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In Vitro Evaluation of Combined Temozolomide and Radiotherapy Using X Raysand High-Linear Energy Transfer Radiation for GlioblastomaAuthor(s): Lara Barazzuol, Raj Jena, Neil G. Burnet, Jonathan C. G. Jeynes, Michael J. Merchant, KarenJ. Kirkby, and Norman F. KirkbySource: Radiation Research, 177(5):651-662. 2012.Published By: Radiation Research SocietyDOI: http://dx.doi.org/10.1667/RR2803.1URL: http://www.bioone.org/doi/full/10.1667/RR2803.1

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RADIATION RESEARCH 177, 651–662 (2012)0033-7587/12 $15.00�2012 by Radiation Research Society.All rights of reproduction in any form reserved.DOI: 10.1667/RR2803.1

In Vitro Evaluation of Combined Temozolomide and Radiotherapy UsingX Rays and High-Linear Energy Transfer Radiation for Glioblastoma

Lara Barazzuol,a,1 Raj Jena,b,c Neil G. Burnet,b,c Jonathan C. G. Jeynes,a Michael J. Merchanta, Karen J. Kirkbya andNorman F. Kirkbyd

a Ion Beam Centre and d Chemical Engineering, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, Surrey GU2 7XH,United Kingdom; b Oncology Centre, Addenbrooke’s Hospital, Cambridge CB2 0QQ, United Kingdom; and c University of Cambridge,

Department of Oncology, Oncology Centre, Addenbrooke’s Hospital, Cambridge CB2 0QQ, United Kingdom

Barazzuol, L., Jena, R., Burnet, N. G., Jeynes, J. C. G.,Merchant, M. J., Kirkby, K. J. and Kirkby, N. F. In VitroEvaluation of Combined Temozolomide and RadiotherapyUsing X Rays and High-Linear Energy Transfer Radiation forGlioblastoma. Radiat. Res. 177, 651–662 (2012).

High-linear energy transfer radiation offers superiorbiophysical properties over conventional radiotherapy andmay have a great potential for treating radioresistanttumors, such as glioblastoma. However, very little pre-clinical data exists on the effects of high-LET radiation onglioblastoma cell lines and on the concomitant application ofchemotherapy. This study investigates the in vitro effects oftemozolomide in combination with low-energy protons and aparticles. Cell survival, DNA damage and repair, and cellgrowth were examined in four human glioblastoma cell lines(LN18, T98G, U87 and U373) after treatment with either Xrays, protons (LET 12.91 keV/lm), or a particles (LET 99.26keV/lm) with or without concurrent temozolomide atclinically-relevant doses of 25 and 50 lM. The relativebiological effectiveness at 10% survival (RBE10) increased asLET increased: 1.17 and 1.06 for protons, and 1.84 and 1.68for a particles in the LN18 and U87 cell lines, respectively.Temozolomide administration increased cell killing in the O6-methylguanine DNA methyltransferase-methylated U87 andU373 cell lines. In contrast, temozolomide provided notherapeutic enhancement in the methylguanine DNA meth-yltransferase-unmethylated LN18 and T98G cell lines. Inaddition, the residual number of c-H2AX foci at 24 h aftertreatment with radiation and concomitant temozolomide wasfound to be lower than or equal to that expected by DNAdamage with either of the individual treatments. Kinetics offoci disappearance after X-ray and proton irradiationfollowed similar time courses; whereas, loss of c-H2AX fociafter a particle irradiation occurred at a slower rate thanthat by low-LET radiation (half-life 12.51–16.87 h). Thecombination of temozolomide with different radiation typescauses additive rather than synergistic cytotoxicity. Never-theless, particle therapy combined with chemotherapy mayoffer a promising alternative with the additional benefit ofsuperior biophysical properties. It is also possible that new

fractionation schedules could be designed to exploit thechange in DNA repair kinetics when MGMT-methylated cellsrespond to high-LET radiation. � 2012 by Radiation Research Society

INTRODUCTION

Glioblastoma (GBM) is the most common and aggressiveprimary brain tumor in adults characterized by its rapidgrowth and infiltration into the surrounding tissues. Atpresent, treatment of GBM is based on a multimodalityapproach including surgery for resection or stereotacticbiopsy, followed by radiotherapy plus concomitant andadjuvant chemotherapy.

Historically, the use of chemotherapy as a standard post-operative treatment has been controversial. Early studies, inthe 1970–1990s, using nitrosourea-based chemotherapy,reported marginal benefit (1). In 2005, an internationalmulticenter randomized phase III trial conducted by theEuropean Organisation for Research and Treatment ofCancer (EORTC) and the National Cancer Institute ofCanada (NCIC) Clinical Trial Group (trial 26981-22981/CE.3) demonstrated the benefit of adding concurrent andadjuvant temozolomide (TMZ), an oral alkylating agent, toradiotherapy (2). Median survival increased from 12.1months with radiotherapy alone, to 14.6 months withradiotherapy plus TMZ.

The therapeutic benefit of TMZ is related to induction ofmethyl adducts to the O6-position of guanine in DNA.Formation of O6-methylguanine (O6-meG) generates incor-rect base pairing ultimately leading to cytotoxicity (3).

Although the use of TMZ has improved outcomessignificantly and is now established as the standard of carein GBM, not all of the patients benefit from the addition ofTMZ. Hegi et al. (4) demonstrated that silencing of the O6-methylguanine DNA methyltransferase (MGMT), mediatedby gene promoter methylation, was an independentprognostic and predictive factor of benefit from TMZ inthe EORTC-NCIC trial. The MGMT promoter wasmethylated in 44.7% of 206 evaluated GBM specimensfrom 576 patients enrolled in that study. The frequency of

1 Address for correspondence: Ion Beam Centre, University ofSurrey, Guildford, Surrey GU2 7XH, UK; e-mail address:l.barazzuol@surrey.ac.uk.

