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BioMed Research International

Volume 2014, Article ID 198697, 5 pages
http://dx.doi.org/10.1155/2014/198697

Research Article
IDH1R132H Mutation Increases U87 Glioma Cell
Sensitivity to Radiation Therapy in Hypoxia

Xiao-Wei Wang,1,2,3 Marianne Labussière,1,2,3 Samuel Valable,4,5,6 Elodie A. Pérès,4,5,6

Jean-Sébastien Guillamo,4,5,6,7 Myriam Bernaudin,4,5,6 and Marc Sanson1,2,3,8,9
1
Université Pierre et Marie Curie-Paris 6, Centre de Recherche de l’Institut du Cerveau et de la Moëlle épinière (CRICM),
UMR-S975, 75013 Paris, France
2
INSERM, U 975, 75013 Paris, France
3
CNRS, UMR 7225, 75013 Paris, France
4
CNRS, UMR 6301 ISTCT, CERVOxy group, GIP CYCERON, Boulevard Henri Becquerel, BP 5229, 14074 Caen cedex, France
5
Université de Caen Basse-Normandie, UMR 6301 ISTCT, 14000 Caen, France
6
CEA, DSV/I2BM, UMR 6301 ISTCT, 14000 Caen, France
7
CHU de Caen, Service de Neurologie, Boulevard Côte de Nacre, 14000 Caen, France
8
AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Service de Neurologie 2, 75013 Paris, France
9
Service de Neurologie 2, Groupe Hospitalier Pitié-Salpêtrière, 75651 Paris Cedex 13, France

Correspondence should be addressed to Marc Sanson; [email protected]

Received 12 February 2014; Accepted 6 April 2014; Published 7 May 2014

Academic Editor: Andrea Pace

Copyright © 2014 Xiao-Wei Wang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Objective. IDH1 codon 132 mutation (mostly Arg132His) is frequently found in gliomas and is associated with longer survival.
However, it is still unclear whether IDH1 mutation renders the cell more vulnerable to current treatment, radio- and chemotherapy.
Materials and Methods. We transduced U87 with wild type IDH1 or 𝐼𝐷𝐻1𝑅132𝐻 expressing lentivirus and analyzed the
radiosensitivity (dose ranging 0 to 10 Gy) under normoxia (20% O2 ) and moderate hypoxia (1% O2 ). Results. We observed that
𝐼𝐷𝐻1𝑅132𝐻 U87 cells grow faster in hypoxia and were more sensitive to radiotherapy (in terms of cell mortality and colony formation
assay) compared to nontransduced U87 and IDH1𝑤𝑡 cells. This effect was not observed in normoxia. Conclusion. These data suggest
that 𝐼𝐷𝐻1𝑅132𝐻 mutation increases radiosensitivity in mild hypoxic conditions.

1. Introduction alpha-ketoglutarate to D-2-hydroxyglutarate (D-2HG) [7].

IDH1/IDH2 mutations result in D-2HG accumulation and
The IDH1 gene encoding the cytoplasmic NADP+-dependent lowering NADPH levels. On one hand D-2HG inhibits
isocitrate dehydrogenase—and more rarely IDH2, encod- various alpha-ketoglutarate dependant reactions, including
ing the mitochondrial isoform—are frequently mutated in histone and DNA demethylation, and is likely to promote—
gliomas, especially low grade gliomas and secondary glioblas- rather than inhibit—HIF1𝛼 degradation [8–11]. On the other
tomas [1]. IDH1/IDH2 mutation is associated with better hand, low NADPH levels might sensitize tumors to oxida-
clinical outcome, whatever the grade, but it is still not clear tive stress, potentiating response to radiotherapy, and may
whether it is merely a prognostic marker or a predictor account for the prolonged survival of patients harboring the
of the response to radiotherapy or chemotherapy [2–6]. mutations.
Recent data IDH1/IDH2 mutation results in a new enzyme Since the majority of gliomas are poorly responsive
function catalyzing the NADPH-dependent reduction of to current treatment regimens, the ability to enhance cell
2 BioMed Research International

15

Quantification of IDH1-mRNA
10

0
U87-control U87-IDH1R132H U87-IDH1wt
Mutant

Figure 1: Real time PCR quantified the expression of the IDH1wt and IDH1R132H transduced genes.

10 10

8 8
Viable cells (AU) ∗
Viable cells (AU)

6 6

4 4

2 2

0 0
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Days after seeding Days after seeding

U87 U87-IDH1mutation U87 U87-IDH1mutation

U87-IDH1wt U87-IDH1wt

Figure 2: Effect of IDH1R132H on U87 cell proliferation. U87, IDH1wt -U87, and IDH1R132H -U87 cells were incubated in normoxia 20% (left) or
hypoxia 1% (right) and cells were counted after 1, 3, and 7 days.

