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.

KeywordsDNA repair, chemotherapy, biomarker, Rad51, -H2AX

Corresponding AuthorChristopher 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 266293Fax: +44(0)1895 269854Author Email Addresschristopher.parris@brunel.ac.uk

Word Count: 4879 (excluding references)No of tables: 1No 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.

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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 co-

workers (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). γ-

H2AX 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 co-

ordinated 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 Fanconi-

associated 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 gμ /mL for NB1-HTERTR and 5.30 gμ /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

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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 γ-H2AX

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

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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

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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 MRC5-

SV1 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-SV1 R , 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-SV1 R 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-HTERT R 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

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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-HTERT R 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-SV1 R 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

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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-SV1 R 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-HTERT R 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-HTERT R 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 cross-

linking 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 NB1-

HTERT 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 γ-

H2AX 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

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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

References

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2. Bourton EC, Plowman PN, Adam Zahir S, Senguloglu GU, Serrai H, Bottley G et al. Multispectral imaging flow cytometry reveals distinct frequencies of gamma-H2AX foci induction in DNA double strand break repair defective human cell lines. Cytometry A 2012;81(2):130-137.

3. Bourton E C, Plowman PN , Smith D, Arlett CF, Parris CN. Prolonged expression of the gamma-H2AX DNA repair biomarker correlates with excess acute and chronic toxicity from radiotherapy treatment. Int J Cancer 2011;129(12):2928-2934.

4. Bellamy WT. P-Glycoprotein and Multidrug Resistance. Annu Rev Pharmacol Toxicol. 1996; 36:161-183.

5. Hartley JA. Alkylating agents. in Souhami RL, Tannock I, Hohenberger P, Horoit JC (Eds.) Oxford Textbook of Oncology, 2nd ed, Oxford University Press 2001;pp.639-654.

6. Palom Y, Suresh KG, Tang LQ, Paz MM, Musser SM, Rockwell S et al. Relative toxicities of DNA cross-links and monoadducts: new insights from studies of decarbamoyl mitomycin C and mitomycin C. Chem Res Toxicol. 2002 Nov;15(11);1398-406.

7. Deans AJ, West SC. DNA interstrand crosslink repair and cancer. Nat Rev Cancer 2011;11(7);467-480.

8. Huang X, Okafuji M, Traganos F, Luther E, Holden E, Darzynkiewicz Z. Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking agent cisplatin. Cytometry A 2004;58(2);99-110.

9. Clingen PH, Wu JY, Miller J, Mistry N, Chin F, Wynne P et al. Histone H2AX phosphorylation as a molecular pharmacological marker for DNA interstrand crosslink cancer chemotherapy. Biochem Pharmacol 2008;76(1);19-27.

10. Boehm T, Folkman J, Browder T, O'Reilly MS Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 1997; 390(6658);404-407.

11. Carvalho H, Garrido LM, Furlan RL, Padilla G, Agnoletto M, Guecheva T et al. DNA damage induced by the anthracycline cosmomycin D in DNA repair-deficient cells. Cancer Chemother Pharmacol 2010;65(5);989-994.

12. Agarwal R and Kaye SB Ovarian cancer: strategies for overcoming resistance to chemotherapy. Nat Rev Cancer 2003;3(7);502-516.

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13. Le Scodan R, Cizeron-Clairac G, Fourme E, Meseure D, Vacher S, Spyratos F et al. DNA repair gene expression and risk of locoregional relapse in breast cancer patients. Int J Radiat Oncol Biol Phys. 2010;78(2);328-36.

14. Arlett CF, Plowman PN, Rogers PB, Parris CN, Abbaszadeh F, Green MH et al. Clinical and cellular ionizing radiation sensitivity in a patient with xeroderma pigmentosum. Br J Radiol 2006; 79(942);510-517.

15. Ulus-Senguloglu G, Arlett CF, Plowman PN, Parnell J, Patel N, Bourton EC et al. Elevated expression of artemis in human fibroblast cells is associated with cellular radiosensitivity and increased apoptosis. Br J Cancer 2012;107(9);1506-1513.

16. Bourton E, Plowman P, Harvey A, Adam Zahir S, Parris CN. The PARP-1 Inhibitor Olaparib Causes Retention of γ-H2AX Foci in BRCA1 Heterozygote Cells Following Exposure to Gamma Radiation JCT 2013;4;44-52

17. Clingen PH, Arlett CF, Hartley JA, Parris CN. Chemosensitivity of primary human fibroblasts with defective unhooking of DNA interstrand cross-links. Exp Cell Res 2007; 313(4); 753-760.

18. Niedernhofer LJ, Odijk H, Budzowska M, Van Drunen E, Maas A, Theil AF et al. The structure-specific endonuclease Ercc1-Xpf is required to resolve DNA interstrand cross-link-induced double-strand breaks. Mol Cell Biol 2004;24(13);5776-5787.

