Báo cáo khoa học: Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells ppt

Expression and functional characterization of P2Y
1
and
P2Y
12
nucleotide receptors in long-term serum-deprived
glioma C6 cells
Patryk Krzemin
´
ski, Dorota Supłat, Rafał Czajkowski, Paweł Pomorski and Jolanta Baran
´
ska
Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland
The focus of this study is glioma C6 cells, which are
chemically transformed glial cells derived from adult
rat brain. They have oligodendrocytic and astrocytic
progenitor properties and are often used as a bio-
chemical model system for studies related to astrocytes
[1]. They are also used as a model for studying glioma
biology [2], e.g. glioblastoma multiforme, which is
pathologically characterized by a marked increase in
cell growth and proliferation. C6 cells can be easily
implanted into rat brains in vivo to test invasiveness.
To date, there is no better model for examining this
process. The increased invasiveness of these cells was
found to be associated with the constitutive up-regula-
tion of phosphatidylinositol 3-kinase (PI3-K) ⁄ protein
Keywords
Akt; glioma C6 cells; mitogen-activated
protein kinase; nucleotide receptors; serum
deprivation

Correspondence
J. Baran
´
ska, Department of Molecular and
Cellular Neurobiology, Nencki Institute of
Experimental Biology, Polish Academy of
Sciences, 3 Pasteur Street, Warsaw PL
02-093, Poland
Fax: +48 22 8225342
Tel: +48 22 5892300
E-mail:
(Received 6 June 2006, revised 7 February
2007, accepted 14 February 2007)
doi:10.1111/j.1742-4658.2007.05741.x
We characterized the expression and functional properties of the ADP-
sensitive P2Y
1
and P2Y
12
nucleotide receptors in glioma C6 cells cultured
in medium devoid of serum for up to 96 h. During this long-term serum
starvation, cell morphology changed from fibroblast-like flat to round, the
adhesion pattern changed, cell-cycle arrest was induced, extracellular sig-
nal-regulated kinase (ERK1 ⁄ 2) phosphorylation was reduced, Akt phos-
phorylation was enhanced, and expression of the P2Y
12
receptor relative to
P2Y
1
was increased. These processes did not reflect differentiation into astro-

cytes or oligodendrocytes, as expression of glial fibrillary acidic protein and
NG2 proteoglycan (standard markers of glial cell differentiation) was not
increased during the serum deprivation. Transfer of the cells into fresh
medium containing 10% fetal bovine serum reversed the changes. This
demonstrates that serum starvation caused only temporary growth arrest
of the glioma C6 cells, which were ready for rapid division as soon as the
environment became more favorable. In cells starved for 72 and 96 h,
expression of the P2Y
1
receptor was low, and the P2Y
12
receptor was the
major player, responsible for ADP-evoked signal transduction. The P2Y
12
receptor activated ERK1 ⁄ 2 kinase phosphorylation (a known cell prolifer-
ation regulator) and stimulated Akt activity. These effects were reduced by
AR-C69931MX, a specific antagonist of the P2Y
12
receptor. On the other
hand, Akt phosphorylation increased in parallel with the low expression of
the P2Y
1
receptor, indicating the inhibitory role of P2Y
1
in Akt pathway
signaling. The shift in nucleotide receptor expression from P2Y
1
to P2Y
12
would appear to be a new and important self-regulating mechanism that

promotes cell growth rather than differentiation and is a defense mechan-
ism against effects of serum deprivation.
Abbreviations
Akt, protein kinase B ⁄ Akt kinase; DIC, differential interference contrast; ERK, Ras extracellular signal-regulated kinase; GFAP, glial fibrillary
acidic protein; IRM, interference reflection microscopy; MEM, minimum essential medium; 2MeSADP, 2-methylthio-ADP; NaCl ⁄ P
i
,
phosphate-buffered saline; PI3-K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog.
1970 FEBS Journal 274 (2007) 1970–1982 ª 2007 The Authors Journal compilation ª 2007 FEBS
kinase B ⁄ Akt kinase (Akt) signaling [3] and with
glioma-induced angiogenesis [4].
Among other functions in the brain, nucleotide re-
ceptors act as sensors of necrotic cell death. The abun-
dance of ATP released from dying cells evokes a
particular response in surviving neighbor cells, which
may result in their transformation from normal to
malignant cells. This is why studies of nucleotide
receptors in transformed cells are so important. The
large family of nucleotide receptors is divided into two
major classes [5]: P1, responding to adenosine; P2,
responding to ATP, ADP, UTP and UDP. P2 recep-
tors are further divided into two groups [6,7]: P2X, the
intrinsic ligand-gated ion channels; P2Y, members of
the G-protein coupled receptor superfamily. To date,
eight genuine mammalian P2Y receptor subtypes have
been cloned and pharmacologically characterized. Of
these, P2Y
2
responds to ATP and UTP, whereas P2Y
1

and P2Y
12
respond to ADP. P2Y
1
and P2Y
2
receptors
are both coupled to phospholipase C [8].
Whereas the presence of the P2Y
2
receptor in glioma
C6 cells is generally accepted, the presence of the P2Y
1
receptor, which stimulates phospholipase C, has been a
subject of serious debate. Studies on glioma C6-2B
and C6 cells have shown the presence of an additional
P2Y
1
-like receptor, termed P2Y-C6 or P2Y
AC
[9–14].
This P2Y receptor, which is negatively coupled to
adenylate cyclase, has been cloned and designated
P2Y
12
[15–17].
Results from our laboratory revealed that both clas-
sic P2Y
1
and P2Y

12
receptors coexist in glioma
C6 cells: P2Y
1
, linked to phospholipase C, and P2Y
12
,
inhibiting adenylate cyclase [18,19]. Observations on
P2Y
1
receptor made by us and other authors
[11,13,14,20] have suggested that its functional inactiv-
ity can be caused by differences in culture conditions.
We have shown that mRNA expression level, as well
as the extent of the response to ADP and ADP ana-
logs such as 2-methylthio-ADP (2MeSADP) depends
on the presence of serum in the cell culture medium
[20]. 2MeSADP has been chosen as a ligand because it
is the agonist of both receptors studied, and ecto-
enzymes present on the glioma C6 cells are unable to
hydrolyze this compound [21]. In cells cultivated in
medium devoid of serum, as well as in a serum-free
chemically defined medium, expression of the P2Y
1
receptor is low [20,22]. We have also found that agon-
ists that stimulate P2Y
1
and P2Y
12
receptors differen-

tially affect the Ras extracellular signal-regulated
kinase (ERK)1 ⁄ 2 and PI3-K signaling pathways and
that it is the P2Y
12
receptor that predominates in
ADP-evoked ERK1 ⁄ 2 activation. These results also
suggest that P2Y
1
and P2Y
12
receptors may regulate
each other’s activities by cross-talk mechanisms
[20,23,24].
In carcinogenesis, several alterations in cell physio-
logy have been proposed, which must cooperate for
malignant growth to occur. In the first place, there
must be self-sufficiency of the growth-promoting signal
[25]. Therefore, in the present study, we used glioma
C6 cells cultured in medium devoid of serum for up to
96 h and examined the effect of such long-term serum
starvation on cellular morphology, adhesion, prolifera-
tive and differentiation state, basal level of ERK1 ⁄ 2
and Akt kinase phosphorylation, and P2Y
1
and P2Y
12
receptor protein expression and functional activity.
Examination of the effects of specific pharmacological
agents (a single agonist and two specific antagonists of
P2Y