radio-chemosensitivity would be of clinical benefit. In this (Invitrogen’s ViraPowerTM HiPerformTM Lentiviral

study, we characterized the impact of IDH1 mutation on U87 Expression Systems; catalog number K5320-00) containing
glioma cell growth and radiosensitivity. the human IDH1 wild type and IDH1R132H cDNA was
generated. The expression clones and the ViraPower
Packaging Mix were cotransfected into the 293FT Cell line
2. Methods and Materials
to produce lentiviral stocks, which were used to transduce
2.1. Cell Culture and Hypoxia Treatment. The human the mammalian U87 cell line. U87-IDH1wt and U87-IDH1R132
glioblastoma cell line U87 MG (HTB14) was obtained from stable cell lines were acquired using EmGFP selection by flow
the American Type Culture Collection (ATCC, Rockville, cytometry. The constructs was verified by DNA sequencing
MD) and maintained in Dulbecco’s modified Eagle’s medium and RT-qPCR analysis.
(DMEM), supplemented with 10% fetal bovine serum (FBS)
and 1% penicillin/streptomycin. Normoxic cells (21% O2 ) 2.3. Cell Proliferation Assay in Normoxia and in Hypoxia. To
were grown in a humidified-air atmosphere incubator evaluate the impact of IDH1 mutation on cell growth in nor-
containing 95% air/5% CO2 at 37∘ C. Hypoxia experiments moxia and in hypoxia by trypan blue dye exclusion method,
were performed in a controlled atmosphere chamber U87, U87-IDH1wt , and U87-IDH1R132H cells (4000/well) plated
(INVIVO2 1000, Ruskinn, Awel, France) set at 1% O2 , 94% in 24-well plates (6 plates in total) were incubated at 37∘ C for
N2 , and 5% CO2 at 37∘ C. six hours in normoxia to adhere; then 3 plates were removed
at 37∘ C in the controlled atmosphere chamber overnight. At
2.2. Production of Recombinant Expression Lentiviruses. 1, 3, and 7 days after exposure to normoxia and hypoxia,
A recombinant pLenti7.3/V5-TOPO expression vector the cells were trypsinized, and the number of viable cells
BioMed Research International 3

Surviving fraction 1 1

Surviving fraction
∗

0.1 0.1

0 2 4 6 8 10 0 2 4 6 8 10
Radiotherapy dose (Gy) Radiotherapy dose (Gy)
U87 U87-IDH1mutation U87 U87-IDH1mutation
U87-IDH1wt U87-IDH1wt

Figure 3: Effect of IDH1R132H on U87 cell viability after irradiation. Transduced cells were plated and then irradiated with doses ranging from
0 to 10 Gy, in normoxia (20%) (left) and in hypoxia (1%) (right). Cells were counted 5 days later.

100 120 h, respectively. Cells were counted with ImageJ software

(Rasband, WS, ImageJ, US NIH).
RT induced cell death (%)

80

60
2.5. Colony-Formation Assay in Normoxia and in Hypoxia.
U87, IDH1wt -U87, and IDH1R132H -U87 cells were plated in 6-
40 well containing 0.3% base agar layer. Six hours later, cells
were either incubated in the hypoxic or normoxic chamber
20 overnight. The next day, the cells were treated by radiotherapy
at the Radiotherapy Department of the Centre de Lutte
0 Contre le Cancer (CLCC) François Baclesse (Caen, France)
U87-IDH1wt

using an X-ray generator with doses ranging 0–8 Gy (Therac

U87

U87
U87-IDH1
U87-IDH1wt

mutation

U87-IDH1
mutation

15-Saturne with a dose rate of 2 Gy/min) and then incubated

again for colony formation. One month later, the colonies
were fixed in 20% ethanol and stained with 0.05% crystal
violet. Colonies that contained more than 50 cells were
Normoxia Hypoxia counted. Survival was calculated as the average number
Figure 4: Cell viability after 8 Gy irradiation. Cells were counted of colonies counted divided by the number of cells plated
before 8 Gy irradiation and 5 days after, in normoxia (20% O2 ) (left) multiplicated by plating efficiency (PE), where PE is the
and in hypoxia (1% O2 ) (right). fraction of colonies counted divided by cells plated without
radiation. The clonogenic survival data were generated using
JMP software. The experiment was performed five times in
triplicate each.
per well was determined by counting with trypan blue. The
experiment was performed three times in triplicate each.
2.6. Statistical Analysis. Results obtained in vitro were
expressed as mean ± SEM Image analysis was performed with
2.4. Comparative Cell Viability Assay after Irradiation, in
in-house macros under the ImageJ Software (Rasband, WS,
Normoxia and in Hypoxia. To evaluate the effect of IDH1R132H ImageJ, US NIH). All statistical analyses were determined
in the response to radiotherapy, U87 cells, IDH1wt -U87, and using post hoc tests after significant ANOVA. Values of 𝑃 <
IDH1R132H -U87 cells were plated (4.103 per well) in 24-well 0.05 were considered statistically significant.
plates. Six hours later at 37∘ C in normoxia, plates were
either kept in normoxia or incubated in the controlled
atmosphere chamber 1% O2 overnight. The next day, cells 3. Results
were irradiated with doses ranging from 0 to 10 Gy in
order to determine the most discriminating dose. Cells were 3.1. Transduced Cells Express High Quantities of IDH1wt and
fixed in paraformaldehyde (PFA) 4%, then stained with IDH1R132H . The presence of IDH1R132H transduced gene was
Hoechst 33342 (10 𝜇g/mL in PBS, Sigma-Aldrich, France) and confirmed by DNA sequencing. Real time PCR showed a high
photographed in a blinded fashion under fluorescence (4 expression of gene IDH1wt and IDH1R132H in transduced U87
wells per condition; 4 photographs per well) at 24 h, 48 h, and cells compared to nontransduced cells (Figure 1).
4 BioMed Research International

