Hypoxia-inducible factor-1a blocks differentiation of
malignant gliomas
Huimin Lu
1,
*, Yan Li
1,2,
*, Minfeng Shu
1
, Jianjun Tang
1
, Yijun Huang
1
, Yuxi Zhou
1
, Yingjie Liang
3
and Guangmei Yan
1
1 Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China
2 Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
3 Department of Pathology, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
Introduction
Deviation from the tissue ⁄ lineage-specific differentia-
tion program is one of the fundamental aspects of
tumorigenesis [1]. The aberrantly differentiated cells
show abnormal growth characteristics and distinct
Keywords
CREB-binding protein (CBP) ⁄ p300; cobalt
chloride; differentiation; HIF-1a; malignant
gliomas
Correspondence

Administration of chetomin in combination with forskolin significantly
suppresses malignant glioma growth in an in vivo xenograft model. Analy-
sis of 95 human glioma tissues revealed an increase of hypoxia-inducible
factor-1a protein expression with progressing tumor grade. Taken together,
these findings suggest a key signal transduction pathway involving
hypoxia-inducible factor-1a that contributes to a differentiation defect in
malignant gliomas and sheds new light on the differentiation therapy of
solid tumors by targeting hypoxia-inducible factor-1a.
Structured digital abstract
l
MINT-7292117: CBP (uniprotkb:Q6JHU9) physically interacts (MI:0915) with Hif1a (uni-
protkb:
O35800)byanti bait coimmunoprecipitation (MI:0006)
Abbreviations
CBP, CREB-binding protein; CREB, cAMP-responsive element binding protein; DFO, deferoxamine; FBS, fetal bovine serum; GFAP, glial
fibrillary acid protein; Glut-1, glucose transporter-1; HIF-1, hypoxia inducible factor-1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide; PCNA, proliferating cell nuclear antigen; pVHL, von Hippel-Lindau tumor suppressor protein; siRNA, small interfering RNA; VHL,
von Hippel-Lindau; VEGF, vascular endothelial growth factor.
FEBS Journal 276 (2009) 7291–7304 ª 2009 The Authors Journal compilation ª 2009 FEBS 7291
invasive and metastatic properties [2]. Treating malig-
nant tumors through the induction of cell differentia-
tion has been an attractive concept, but clinical
development of differentiation-inducing agents, espe-
cially for solid tumors, has been limited to date [3]. A
successful example of differentiation therapy is the clin-
ical use of all-trans retinoic acid for the treatment of
acute promyelocytic leukemia [4]. Several cell lines
established from solid tumors were also reported to be
differentiated by respective differentiation-inducers
in vitro [5–8]. However, this differentiation has not been

binding protein (CREB)-binding protein (CBP) ⁄ p300
co-activator, binds to hypoxia-response elements and
transcriptionally modulates target genes [17]. HIF-1
has been shown to play critical roles in tumor angio-
genesis, glucose metabolism, invasion ⁄ metastasis, and
response to radiation and chemotherapy [18–22].
However, little is known about its possible role in the
process of cellular differentiation in solid tumors.
Gliomas are the most common and malignant pri-
mary brain tumors in humans and are among the most
hypoxic tumors known [23,24]. Glioblastoma multi-
forme, the highest-grade glioma, is characterized by
large necrotic areas within the tumor mass, which cor-
relates with enhanced resistance to therapy, increased
invasiveness and a poor prognosis for the patient [24].
In addition, malignant glioma cells could be induced
to undergo differentiation towards their normal coun-
terparts and thus serve as a faithful model to study
molecular mechanisms underlying differentiation
defects in solid tumors [5,25].
In the present study, cobalt chloride and defer-
oxamine (DFO) were used to mimic an intratumoral
mild hypoxia condition, and we found that differentia-
tion induced by forskolin in rat C6 and primary cultured
human malignant glioma cells was reversibly inhibited.
Deletion of the endogenous HIF-1a gene restores the
differentiation capacities, even in the presence of cobalt
chloride. In contrast, stabilization of HIF-1 with small
interfering RNA (siRNA) against VHL, which leads to
proteosomal degradation of HIF-1a, shows differentia-

dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
(MTT) assay (Fig. 1C). Additionally, cell cycle analysis
HIF-1a modulates malignant glioma differentiation H. Lu et al.
7292 FEBS Journal 276 (2009) 7291–7304 ª 2009 The Authors Journal compilation ª 2009 FEBS
showed an accumulation in the G
1
-phase fraction to
91.2% and a remarkable decrease in the S-phase frac-
tion to 4.4% (compared with 82.6% and 13.8%
respectively in the vehicle control group) (Fig. 1D).
Together, these results agree with those of a previ-
ous report [28] and indicate that forskolin has the abil-
ity to induce the differentiation of malignant glioma
cells into maturate astrocytes and may be an appropri-
ate differentiation-inducer in our research.
Cobalt chloride and DFO inhibit differentiation
induced by forskolin in C6 glioma cells
Cobalt chloride has been widely used as a hypoxia-
mimicking agent in both in vivo and in vitro studies as
a result of its inhibitory effects on HIF-1 degradation,
producing biochemical effects similar to those of low
(1–3%)-oxygen hypoxia [29,30]. Here we found that
forskolin-induced astrocyte-like morphological trans-
formation and GFAP increase was blocked by co-
incubation with cobalt chloride, while 100 lm cobalt
chloride alone caused neither morphological nor
GFAP changes (Fig. 2A,B). However, cobalt chloride
showed a synergistic effect with forskolin on PCNA
levels. In agreement with data on PCNA expression,
the forskolin-induced decrease of the S-phase fraction

cobalt chloride at this time point caused a dynamic
reduction of HIF-1a expression (Fig. 3A). Concomi-
tantly, 24 h after withdrawing cobalt chloride from
co-incubation with forskolin, forskolin has resumed its
AB
C
D
Fig. 1. Forskolin induces differentiation of C6 glioma cells. C6 cells were incubated with or without forskolin for 24 h or for the time indi-
cated. (A) Morphological transformation (original magnification, ·200). (B) Dose-dependent effect of forskolin on GFAP and PCNA expression.
(C) Inhibition of cell proliferation. (D) Cell cycle distributions.
H. Lu et al. HIF-1a modulates malignant glioma differentiation
FEBS Journal 276 (2009) 7291–7304 ª 2009 The Authors Journal compilation ª 2009 FEBS 7293
capacity to induce morphological changes and altera-
tions of GFAP and PCNA expression (Fig. 3B,C).
These results suggest the essential role of persistent
up-regulation of HIF-1a for the differentiation block-
age in glioma cells.
HIF-1a is required for the differentiation-
inhibitory effect of cobalt chloride in C6 cells
We next used siRNA, targeting HIF-1a, to further
confirm the role of HIF-1a in differentiation blockage
caused by cobalt chloride. As shown in Fig. 4A, cobalt
chloride failed to induce HIF-1a accumulation in C6
cells transfected with siHIF-1a. Meanwhile, forskolin-
induced morphological transformation and GFAP
up-regulation was not inhibited, despite sustained
exposure to cobalt chloride (Fig. 4B,C). These data
indeed indicate the necessity of HIF-1a accumulation
for differentiation blockage induced by cobalt chloride
in glioma cells.

effect of cobalt chloride in vitro
Chetomin disrupts the structure of the CH1 domain of
CBP ⁄ p300, the transcriptional co-activator, thereby
precluding its interaction with HIF-1 and attenuating
hypoxia-inducible transcription [33]. We first examined
the binding of HIF-1a to CBP ⁄ p300 by immunoprecip-
itation. Figure 6A shows that chetomin significantly
hampers the binding of HIF-1a to CBP ⁄ p300. We also
found that cobalt chloride effectively blocks the mor-
phological and GFAP changes induced by forskolin
(data not shown), while in the presence of chetomin,
this inhibitory effect of cobalt chloride is removed
(Fig. 6B,C). However, chetomin alone showed no
effects, either on the basal and cobalt chloride-induced
HIF-1a protein levels, or on the morphology and
A
B
C
a b
a b
Fig. 3. Cobalt chloride (CoCl
2
) reversibly blocks differentiation in C6
cells. (A) Cells were incubated with 100 l
M CoCl
2
for 24 h and then
CoCl
2
was withdrawn. Twenty-four hours later, the HIF-1a protein