651

MGMT promoter methylation varies widely across clinicalstudies ranging from 35% to 73% (5).

Epigenetic changes, including DNA methylation, havealso been reported after exposure to ionizing radiation. Todate, studies have focused on low-linear energy transfer(LET) radiation effects on DNA methylation reportingglobal hypomethylation (6). However, this effect is notspecific to the formation of O6-meG. Little is known aboutthe occurrence of epigenetic alterations with high-LETradiation. In one study, Goetz et al. (7) reported a generaltrend for hypermethylation in cells exposed to protons andiron ions. While there is evidence for radiation-inducedepigenetic changes, LET-dependence remains to be clari-fied. Interestingly, no significant change in the MGMTpromoter methylation level after both low- and high-LETradiation has been reported (7, 8).

Survival advantage of TMZ was maintained for up to 5years of follow-up, however, most patients eventuallydeveloped tumor recurrence within a few centimeters ofthe treated site, and died (9). Clearly, there is a strongclinical need for novel treatment approaches to improveoutcomes in both unmethylated and methylated MGMTtumor groups.

Standard radiotherapy for GBM delivers a dose of 60 Gyin 30 fractions (10). Theoretically, very high doses ofradiation (up to 90 Gy) may eradicate the tumor, but anincrease in dose is generally associated with an increasedrisk of radiation necrosis (11).

Particle therapy is a radiotherapeutic modality character-ized by a better physical dose distribution compared toconventional photons. A photon beam dose profile exhibitsmaximal energy deposition in the first few centimeters oftissue followed by an exponential drop with depth. Unlikephotons, charged particles, such as protons or heavier ions,deposit low levels of energy as they pass through the body,followed by high-energy deposition in the Bragg peak, andvirtually no dose beyond. These physical characteristics canreduce dose to healthy tissues surrounding the target andspare neighboring organs at risk (12).

High-LET radiation has also been of particular interest inthe treatment of GBM because of their increased potential ofkilling hypoxic tumor cells compared to X rays. Previousstudies have shown an oxygen enhancement ratio (OER) ofapproximately 1 for high-LET radiation, as compared to Xrays, where the OER is 2.5–3 (12, 13).

Neutrons were the first high-LET radiation clinicallyinvestigated on high-grade gliomas. However, in the early1940s, a number of studies reported disappointing resultsattributed to the lack of the Bragg Peak in the dosedeposition, together with high absorption in neural tissueswith elevated hydrogen content. These factors led toincreased toxicity to normal brain tissues due to thespreading of the high-LET component along the wholeparticle range (14, 15).

Later clinical studies at the University of CaliforniaLawrence Berkeley National Laboratory (LBNL) investi-

gated the use of other charged particles characterized by abetter dose distribution. Thirty-nine patients with gliomas,of which 17 patients had primary GBM, received either aparticles alone, or photon irradiation with either a particles,or carbon, or neon ions as a boost, or neon ions alone. Themedian survival for GBM was 13.9 months and radio-necrosis of the brain was minimal (16).

Fitzek et al. (17) reported a very low local tumorrecurrence rate (in only one case out of 23 patients) and amedian survival of 20 months in patients with GBM aftertreatment with a dose of 90 Gray equivalent (GyE) usingaccelerated fractionated proton therapy. However, despitethe lengthened median survival, this dose led to a very highrate of radiation necrosis.

More recently, Mizoe et al. (18) treated 48 patients withmalignant gliomas, of which 32 were GBM, with carbon ionboosts (8 fractions/2 weeks). The total dose was increasedfrom 16.8 to 24.8 GyE. No grade 3 or higher toxicity wasobserved; the median survival time of GBM patients was 17months and increased to 26 months for the high-dosetreatment group. However, this study was relatively smalland standard TMZ chemotherapy was not administered.

Although some clinical studies have been published, norandomized controlled trials have been carried out toprovide high-level evidence to support the use of particletherapy for GBM (19).

Little pre-clinical data exists on high-LET radiation onGBM cell lines including the concomitant application ofchemotherapy. Benzina et al. (20) examined the combina-tion of oxaliplatin, a third-generation platinum anticanceranalogue, with p(65) þ Be neutrons, and showed enhancedcytotoxicity. Combs et al. (21) investigated the cytotoxiceffect of TMZ in addition to carbon ions on two GBM celllines, reporting independent additive effects. Neither ofthese studies was able to stratify treatment responseaccording to the MGMT methylation status. Moreover,there are conflicting preclinical data in the literature onwhether TMZ acts synergistically with radiation or isindependently killing tumor cells (22, 23). However, this isa key concept behind efforts to optimize the combination oftreatment modalities for this aggressive tumor type.

In this study, we first assessed the response of TMZ andconventional photon radiotherapy in two MGMT-methylat-ed cell lines and two MGMT-unmethylated cell lines. Thesecond part of the study investigated the combination ofTMZ with low-energy 3 MeV protons and 6 MeV aparticles, corresponding to clinically-significant LET valuesof 12.91 and 99.26 keV/lm, respectively, in both anMGMT-methylated cell line and an MGMT-unmethylatedcell line. While particles with these low energies (, 10MeV) are not clinically applicable, they are useful for invitro studies because they allow high-LET ionization tooccur within the intra-cellular target. Clinical accelerators,either cyclotrons or synchrotrons, produce particle beams ofenergy up to 250 MeV for protons and 430 MeV per

652 BARAZZUOL ET AL.

nucleon for carbon ions for the treatment of deep-seatedtumors (24).