1
and P2Y
12
receptors) on Akt and ERK1 ⁄ 2 phos-
phorylation should help to clarify the effect of cross-
talk between the two receptors on cell physiology.
Our data show that the P2Y
12
receptor primarily
contributes to glioma C6 cell growth and proliferation,
as long-term serum deprivation strongly decreases
expression of the P2Y
1
receptor, and under these con-
ditions the concentration of the P2Y
12
receptor protein
predominates. We also observed a simultaneous
increase in Akt kinase phosphorylation. We suggest
that this high level of Akt phosphorylation is an
important, self-sufficient mechanism for survival under
inhospitable conditions. These results may be useful in
the development of therapeutic strategies targeting
brain tumor angiogenesis.
Results
Glioma C6 cells are characterized by a typical fibro-
blast-like flat morphology clearly visible by differential
interference contrast (DIC) microscopy (also known as
Nomarski contrast microscopy). As shown in Fig. 1A
(control), the cells are flattened and their adhesion

spots, visualized by interference reflection microscopy
(IRM), are distributed under the periphery of the cell
body (Fig. 2A, arrows) in a manner characteristic of
motile cells. Vinculin staining reveals adhesion spots
distributed evenly on the whole basolateral surface of
the cell (Fig. 2E). However, during prolonged (48, 72
and 96 h) serum deprivation, changes in cell morphol-
ogy from an epithelial, bipolar, to a rounded shape
can be observed (Fig. 1B–D). The cells change from a
fibroblast-like flat morphology to a rounded appear-
ance characterized by numerous long extrusions. These
particular changes were clearly visible when single
enlarged cells are compared (Fig. 1H versus Fig. 1I).
Figure 1G shows that, whereas the percentage of
altered, rounded cells in the control amounted only
P. Krzemin
´
ski et al. P2Y
1
and P2Y
12
in serum-deprived glioma C6
FEBS Journal 274 (2007) 1970–1982 ª 2007 The Authors Journal compilation ª 2007 FEBS 1971
to 8%, the percentage in cells cultivated for 96 h in
serum-free medium rose to 75%. Also, cell adhesion
was generally increased and adhesion regions were pre-
sent under the center of cell body (Fig. 2B,F, arrows).
Replacement of the medium with a fresh, but still
serum-free, medium only partially reversed the mor-
phological alterations (Fig. 1E) and adhesion pattern

(Fig. 2C,G). However, replacement with complete
medium containing 10% (v ⁄ v) fetal bovine serum led
to fibroblast-like morphology and typical C6 cell body
shape (Fig. 1F) and distribution of adhesion spots
(Fig. 2D,H). Under these conditions, the percentage of
altered cells decreased to  25%. These results suggest
that serum deprivation, forcing glioma C6 cells to
change their morphology, did not cause permanent
alterations, and, even after 96 h of starvation, the cells
were still ready to be activated. Serum deprivation did
not cause significant loss of cell viability and the
majority of cells remained adherent (not shown).
Serum withdrawal can induce cell cycle arrest in
many cell lines, including astrocytes [26,27]. In the pre-
sent study, we used propidium iodide staining and flow
ABC
DEF
GHI
Fig. 1. Induction and reversal of a morphological change in serum-starved glioma cells. Cells were seeded at low density on Petri dishes
(8 cm diameter) and allowed to recover for 24 h in MEM with 10% fetal bovine serum. (A) Nonstarved cells; (B) cells cultivated for 48 h in
serum-free MEM; (C) cells cultivated for 72 h in serum-free MEM; (D) cells cultivated for 96 h in serum-free MEM; (E) cells cultivated for
96 h in serum-free MEM followed by 24 h recovery in fresh MEM without serum; (F) cells cultivated for 96 h in serum-free MEM followed
by 24 h recovery in fresh MEM with serum. (G) Percentage changes in morphology during serum deprivation. Control, see A; 48 h, see B;
96 h, see D; 96 h +24 h M, see E; 96 h +24 h M+, see F. Cells were counted in five independent images for every experimental setup. In
each image, 100–150 total cells were counted. (H) Single glioma C6 cell cultivated in the presence of serum. (I) Single glioma C6 cell cultiva-
ted for 96 h in the absence of serum. Note appearance of cell extrusions indicated by arrows. The micrographs were taken with a Nikon
Diaphot inverted microscope, equipped with 40· DIC Nomarski objective lens. A–D, bars ¼ 50 lm; H-I, bars ¼ 15 lm.
P2Y
1
and P2Y

12
in serum-deprived glioma C6 P. Krzemin
´
ski et al.
1972 FEBS Journal 274 (2007) 1970–1982 ª 2007 The Authors Journal compilation ª 2007 FEBS
cytometry analysis to determine how long serum dep-
rivation was needed to cause full cell cycle arrest.
Glioma C6 cells growing in medium supplemented
with 10% (v ⁄ v) fetal bovine serum were used as a con-
trol. Figure 3 shows that  60% could be synchron-
ized in G
0
⁄ G
1
, 25% in G
2
⁄ M, and 15% in S phase.
After 24 and 48 h of serum starvation,  80%
and 90% of cells were growth arrested, respectively.
However, there were still a number of unsynchronized
cells, making it impossible to consider them as one
population. However, incubation in serum-free condi-
tions for 96 h resulted in 95% of cells arrested in
G
0
⁄ G
1
phases, with less than 5% of the cells in other
cell cycle phases. Figure 3 shows that the addition of
fresh serum-free medium to cells starved for 96 h and