19. Kuraoka I, Kobertz WR, Ariza RR, Biggerstaff M, Essigmann JM, Wood RD. Repair of an interstrand DNA cross-link initiated by ERCC1-XPF repair/recombination nuclease. J Biol Chem 2000;275(34);26632-26636.

20. De Silva IU, McHugh PJ, Clingen PH, Hartley JA Defects in interstrand cross-link uncoupling do not account for the extreme sensitivity of ERCC1 and XPF cells to cisplatin. Nucleic Acids Res 2002;30(17);3848-3856.

21. Al-Minawi AZ, Lee YF, Hakansson D, Johansson F, Lundin C, Saleh-Gohari N et al. The ERCC1/XPF endonuclease is required for completion of homologous recombination at DNA replication forks stalled by inter-strand cross-links. Nucleic Acids Res 2009;37(19);6400-6413.

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23. Crossan, GP and Patel KJ The Fanconi anaemia pathway orchestrates incisions at sites of crosslinked DNA. J Pathol 2012;226(2);326-337.

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

Developed through continual exposure of parental cell line MRC5-SV1 to gradually incremented doses of HN2.

NB1-HTERTR Resistant to HN2, Pt

Developed through continual exposure of parental cell line NB1-HTERT to gradually incremented doses of HN2.

GM08437B (Coriell Institute, New Jersey, USA)

Defective nucleotide excision repair

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.

A2780 (European Collection of Cell Cultures, Porton Down)

DNA repair normal

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 MRC5-

SV1 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-SV1 R 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 γ-

H2AX foci at 24 hrs was seen in the GM08437B cell line. Foci numbers for the NB1-HTERT R 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 γ-

H2AX 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

0

1

10

100

0 0.5 1 1.5 2 2.5

Surv

ival

(%)

Dose (µg/mL)

Comparison of cytotoxicity post 1hr treatment with HN2 MRC5-SV1

MRC5-SV1 R

NB1-HTERT

NB1-HTERT R

GM08437B

0

1

10

100

0 5 10 15 20 25 30 35

Surv

ival

(%)

Dose (µg/mL)

Comparison of cytotoxicity post 1hr treatment with Pt MRC5-SV1

MRC5-SV1 R

NB1-HTERT

NB1-HTERTR

GM08437B

A B

27

Figure 2

2.00

4.00

6.00

8.00

10.00

12.00

14.00

0 3 5 24 30 48

Foci

Num

ber

Time (hours)

Average γ-H2AX Foci counts in cells post 1 hour HN2 treatment MRC5-SV1MRC5-SV1 RA2780A2780 CIS

2.00

7.00

12.00

17.00

22.00

0 6 24 30 48

Foci

Num

ber

Time (hours)

Average Rad51 Foci counts in cells post 1 hour HN2 treatment MRC5-SV1MRC5-SV1RA2780A2780Cis

28

Figure 3

0

2

4

6

8

10

12

14

16

0 6 24 30 48

Foci

Num

ber

Time (hrs)

Average Rad51 Foci numbers post 1hr HN2 treatment NB1-HTERTNB1-HTERTRGM08437B

B

4

6

8

10

12

14

0 3 5 24 30 48

Foci

Num

ber

Time (hrs)

Average γ-H2AX Foci numbers post 1hr HN2 treatmentNB1-HTERTNB1-HTERT RGM08437B

A

29

Figure 4

0

2

4

6

8

10

12

14

16

18

0 6 12 24 30 48 72

Aver

age

Foci

Num

ber

Time (hrs)

Average Rad51 foci numbers post 1 hr treatment with Pt MRC5-SV1

MRC5-SV1 R

0

5

10

15

20

0 6 24 30 48 72

Aver

age

Foci

Num

ber

Time (hrs)

Average γ-H2AX foci numbers post 1 hr treatment with Pt MRC5-SV1MRC5-SV1 RA

B

30

Figure 5

0

2

4

6

8

10

12

14

16

18

0 6 12 24 30 48 72

Foci

Num

ber

Time (hrs)

Average Rad51 foci numbers post 1 hr treatment with Pt NB1-HTERTNB1-HTERTRGM08437B

B

456789

1011121314

0 6 24 30 48 72

Foci

Num

ber

Time (hrs)

Average γ-H2AX foci numbers post 1 hr treatment with Pt NB1-HTERTNB1-HTERTRGM08437B

31

Figure 6

Ch01 Ch02 Ch02 Ch054665

Ch01 Ch02 Ch02 Ch051031

Ch01 Ch02 Ch02 Ch05462

Ch01 Ch02 Ch02 Ch051410

5

20

15

10

Increasing foci numbers

5

10

15

20

A

B

Ch01 Ch02 Ch02 Ch053941

Ch01 Ch02 Ch02 Ch05643

Ch01 Ch02 Ch02 Ch051724

Ch01 Ch02 Ch02 Ch053919

32