METHODS AND MATERIALS

Cell Cultures and Reagents

Four human glioma cell lines, U373, U87, T98G, and LN18, wereused in this study. U373 and T98G cells were provided as a gift byMick Woodcock, Gray Institute for Radiation Oncology and Biology,Oxford, UK; U87 and LN18 cells were obtained from the HealthProtection Agency Culture Collections (HPACC, Wiltshire, UK) andthe American Type Culture Collection (ATCC, Middlesex, UK),respectively. All cell lines were confirmed Mycoplasma-free beforeuse. The cells were cultured in Eagle’s Minimum Essential Medium(EMEM) containing 10% fetal bovine serum, 1% penicillin/strepto-mycin, 4 mM L-glutamine, 1 mM sodium pyruvate, 1500 mg/L sodiumbicarbonate, and 1% MEM eagle nonessential amino acids (Lonza,Berkshire, UK). Cells were maintained at 378C with 5% CO2 and 80%humidity, and passaged weekly by exposing them in 0.25% trypsin/versene and then resuspended in growth medium. As previouslydetermined by Hermisson et al. (25), T98G and LN18 express highlevels of MGMT activity, whereas U373 and U87 show very lowlevels.

Irradiation with Conventional X Rays

X-ray irradiation was performed using a Pantak machine (RoyalSurrey County Hospital, Guildford, UK) operating at 300 kVp with adose rate of 1 Gy/min. Cells were grown in 6-well plates andincubated for 5 h before irradiation. Cells were then exposed at roomtemperature to doses between 1–6 Gy.

Proton- and Alpha-Particle Irradiation

The Wolfson vertical beam line at the University of Surrey, IonBeam Centre (26), with a 2 MV Tandem accelerator was used toproduce protons and a particles at energies of 3 and 6 MeV,respectively, with a volume-averaged LET of 12.91 and 99.26 keV/lm (assuming that the cell nucleus can be approximated to a sphere,10 lm in diameter). Table 1 summarizes the spread of energies andLETs within the cell nucleus. We applied doses similar to the X rayexperiments between 0.4 and 6.57 Gy using a particle fluence of 1.273 106 particles/cm2. Cells were plated on custom-designed petri dishesand irradiated through a polypropylene foil (4 lm thick). At first, cellswere diluted to a final concentration of 1 3 106 cells/ml and pipetted ina droplet onto the polypropylene dish. The dish can contain a numberof droplets, including the control, each of which receives a differentradiation dose. Our system possesses a computer-controlled XY stage(Marhauser, Wetzlar, Germany) that is able to precisely move thestage to each required location of the dish relative to the fixed nozzle

position. Immediately after irradiation, cells were replated at lowerconcentrations and 3 wells per dose were used. The particle fluencemeasurement was based on single-particle counting using a PiN diodemounted into the camera objective located over the beam exit windowin the same plane as the sample to be irradiated. The final fluence to bedelivered was measured and pre-set before each experiment. When thefixed number of particles was delivered, electrostatic deflectorsstopped the beam with a response time of 10 ns, and then the stagewas moved to the next irradiation position. The delivered dose wascalculated according to the equation:

DðGyÞ ¼ 1:6 3 10�9 F L 1=q

where D is the dose in gray, F is the particle fluence in particles/cm2; Lis the LET in keV/lm calculated at 4 lm into the cell nucleus by thestopping power and range of ions in matter (SRIM) program (27); andq is the cell density in g/cm3 that is assumed to be equal to 1 g/cm3 asthe reference density of liquid water. In addition, discs of radio-chromic film (GafChromic, Harpell Associates Inc., Ontario, Canada)and CR39 plastic (TASL Ltd, Bristol, UK) were irradiated in thesample wheel as an independent verification of dosimetry. Details ofthe irradiation procedure and dosimetry have been describedelsewhere (submitted for publication).

Temozolomide Treatment

Temozolomide was provided by Fluka (Sigma-Aldrich, Dorset,UK) and reconstituted in dimethyl-sulfoxide (DMSO) to a finalconcentration not exceeding 0.1% (at this concentration, DMSO alonehad no effect on cell viability). Temozolomide was administered indoses of 25 and 50 lM in accordance to the TMZ populationpharmacokinetic values in plasma and cerebrospinal fluid (CSF)reported by Ostermann et al. (28). Explicitly, the TMZ concentrationof 25 lM corresponded to the in vivo plasma concentration of 75 mg/m2 (concomitant phase) and 50 lM of 150 mg/m2 (adjuvant phase).For the X-ray experiments, after allowing time to attach (5 h), cellsreceived TMZ in doses of 25 and 50 lM for a total exposure time of 2h, including 1 h before irradiation. After 2 h with TMZ, the mediumwas removed and fresh medium was added. In the case of protons anda particles, cells were incubated with TMZ 2 h before irradiation, andthen trypsinized, counted, and transferred into custom-designeddishes.

Clonogenic Assay

After irradiation with X rays, protons, and a particles with orwithout TMZ at 25 and 50 lM, the cells were incubated for up to 14days. Colonies were fixed with 50% ethanol in PBS and then stainedwith 5% crystal violet in PBS (Sigma-Aldrich, Dorset, UK). Colonieswith more than 50 cells were counted and survival fractions weredetermined taking into consideration the plating efficiency for alltreatment modalities based on three separate experiments.

TABLE 1

ParticleIncident

energy (MeV)CSDA rangein water (lm)

At entrance (0 lm) At middle (5 lm) At exit (10 lm)

Volume-averagedLET (keV/lm)

Energy(MeV)

LET(keV/lm)

Energy(MeV)

LET(keV/lm)

Energy(MeV)

LET(keV/lm)

Hþ 3 145.44 2.83 12.68 2.76 12.91 2.70 13.14 12.91He2þ 6 48.08 4.93 92.3 4.46 98.83 3.97 107.1 99.26

Notes. The incident energy, the continuous slowing down approximation (CSDA) range in water, the energies and LETs at the entrance surface,the middle, and the exit surface of the cell nucleus, and hence the volume-averaged LET within the cell nucleus for the particle beams used in thisstudy. These values were calculated in SRIM (27), taking into account the energy loss through each component of the beam path, and assumingthat the cell nucleus can be approximated to a sphere, 10 lm in diameter.