1 1

Surviving fraction
Surviving fraction

0.1 0.1

∗∗

0.01 0.01

0 2 4 6 8 0 2 4 6 8
Radiotherapy dose (Gy) Radiotherapy dose (Gy)
U87 U87-IDH1mutation U87 U87-IDH1mutation
U87-IDH1wt U87-IDH1wt

Figure 5: IDH1R132H -U87 cells have a reduced colony forming cell ability after irradiation in hypoxia. U87, IDH1wt -U87, and IDH1R132H -U87
cells were plated 24 h before irradiation (0-2-4-6-8 Gy) in agar and incubated for one month in normoxia (20% O2 ) and in hypoxia (1%
O2 ). The colonies were fixed in ethanol, stained with 0.05% crystal violet, and counted. Survival rate was estimated by the ratio between the
colonies count and the number of cells plated, multiplicated by the plating efficiency.

3.2. IDH1R132H Expressing U87 Glioma Cells Grow Faster 3.5. Radiosensitivity of U87- IDH1R132H in Hypoxia Is Con-
in Hypoxia. We determined whether IDH1R132H expression firmed by Colony-Formation Assay. A colony-formation
directly influences cell growth in normoxia and in hypoxia. assay was used to confirm the effect of IDH1R132H on the
The viable cell number per well was determined by counting response to radiotherapy. Cells were treated with graded
with trypan blue at 1, 3, and 7 days after incubation in doses of radiation (0, 2, 4, 6, and 8 Gy). Colony-forming
normoxia and in hypoxia. Proliferation rate of U87-IDH1R132H efficiency was determined 1 month later and surviving
cells was significantly higher in normoxia than in hypoxia fractions were calculated. In normoxia, U87, U87-IDH1wt ,
for all the three cell lines. In normoxia, U87, U87-IDH1wt , and U87-IDH1R132H had the same colony-formation capacity
and U87-IDH1R132H cells grew at the same rate, whereas U87- after radiotherapy. In hypoxia, the colony number of U87-
IDH1R132H grew faster than U87 and U87-IDH1wt in hypoxia IDH1R132H after radiotherapy was significantly lower than
(Figure 2). U87 and U87-IDH1wt (Figure 5). Thus, U87-IDH1R132H signif-
icantly sensitized U87 glioma cells to radiation.
3.3. Effect of Transduced IDH1R132H on Cell Viability upon
Exposure to Doses Ranging 0 to 10 Gy in Normoxia and in 4. Discussion
Hypoxia. To evaluate the role of IDH1R132H in the response
to radiotherapy, U87, U87-IDH1wt , and U87-IDH1R132H were We observed here that IDH1 mutated U87 grew faster in
exposed to different doses (range: 0–10 Gy): in normoxia moderate hypoxic conditions (1% O2 ) than in normoxia
the three cell lines showed the same radiosensitivity profile, (21% O2 ). This contrast with data obtained in normoxia,
whereas in hypoxia, the viability of U87-IDH1R132H cells was IDH1R132H overexpression in established glioma cell lines in
significantly lower after 5 days compared to control cells and vitro, resulted in a marked decrease in proliferation and mice
IDH1wt cells (Figure 3) (13% versus 23% and 22% for a dose injected with IDH1R132H -U87 cells had prolonged survival
of 10 Gy, 𝑃 < 0.001), respectively. This result suggests that compared to mice injected with IDH1wt -U87 cells [12].
IDH1R132H makes the cells more radiosensitive in hypoxic, but We found then that IDH1R132H -U87 were more sensitive
not in normoxic conditions. to radiotherapy in hypoxic condition. Indeed a high rate of
cell proliferation is per se a sensitive factor of the radiation
therapy response. But on the other hand, IDH1/IDH2 mutated
3.4. Effect of Transduced IDH1R132H on Cell Mortality over Time cells may be more sensitive to oxidative stress. The role of
following 8 Gy Irradiation in Normoxia and in Hypoxia. We isocitrate dehydrogenase in cellular defense against oxidative
quantified then cell death at 24 h, 48 h, and 120 h after 8 Gy stress has been suggested [13]. Indeed, IDH1/IDH2 serves as
irradiation. There was no substantial cell death after 24 h. The a major source of cytosolic and mitochondrial NADPH pro-
effect appeared at 48 h in both normoxia and in hypoxia (data duction necessary to regenerate reduced glutathione (GSH)
not shown) and was maximal after 5 days. Cell death was by glutathione reductase and for the activity of NADPH-
significantly higher for IDH1R132H transduced cells in hypoxia dependent thioredoxin system, both are important in the
but not in normoxia (Figure 4). protection of cells from oxidative damage [14, 15]. Thus, the
BioMed Research International 5

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