Tài liệu Báo cáo khoa học: Hypoxia-inducible factor-1a blocks differentiation of malignant gliomas pdf

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

G. Yan, Department of Pharmacology,
Zhongshan School of Medicine, Sun Yat-Sen
University, 74 Zhongshan Road II,
Guangzhou, 510080, China
Fax: (86) 20-87330578
Tel: (86) 20-87333258
E-mail:
*These authors contributed equally to this
work
(Received 26 August 2009, revised
2 October 2009, accepted 14 October
2009)
doi:10.1111/j.1742-4658.2009.07441.x
Aberrant differentiation is a characteristic feature of neoplastic transforma-
tion, while hypoxia in solid tumors is believed to be linked to aggressive
behavior and poor prognosis. However, the possible relationship between
hypoxia and differentiation in malignancies remains poorly defined. Here
we show that rat C6 and primary human malignant glioma cells can be
induced to differentiate into astrocytes by the well-known adenylate cyclase
activator forskolin. However, hypoxia-inducible factor-1a expression stimu-
lated by the hypoxia mimetics cobalt chloride or deferoxamine blocks this
differentiation and this effectiveness is reversible upon withdrawal of the
hypoxia mimetics. Importantly, knockdown of hypoxia inducible factor-1a
by RNA interference restores the differentiation capabilities of the cells,
even in the presence of cobalt chloride, whereas stabilization of hypoxia-
inducible factor-1a through retarded ubiquitination by von Hippel-Lindau
tumor suppressor gene silence abrogates the induced differentiation. More-
over, targeting of HIF-1 using chetomin, a disrupter of HIF-1 binding to
its transcriptional co-activator CREB-binding protein (CBP) ⁄ p300, abol-
ishes the differentiation-inhibitory effect of hypoxia-inducible factor-1a.

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

verified in in vivo animal models or clinically, and there
exists little convincing explanation for this finding.
Solid tumors frequently develop in regions with
hypoxia because of an imbalance in oxygen supply and
consumption. Recent reports indicate that hypoxic
microenvironments contribute to cancer progression by
activating adaptive transcriptional programs that pro-
mote cell survival, motility and tumor angiogenesis
[9,10]. Histopathological analyses frequently reveal the
spatial overlap of hypoxia and dedifferentiation within
solid tumors, suggesting the role of hypoxia in tumor
cell differentiation [11,12]. However, it is unclear
whether hypoxia plays a causal role in this relation-
ship.
Cells within hypoxic regions adapt to this environ-
ment by altering their gene-expression program, and
thus their phenotype [3]. One of the transcription fac-
tors primarily responsible for this change is the
hypoxia-inducible factor-1 (HIF-1) [13]. HIF-1 is a
heterodimer that consists of a constitutively expressed
b subunit (HIF-1b or ARNT) and a catalytic a subunit
(HIF-1a) [10,13]. At normoxia, HIF-1a is hydroxyl-
ated at specific proline residues by oxygen-dependent
prolyl hydroxylases, leading to an interaction with
the von Hippel-Lindau tumor suppressor protein
(pVHL) ⁄ E3 ligase complex and subsequent ubiquitin-
mediated destruction [14–16]. Under hypoxic
conditions, HIF-1a escapes from hydroxylation and
tranlocates to the nucleus, where it forms a complex
with HIF-1b and the cAMP-responsive element

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-

tion blockage similar to that induced by cobalt chloride.
We also demonstrated that inhibition of HIF-1a binding
to its transcriptional co-activator, CBP ⁄ p300, abolishes
the differentiation-inhibitory effect of HIF-1. Analyses
of human glioma tissues have suggested a strong
correlation between the expression of HIF-1a and
malignancy (World Health Organization grade). Taken
together, our results indicate that HIF-1 negatively
regulates the differentiation of malignant gliomas and
provide new insights into the differentiation therapy by
targeting the HIF-1 pathway in solid tumors.
Results
Forskolin induces differentiation of C6 glioma
cells
Microscopic observation of C6 glioma cells treated for
24 h with 10 lm forskolin revealed major alterations in
morphology. Unlike the mainly polygonal morphology
of the vehicle control, the shape of forskolin-treated
cells was similar to that of mature astrocytes, with
smaller round cell bodies and much longer, fine, taper-
ing processes (Fig. 1A). Western blotting analysis
further confirmed a significant, dose-dependent,
up-regulation of glial fibrillary acid protein (GFAP)
protein, an established marker of mature astrocytes
[26] (Fig. 1B). Meanwhile, the level of proliferating cell
nuclear antigen (PCNA), a well-accepted marker of
proliferation that facilitates the rapid processing of
DNA [27], was markedly reduced (Fig. 1B).
Forskolin also caused a statistically significant sub-
dued proliferation, as demonstrated by the 3-(4,5-