TEMOZOLOMIDE AND HIGH-LINEAR-ENERGY TRANSFER RADIATION 653

Growth Curve Assay

To characterize cell proliferation and doubling time in response toTMZ, growth curves were performed with or without TMZconcentrations of 25 and 50 lM. Cells were diluted to 1 3 104 cells/ml and seeded in 24-well plates with 1 ml of the appropriate cellsuspension per well. After 4 h, cells from 3 wells per plate weretrypsinized with 100 ll of 1:10 trypsin in versene (Sigma-Aldrich,Dorset, UK) and counted using a hemocytometer. Cells were thencounted every 24 h for 6–14 days. Each set of experiments wasperformed in duplicate.

Immunofluorescence Detection of DSB Induction and Repair

U87 and LN18 cells were grown on glass slides at a concentrationof 1 3 106 cells/ml, irradiated with a 2 Gy dose of X rays with orwithout TMZ at concentrations of 25 and 50 lM. In the case of proton-and a-particle irradiation, dishes were treated for 1 h at 378C with 10lg/ml fibronectin (Invitrogen, Eugene, OR) to promote cell attach-ment to the polypropylene foil. After excess fibronectin was removedby aspiration, 5 3 103 cells were added to each irradiation positionincluding the control, and attachment was allowed to proceed for 1 h.Cells were then irradiated with a 2 GyE dose of 3 MeV protons aloneor in combination with a single concentration of 25 lM TMZ. After atotal exposure time to TMZ of 2 h, the medium was aspirated, cellswere incubated for various post-recovery time points (1, 4 and 24 h)and fixed in 2% paraformaldehyde in PBS for 15 min, and then with0.5% triton (Sigma-Aldrich, Dorset, UK) in PBS for 10 min at roomtemperature. Afterwards, cells were washed in PBS twice beforeadding a dilution of 0.4% bovine serum albumin (BSA; Sigma-Aldrich, Dorset, UK) in PBS for 20 min. Then anti-phospho-histoneH2AX (Millipore, Watford, UK) was added at a dilution of 1:500 in0.4% BSA in PBS for 45 min. The cells were then washed again threetimes with PBS before placing them in a darkened environment withan FITC-conjugated goat anti-mouse IgG secondary antibody

(Millipore, Watford, UK) at a dilution of 1:400 in 0.4% BSA inPBS for another 45 min. Cells were washed once with PBS and thenuclei were stained with 3 lM propidium-iodide (Invitrogen) in PBSfor 15 min. The cells were finally washed twice with PBS beforemounting them on glass coverslips with Vectashield hard-setmounting medium (Vector Laboratories, Peterborough, UK). Theslides were examined on a LSM 510 META laser scanning confocalmicroscope; images were captured by a camera and imported into theZeiss LSM image analysis software package. For each treatmentcondition, two slides were evaluated and c-H2AX foci weredetermined by eye in at least 100 randomly selected cells per samplefrom two independent experiments.

Statistical Analysis and Model Calculation

All experiments were performed in either duplicate or triplicate.Error bars represent either the standard deviation or the standard error(i.e., standard deviation divided by the square root of the sample size)among the different experiments.

The linear quadratic (LQ) model was used to evaluate and comparethe clonogenic survival curves. The method of weighted least squareswas employed to fit the survival data via a trust-region reflectiveNewton algorithm implemented in Matlab (R2010a, The Mathworks,Natick, MA). Relative biological effectiveness (RBE) values werepredicted mathematically from the LQ fittings of the clonogenic dataand propagation of uncertainty was considered in the RBE erroranalysis.

RESULTS

Cell Growth Analysis in the Presence of Temozolomide

Growth curves for the MGMT-unmethylated LN18 andT98G cell lines and the MGMT-methylated U87 and U373

FIG. 1. Growth curves of MGMT-unmethylated cells, LN18 (panel a) and T98G (panel b), and MGMT-methylated cells, U87 (panel c) and U373 (panel d). Cells were incubated with medium alone (solid line), or with25 lM (dashed line) and 50 lM TMZ (dash-dot line). Error bars represent the standard error of the mean of twoindependent experiments.

654 BARAZZUOL ET AL.

cell lines are shown in Fig. 1. There was no significantdifference in the growth rate when MGMT-unmethylatedcells were incubated with or without 25 and 50 lM TMZ.Similarly, TMZ had no effect on the cell population doublingtime of MGMT-unmethylated T98G cells (relative percentdifference ,1.2%) and a modest effect on the LN18 cells bydecreasing the doubling time from 23.96 to 23.24 and 22.05h in the presence of 25 and 50 lM TMZ, respectively. Incontrast, the MGMT-methylated cell lines showed a cleardecrease in growth rate after 48 h when incubated with TMZ,and then a plateau-like region over 14 days (data not shown).Accordingly, this decrease was accompanied by longerpopulation doubling times in both U87 and U373 cell lines.Data also indicate that TMZ effects on growth curves aredose-dependent, and that doubling times increase with TMZdoses of 25 and 50 lM in a linear way. Viability assays bydye exclusion were also performed, and no significantdifference was detectable between the control and TMZ-treated cells over the first 5 days.