incubation for the next 24 h (Fig. 3, M-) increased the
Fig. 3. Cell cycle analysis performed on propidium iodide-stained glioma C6 cells. Each phase (G
o
⁄ G
i
, S and G
2
⁄ M) is indicated. Control: cells
were grown in MEM containing 10% fetal bovine serum. 24, 48, 72, 96 h: for serum deprivation, medium was changed to serum-free MEM
and cells were cultivated without medium exchange for up to 96 h. M-: cells were cultivated for 96 h in serum-free MEM followed by 24 h
recovery in fresh MEM without serum. M+: cells were cultivated for 96 h in serum-free MEM followed by 24 h recovery in fresh medium
with serum. The data represent one typical experiment, similar to three other repetitions.
ABCD
EFGH
Fig. 2. Changes in adhesion pattern during starvation and recovery of glioma cells. (A, E) Nonstarved cells, adhesion regions on the cell edge
indicated by arrows (A). Vinculin staining on whole cell surface (green, E). (B, F) Cells cultivated for 96 h in serum-free MEM; arrows point
to strongly adherent central region of the cell (B). Vinculin staining concentrated in the central part of the cell (green, F). (C, G) Cells cultiva-
ted for 96 h in serum-free MEM followed by 24 h recovery in fresh MEM without serum; arrow points to fragmented adhesion region in the
cell center. Vinculin staining on the whole cell surface in most of the cells (green, G); some cells still have vinculin staining similar to starved
cells (arrow). (D, H) Cells cultivated for 96 h in serum-free MEM followed by 24 h recovery in fresh MEM with serum; arrows point to recov-
ering adhesion regions on the cell edges. Vinculin staining similar to control cells (E). (A–D) The IRM micrographs were taken with a Nikon
Diaphot inverted microscope, equipped with a 100· DIC Nomarski objective lens and IRM epifluorescence illumination. Bar ¼ 15 lm. (E–H)
Fluorescence microphotographs were taken with a Nikon Diaphot inverted microscope, equipped with a 63· fluo lens. Specimens stained
with antibodies against vinculin visualized by secondary Alexa Fluor 488-conjugated secondary antibodies (green) and phalloidin conjugated
with TRITC (red) were closed with 4¢,6¢-diamino-2-phenylindole (DAPI)-containing mounting medium (blue). Bar ¼ 30 lm.
P. Krzemin
´
ski et al. P2Y
1
and P2Y

12
in serum-deprived glioma C6
FEBS Journal 274 (2007) 1970–1982 ª 2007 The Authors Journal compilation ª 2007 FEBS 1973
number of cells in the S phase. On the other hand,
addition of serum-containing medium (Fig. 3, M+)
almost completely restored the control pattern of the
cell cycle phases, with  70% of cells in G
0
⁄ G
1
phases
and  20% in S phase. In addition, no significant
amounts of apoptotic sub-G
1
cells appeared in any of
the experiments (not shown).
Similar morphological changes concomitant with cell
growth arrest and induction of synthesis of glial fibril-
lary acidic protein (GFAP) were observed when glioma
C6 cells were treated with agents that increase the
intracellular concentration of cAMP [28,29]. GFAP is
widely expressed in astrocytes and is used as a marker
for the induction of differentiation into an astrocyte
type II [30,31]. Therefore, in further experiments we
measured the expression of this protein in serum-
deprived cells. However, long-term serum starvation
did not increase the low GFAP expression (Fig. 4)
characteristic of transformed glioma C6 cells [31,32].
In the control glioma C6 cells cultivated in medium
supplemented with serum, the GFAP concentration

was about 1000-fold lower than in astrocytes
(Fig. 4A). Similarly, the changes in glioma C6 cell
morphology were not caused by differentiation of the
cells into the oligodendrocytic lineage. In this study,
the low expression of NG2 proteoglycan (a marker of
oligodendrocyte progenitors [33]) was examined by im-
munocytochemistry. No statistically significant change
in staining intensity was found during serum starvation
(not shown). Moreover, Messens & Slegers [31] des-
cribed changes in glioma C6 cell morphology from
bipolar to more round when cells grown in 10% fetal
calf serum were cultivated in chemically defined serum-
free medium. As, under these conditions, the concen-
tration of P2Y
1
receptor mRNA greatly decreased [22],
these data and those presented in this study show that
the differentiation-like morphological changes are not
connected with differentiation of the cells into astro-
cytes or oligodendrocytes, but are connected with the
lack of serum in the culture medium. Furthermore,
these data indicate that growth arrest and GFAP
expression proceed via different pathways.
We have previously confirmed the presence of P2Y
1
,
P2Y
2
and P2Y
12

receptors in glioma C6 cells using
PCR on reverse-transcribed total mRNA [20,22]. We
have found that, in cells serum-deprived for 48 h, the
concentration of P2Y
1
mRNA decreased, whereas
P2Y
12
mRNA expression slightly increased. In con-
trast, serum starvation was without effect on P2Y
2
mRNA receptor expression [20]. To explore the effects
of prolonged serum starvation on ADP-sensitive recep-
tor expression, we measured changes in protein con-
centration using Western blot analysis. The cells were
cultivated for 24, 48, 72 and 96 h in serum-free med-
ium in exactly the same conditions as used for the
experiments on cell cycle, adhesion and morphology.
Figure 5 shows that the concentration of P2Y
1
recep-
tor protein gradually decreased. Changes in the con-
centration of the P2Y
12
receptor protein were not so
evident, although densitometry analysis of the blots
from several experiments revealed a slight increase,
particularly after 72 and 96 h of serum deprivation.
However, given the fact that, in cells growing in
serum-free medium for 72 and 96 h, the concentration

of P2Y
1
receptor protein was low, under such condi-
tions, P2Y
12
receptor expression must strongly pre-
dominate (Fig. 5). These results suggest that, during
prolonged serum starvation, the P2Y
12
receptor is pri-
marily responsible for ADP-evoked signal transduc-
tion. The addition of fresh medium for the next 24 h
restored low expression of the P2Y
1
receptor (Fig. 5,
M-). The increase in P2Y
1
receptor expression was
especially noticeable when, after 96 h of serum starva-
tion, the medium was replaced with complete medium
containing 10% (v ⁄ v) fetal bovine serum (Fig. 5,
M+). The serum-free medium also increased the
B
A
Astrocytes
Fig. 4. GFAP expression in serum-deprived glioma C6 cells. Total
protein isolated from glioma cells was subjected to SDS ⁄ PAGE on
10% polyacrylamide gel and transferred to nitrocellulose mem-
branes (A). Filters were probed with the indicated GFAP antiserum.
Protein bands were detected with secondary antisera coupled to

horseradish peroxidase by enhanced chemiluminescence, and then
densitometry analysis was performed (B). Bars represent mean
integrated band intensity taken from three independent experi-
ments. Control cells cultivated in medium with 10% fetal bovine
serum displayed as 100%; 48 h-, cells cultivated in medium with-
out fetal bovine serum for 48 h; 96 h-, cells cultivated in medium
without fetal bovine serum for 96 h. The results are mean ± SD
and are representative of three separate experiments.
P2Y
1
and P2Y
12
in serum-deprived glioma C6 P. Krzemin
´
ski et al.
1974 FEBS Journal 274 (2007) 1970–1982 ª 2007 The Authors Journal compilation ª 2007 FEBS
concentration of P2Y
1
but to a much lesser extent. No
such difference was observed in the case of the P2Y
12
receptor. Multiple bands observed for P2Y receptors
are probably the effect of protein glycosylation. We
have investigated this phenomenon in glioma C6 cells
and have recently shown that preincubation of cell
lysates with N-glycosidase F resulted in the reduction
of bands detected by P2Y antibodies [34].
We have previously shown that glioma C6 cells
growing in medium supplemented with serum exhibit
high, constitutive concentrations of active, phosphoryl-