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

was accentuated by cobalt chloride, while cobalt
chloride alone only resulted in a minor reduction in
the S-phase fraction.
We next clarified whether the blockage in differenti-
ation is a cobalt chloride-specific phenomenon. DFO is
commonly used as an iron chelator and is completely
different from cobalt chloride in its molecular structure
and chemical formula [31]. The assays described above
were repeated using 100 l m DFO instead of cobalt
chloride, and similar results were obtained (Fig.
2A,C,D). Furthermore, both cobalt chloride and DFO
caused remarkable accumulation of HIF-1a protein,
while HIF-1a expression remained unaltered by expo-
sure to forskolin (Fig. 2B,C), suggesting the involve-
ment of HIF-1 a in the described differentiation
blockage.
Therefore, chemical hypoxia is likely to block differ-
entiation in C6 glioma cells, causing them to remain in
an undifferentiated quiescent state after exiting from
the cell cycle.
Cobalt chloride reversibly blocks differentiation
induced by forskolin
Cobalt chloride stabilizes HIF-1a by inhibition of spe-
cific prolyl hydroxylase activity, which is reversible
upon the withdrawal of cobalt chloride [32]. To eluci-
date the role of HIF-1 a more fully, we examined
whether withdrawal of cobalt chloride releases C6 cells
from differentiation blockage. Significant induction of
HIF-1a protein was observed in response to 24 h of
exposure to cobalt chloride, while the withdrawal of

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.

pVHL is a component of a ubiqitin ligase complex
(or E3) that polyubiquitinates HIF-1a in the presence
of oxygen. Loss of pVHL function leads to constitu-
tive HIF-1a stabilization and activity [14]. To further
confirm the role of HIF-1a in the differentiation
blockage, gene knockdown of VHL was used to acti-
B
AC
D
Fig. 2. Cobalt chloride (CoCl
2
) and DFO inhibit differentiation induced by forskolin in C6 glioma cells. Cells were pretreated with 100 lM
CoCl
2
or 100 lM DFO for 2 h and then treated with 10 lM forskolin for a further 24 h for morphology analyses (A) (original magnification:
·200), western blotting to evaluate HIF-1a, GFAP and PCNA expression (B and C), and cell cycle distributions (D).
HIF-1a modulates malignant glioma differentiation H. Lu et al.
7294 FEBS Journal 276 (2009) 7291–7304 ª 2009 The Authors Journal compilation ª 2009 FEBS
vate HIF-1. Thirty-six hours after transfection, the
pVHL level was greatly reduced compared with the
scrambled control (negative control) group; accord-
ingly, HIF-1a was significantly accumulated in C6 cells
(Fig. 5A,C). Similarly to chemical hypoxia induced by
cobalt chloride or DFO, silence of VHL abrogated the
morphological changes and up-regulation of GFAP
expression induced by forskolin (Fig. 5B,C). These
observations demonstrate that the expression of
HIF-1a is necessary and sufficient to abrogate the
differentiation capabilities of glioma cells.
Chetomin abrogates the differentiation-inhibitory

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

levels were examined. (B, C) C6 cells were treated with 100 l
M
CoCl
2
and 10 lM forskolin for 24 h, and then CoCl
2
was withdrawn
(b) or not withdrawn (a). Twenty-four hours later, the cell morphol-
ogy was analyzed (B) and western blotting was performed to
evaluate GFAP and PCNA expression (C).
A
B
C
Fig. 4. HIF-1a is required for the differentiation-inhibitory effect of
cobalt chloride (CoCl
2
) in C6 cells. (A) Cells transfected with 30 nM
HIF-1a or scrambled siRNAs (Scram) for 24 h were treated with
100 l
M CoCl
2
for 2 h. Efficacy of HIF-1a silencing was evaluated
using western blotting for HIF-1a accumulation induced by CoCl
2
.
(B, C) Cells transfected with 30 n
M HIF-1a or Scram siRNAs for
24 h were pretreated with 100 l
M CoCl
2