Evaluation of DNA Damage and Repair after X Rays,Protons, Alpha Particles, and Temozolomide

As phosphorylation of a histone H2A variant, H2AX, atthe sites of DSBs, is one of the earliest events in the DNAdamage response to radiation. We investigated in LN18 and

U87 cells whether the addition of TMZ to a clinical dose of2 Gy X rays may enhance the number of c-H2AX foci atdifferent time points after treatment. Particular attention wasgiven to the number of c-H2AX foci left unrepaired after 24h, which is often related to radiosensitivity (29). Thekinetics of c-H2AX foci formation and resolution is shownin Fig. 2. For the MGMT-unmethylated LN18 cells, thenumber of DSBs at baseline slightly increased when TMZwas added to X rays. However, 24 h after treatment therewas little or no difference in the residual number of fociwhen cells received X rays and TMZ compared with X raysalone. In contrast, in MGMT-methylated U87 cells thenumber of c-H2AX foci increased with TMZ addition to Xrays and persisted up to 24 h after treatment. Quantitativeevaluation of the foci number 24 h after treatment showedan additive effect between radiation and TMZ: 3.36 foci/cell(25 lM TMZ alone) or 3.56 foci/cell (50 lM TMZ alone)plus 2.5 foci/cell (2 Gy alone) compared to 4.58 foci/cell (2Gy plus 25 lM TMZ) or 5.14 foci/cell (2 Gy plus 50 lMTMZ).

The number of c-H2AX foci was also evaluated afterirradiation with 3 MeV protons alone or in combinationwith 25 lM TMZ (Fig. 2). Kinetics of DSB repair were verysimilar between X rays and protons. The initial number ofDSBs induced by protons was only slightly higher thaninduced by X rays. In contrast, after 4 h the number of c-

FIG. 2. Time-course kinetics of c-H2AX foci in MGMT-unmethylated LN18 cells and MGMT-methylated U87 cells exposed to either 25 or 50lM TMZ and 2 Gy X rays (panels a, d), protons (panels b, e), and a particles (panels c, f), either alone or in combination. Bars represent thestandard error of the mean of two independent experiments. Please note the scale change on the two right-hand graphs (panels c, f).

TEMOZOLOMIDE AND HIGH-LINEAR-ENERGY TRANSFER RADIATION 655

H2AX foci was lower than that induced by X rays.However, by 24 h the number of residual c-H2AX foci wascomparable: in LN18 cells 3.48 and 3.82 foci/cell, and inU87 cells 2.58 and 2.62 foci/cell after 2 Gy X rays andprotons, respectively. Although at 1 h from treatment theDSB induction ratio of protons over X rays was 1.32 and1.33 for LN18 and U87, respectively; after 24 h it wascloser to 1 (1.1 for LN18 and 1.02 for U87).

The addition of 25 lM TMZ to protons gave equivalentresults to protons alone for MGMT-unmethylated LN18cells. In contrast, in MGMT-methylated U87 cells thenumber of DSBs was moderately higher at each repair timepoint than for protons alone. The pattern of additional c-H2AX induced by TMZ was analogous to that with X rays.At 24 h from proton irradiation in MGMT-methylated U87cells, we found 2.62 and 4.96 foci/cell at 2 Gy and at 2 Gyplus 25 lM TMZ, respectively, that corresponded to 16.27and 29.81% of the number induced at 1 h after treatment.Equally, at 24 h after X rays we counted 2.58 and 4.58 foci/cell that are 21.32 and 34.28% of those at 1 h, respectively.The additional percentage of DSBs induced by 25 lM TMZwas very similar when combined with both protons(13.54%) and X rays (12.96%).

When cells were irradiated with 2 Gy doses of 6 MeV aparticles (Fig. 2), the initial number of c-H2AX foci waslower than with both X rays and 3 MeV protons. Despitethis, the residual numbers of DSBs at 24 h after treatmentwere very similar between the different radiation forms.

Once again, the addition of 25 lM TMZ showed similarvalues to a particles alone for MGMT-unmethylated LN18cells, and additivity for the MGMT-methylated U87 cells.For 6 MeV a particles we found higher percentages of c-H2AX foci at 24 h than with X rays and protons: 2.82 and4.3 foci/cell that corresponded to 35.25% and 56.28% ofthose at 1 h, respectively.

DNA repair kinetics can also be described by a first-orderexponential decay process. The estimated DSB half-liveswere in LN18 cells 12.56 6 1.95, 10.66 6 1.53, and 12.546 1.40 h for X rays, protons, and a particles, respectively,and in U87 cells 10.37 6 1.41, 8.24 6 0.83 and 16.92 6

2.74 h. In MGMT-unmethylated LN18 cells; time coursesafter different treatments were largely similar.

Clonogenic Cell Survival with X Rays and Temozolomide

Survival curves for the four cell lines irradiated with Xrays alone, and concomitantly with 25 and 50 lM TMZ, arepresented in Fig. 3. Graphs show that the MGMT-unmethylated LN18 and T98G cell lines reported nosignificant difference when treated with radiation aloneand radiation in combination with TMZ at different doses.Similar a and b parameters were also found in LN18 andT98G cells, with, in general, minor increases in a andequivalent b values (Table 2). In addition, no significantTMZ-induced cytotoxicity could be observed in these twocell lines (equivalent plating efficiencies).

FIG. 3. Cell survival curves of MGMT-unmethylated LN18 (panel a) and T98G cells (panel b), and MGMT-methylated U87 (panel c) and U373 cells (panel d). Cells received X rays only (solid line), X rays plus 25 lMTMZ (dashed line), and X rays plus 50 lM TMZ (dash-dot line). Symbols represent mean 6 standard error of atleast three independent experiments.

656 BARAZZUOL ET AL.

The behavior of MGMT-methylated U87 and U373 cells

was dissimilar, giving evident dose-dependent cytotoxicity

after TMZ treatment (Fig. 3). Plating efficiencies were

reduced from 20% to 9% (25 lM) and 4% (50 lM) for U87

cells, and from 41% to 18% (25 lM) and 10% (50 lM) for

U373 cells. When combined, TMZ and X rays yielded

independent and additive cell killing. This was apparent

when cell survival curves were normalized for the plating

efficiency induced by TMZ alone, showing no specific

interactions between radiation and TMZ. Moreover, no

significant difference was found in the a and b parameters

when cells received X rays plus 25 and 50 lM TMZ

compared to X rays alone, with the exception of U373 cells

exposed to X rays plus 50 lM TMZ that reported a slight

reduction of a and no change in b (Table 2). The relative

survival curves for U373 cells seem to slightly converge at

radiation doses between 3 and 6 Gy.

