs. Error bars represent the standard
error of the mean derived from approximately 10000 cells analysed at each time point for each cell line.
Figure 6
Fig. 6 shows example images of cells with increasing numbers of γ-H2AX (A) and Rad51 (B) foci. Ch01
shown in the first column depicts the BF image of these cells with the morphology mask shown in blue
which highlights the nuclei of the cells. Ch02 shown in the second column (representing AF488 staining)
25
depicts the unmasked cells whilst the third column depicts the same images in Ch02 masked with either
the γ-H2AX or Rad51 foci spot mask. Ch05 shows the morphology of the nucleus of these cells
represented by Draq5 staining.
26
Figure 1
Comparison of cytotoxicity post 1hr treatment
MRC5-SV1
with HN2
A
Comparison of cytotoxicity post 1hr treatment with
MRC5-SV1
Pt
B
MRC5-SV1 R
100
NB1-HTERT
MRC5-SV1 R
100
NB1-HTERT
NB1-HTERT R
NB1-HTERTR
GM08437B
GM08437B
10
Survival (%)
Survival (%)
10
1
1
0
0
0
0.5
1
1.5
Dose (µg/mL)
2
0
2.5
27
5
10
15
20
Dose (µg/mL)
25
30
35
Figure 2
Average γ-H2AX Foci counts in cells post 1 hour HN2 treatment
14.00
MRC5-SV1
MRC5-SV1 R
A2780
A2780 CIS
Foci Number
12.00
10.00
8.00
6.00
4.00
2.00
0
3
5
24
30
48
Time (hours)
Average Rad51 Foci counts in cells post 1 hour HN2 treatment
Foci Number
22.00
MRC5-SV1
MRC5-SV1R
A2780
A2780Cis
17.00
12.00
7.00
2.00
0
6
24
Time (hours)
28
30
48
Figure 3
A
Average γ-H2AX Foci numbers post 1hr HN2 treatment
14
NB1-HTERT
NB1-HTERT R
GM08437B
Foci Number
12
10
8
6
4
0
B
3
5
Time (hrs)
24
30
Average Rad51 Foci numbers post 1hr HN2 treatment
16
48
NB1-HTERT
NB1-HTERTR
GM08437B
14
Foci Number
12
10
8
6
4
2
0
0
6
24
Time (hrs)
29
30
48
Figure 4
Average γ-H2AX foci numbers post 1 hr treatment with Pt
A
MRC5-SV1
MRC5-SV1 R
Average Foci Number
20
15
10
5
0
0
6
24
30
48
72
Time (hrs)
B
MRC5-SV1
Average Rad51 foci numbers post 1 hr treatment with Pt
18
MRC5-SV1 R
Average Foci Number
16
14
12
10
8
6
4
2
0
0
6
12
24
Time (hrs)
30
30
48
72
Figure 5
Average γ-H2AX foci numbers post 1 hr treatment with Pt
NB1-HTERT
NB1-HTERTR
14
GM08437B
13
12
Foci Number
11
10
9
8
7
6
5
4
0
B
6
24
Time (hrs)
30
48
72
NB1-HTERT
Average Rad51 foci numbers post 1 hr treatment with Pt
18
NB1-HTERTR
GM08437B
16
Foci Number
14
12
10
8
6
4
2
0
0
6
12
24
Time (hrs)
31
30
48
72
Figure 6
A
5
Increasing foci numbers
10
15
20
B
5
10
15
20
32