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-

ous effects on body weight over the treatment period
(data not shown), revealing a synergistic effect between
the HIF-1 pathway suppressor and the in vitro differ-
entiation agent in malignant glioma xenografts.
HIF-1a expression increases with tumor grade
and inhibits differentiation induced by forskolin
in primary human malignant glioma cells
Malignant gliomas are a spectrum of tumors of
varying differentiation and malignancy grades. To
study HIF-1a expression in human glioma tissues of
different grades (World Health Organization I–IV),
we performed immunohistochemical staining with
paraffin-embedded specimens and analyzed exclusively
the non-necrotic region. Expression of HIF-1a protein
was found in all 95 samples (representative immuno-
staining images are shown in Fig. 7A and the results
of HIF-1a immunohistochemistry analyses in patients
are summarized in Table S1), and no obvious staining
was observed in the five normal brain samples (a
representative image is shown in Fig. 7A). Statistical
evaluation revealed that the amount of HIF-1a was
significantly increased in parallel with increasing glioma
grade (Fig. 7B). The percentage of HIF-1a-positive
cells in Grade I averaged 19.4%, while those in
Grades II, III and IV were 32.5%, 46.1% and 70.5%
respectively. Thus, HIF-1a was demonstrated to be
broadly accumulated in glioma cells and its over-
expression was correlated with glioma malignance
grading, in other words, higher levels of expression of
HIF-1a suggest a greater degree of differentiation

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

HIF-1a activity with differentiation in malignant
glioma cells.
Discussion
Gliomas derived from astrocytes or astroglial precur-
sors are the most common malignant cancers affecting
the central nervous system, accounting for > 60% of
primary brain tumors [23]. Despite modern treatments
with neurosurgical resection, radiotherapy and chemo-
therapy, the median life expectancy for patients with
malignant gliomas is approximately 12 months [34,35].
Rat C6 glioma cells are one of the well-established gli-
oma cell lines with an undifferentiated phenotype and
oligodendrocytic, astrocytic and neuronal properties,
constituting a useful model in studies of glial-cell
A

D

B E
C F
Fig. 6. Targeting HIF-1 by chetomin abolishes the differentiation-inhibitory effect of cobalt chloride (CoCl
2
) in vitro and cooperates with fors-
kolin to attenuate glioma growth in vivo. (A) The binding activity of HIF-1a to CBP. Cells stimulated with 1 n
M chetomin for 24 h in the pres-
ence of 100 l
M CoCl
2
were immunoprecipitated with a CBP antibody followed by western blot analysis using an HIF-1a antibody. (B–D) C6
cells were pretreated with 1 n

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].

Our results, showing that some malignant glioma cells
exit from the proliferating cell cycle and then may be
A C
D
I
II
IV
B
E
III
Fig. 7. HIF-1a expression increases with tumor grade and blocks differentiation in human gliomas. (A) HIF-1a immunohistochemistry of
stained sections from representative tissues of grade I–IV primary gliomas and of normal brains. (B) Statistical analysis of HIF-1a expression
indicated that HIF-1a levels are significantly higher in high-grade gliomas than in low-grade gliomas (*, P < 0.01, compare with grade I).
(#, HIF-1a is not detected). (C–E) Human malignant glioma cells were treated with 100 l
M CoCl
2
for 2 h and then with 10 lM forskolin for a
further 24 h. (C) Morphology of cells. (D) Immunocytochemistry of GFAP levels. (E) Quantification of the percentage of GFAP-expressing
cells (*, P < 0.01, compared with the control; #, P < 0.01, compared with the forskolin group).
HIF-1a modulates malignant glioma differentiation H. Lu et al.
7298 FEBS Journal 276 (2009) 7291–7304 ª 2009 The Authors Journal compilation ª 2009 FEBS
induced to differentiate by forskolin, indicate that this
model may be appropriate for the subsequent investi-
gation on differentiation.
Contrary to the development of differentiation-
induction in in vitro cell lines, successful differentia-
tion-inducing therapy for in vivo animal models and
for patients with malignant gliomas, as well as other
solid tumors, has not been reported to date. This sug-
gests that the solid tumor microenvironment may

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

HIF-1a in glioma differentiation.
HIF-1a is overexpressed in more than 70% of
human cancers and regulates multiple steps of tumori-
genesis, including tumor formation, progression and
response to therapy [18]. However, the precise role of
HIF-1a in tumor differentiation is unknown and
highly controversial as a result of the conflicting results
of several tumor models. While some studies found
that HIF-1a mediates differentiation triggered by
cobalt chloride or low oxygen tension in acute myeloid
leukemic cells and pheochromocytoma PC12 cells [42–
44], other studies have shown that HIF-1a promotes a
dedifferentiation phenotype in breast carcinoma and
neuroblastoma cells [11,12] and that HIF-1a represses
differentiation in lung carcinoma cells and high-grade
glioma-derived precursors [45,46]. Here we find that
HIF-1a induced by cobalt chloride ⁄ DFO or VHL
knockdown abrogates the differentiation-induced
potential of C6 malignant glioma cells, while silence or
restrained function of HIF-1a restores the susceptibil-
ity of C6 cells to forskolin-induced differentiation.
These phenomena all indicate the essential role of
HIF-1a in the negative regulation of differentiation in
malignant gliomas.
Interestingly, in contrast to their differentiation-
inhibiting effect, cobalt chloride or DFO synergized
with forskolin to suppress C6 cell proliferation and
retained C6 cells in a neither differentiating nor prolif-
erating stage. Although a poorly differentiated pheno-
type is usually associated with rapid proliferation, it is