for 2 h and then treated
with 10 l
M forskolin for a further 24 h before morphology analyses
(original magnification: ·200) (B) and western blotting to evaluate
GFAP expression (C).
H. Lu et al. HIF-1a modulates malignant glioma differentiation
FEBS Journal 276 (2009) 7291–7304 ª 2009 The Authors Journal compilation ª 2009 FEBS 7295
GFAP expression in C6 cells (Fig. 6B,C). The mRNA
levels of vascular endothelial growth factor (VEGF)
and glucose transporter-1 (Glut-1), two well-known
HIF-1 target genes, were further evaluated to measure
HIF-1 transcriptional activity. Figure 6D shows that
the elevated mRNA levels of VEGF and Glut-1
induced by cobalt chloride were significantly alleviated
by exposure to chetomin. Taken together, these data
suggest an involvement of HIF-1a transactivation with
the co-activator CBP ⁄ p300 in the differentiation-inhibi-
tory efficacy of cobalt chloride.
Combination of chetomin and forskolin
attenuates tumor growth in vivo
Besides its in vitro effectiveness, chetomin also affects
the HIF-1 pathway and disrupts the interaction of
HIF and CBP in vivo [33]. We therefore utilized xeno-
graft tumor models to evaluate the effect of chetomin
plus forskolin on tumor growth. In comparison to
mice treated with vehicle control, neither chetomin nor
forskolin alone had any remarkable influences on the
growth of tumor xenografts (Fig. 6E,F). However,
simultaneous exposure to chetomin and forskolin had
significant antitumor activity (Fig. 6E,F) without obvi-

defects.
Then, we sought to test the generality of the inhibi-
tory effects of HIF-1a in primary cultured human
glioma cells. Exposure to the differentiation agent fors-
kolin also resulted in differentiated characteristics, of a
stellar shape with filamentous processes and increased
GFAP expression in the primary glioma cells
(Fig. 7C,D). However, co-incubation with cobalt chlo-
ride blocked the morphological alterations and
increased the amount of GFAP induced by forskolin
(Fig. 7C,D). Quantitative analysis indicated that the
percentage of GFAP-expressing cells was significantly
up-regulated upon treatment with forskolin. Moreover,
the up-regulation was reversed by cobalt chloride
B
C
A
Fig. 5. VHL knockdown blocks differentiation of C6 cells. (A) Wes-
tern blotting was used to estimate the levels of pVHL after trans-
fection with 30 n
M VHL or scrambled siRNAs (Scram) for 36 h. (B,
C) C6 transfected with siVHL for 36 h were treated with 10 l
M
forskolin for a further 24 h before morphology analyses (B) (original
magnification, ·200) and western blotting to evaluate the expres-
sion of HIF-1a and GFAP (C).
HIF-1a modulates malignant glioma differentiation H. Lu et al.
7296 FEBS Journal 276 (2009) 7291–7304 ª 2009 The Authors Journal compilation ª 2009 FEBS
(Fig. 7E). These results confirm our findings in C6
cells and, moreover, suggest a general correlation of

M chetomin for 2 h and then treated with 10 lM forskolin and ⁄ or 100 lM CoCl
2
for 24 h. (B) Morphology analy-
ses (original magnification, ·200). (C) HIF-1a and GFAP protein levels were evaluated by western blotting. (D) mRNA levels of VEGF and
Glut-1, as measured by RT-PCR. (E–F) The effects of chetomin and forskolin on tumor growth (E) and weight (F) in mice with C6 xenografts.
Animals with established tumors after 1 week of growth were divided into groups treated with 5 mgÆkg
)1
of forskolin, 0.2 mgÆkg
)1
of cheto-
min, 5 mgÆkg
)1
of forskolin plus 0.2 mgÆkg
)1
chetomin, or vehicle control. Data represent means ± standard error of the mean; n = 6 per
group; *P < 0.05; **P < 0.01, compared with vehicle.
H. Lu et al. HIF-1a modulates malignant glioma differentiation
FEBS Journal 276 (2009) 7291–7304 ª 2009 The Authors Journal compilation ª 2009 FEBS 7297
differentiation [26]. They exhibit reversible defects in
differentiation, which upon certain types of stimula-
tion, allow them to differentiate normally. GFAP, the
50-kDa type III intermediate filament protein, may
serve as a reliable marker of differentiation for normal
astrocytes, while PCNA can be considered to be a
marker of malignant proliferation and higher expres-
sion, which is associated with a higher risk of malig-
nancy. Forskolin, a small lipophilic molecule easily to
be absorbed and distributed, is a well-known adenylate
cyclase activator and a widely reported differentiating
agent in various tumors, including glioma cells [36,37].