Clonogenic Cell Survival with Low-Energy Protons, AlphaParticles, and Temozolomide

The experiments shown in Fig. 3 were also performed

with LN18 and U87 cell lines using low-energy protons and

TABLE 2Mean Values and Standard Deviations of the a and b Parameters Estimated by Fitting the Cell Survival to the

LQ Model

Treatment

LN18 T98G U87 U373

a (Gy�1) b (Gy�2) a (Gy�1) b (Gy�2) a (Gy�1) b (Gy�2) a (Gy�1) b (Gy�2)

X rays 0.22 6 0.09 0.04 6 0.02 0.11 6 0.04 0.03 6 0.01 0.16 6 0.05 0.05 6 0.01 0.17 6 0.05 0.04 6 0.01X rays þ 25 lM TMZ 0.24 6 0.02 0.04 6 0.01 0.18 6 0.06 0.03 6 0.01 0.16 6 0.05 0.05 6 0.01 0.18 6 0.06 0.04 6 0.01X rays þ 50 lM TMZ 0.26 6 0.06 0.03 6 0.01 0.21 6 0.03 0.02 6 0.01 0.19 6 0.11 0.05 6 0.02 0.12 6 0.04 0.04 6 0.013 MeV protons 0.31 6 0.04 0.04 6 0.01 - - 0.36 6 0.04 0.02 6 0.01 - -3 MeV protons þ

25 lM TMZ 0.41 6 0.03 0.02 6 0.01 - - 0.39 6 0.05 0.01 6 0.01 - -6 MeV a 0.77 6 0.03 2e-05 6 0.01 - - 0.65 6 0.08 0.02 6 0.02 - -6 MeV a þ

25 lM TMZ 0.84 6 0.02 2e-05 6 0.01 - - 0.71 6 0.07 2e-05 6 0.01 - -

FIG. 4. Cell survival curves of MGMT-unmethylated LN18 cells (panels a, b) and MGMT-methylated U87cells (panels c, d) irradiated with 3 MeV protons (panels a, c) and 6 MeV alpha particles (panels b, d). Cells alsoreceived concomitant TMZ (dashed lines). Survival curves with 300 kV X rays were used as comparison (filledmarkers). Error bars indicate the standard error of at least three independent experiments.

TEMOZOLOMIDE AND HIGH-LINEAR-ENERGY TRANSFER RADIATION 657

a particles with or without 25 lM TMZ (Fig. 4). No major

difference between the proton survival curves and the X ray

curves could be observed in both cell lines, with an RBE at

10% survival (RBE10) of 1.17 6 0.49, and of 1.06 6 0.35

for LN18 and U87 cells irradiated with 3 MeV protons,

respectively (Table 3). RBE was also mathematically

estimated from the ratio atest radiation/areference radiation (RBEa)

and at the 3 GyE dose level (RBE3 GyE). These values were

higher than the clinically-relevant RBE10, ranging from 1.41

to 2.25 for RBEa, and 1.35 to 1.37 for RBE3 GyE. The

addition of concurrent TMZ caused additive cell killing, as

reported earlier for X rays. For MGMT-unmethylated LN18

cells TMZ did not seem to affect cell survival. In contrast,

for MGMT-methylated U87 cells TMZ significantly

reduced the plating efficiency without changing the survival

curve slope (a value of 0.36 compared to 0.39).

Cells were more sensitive to 6 MeV a particles compared

to X rays and 3 MeV protons. Figure 4b and d show a

reduction of the shoulder effect after a-particle irradiation in

both cell lines. As reported in Table 3, the RBE10 for LN18

and U87 cells were 1.84 6 0.67 and 1.68 6 0.28,

respectively. Like protons, the RBEa and RBE3 GyE signif-

icantly increased to values in the range of 3.38 to 4.06.

Again, the addition of TMZ had no apparent effect on the

MGMT-unmethylated LN18 cells and only additive cyto-

toxicity on the MGMT-methylated U87 cells (Fig. 5). As for

the survival parameters, the addition of 25 lM TMZ

marginally altered only the a component (Table 2).

DISCUSSION

As reported by the EORTC-NCIC trial (2, 9), the survivaladvantage of radiotherapy combined with TMZ comparedto radiotherapy alone in newly diagnosed GBM has led to asignificant change in the treatment of this tumor towards acombined-modality approach. On this basis, in the presentstudy we assessed the combination of low- and high-LETradiation with TMZ on glioma cells.

A number of pre-clinical studies have reported conflictingresults on whether TMZ increases the sensitivity of tumorcells to X rays. Only one study has looked at thecombination of high-LET radiation (carbon ions) andTMZ, reporting independent cytotoxicity (21). Our findingscorroborate the hypothesis that the cytotoxic effects of TMZand X rays are not likely to be correlated. Independentcytoxicity has also been shown when TMZ was added toprotons and a particles. These observations were indepen-dent of the tumor MGMT promoter status.

Administration of TMZ Reduces Growth Rate in MGMT-Methylated Cells

TMZ-independent cytotoxicity was observed in thegrowth curve experiments where, after 2 days, a significantdecrease in growth rate was measured in MGMT-methyl-ated cell lines. This observation agrees with the initialfindings of Newlands et al. (30), that TMZ cytotoxicityneeds one or two cell divisions before DNA damagerecognition.