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

remarkable anti-glioma activity in vivo
. We thus pro-
pose that the differentiation-induction is highly depen-
dent on the expression of HIF-1a and that
stabilization of HIF-1a may abrogate the differentia-
tion-induced potential of malignant glioma cells, at
least in part through competition with CREB for
binding to CBP ⁄ p300.
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 7299
It is noteworthy that chetomin has also been previ-
ously characterized with potent immunosuppressive
activity [55]. However, to date, few studies have
reported the potential involvement of this feature in
the anti-tumor effect of chetomin. In this context, it
will be interesting to establish whether the immunosup-
pressive effect participates in the in vivo action of
chetomin in malignant gliomas.
More than 100 direct HIF target genes have been
identified that regulate a number of cellular processes,
including glucose metabolism, angiogenesis, erythro-
poiesis, survival and invasion [18]. It has also been
documented that HIF indirectly regulates proliferation
and differentiation through interactions with other sig-
naling proteins, such as Notch and Myc. Gustafsson
[56] has provided evidence that hypoxia and HIF-1a
lead to the inhibition of differentiation in cortical neu-
ral stem cells, myogenic satellite cells and C2C12 cells
by interacting with and stabilizing the Notch ICD
domain. In contrast, Koshiji [57] reported that HIF-1a

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

provide evidence that HIF-1a exerts this differentia-
tion-inhibitory efficacy by binding to its co-activator
CBP ⁄ p300. Collectively, we identify HIF-1a as a nega-
tive regulator of the differentiation in malignant glio-
mas, suggesting a novel therapeutic strategy by
targeting the HIF-1 pathway in the differentiation-
inducing therapy in solid tumors. However, the precise
mechanism by which HIF-1a competes with CREB for
CBP ⁄ p300 and then blocking differentiation is still to
be investigated.
Experimental procedures
Cell culture and drug treatment
C6 rat glioma cells were obtained from the American Type
Culture Collection (Manassas, VA, USA) and maintained
in Dulbecco’s modified Eagle’s medium (DMEM) (Invitro-
gen, Grand Island, NY, USA), supplemented with 10%
fetal bovine serum (FBS) (Hyclone Laboratories, Logan,
UT, USA), in a humidified atmosphere of 5% CO
2
at
37 °C. Human malignant glioma tissues were obtained
immediately after surgical removal with approval of the
Ethical Committee of Sun Yat-Sen University. Primary cul-
tures of human glioma cells were prepared as previously
described [5]. Differentiation was induced by 24 h of expo-
sure to 10 lm forskolin (Sigma, St Louis, MO, USA) in
DMEM containing 1% FBS. For primary human glioma
cells, forskolin was added to DMEM containing 10% FBS.
The mimicked hypoxia condition was achieved by stimula-
tion with 100 lm cobalt chloride or DFO (Sigma) 2 h

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

) and
RNase (1.0 mgÆmL
)1
) for 30 min at 37 °C in the dark.
Finally, cells were washed and red fluorescence was ana-
lyzed by a FACSCalibur flow cytometer (BD, Heidelberg,
Germany) using a peak fluorescence gate to discriminate
aggregates.
siRNA-mediated knockdown of HIF-1a and VHL
expression
The DNA sequence corresponding to the targeting siRNAs
is: HIF-1a:5¢-TCGACAAGCTTAAGAAAGA-3¢; VHL:
5¢-CCAAGACACCTCGAGAAT-3¢ (Ribobio, CN). The
scrambled siRNA sequence used as negative control is
5¢-GAGUAGAAGAUUCAAGCUU-3¢ (Ribobio). C6 cells
grown to 30–50% confluence were transfected using Lipo-
fectamine 2000 (Invitrogen), according to the manufac-
turer’s protocol. Inhibition of protein expression was
assessed by immunoblot analysis. To assess the efficacy of
siHIF-1a, an additional 24 h of treatment with cobalt chlo-
ride was used to induce HIF-1a expression. Differentiation
experiments were carried out 24 or 36 h after siRNA trans-
fection.
Western blot analysis
After lysis of cells and measurement of protein concentra-
tion, the cells were dissolved in SDS sample buffer
[62.5 mm Tris ⁄ HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mm
dithiothreitol and 0.1% Bromophenol Blue]. Equal
amounts of proteins were analyzed by SDS ⁄ PAGE on 12%
polyacrylamide gels. Proteins were electroblotted onto a