ated ERK1 ⁄ 2 and PI3-K [22]. In the present study,
we performed experiments with cells incubated with
medium devoid of serum for up to 96 h. Figure 6A
shows that, in the absence of serum stimulation, the
level of p42 and p44 ERK1 ⁄ 2 phosphorylation was
greatly reduced, as monitored by Western blot analysis
with antibodies to p42 ⁄ p44 ERK. In contrast, pro-
longed serum deprivation did not decrease Akt kinase
phosphorylation. In fact, 24–72 h of serum starvation
resulted in its increase, showing that exhibition of a
high basal level of Akt activity is part of an important
self-regulatory mechanism. This process was only
stopped, and Akt phosphorylation decreased, in cells
after very prolonged (96 h) serum starvation (Fig. 6A),
suggesting that the stimulatory role of the P2Y
12
Fig. 5. Expression level of P2Y
1
and P2Y
12
receptor protein in nonstarved and serum-
starved cells. Total proteins isolated from
whole glioma cell lysates were subjected to
SDS ⁄ PAGE on 10% polyacrylamide gel and
transferred to nitrocellulose membranes. Fil-
ters were probed with the indicated P2Y
antisera. Protein bands were detected with
secondary antisera coupled to horseradish
peroxidase by enhanced chemilumines-
cence. Equal loading of proteins was

ensured by b-actin concentration. Control:
cells were growing in MEM containing 10%
fetal bovine serum. 24, 48, 72, 96 h: cells
were cultivated in MEM without serum for
24, 48, 72 and 96 h, respectively. M-: cells
were cultivated for 96 h in serum-free MEM
followed by 24 h recovery in fresh MEM
without serum. M+: cells were cultivated
for 96 h in serum-free MEM followed by
24 h recovery in fresh medium with serum.
The blot represents one typical experiment.
Plots are integrated densitometry analysis
results and are mean ± SD from three
repetitions.
P. Krzemin
´
ski et al. P2Y
1
and P2Y
12
in serum-deprived glioma C6
FEBS Journal 274 (2007) 1970–1982 ª 2007 The Authors Journal compilation ª 2007 FEBS 1975
receptor is only one of the factors that affect the mul-
tiple and complex process of Akt activation. However,
replacement of the medium with fresh but still serum-
free medium (Fig. 6B, M-), and especially with fresh
medium containing 10% (v ⁄ v) fetal bovine serum
(Fig. 6B, M+), and subsequent incubation for the
next 24 h restored ERK1 ⁄ 2 phosphorylation. In con-
trast, fresh serum-free medium restored Akt phos-

phorylation (Fig. 6B, M-), whereas fresh medium
supplemented with serum had the opposite effect
(Fig. 6B, M+). Thus, phosphorylation of Akt
(Fig. 6A, p-Akt) increased up to 72 h, in parallel with
the decrease in P2Y
1
receptor protein concentration
(Fig. 5), and decreased (Fig. 6B, M+) when P2Y
1
receptor protein concentration increased after replace-
ment with medium containing serum (Fig. 5).
To check whether, in the cells adapted to serum-free
medium for 96 h, P2Y
12
receptor is still functionally
active and able to respond to agonist affecting ERK1 ⁄ 2
as well as Akt activity, in the next experiments the
cells were stimulated with 2MeSADP. Within 15 min,
2MeSADP had induced a rapid,  35-fold increase in
the concentration of p44 ⁄ p42 ERK1 ⁄ 2 (Fig. 6C) and
25-fold increase in Akt phosphorylation (Fig. 6D).
Within 30 min of the addition of the agonist, phos-
phorylation had declined; in the case of p-ERK1 ⁄ 2,
concentrations fell to basal, whereas in the case of p-
Akt, concentrations declined to half the observed
increase and reached a plateau. Figure 6D shows that
AR-C69931MX (a P2Y
12
-specific antagonist) almost
completely inhibited Akt phosphorylation, whereas

ERK1 ⁄ 2 phosphorylation was inhibited by  70%,
returning to the level observed after the addition of
fresh medium in the absence of agonist (Fig. 6C, inset).
Incubation in media containing specific receptor antag-
onists and in the absence of agonists shows that
AR-C69931MX, the P2Y
12
receptor antagonist, greatly
reduces the level of active ERK1 ⁄ 2 phosphorylation,
whereas MRS2179, the P2Y
1
receptor antagonist, had a
much weaker effect (Fig. 6E). These results again high-
light the stimulatory role of P2Y
12
in mitogen-activated
protein kinase and Akt kinase signaling pathways.
On the other hand, the functional activity of the
P2Y
1
receptor in long-term serum-starved cells was
recently confirmed by us by their ability to generate
calcium signals. We showed that, in glioma C6 cells
serum- starved for 96 h, there was a tight correlation
between low expression of this receptor and low
C
AB
D
E
Fig. 6. Changes in constitutive ERK1 ⁄ 2 and Akt phosphorylation

during long-term serum starvation (A, B) and the effect of P2Y
12
receptor stimulation on ERK1 ⁄ 2 (C, E) and Akt (D) phosphorylation
in cells adapted for 96 h to serum-free medium. (A) p44 ⁄ p42
ERK1 ⁄ 2 and Akt-Ser473 phosphorylation in glioma C6 cells growing
in MEM with 10% fetal bovine serum (control) or after incubation
in serum-free MEM for 24, 48, 72 and 96 h. (B) Cells cultivated in
serum-free MEM for 96 h followed by long-term recovery (24 h) in
fresh MEM with 10% fetal bovine serum (M+) or in MEM without
serum (M-). (C) p44 ⁄ p42 ERK1 ⁄ 2 phosphorylation in cells cultivated
in serum-free MEM for 96 h and then stimulated by addition of
2MeSADP (10 l
M) up to 180 min. Inset shows inhibition of
2MeSADP-dependent phosphorylation of ERK1 ⁄ 2by10l
M AR-
C69931MX, a P2Y
12
receptor antagonist. The cells were preincubat-
ed for 2 min with the above receptor antagonists and, still in their
presence, stimulated for 15 min with 2MeSADP. (D) Akt-Ser473
phosphorylation in glioma C6 cells cultivated in serum-free MEM
for 96 h and then stimulated by addition of 2MeSADP (10 l
M)or
cultivated with 2MeSADP and 10 l
M AR-C69931MX for up to
180 min (E), cells cultivated in serum-free MEM for 96 h followed
by long-term recovery (24 h) in fresh serum-free medium with addi-
tion of 10 l
M AR-C69931MX [the P2Y
12