counteract the actions of differentiation inducers in
ways not present under in vitro culture conditions. This
presumption is further strengthened by our data that
forskolin alone failed to inhibit the growth of glioma
xenografts.
One aspect of the microenvironment that differs in
tumor tissue versus normal tissue is oxygen tension.
Normal oxygen tensions in cortical grey matter gener-
ally range from 2.5 to 5.3%, with readings as high as
13% [38]. In contrast, oxygen tensions in solid tumors
can range from physiological levels to below 0.1% in
necrotic regions [39]. Ample experimental evidence
revealed that the growth of malignant cells in vivo
requires a hypoxic response and that this occurs pri-
marily through the action of HIF-1a [13]. Recent data
have also demonstrated that aberrant differentiation
typically shows characteristics of abnormal growth and
distinct invasion and metastasis, leading to the tumori-
genic progression. The data presented here argue that
targeting the HIF-1a response for the differentiation
defects in solid tumors also has to be taken into
account.
It is well established that cobalt chloride stabilizes
HIF-1a with kinetics similar to that of hypoxia [40].
However, a high concentration of cobalt chloride was
previously reported to be antiproliferative, pro-apopto-
tic and cytotoxic in diverse established cell lines [41].
Here we found that the use of a non-toxic concentra-
tion of cobalt chloride (Fig. S1) provided a mild
hypoxic model suitable for investigating the role of

well known that the proliferation rate of cells in a hyp-
oxic environment is reduced [47], which in tumor
lesions could be explained by a shortage of nutrients
and growth factors, in addition to low oxygen levels.
This suggests that the hypoxic environment as such
might have both growth-inhibiting and differentiation-
inhibiting efficacy.
The coordinated transcriptional response mediated
by the HIF-1 pathway requires co-activation by the
CBP ⁄ p300 transcriptional co-activators. CBP ⁄ p300
are functional integrators of multiple signal-transduc-
tion pathways because diverse transcription factors,
among which cAMP-responsive element binding pro-
tein (CREB) is a principal one, compete with each
other to interact with a limited amount of CBP ⁄ p300
within the cell [48–51]. Competition between different
transcription factors for CBP ⁄ p300 has been proposed
to play roles in the coordination of gene expression
in response to signaling [52]. One of the most
remarkable examples of this phenomenon is the reci-
procal functional antagonism between p53 and
nuclear factor-jB through competition for CBP ⁄ p300
[53,54]. We have previously identified CREB as a req-
uisite regulator of cellular differentiation in malignant
glioma cells [5]. The present data show that the small
molecule chetomin, which disrupts the structure of
the CH1 domain of CBP ⁄ p300 and thus precludes its
interaction with HIF-1, interferes with the differentia-
tion blockage efficacy of HIF-1a. More importantly,
in combination with chetomin, forskolin shows

induces cell cycle arrest in HCT-116 colon cancer cells
by functionally counteracting Myc, the inactivation of
which results in the differentiation of tumor cells [58–
60]. HIF-1a-induced cell cycle arrest, and thus differ-
entiation, in colon carcinomas seems paradoxical to its
role in the present study. As discussed above, however,
the exact role of HIF-1a in tumor differentiation
remains highly controversial as a result of the different
tumor models. Undoubtedly, further investigation is
warranted for a better understanding of the divergent
role and target genes of HIF-1a in different types of
cancer.
Malignant gliomas constitute a spectrum of brain
tumors with varying differentiation and malignancy
grades, and with clinical courses that range from indo-
lent to highly malignant. Glioblastoma multiforme, the
most common and lethal subtype of the malignant gli-
omas, is characterized by poorly differentiated cells
with intense proliferation and widespread invasion
[61]. Our data, showing that an increased amount of
HIF-1a protein accompanies progressing malignant gli-
oma grade, provide further evidence in support of the
correlation between HIF-1a and differentiation defects
in solid tumors.
In summary, we have shown that HIF-1a protein,
inducible by the hypoxia mimicker cobalt chloride, or
by DFO, blocks induced differentiation in rat C6 and
primary cultured human malignant glioma cells. Loss
of HIF-1a abrogates this blockage, whereas forced
expression of HIF-1a stimulates this blockage. We also