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

transcription with a commercial kit (Invitrogen). PCR was
performed using an initial step of denaturation (5 min at
95 °C), 30 cycles of amplification (95 °C for 30 s, 55 °C for
1 min and 72 °C for 1 min) and an extension (72 °C for
2 min). PCR products were analyzed on 2% agarose gels.
The oligonucleotide primers used were as follows. GLUT1:
forward, 5¢-CCCGCTTCCTGCTCATCAA-3¢; and reverse,
5¢-GACCTTCTTCTCCCGCATCATC-3¢. VEGF: forward,
5¢-ACGAAAGCGCAAGAAATCCC-3¢; and reverse, 5¢-
TTAACTCAAGCTGCCTCGCC-3¢. Beta-actin: forward,
5¢-AGGCTCTTTTCCAGCCTTCCT-3¢; and reverse, 5¢-
GTCTTTACGGATGTCAACGTCACA-3¢.
Xenograft tumor model
Xenografts were generated by injecting 1 · 10
6
C6 cells
subcutaneously into the backs of male BALB ⁄ c nude mice
(4 weeks old, n = 24, obtained from Vital River, Bejing,
China). After 1 week of growth, mice with measurable
tumors were segregated into four treatment groups (n =6
in each group). Forskolin, chetomin, forskolin plus cheto-
min or vehicle control (dimethylsulfoxide) was administered
every day via intraperitoneal injection. Tumors were mea-
sured using calipers, and the tumor volume was calculated
as 0.5 · length · width
2
[33]. After 12 days of treatment,
the animals were killed and the tumor weight was deter-
mined. All animal procedures were performed under the
guidelines of the National Institutes of Health.

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

P-value of less than 0.05 was considered statistically signifi-
cant.
Acknowledgements
We thank Professor Ying Guo, Professor Chunkui
Shao and Professor Jianyong Shao for providing
human primary glioma tissues, and Professor Huilan
Rao for technical assistance in pathological analysis.
This work was supported by grants from National
Natural Science Foundation of China (30801408),
National Natural Science Foundation of Guangdong
Province (8451008901000297) and Chinese Postdoc-
toral Science Foundation (20080430801).
References
1 Scott RE (1997) Differentiation, differentiation ⁄ gene
therapy and cancer. Pharmacol Ther 73, 51–65.
2 Sell S (2004) Stem cell origin of cancer and differentia-
tion therapy. Crit Rev Oncol Hematol 51, 1–28.
3 Leszczyniecka M, Roberts T, Dent P, Grant S & Fisher
PB (2001) Differentiation therapy of human cancer:
basic science and clinical applications. Pharmacol Ther
90, 105–156.
4 Wang ZY & Chen Z (2000) Differentiation and apopto-
sis induction therapy in acute promyelocytic leukaemia.
Lancet Oncol 1, 101–106.
5 Li Y, Yin W, Wang X, Zhu W, Huang Y & Yan G
(2007) Cholera toxin induces malignant glioma cell dif-
ferentiation via the PKA ⁄ CREB pathway. Proc Natl
Acad Sci U S A 104, 13438–13443.
6 Choi EJ, Oh HM, Wee H, Choi CS, Choi SC, Kim
KH, Han WC, Oh TY, Kim SH & Jun CD (2009)

Eupatilin exhibits a novel anti-tumor activity through
the induction of cell cycle arrest and differentiation of
gastric carcinoma AGS cells. Differentiation 77, 412–
423.
7 Chintharlapalli S, Smith R III, Samudio I, Zhang W &
Safe S (2004) 1,1-Bis(3¢-indolyl)-1-(p-substitutedphe-
nyl)methanes induce peroxisome proliferator-activated
receptor gamma-mediated growth inhibition, transacti-
vation, and differentiation markers in colon cancer cells.
Cancer Res 64, 5994–6001.
8 El-Metwally TH, Hussein MR, Pour PM, Kuszynski CA
& Adrian TE (2005) High concentrations of retinoids
induce differentiation and late apoptosis in pancreatic
cancer cells in vitro. Cancer Biol Ther 4, 602–611.
9 Pouyssegur J, Dayan F & Mazure NM (2006) Hypoxia
signalling in cancer and approaches to enforce tumour
regression. Nature 441, 437–443.
10 Harris AL (2002) Hypoxia–a key regulatory factor in
tumour growth. Nat Rev Cancer 2, 38–47.
11 Jogi A, Ora I, Nilsson H, Lindeheim A, Makino Y,
Poellinger L, Axelson H & Pahlman S (2002) Hypoxia
alters gene expression in human neuroblastoma cells
toward an immature and neural crest-like phenotype.
Proc Natl Acad Sci U S A 99, 7021–7026.
12 Helczynska K, Kronblad A, Jogi A, Nilsson E,
Beckman S, Landberg G & Pahlman S (2003) Hypoxia
promotes a dedifferentiated phenotype in ductal breast
carcinoma in situ. Cancer Res 63, 1441–1444.
13 Semenza GL (2003) Targeting HIF-1 for cancer ther-
apy. Nat Rev Cancer 3, 721–732.