receptor antagonist (ARC)],
30 l
M MRS2179 [the P2Y
1
receptor antagonist (MRS)] or both
(ARC + MRS).The cell growing conditions and Western blot analy-
sis were as described in Experimental procedures. b-actin was
used as a control of equal protein loading. The results are represen-
tative of three separate experiments and are mean ± SD.
P2Y
1
and P2Y
12
in serum-deprived glioma C6 P. Krzemin
´
ski et al.
1976 FEBS Journal 274 (2007) 1970–1982 ª 2007 The Authors Journal compilation ª 2007 FEBS
calcium response [24]. However, to eliminate the possi-
bility that ATP secreted by glioma C6 cells, as well as
products of its hydrolysis, might specifically desensitize
P2Y
1
which could eventually modulate its expression
at the transcriptional level, we measured the cons-
titutive release of ATP into the extracellular medium.
Figure 7 shows that, after careful exchange of the med-
ium for one without serum, the ATP concentration
increased slightly, reaching the low nanomolar range.
It then increased to more than 100 nm at 24 h after
the medium exchange. It then slowly decreased and

stayed at  10 nm from 48 h until 96 h. As this value
is low and similar to that observed in the presence of
serum, possible receptor desensitization occurring
through its continuous stimulation seems to be doubt-
ful. What’s more, the ATP concentration in the med-
ium fell over time, reaching control levels after 48 h. A
similar low concentration of ATP released into the
medium by glioma C6 cells and stable for up 12 h was
described by Lazarowski et al. [35].
Discussion
This study shows that long-term serum-starved glioma
C6 cells is characterized by increased expression of the
P2Y
12
receptor and low expression of the P2Y
1
recep-
tor. In cells grown in medium containing 10% (v ⁄ v)
fetal bovine serum, the expression ratio of the two
receptors is reversed.
The different expression of P2Y
1
and P2Y
12
recep-
tors in serum-starved and nonstarved cells seems to
have an important biological significance. Given the
shift in receptor expression, there is simultaneous cell
growth arrest and changes in the adhesion pattern and
the cell morphology. The addition of fetal bovine

serum to cells serum-deprived for 96 h that have
become morphologically altered causes reversion to the
normal fibroblast-like, flat morphology, restores the
expression of the P2Y
1
receptor, and halts growth
arrest. These data indicate that, during very prolonged
serum starvation, glioma C6 cells enter a transient
physiological state in which they stay until there is an
improvement in the inhospitable environment. The
P2Y
1
and P2Y
12
receptors appear to play an important
role in this process. We believe that the shift in expres-
sion of these receptors underlies a new defense mech-
anism of transformed cells, which allows maintenance
of self-sufficiency in growth signal and proliferation
even in unfavorable conditions.
We have previously reported that the P2Y
12
receptor
in glioma C6 cells is negatively coupled to adenylate
cyclase [18,19] and is predominantly involved in
ERK1 ⁄ 2 activation. Furthermore, antagonizing the
P2Y
12
receptor with AR-C69931MX counteracted its
activation by ADP or 2MeSADP [18,22]. Moreover, it

has a stimulatory effect on PI3-K activation, whereas
the effect of P2Y
1
on this kinase has been charac-
terized as inhibitory [22]. Antagonists of the P2Y
1
receptor (pyridoxal phosphate-6-azophenyl-2,4-disulfo-
nic acid and MRS2179) inhibit calcium mobilization
[18,19], but stimulate PI3-K activity [22]. Similar
opposite effects of P2Y
1
and P2Y
12
receptors on
PI3-K activity have been described in human platelets
[36]. It is noteworthy that glioma C6 cells are charac-
terized by a lack of measurable lipid phosphatase
[phosphatase and tensin homolog (PTEN)] [3]. PTEN
dephosphorylates the 3¢ position of phosphoinositides
and acts as an antagonist of PI3-K, resulting in a
reduction of the concentration of this kinase product.
As a high concentration of phosphatidylinositol 3,4,
5-trisphosphate is necessary to increase tumor prolifer-
ation and motility [3], PTEN phosphatase can be
treated as a tumor suppressor [37] and PI3-K as a
tumor enhancer.
In primary astrocytes and glioma C6 cells, an
increase in intracellular cAMP concentration inhibits
ERK1 ⁄ 2 activity, blocks cell growth, and promotes
differentiation [38–42]. Differentiation into an astro-

cyte type II may be induced upon stimulation of
b-adrenergic receptors, positively coupled to adenylate
cyclase. In this case, glioma C6 cells undergo morpho-
logical changes from a bipolar to a round shape,
Time (hours)
0 24487296
nM)( noitartnecnoc PTA
1
10
100
1000
Fig. 7. ATP concentration in medium above serum-starved glioma
C6 cells. ATP concentration was measured by the luciferin–lucif-
erase assay (see Experimental procedures for details). Each time
point is the mean ± SD consisting of six measurements repeats
from three experiments. Zero time represents the ATP concentra-
tion in the culture medium containing serum, before the experi-
ment. Broken line, observed increase in ATP concentration after
exchange of the medium for a serum-free one. Data are shown in
logarithmic scale.
P. Krzemin
´
ski et al. P2Y
1
and P2Y
12
in serum-deprived glioma C6
FEBS Journal 274 (2007) 1970–1982 ª 2007 The Authors Journal compilation ª 2007 FEBS 1977
concomitant with induction of GFAP synthesis, the
astrocytic marker [28]. Furthermore, Claes et al. [28]

indicated that, in glioma C6, the P2Y
12
receptor negat-
ively coupled to adenylate cyclase and-stimulated by
2MeSADP abolished b-adrenergic receptor induced
differentiation. Antagonists that inhibit PI3-K ⁄ Akt
activity also induced C6 glioma differentiation [43].
Thus, serum starvation and the increase in intracellular
cAMP concentration produce similar morphological
changes, whereas serum withdrawal does not increase
GFAP concentration, as shown above.
Coyle [44] reported that glioma C6 cells growing in
the presence of serum may be characterized by two popu-
lations of cells (young oligodendrocyte phenotype and
mixed astrocyte–oligodendrocyte phenotype), whereas
cells adapted to serum-free conditions exhibit more
mature morphology with only one population of cells,
the mixed astrocyte–oligodendrocyte phenotype. Thus,
serum withdrawal changed the phenotype of the cells
but, as we also observed in this study, did not affect
their differentiation, as investigated by oligodendrocyte
progenitor or astrocyte markers.
Concern arises if a low concentration of P2Y
1
can-
not be interpreted as the result of receptor desensitiza-
tion by ATP released by serum-starved cells
themselves. We doubt whether it can, as, first of all, a
raised extracellular concentration of ATP should first
influence the P2Y