TABLE 3Resulting RBE Values Calculated at 10% Survival (RBE10), at the Initial Slope of the Survival Curve a

(RBEa ¼ atest radiation/areference radiation), and at the Survival Level after 3 GyE (RBE3 GyE)

Treatment

LN18 U87

RBE10 RBEa RBE3 GyE RBE10 RBEa RBE3 GyE

3 MeV protons 1.17 6 0.49 1.41 6 0.35 1.35 6 0.26 1.06 6 0.35 2.24 6 0.46 1.37 6 0.186 MeV a 1.84 6 0.67 3.5 6 0.83 3.79 6 0.73 1.68 6 0.28 4.03 6 0.85 3.38 6 0.71

FIG. 5. Cell survival curves of MGMT-methylated U87 cells irradiated with 3 MeV protons (panel a) and 6MeV alpha particles (panel b) normalized for the cytotoxicity induced by TMZ alone (dashed lines). Survivalcurves with 300 kVp X rays were used as comparison (filled markers). Error bars indicate the standard error.

658 BARAZZUOL ET AL.

Previous studies with U87 cells have reported similar

results. Trog et al. (31) studied the growth of U87 cellsexposed to 10 and 30 lg/ml TMZ, equivalent to 51.5 and

154.52 lM, respectively, and found a decrease in growth

rate only after 48 h from treatment. Similarly, Hirose et al.(32) showed a growth arrest 2 days after TMZ treatment (3h incubation with 100 lM) in U87 cells and reported a

constant cell number over the following 12 days. Combs etal. (21) also performed a cell-growth analysis of U87 cells

after treatment with 20 lM TMZ for a period of 4 h. Therelative growth curves (Fig. 1) showed a decrease in growth

rate after 20 h from treatment, and a transient drop in cell

number at 45 h.

An expected, synergistic increase of cell killing might bereached by irradiation after 48 h from TMZ exposure.

However, Combs et al. (21) reported no significance

difference in survival when cells were irradiated immedi-

ately after treatment or 48 h later.

Co-Treatment with TMZ and X-Rays Induces IndependentCytotoxicity

Results of the present work suggest that TMZ has anindependent additive effect to X rays. Cell killing was

significantly more pronounced in those cells lacking a

functional MGMT (U87 and U373). In contrast, for the

MGMT-unmethylated cell lines (LN18 and T98G) therewas no significant difference in clonogenic survival with the

addition of TMZ at different clinically-equivalent concen-

trations. These data are in agreement with the in vivofindings of Hegi et al. (4), that MGMT is an importantpredictor of sensitivity to TMZ. The cell survival curves of

combined TMZ and radiation were generally parallel to

those of radiation alone (Fig. 3). In regard to the LQ model

fittings, all cell lines exposed to X rays plus 25 and 50 lMTMZ reported similar values for the b parameter. Chalmerset al. (23) reported little or no change in b when T98G and

U87 cells received 10 lM TMZ 1 h pre-irradiation.

However, they observed a decrease of b when cells received

10 lM TMZ 72 h before being irradiated. We also observeda general increase of a, with the exception of U373 cells

exposed to 50 lM TMZ. Likewise, Chalmers et al. (23)

reported a similar increase in a particles when cells were

treated with 10 lM TMZ 1 and 72 h before irradiation. Theyalso observed a decrease in a particles only in U373 cells

when exposed to 10 lM TMZ.

The survival curve for the MGMT-methylated U373 cell

line treated with X rays plus 50 lM TMZ, appears toconverge toward the other curves in the dose range beyond

2 Gy. The importance of this observation is unclear, but it is

supported by a number of other studies (21, 23, 33).

Chalmers et al. (23) proposed that this may be due to an up-

regulation of the MGMT activity in response to higherradiation doses, whereas Combs et al. (21) hypothesized

that this may be due to a kind of feeder effect (i.e.,

nonproliferating cells provide support, similar to a matrix, toundamaged cells).

TMZ Yields Additive Cell Killing When Combined WithHigh-LET Radiation

We evaluated the clonogenic radiosensitivity of LN18 andU87 cell lines to proton- and a-particle radiation. Despitediverse p53 and MGMT status, the RBE10 values differ onlymodestly between both cell lines with values on the order of1.06–1.17 and 1.68–1.84 for 3 MeV protons and 6 MeV aparticles, respectively. The RBE10 values for protons are inagreement with an RBE10 value of 1.1 that are generallyused in clinical proton therapy (34). Significant increases inRBE10 values were observed with increasing LET irradia-tion employing a particles with an initial energy of 6 MeV.A limited number of in vitro studies have examined theeffects of a particles on GBM cells. Takahashi et al. (35)examined A172 and T98G cells after a-particle irradiationwith a dose-averaged LET of 156 keV/lm at 2.24 MeVincident energy, reporting an RBE10 ranging from 1.19(A172) to 1.33 (T98G). It should be noted, however, thatthese cells were irradiated using a 241Am source in an He gaschamber.

The p53 wild-type U87 cell line was slightly moresensitive to X rays with a D10 (dose required to reduce theviable number to 10%) of 5.31 Gy compared to the p53mutant LN18 cell line with a D10 of 5.49 Gy. The role ofthe tumor suppressor gene p53 in radiation response hasbeen controversial. Some papers have associated mutationor loss of p53 with resistance to conventional radiotherapy(36). In contrast, in some other studies, radiosensitivity wasreported to be p53-independent in particular when cellswere irradiated with high-LET radiation (35, 37, 38).

Proton- and a-particle irradiation was also assessed incombination with 25 lM TMZ. Once again, no significantinteraction between protons, a particles, and TMZ could beobserved in both MGMT-unmethylated LN18 and MGMT-methylated U87 cell lines (Fig. 4). Combs et al. (21)observed converging survival curves at higher radiationdoses with both X rays and carbon ions when combinedwith 10 and 20 lM TMZ. This effect was observed to alimited degree in our study in MGMT-methylated U87 cellsexposed to a particles plus 25 lM TMZ at doses of 3 Gyand upward.