14 Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh
M, Salic A, Asara JM, Lane WS & Kaelin WG . Jr
(2001) HIFalpha targeted for VHL-mediated destruc-
tion by proline hydroxylation: implications for O2
sensing. Science 292, 464–468.
15 Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert
J, Gaskell SJ, Kriegsheim A, Hebestreit HF, Mukherji
M, Schofield CJ et al. (2001) Targeting of HIF-alpha to
the von Hippel-Lindau ubiquitylation complex by
O2-regulated prolyl hydroxylation. Science 292,
468–472.
16 Maxwell PH, Wiesener MS, Chang GW, Clifford SC,
Vaux EC, Cockman ME, Wykoff CC, Pugh CW,
Maher ER & Ratcliffe PJ (1999) The tumour suppres-
sor protein VHL targets hypoxia-inducible factors for
oxygen-dependent proteolysis. Nature 399, 271–275.
17 Semenza G (2002) Signal transduction to hypoxia-
inducible factor 1.
Biochem Pharmacol 64, 993–998.
18 Rankin EB & Giaccia AJ (2008) The role of hypoxia-
inducible factors in tumorigenesis. Cell Death Differ 15,
678–685.
19 Fulda S & Debatin KM (2007) HIF-1-regulated glucose
metabolism: a key to apoptosis resistance? Cell Cycle 6,
790–792.
20 Lin MT, Kuo IH, Chang CC, Chu CY, Chen HY, Lin
BR, Sureshbabu M, Shih HJ & Kuo ML (2008)
Involvement of hypoxia-inducing factor-1alpha-depen-
dent plasminogen activator inhibitor-1 up-regulation in
Cyr61 ⁄ CCN1-induced gastric cancer cell invasion.

J Biol Chem 283, 15807–15815.
HIF-1a modulates malignant glioma differentiation H. Lu et al.
7302 FEBS Journal 276 (2009) 7291–7304 ª 2009 The Authors Journal compilation ª 2009 FEBS
21 Yang MH, Wu MZ, Chiou SH, Chen PM, Chang SY,
Liu CJ, Teng SC & Wu KJ (2008) Direct regulation of
TWIST by HIF-1alpha promotes metastasis. Nat Cell
Biol 10, 295–305.
22 Moeller BJ, Dreher MR, Rabbani ZN, Schroeder T,
Cao Y, Li CY & Dewhirst MW (2005) Pleiotropic
effects of HIF-1 blockade on tumor radiosensitivity.
Cancer Cell 8, 99–110.
23 DeAngelis LM (2001) Brain tumors. N Engl J Med 344,
114–123.
24 Rong Y, Durden DL, Van Meir EG & Brat DJ (2006)
‘Pseudopalisading’ necrosis in glioblastoma: a familiar
morphologic feature that links vascular pathology,
hypoxia, and angiogenesis. J Neuropathol Exp Neurol
65, 529–539.
25 Takanaga H, Yoshitake T, Hara S, Yamasaki C &
Kunimoto M (2004) cAMP-induced astrocytic differen-
tiation of C6 glioma cells is mediated by autocrine
interleukin-6. J Biol Chem 279, 15441–15447.
26 Roymans D, Vissenberg K, De Jonghe C, Grobben B,
Claes P, Verbelen JP, Van Broeckhoven C & Slegers H
(2001) Phosphatidylinositol 3-kinase activity is required
for the expression of glial fibrillary acidic protein upon
cAMP-dependent induction of differentiation in rat C6
glioma. J Neurochem 76, 610–618.
27 Krishna TS, Kong XP, Gary S, Burgers PM & Kuriyan
J (1994) Crystal structure of the eukaryotic DNA poly-