2
receptor, and furthermore its meta-
bolite should influence the P2Y
12
receptor as strongly
as P2Y
1
. We do know that the concentration of the
P2Y
2
receptor does not change during long-term serum
starvation [24], and the concentration of the P2Y
12
receptor even increases. What’s more, the observed
changes in protein concentration follow parallel chan-
ges in mRNA concentration [20,22], so these transcrip-
tional changes are probably the result of growth factor
deprivation, not receptor desensitization. Nevertheless,
we cannot exclude the possibility that the ATP
released as the result of the mechanical stress accom-
panying medium exchange [35] is responsible for the
effects observed after replacement of serum-free med-
ium with fresh medium.
Taken together, the data presented here indicate that
the P2Y
12
receptor acts in favor of cell growth and
proliferation because of (a) the reduction in cAMP
concentration, (b) the stimulation of ERK1 ⁄ 2, and (c)
the stimulation of PI3-K ⁄ Akt signaling cascades. As

the P2Y
1
receptor inhibits PI3-K ⁄ Akt signaling, a
decrease in its expression has a similar effect. We pos-
tulate therefore that the shift in P2Y nucleotide recep-
tor expression from P2Y
1
to P2Y
12
observed during
prolonged serum starvation, which leads to a high, sta-
ble basal level of Akt phosphorylation, is part of the
self-sufficiency mechanism of glioma C6 cells, enabling
survival in unfavorable metabolic condition and sta-
ving off cell death.
In conclusion, these data reveal the role of the
P2Y
12
receptor in cell survival. Sak & Illes [8] have
recently reported that a general property of neuronal
malignant cells appears to be the absence of P2Y
1
receptor, which is present in healthy cells. Our results
are consistent with this. Our findings suggest that, in
malignant cells, the lack of the P2Y
1
receptor inhibit-
ing PI3-K ⁄ Akt signaling may play an important role
in maintaining intense pathological proliferation and
invasiveness. Our results also demonstrate that the

change in glioma C6 cell morphology is not part of the
differentiation process to astrocytes or oligodendro-
cytes. One can speculate that the observed changes
may be malignant-like behavior. As, even after 96 h of
serum starvation we can still observe functional cross-
talk between P2Y
1
and P2Y
12
calcium signaling [24], it
would be extremely interesting if similar cross-talk
between P2Y
12
and P2Y
2
receptors exists in malignant
cells devoid of P2Y
1
, or if P2Y
12
receptor function is
limited in those cells to cAMP inhibition.
Our data show that even long-term serum starvation
can be readily reversed. Prasad et al. [45] suggested
that most glioma cells do exhibit mutation, only down-
regulation, of the differentiation gene and therefore
they are capable of differentiating in the presence of
experimental chemical stimuli, e.g. cAMP. However, in
contrast with neuroblastoma, glioma cell s, owing to a de-
fect in the regulatory genes, are unable to maintain a

differentiated phenotype upon removal of the stimulus.
Therefore, their differentiation into astrocyte type II
induced by cAMP is reversible. According to the
authors, such defects may have occurred at a later
stage of tumor development [45].
As P2Y
1
inhibits PI3-K, a tumor enhancer, we
assume that our findings will provide the rationale for
the development of therapeutic strategies specifically
targeting this nucleotide receptor. These data may also
provide a new approach to the current use of glioma
C6 cells as a model for glioma antiangiogenic therapies
[46,47].
Experimental procedures
Materials
MEM, fetal bovine serum, antibiotics, MEM vitamins,
MEM nonessential amino acids, and phosphate buffered
saline (NaCl ⁄ P
i
) were from Sigma Chemical Co. (St Louis,
MO, USA). Complete EDTA-free protease inhibitors
were from Roche Molecular Biochemicals (Mannheim,
P2Y
1
and P2Y
12
in serum-deprived glioma C6 P. Krzemin
´
ski et al.

1978 FEBS Journal 274 (2007) 1970–1982 ª 2007 The Authors Journal compilation ª 2007 FEBS
Germany). Propidium iodide was from Molecular Probes
(Leiden, the Netherlands). Antibodies recognizing the
C-terminus of P2Y
1
and P2Y
12
receptors, Tween 20 and
cell-dissociation solution were obtained from Alomone
Laboratories (Jerusalem, Israel) and Sigma Chemical Co.
Antibodies against Akt and phosphorylated Akt
(Ser473 ⁄ Thr308), phosphorylated p44 ⁄ 42 mitogen-activated
protein kinase (Erk1 ⁄ 2; Thr202 ⁄ Tyr204) and horseradish
peroxidase-conjugated anti-rabbit IgG came from Cell Sig-
nalling (New England Biolabs, Hitchin, UK). Monoclonal
antibodies against GFAP were from Calbiochem (Meudon,
France). Polyclonal antibodies against NG2 proteoglycan,
horseradish peroxidase-conjugated anti-mouse IgG, secon-
dary antibodies against rat IgG and antibodies against
b-actin were from Oncogene Research Products (Darms-
tadt, Germany). Nitrocellulose membrane and enhanced
chemiluminescence detection system came from Amersham
Pharmacia Biotech (Little Chalfont, UK). AR-C69931MX
was a gift from AstraZenca (Wilmington, DE, USA).
MRS2179 tetra-ammonium salt was from Tocris (Ellisville,
MO, USA). 2MeS-ADP and all other reagents were pur-
chased from Sigma Chemical Co.
Cell culture and morphology
Rat glioma C6 cells (passages 40–55) were obtained from
American Type Culture Collection and cultured in MEM

supplemented with 10% (v ⁄ v) fetal bovine serum and
penicillin (50 IUÆmL
)1
), streptomycin (10 lgÆmL
)1
), ampho-
tericin B (25 lgÆmL
)1
) and 2 mml-glutamine in a
humidified atmosphere of 5% CO
2
at 37 °C. For experi-
ments, cells were cultivated in MEM supplemented with
10% (v ⁄ v) fetal bovine serum to reach 80% confluence.
In the case of serum-starved cells, the medium was chan-
ged to MEM without fetal bovine serum for up to 96 h
before the experiment. For morphology changes, the cells
were cultured in MEM with 10% (v ⁄ v) fetal bovine
serum, or for 48, 72 and 96 h in MEM without fetal
bovine serum, and finally allowed to recover for 24 h in
MEM with or without fetal bovine serum. For statistical
analysis of morphology changes during serum deprivation,
altered and control cells were counted from five independ-
ent images taken at each time point. The observations
were performed with a Nikon Diaphot inverted micro-
scope (DIC Nomarski optics equipped with 40 ⁄ 0.55 LWD
objective lens). All experiments were performed at least
twice.
Flow cytometric cell cycle analysis
After treatment, floating cells detached by treatment with

cell-dissociation solution were pooled, centrifuged for
10 min at 200 g, washed with NaCl ⁄ P
i
, and fixed in 70%
(v ⁄ v) ethanol at )20 °C overnight. Before analysis, cells
were centrifuged, washed twice in NaCl ⁄ P
i
, subsequently
suspended in a DNA staining solution (75 lgÆmL
)1
propi-
dium iodide and 50 lgÆmL
)1
RNase A in NaCl ⁄ P
i
), and
incubated for 30 min at room temperature in the dark.
Cells were then analyzed for DNA content with a FAC-
Scalibur flow cytometer using cellquest software (Becton
Dickinson, Warsaw, Poland).
Western blot analysis
For the experiment, cells were grown up to 80% confluency
on 10 mm dishes in MEM containing 10% (v ⁄ v) fetal
bovine serum. At the end of the experiment, cells were
washed in NaCl ⁄ P
i
, detached with cell-dissociation solution
(Sigma), centrifuged at 1500 g for 5 min, and then
resuspended in lysis buffer [50 mm Tris ⁄ HCl, pH 7.5,
120 mm NaCl, 1% Nonidet P40 (Boehringer, Mannheim,