before treatment with forskolin. Chetomin (Alexis Biochem-
icals, San Diego, CA, USA) was dissolved in dimethysulf-
oxide and added 2 h before treatment with forskolin at a
concentration of 1 nm. For the vehicle control group, 0.1%
dimethysulfoxide was used.
Morphological evaluation
The cell morphologies were studied during the indicated
time course using an Olympus (Melville, NY, USA) IX71
inverted microscope and a DP70 CCD camera.
MTT assay
Cell proliferation was evaluated with the 4-[3-(4-iodophe-
nyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfo-
nate (WST-8) assay using a Cell Counting Kit (CCK-8;
Dojindo Molecular Technologies, Gaitherburg, MD,
USA).
Cell cycle analysis
A flow cytometry analysis of the DNA content of cells was
performed to assess the cell-cycle phase redistributions, as
described previously [62]. In brief, the cells were collected
by trypsinization, washed in phosphate-buffered saline
HIF-1a modulates malignant glioma differentiation H. Lu et al.
7300 FEBS Journal 276 (2009) 7291–7304 ª 2009 The Authors Journal compilation ª 2009 FEBS
(NaCl ⁄ P
i
) and fixed in 70% ethanol for 30 min at 4 °C.
After washing with NaCl ⁄ P
i
, cells were incubated with the
DNA-binding dye propidium iodide (50 lgÆmL
)1

nitrocellulose membrane. Membranes were incubated in 5%
nonfat dry milk in TBST [Tris-buffered saline (NaCl ⁄ Tris)
containing 0.05% Tween-20] and then overnight at 4 °C
with antibodies against GFAP, VHL, PCNA (1 : 1000 dilu-
tions; Cell Signaling Technology, Beverly, MA, USA),
HIF-1a (1 : 1000 dilution; Chemicon International Inc.,
Temecula, CA, USA) and b-actin (1 : 2000 dilution; New
England Biolabs, Ipswich, MA, USA). After incubation
with horseradish peroxidase-labelled secondary antibody
(1 : 1000 dilution; Cell Signaling Technology), visualization
was achieved with enhanced chemiluminescence (Amersham
Pharmacia Biotech, Piscataway, NJ, USA) using a Gene-
Gnome chemiluminescence imaging and analysis system
(Syngene Bio Imaging, Cambridge, UK).
Immunoprecipitation
Immunoprecipitation was conducted as previously
described [63]. Cellular protein was immunoprecipitated
with a CBP rabbit antibody (2 lg per 10
6
cells; Santa
Cruz) followed by western blot analysis with HIF-1a
mouse antibody (1 : 1000 dilution; Chemicon Inter-
national Inc.). Nonspecific IgG (6 lg per 10
6
cells;
Upstate Biotechnology Inc., Lake Placid, NY, USA) was
used as a negative control.
Reverse transcription–polymerase chain reaction
Total RNA of treated cultures was extracted using a
TRIzol kit (Invitrogen). RNA (1 lg) was used for reverse

Tissue samples and immunohistochemistry
Tumor tissues from a total of 95 adult patients, 53 men
and 45 women (18 to 73 years of age; average 42 years)
diagnosed with gliomas (World Health Organization grades
I–IV), consecutively operated on between 2004 and 2007,
were collected from the Cancer Center, First and Third
Affiliated Hospitals, Sun Yat-Sen University (Guangzhou,
China) after obtaining informed consent from each patient.
In all cases, the diagnoses and grading were peer-reviewed
by an experienced pathologist according to the principles
laid down in the latest World Health Organization classifi-
cation. The investigation of these tissues was in accordance
with the rules of the Declaration of Helsinki and the
Ethical Committee of Sun Yat-Sen University.
H. Lu et al. HIF-1a modulates malignant glioma differentiation
FEBS Journal 276 (2009) 7291–7304 ª 2009 The Authors Journal compilation ª 2009 FEBS 7301
Immunohistochemistry staining of primary cells and of
4-lm sections of paraffin-embedded samples were per-
formed as described previously [64]. Sections were stained
with primary antibodies (1 : 100 dilution) overnight at
4 °C, with bio-anti mouse IgG for 1 h and were then incu-
bated with avidin–biotin–peroxidase complex diluted in
NaCl ⁄ P
i
. To evaluate the HIF-1a levels, immunostained
slides were scored using the image pro plus 6.0 software.
Statistical analysis
Data are presented as mean ± standard error of the mean
of three separate experiments. Statistical significance was
determined using the Student’s t-test. A result with a

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Supporting information
The following supplementary material is available:
Fig. S1. Influence of cobalt chloride on HIF-1a stabil-
ity and cell survival and proliferation of C6 cells.
Table S1. Summary of HIF-1a immunohistochemistry
in human malignant glioma tissues.
This supplementary material can be found in the
online version of this article.
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