merase processivity factor PCNA. Cell 79, 1233–1243.
28 Chen TC, Hinton DR, Zidovetzki R & Hofman FM
(1998) Up-regulation of the cAMP ⁄ PKA pathway
inhibits proliferation, induces differentiation, and leads
to apoptosis in malignant gliomas. Lab Invest 78, 165–
174.
29 Badr GA, Zhang JZ, Tang J, Kern TS & Ismail-Beigi F
(1999) Glut1 and glut3 expression, but not capillary
density, is increased by cobalt chloride in rat cerebrum
and retina. Brain Res Mol Brain Res 64, 24–33.
30 Grasselli F, Basini G, Bussolati S & Bianco F (2005)
Cobalt chloride, a hypoxia-mimicking agent, modulates
redox status and functional parameters of cultured
swine granulosa cells. Reprod Fertil Dev 17, 715–720.
31 Bruick RK & McKnight SL (2001) A conserved family
of prolyl-4-hydroxylases that modify HIF. Science 294,
1337–1340.
32 Yang YT, Ju TC & Yang DI (2005) Induction of
hypoxia inducible factor-1 attenuates metabolic insults
induced by 3-nitropropionic acid in rat C6 glioma cells.
J Neurochem 93, 513–525.
33 Kung AL, Zabludoff SD, France DS, Freedman SJ,
Tanner EA, Vieira A, Cornell-Kennon S, Lee J, Wang
B, Wang J et al. (2004) Small molecule blockade of
transcriptional coactivation of the hypoxia-inducible
factor pathway. Cancer Cell 6, 33–43.
34 Stupp R, Mason WP, van den Bent MJ, Weller M,
Fisher B, Taphoorn MJ, Belanger K, Brandes AA,
Marosi C, Bogdahn U et al. (2005) Radiotherapy
plus concomitant and adjuvant temozolomide for

glioblastoma. N Engl J Med 352, 987–996.
35 Maher EA, Furnari FB, Bachoo RM, Rowitch DH,
Louis DN, Cavenee WK & DePinho RA (2001) Malig-
nant glioma: genetics and biology of a grave matter.
Genes Dev 15, 1311–1333.
36 Salero-Coca E, Vergara P & Segovia J (1995) Intracellu-
lar increases of cAMP induce opposite effects in glu-
tamic acid decarboxylase (GAD67) and glial fibrillary
acidic protein immunoreactivities in C6 cells. Neurosci
Lett 191, 9–12.
37 Ammer H & Schulz R (1997) Regulation of stimulatory
adenylyl cyclase signaling during forskolin-induced dif-
ferentiation of mouse neuroblastoma x rat glioma
(NG108-15) cells. Neurosci Lett
230, 143–146.
38 Erecinska M & Silver IA (2001) Tissue oxygen tension
and brain sensitivity to hypoxia. Respir Physiol 128,
263–276.
39 Ljungkvist AS, Bussink J, Kaanders JH & van der
Kogel AJ (2007) Dynamics of tumor hypoxia measured
with bioreductive hypoxic cell markers. Radiat Res 167,
127–145.
40 Wang GL & Semenza GL (1993) General involvement
of hypoxia-inducible factor 1 in transcriptional response
to hypoxia. Proc Natl Acad Sci U S A 90, 4304–4308.
41 Lee M, Lapham A, Brimmell M, Wilkinson H & Pack-
ham G (2008) Inhibition of proteasomal degradation of
Mcl-1 by cobalt chloride suppresses cobalt chloride-
induced apoptosis in HCT116 colorectal cancer cells.
Apoptosis 13, 972–982.

42 Huang Y, Du KM, Xue ZH, Yan H, Li D, Liu W,
Chen Z, Zhao Q, Tong JH, Zhu YS et al. (2003) Cobalt
chloride and low oxygen tension trigger differentiation
of acute myeloid leukemic cells: possible mediation of
hypoxia-inducible factor-1 alpha. Leukemia 17, 2065–
2073.
43 Pacary E, Tixier E, Coulet F, Roussel S, Petit E & Ber-
naudin M (2007) Crosstalk between HIF-1 and ROCK
pathways in neuronal differentiation of mesenchymal
stem cells, neurospheres and in PC12 neurite outgrowth.
Mol Cell Neurosci 35, 409–423.
44 Pacary E, Petit E & Bernaudin M (2008) Concomitant
inhibition of prolyl hydroxylases and ROCK initiates
differentiation of mesenchymal stem cells and PC12
towards the neuronal lineage. Biochem Biophys Res
Commun 377, 400–406.
45 Pistollato F, Chen HL, Rood BR, Zhang HZ, D’Avella
D, Denaro L, Gardiman M, te Kronnie G, Schwartz
PH, Favaro E et al. (2009) Hypoxia and HIF1alpha
repress the differentiative effects of BMPs in high-grade
glioma. Stem Cells 27, 7–17.
46 Galluzzo M, Pennacchietti S, Rosano S, Comoglio PM
& Michieli P (2009) Prevention of hypoxia by
myoglobin expression in human tumor cells promotes
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 7303
differentiation and inhibits metastasis. J Clin Invest 119,
865–875.
47 Carmeliet P, Dor Y, Herbert JM, Fukumura D,
Brusselmans K, Dewerchin M, Neeman M, Bono F,