Germany)] containing freshly added proteinase inhibitors
(Roche). Proteins were separated by SDS ⁄ PAGE [10%
(v ⁄ v) polyacrylamide gels]. They were then transferred to
nitrocellulose membranes (Hybond C; Amersham Pharma-
cia Biotech, Milan, Italy), blocked for 1 h at room tempera-
ture with 5% milk in NaCl ⁄ P
i
⁄ Tris-buffered solution
(pH 7.6) ⁄ 0.005% Tween 20 (NaCl ⁄ P
i
-T) and incubated
overnight at 4 °C with the following anti-rabbit sera diluted
in 1% non-fat milk in NaCl ⁄ P
i
-T: P2Y
1
, P2Y
12
(both dilu-
ted 1 : 2500) (Alomone and Sigma Chemical Co); Akt,
phospho-Ser473 Akt, phospho-ERK1 ⁄ 2 (all diluted
1 : 2500) (Cell Signalling); GFAP (Calbiochem). The pri-
mary antibody reaction was followed by 2 h incubation
with the relevant secondary antibodies against rabbit IgG
(1 : 2500) conjugated with horseradish peroxidase, or, in
the case of GFAP, secondary antibody against rat IgG
(1 : 2500) conjugated with horseradish peroxidase (Onco-
gene Research Products). Immunoreactions were analyzed
using enhanced chemiluminescence (Amersham Pharmacia
Biotech) and membrane exposure to X-ray film (Foton,

Warsaw, Poland). Protein molecular mass was determined
with a prestained protein ladder (Fermentas Life Sciences,
Vilnius, Lithuania). Band intensities were determined by
densitometric analysis using ingenius genesnap genetools
software (Syngene Bioimaging, Cambridge, UK). Only sta-
tistically significant immunoblot band intensity data are
reported.
Adhesion measurements
Distribution of the cell adhesion regions was assessed by
the IRM technique at 37 °C. A Nikon Diaphot inverted
microscope equipped with planapochromat 60·⁄1.4 NA
strainless DIC and fluor 40·⁄1.3 NA oil immersion lenses
was used. Interference images were formed by two perpen-
dicular polarizers and 50 ⁄ 50 beamsplitter (Chroma, Rock-
ingham, VT, USA). Images were acquired with a
Retiga 1300 cooled digital camera and aqm advance 6
software (Kinetic Imaging, Rockingham, VT, USA).
P. Krzemin
´
ski et al. P2Y
1
and P2Y
12
in serum-deprived glioma C6
FEBS Journal 274 (2007) 1970–1982 ª 2007 The Authors Journal compilation ª 2007 FEBS 1979
Immunocytochemistry analysis of f-actin
and vinculin distribution
The distribution of vinculin was assessed with a primary
antibody against vinculin (Sigma) detected by secondary
antibody conjugated with Alexa Fluor 488 (Invitrogen, Lei-

den, the Netherlands). At the end of the experiment, cells
were washed in NaCl ⁄ P
i
and fixed in freshly prepared
3.87% paraformaldehyde in NaCl ⁄ P
i
for 30 min at room
temperature. After the NaCl ⁄ P
i
wash, cells were permeabi-
lized in 0.01% (volume) Triton X-100 for 30 s and washed
again in NaCl ⁄ P
i
. Before overnight incubation with primary
anti-rabbit IgG against vinculin at 4 °C, cells were blocked
in 2.5% fetal bovine serum for 1 h at room temperature.
After overnight incubation with antibody against vinculin
(1 : 1000 dilution), cells were washed three times in NaCl ⁄ P
i
and subjected to secondary antibody against rabbit IgG
(1 : 500 dilution) together with phalloidin (1 : 500 dilution)
conjugated with tetramethyl rhodamine isothiocyanate
(TRITC; Sigma). After 2 h incubation at room temperature,
cells were washed with NaCl ⁄ P
i
and mounted in mounting
medium (as described previously). For NG2 proteoglycan
staining, Oncogene Research Products primary polyclonal
antibodies were used together with Alexa Fluor 546 from
Invitrogen. Observations were made using a Nikon Diaphot

inverted microscope equipped with Chroma Technology
Corp. filter sets and PlanApo 60·⁄1.4 oil immersion lens.
Measurement of ATP concentration in
extracellular medium
Medium from above the cells was collected very delicately
and without moving the Petri dish from the incubator. The
ATP concentration in the medium was measured using the
luciferin–luciferase assay (ATP Determination Kit; Biaffin
GmbH, Kassel, Germany) according to the manufacturer’s
protocol. For bioluminescence measurement, the TD-20 ⁄ 20
luminometer (Turner Designs, Sunnyvale, CA, USA) was
used. Standard curves were performed routinely using
molecular biology grade ATP, which was diluted in the
same buffer and processed in parallel with cell samples. The
threshold value for ATP detection was 0.1 pm, and log
luminescence intensity was exponentially associated with
ATP concentration to 100 pm. Appropriate dilutions of
medium were used when necessary.
Statistical analysis
For al l densitometry assays, standard deviation was calculated.
Acknowledgements
This study was supported by grant no. 401 085
32 ⁄ 1945 from the Ministry of Science and Higher Edu-
cation, Poland.
References
1 Brismar T (1995) Physiology of transformed glial cells.
Glia 15, 231–243.
2 Grobben B, De Deyn PP & Slegers H (2002) Rat C6
glioma as experimental model system for the study of
glioblastoma growth and invasion. Cell Tissue Res 310,