It would be of interest to determine the underlyingmechanisms of cell death and whether TMZ affects celldeath kinetics and response to high-LET radiation. To date,however, only pre-clinical data exist on apoptosis with Xrays in combination with TMZ. Chackravarti et al. (39)observed a biphasic apoptic response in two MGMT-methylated cell lines treated with 6 Gy X rays and 100 lMTMZ, and speculated that TMZ acts as a catalyst inpromoting the second delayed wave of apoptosis (beyond36 h of treatment). In contrast, Kil et al. (22) reported noeffect of TMZ on apoptosis in an MGMT-methylated cell

TEMOZOLOMIDE AND HIGH-LINEAR-ENERGY TRANSFER RADIATION 659

line treated with 2 Gy X rays and 50 lM TMZ. After high-LET radiation, there is evidence that, in addition toapoptosis, other types of cell death, such as mitoticcatastrophe or senescence, may play an important role (37).

Influence of TMZ and LET on the Yield of c-H2AX Fociafter Proton- and Alpha-Particle Irradiation

Only a few studies have investigated the induction andrepair kinetics of DSBs following high-LET radiation onglioma cell lines. Presently, no data exist on the DNAdamage and repair from concomitant TMZ chemotherapyand high-LET radiation. In this study, we have evaluated thenumber of c-H2AX foci as an indicator of DNA damageinduced by a 2 Gy clinically-relevant radiation dose of 3MeV protons (LET 12.91 keV/lm) and 6 MeV a particles(LET 99.26 keV/lm) with or without 25 lM TMZ. Fociresolution should also follow DSB repair kinetics; however,several studies showed that dephosphorylation of the H2AXhistone, due to the process of protein dissociation from thechromatin structure, takes place with a significant delaycompared to the actual repair of DSBs (40).

We found that per GyE, protons induce a similar numberof foci compared to low-LET X rays. In contrast, when cellswere irradiated with a particles, the number of foci per graywas lower than that of X rays and protons. The yield of c-H2AX foci at 1 h after a irradiation was 6.18 and 4 foci/cell/Gy for LN18 and U87 cells, respectively. This numberclosely corresponds to the number of alpha tracks thatshould traverse the nucleus of a 10-lm diameter cell.Indeed, the average size of the foci appeared to be greaterthan those induced by X ray and proton irradiation. Thesefoci might represent clustered lesions at the entrance-exitsites of the linear particle trajectory across the cell nucleus.Similarly, Leatherbarrow et al. (41) reported 3.8 foci/cell/Gy at 20 min following irradiation with 3.31 MeV aparticles. The average number of tracks per cell was 6 per 2Gy, while in our study this number was closer to 10. This,together with the different observation times after treatment(20 min compared to 1 h), might explain the slightlydifferent yield values. Nevertheless, the kinetics of lost offoci was similar following X ray, proton and a irradiationwith comparable values of residual c-H2AX foci at 24 h.

The comparison of DSBs between X rays and protons dueto different support materials (i.e., glass cover slips for Xrays and polypropylene foil for protons) has been recentlyevaluated. Kegel et al. (42) reported a twofold increase in c-H2AX foci number in cells irradiated with X rays on glasscompared to a plastic surface. Furthermore, Antoccia et al.(43) reported an excess of DSBs in cells that received Xrays on glass slides compared to low-energy protons (28.5keV/lm). These findings might explain the higher numberof c-H2AX foci at 4 h after X rays compared to protonsshown in this study.

We have also assessed the resolution of DSBs afterirradiation with protons and a particles in the presence of 25

lM TMZ. Exposure to TMZ, protons, and a particlesresulted in a greater number of foci at 24 h; however, theincrease was less than additive in terms of individualtreatments as was reported for X rays.

Fitting of the repair kinetics, after proton- and a-particle-irradiation, to an exponential decay process suggested repairhalf-lives of 8.24–12.56 h. These findings are in line withthe proposed model of repair of radiation-induced DSBs byIliakis et al. (44). According to this model, the process ofDNA repair involves a slow and a fast component. The firstcomponent takes place 1–30 min after irradiation and is dueto the DNA protein kinase-dependent nonhomologous endjoining (D-NHEJ). The second component is responsible forthe slow repair (half-life 2–10 h) and is known as backupNHEJ (B-NHEJ). Based on our study, our repair kinetics arelikely to represent the slower component. Indeed, we startedto analyze the foci formation only at 1 h after irradiation.

CONCLUSIONS

These results suggest that TMZ causes reproducibleadditive cytotoxicity when combined with radiation,regardless of the radiation types. This effect is only evidentin MGMT-methylated cell lines. It is possible that newfractionation schedules could be designed to exploit thechange in the DNA repair kinetics when MGMT-methyl-ated cells respond to high-LET radiation.

In terms of tumor control for GBM patients, predictableeffects should be observed for the combination of TMZ withparticle therapy, with the significant potential benefits beingthat high-LET radiation could spare normal tissues andallow the integration of concomitant chemotherapy withoverlapping toxicities, while increasing tumor cell kill inhypoxic regions of the tumor. In the immediate future,further evaluations of the combined efficacy of TMZ andhigh-LET radiation in hypoxic condition will be performedby our group.

ACKNOWLEDGMENTS

We are grateful to the Royal Surrey County Hospital for providing the

time and supervision in the X-ray experiments. The research leading to

these results has received funding from the European Community’s

Seventh Framework Programme [(FP7/2007–2013) under grant agreement

no. 215840-2]. Raj Jena is supported by the Health Foundation, UK. Neil

G Burnet is supported by the National Institute of Health Research

Cambridge Biomedical Research Centre, UK. This work has also been

supported by the European Community as an Integrating Activity ‘Support

of Public and Industrial Research Using Ion Beam Technology (SPIRIT)’

under EC contract no. 227012.

Received: September 19, 2011; accepted: December 12, 2011; published

online: April 2, 2012

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