Abramovitch R, Maxwell P et al. (1998) Role of HIF-
1alpha in hypoxia-mediated apoptosis, cell proliferation
and tumour angiogenesis. Nature 394, 485–490.
48 Fronsdal K, Engedal N, Slagsvold T & Saatcioglu F
(1998) CREB binding protein is a coactivator for the
androgen receptor and mediates cross-talk with AP-1.
J Biol Chem 273, 31853–31859.
49 Horvai AE, Xu L, Korzus E, Brard G, Kalafus D,
Mullen TM, Rose DW, Rosenfeld MG & Glass CK
(1997) Nuclear integration of JAK ⁄ STAT and Ras ⁄
AP-1 signaling by CBP and p300. Proc Natl Acad Sci
USA94, 1074–1079.
50 Lemasson I & Nyborg JK (2001) Human T-cell leuke-
mia virus type I tax repression of p73beta is mediated
through competition for the C ⁄ H1 domain of CBP.
J Biol Chem 276, 15720–15727.
51 McKay LI & Cidlowski JA (2000) CBP (CREB binding
protein) integrates NF-kappaB (nuclear factor-kappaB)
and glucocorticoid receptor physical interactions and
antagonism. Mol Endocrinol 14, 1222–1234.
52 Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R,
Gloss B, Lin SC, Heyman RA, Rose DW, Glass CK
et al. (1996) A CBP integrator complex mediates tran-
scriptional activation and AP-1 inhibition by nuclear
receptors. Cell 85, 403–414.
53 Ravi R, Mookerjee B, van Hensbergen Y, Bedi GC,
Giordano A, El-Deiry WS, Fuchs EJ & Bedi A (1998)
p53-mediated repression of nuclear factor-kappaB RelA
via the transcriptional integrator p300. Cancer Res 58,
4531–4536.

54 Yuan LW & Gambee JE (2000) Phosphorylation of
p300 at serine 89 by protein kinase C. J Biol Chem 275,
40946–40951.
55 Fujimoto H, Sumino M, Okuyama E & Ishibashi M
(2004) Immunomodulatory constituents from an Asco-
mycete, Chaetomium seminudum. J Nat Prod 67, 98–102.
56 Gustafsson MV, Zheng X, Pereira T, Gradin K, Jin S,
Lundkvist J, Ruas JL, Poellinger L, Lendahl U &
Bondesson M (2005) Hypoxia requires notch signaling
to maintain the undifferentiated cell state. Dev Cell 9,
617–628.
57 Koshiji M, Kageyama Y, Pete EA, Horikawa I, Barrett
JC & Huang LE (2004) HIF-1alpha induces cell cycle
arrest by functionally counteracting Myc. EMBO J 23,
1949–1956.
58 Acosta JC, Ferrandiz N, Bretones G, Torrano V,
Blanco R, Richard C, O’Connell B, Sedivy J, Delgado
MD & Leon J (2008) Myc inhibits p27-induced ery-
throid differentiation of leukemia cells by repressing
erythroid master genes without reversing p27-mediated
cell cycle arrest. Mol Cell Biol 28, 7286–7295.
59 Shachaf CM, Kopelman AM, Arvanitis C, Karlsson
A, Beer S, Mandl S, Bachmann MH, Borowsky AD,
Ruebner B, Cardiff RD et al. (2004) MYC inactivation
uncovers pluripotent differentiation and tumour
dormancy in hepatocellular cancer. Nature 431,
1112–1117.
60 Zhou ZQ & Hurlin PJ (2001) The interplay between
Mad and Myc in proliferation and differentiation.
Trends Cell Biol 11, S10–14.

61 Bachoo RM, Maher EA, Ligon KL, Sharpless NE,
Chan SS, You MJ, Tang Y, DeFrances J, Stover E,
Weissleder R et al. (2002) Epidermal growth factor
receptor and Ink4a ⁄ Arf: convergent mechanisms gov-
erning terminal differentiation and transformation along
the neural stem cell to astrocyte axis. Cancer Cell 1,
269–277.
62 Roz L, Gramegna M, Ishii H, Croce CM & Sozzi G
(2002) Restoration of fragile histidine triad (FHIT)
expression induces apoptosis and suppresses tumorige-
nicity in lung and cervical cancer cell lines. Proc Natl
Acad Sci U S A 99, 3615–3620.
63 Huang WC, Ju TK, Hung MC & Chen CC (2007)
Phosphorylation of CBP by IKKalpha promotes cell
growth by switching the binding preference of CBP
from p53 to NF-kappaB. Mol Cell 26, 75–87.
64 Deng J, Miller SA, Wang HY, Xia W, Wen Y, Zhou
BP, Li Y, Lin SY & Hung MC (2002) beta-catenin
interacts with and inhibits NF-kappa B in human colon
and breast cancer. Cancer Cell 2, 323–334.
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.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied

by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
HIF-1a modulates malignant glioma differentiation H. Lu et al.
7304 FEBS Journal 276 (2009) 7291–7304 ª 2009 The Authors Journal compilation ª 2009 FEBS