257–270.
3 Kubiatowski T, Jang T, Lachyankar MB, Salmonsen R,
Nabi RR, Quesenberry PJ, Litofsky NS, Ross AH &
Recht LD (2001) Association of increased phosphatidy-
linositol 3-kinase signaling with increased invasiveness
and gelatinase activity in malignant gliomas. J Neuro-
surg 95, 480–488.
4 Vajkoczy P, Goldbrunner R, Farhadi M, Vince G,
Schilling L, Tonn JC, Schmiedek P & Menger MD
(1999) Glioma cell migration is associated with glioma-
induced angiogenesis in vivo. Int J Dev Neurosci 17,
557–563.
5 Burnstock G, Cocks T, Kasakov L & Wong HK (1978)
Direct evidence for ATP release from non-adrenergic,
non-cholinergic (‘purinergic’) nerves in the guinea-pig
taenia coli and bladder. Eur J Pharmacol 49, 145–149.
6 Abbracchio MP & Burnstock G (1994) Purinoceptors:
are there families of P2X and P2Y purinoceptors?
Pharmacol Ther 64, 445–475.
7 Boeynaems JM, Communi D, Savi P & Herbert JM
(2000) P2Y receptors: in the middle of the road. Trends
Pharmacol Sci 21, 1–3.
8 Sak K & Illes P (2005) Neuronal and glial cell lines as
model systems for studying P2Y receptor pharmacology.
Neurochem Int 47, 401–412.
9 Boarder MR & Hourani SM (1998) The regulation of
vascular function by P2 receptors: multiple sites and
multiple receptors. Trends Pharmacol Sci 19, 99–107.
10 Boyer JL, Lazarowski ER, Chen XH & Harden TK
(1993) Identification of a P2Y-purinergic receptor that

inhibits adenylyl cyclase. J Pharmacol Exp Ther 267,
1140–1146.
11 Grobben B, Claes P, Van Kolen K, Roymans D,
Fransen P, Sys SU & Slegers H (2001) Agonists of the
P2Y (AC)-receptor activate MAP kinase by a ras-inde-
pendent pathway in rat C6 glioma. J Neurochem 78,
1325–1338.
12 Pianet I, Merle M & Labouesse J (1989) ADP and,
indirectly, ATP are potent inhibitors of cAMP produc-
tion in intact isoproterenol-stimulated C6 glioma cells.
Biochem Biophys Res Commun 163, 1150–1157.
13 Schachter JB, Boyer JL, Li Q, Nicholas RA & Harden
TK (1997) Fidelity in functional coupling of the rat
P2Y1 receptor to phospholipase C. Br J Pharmacol 122,
1021–1024.
14 Schachter JB, Li Q, Boyer JL, Nicholas RA & Harden
TK (1996) Second messenger cascade specificity and
P2Y
1
and P2Y
12
in serum-deprived glioma C6 P. Krzemin
´
ski et al.
1980 FEBS Journal 274 (2007) 1970–1982 ª 2007 The Authors Journal compilation ª 2007 FEBS
pharmacological selectivity of the human P2Y1-purino-
ceptor. Br J Pharmacol 118, 167–173.
15 Communi D, Gonzalez NS, Detheux M, Brezillon S,
Lannoy V, Parmentier M & Boeynaems JM (2001)
Identification of a novel human ADP receptor coupled

to G (i). J Biol Chem 276, 41479–41485.
16 Hollopeter G, Jantzen HM, Vincent D, Li G, England
L, Ramakrishnan V, Yang RB, Nurden P, Nurden A,
Julius D & Conley PB (2001) Identification of the plate-
let ADP receptor targeted by antithrombotic drugs.
Nature 409, 202–207.
17 Savi P, Labouret C, Delesque N, Guette F, Lupker J &
Herbert JM (2001) P2y (12), a new platelet ADP recep-
tor, target of clopidogrel. Biochem Biophys Res Commun
283, 379–383.
18 Czajkowski R, Lei L, Sabala P & Baranska J (2002)
ADP-evoked phospholipase C stimulation and adenylyl
cyclase inhibition in glioma C6 cells occur through two
distinct nucleotide receptors, P2Y (1) and P2Y (12).
FEBS Lett 513, 179–183.
19 Sabala P, Czajkowski R, Przybylek K, Kalita K,
Kaczmarek L & Baranska J (2001) Two subtypes of G
protein-coupled nucleotide receptors, P2Y (1) and P2Y
(2) are involved in calcium signalling in glioma C6 cells.
Br J Pharmacol 132, 393–402.
20 Baranska J, Czajkowski R & Sabala P (2004) Cross-
talks between nucleotide receptor-induced signaling
pathways in serum-deprived and non-starved glioma C6
cells. Adv Enzyme Regul 44, 219–232.
21 Grobben B, Anciaux K, Roymans D, Stefan C, Bollen
M, Esmans EL & Slegers H (1999) An ecto-nucleotide
pyrophosphatase is one of the main enzymes involved in
the extracellular metabolism of ATP in rat C6 glioma.
J Neurochem 72, 826–834.
22 Czajkowski R, Banachewicz W, Ilnytska O, Drobot LB

& Baranska J (2004) Differential effects of P2Y1 and
P2Y12 nucleotide receptors on ERK1 ⁄ ERK2 and phos-
phatidylinositol 3-kinase signalling and cell proliferation
in serum-deprived and nonstarved glioma C6 cells. Br J
Pharmacol 141, 497–507.
23 Czajkowski R & Baranska J (2002) Cross-talk between
the ATP and ADP nucleotide receptor signalling path-
ways in glioma C6 cells. Acta Biochim Pol 49, 877–
889.
24 Supłat D, Krzemin
´
ski P, Pomorski P & Baran
´
ska J
(2007) P2Y1 and P2Y12 receptors cross-talk in calcium
signalling: evidence from non-starved and long-term
serum-deprived glioma C6 cells. Purinergic Signalling in
press.
25 Hanahan D & Weinberg RA (2000) The hallmarks of
cancer. Cell 100, 57–70.
26 Chou RC & Langan TJ (2003) In vitro synchronization
of mammalian astrocytic cultures by serum deprivation.
Brain Res Brain Res Protoc 11, 162–167.
27 Tu MT, Luo SF, Wang CC, Chien CS, Chiu CT,
Lin CC & Yang CM (2000) P2Y (2) receptor-mediated
proliferation of C (6) glioma cells via activation of
Ras ⁄ Raf ⁄ MEK ⁄ MAPK pathway. Br J Pharmacol 129,
1481–1489.
28 Claes P, Van Kolen K, Roymans D, Blero D,
Vissenberg K, Erneux C, Verbelen JP, Esmans EL &

Slegers H (2004) Reactive blue 2 inhibition of cyclic
AMP-dependent differentiation of rat C6 glioma cells
by purinergic receptor-independent inactivation of
phosphatidylinositol 3-kinase. Biochem Pharmacol 67,
1489–1498.
29 Oey J (1975) Noradrealine induces morphological alter-
ations in nucleated and enucleated rat C6 glioma cells.
Nature 257, 317–319.
30 Dahl D (1981) The vimentin-GFA protein transition in
rat neuroglia cytoskeleton occurs at the time of myelina-
tion.
J Neurosci Res 6, 741–748.
31 Messens J & Slegers H (1992) Synthesis of glial fibrillary
acidic protein in rat C6 glioma in chemically defined
medium: cyclic AMP-dependent transcriptional and
translational regulation. J Neurochem 58, 2071–2080.
32 Lee K, Kentroti S, Billie H, Bruce C & Vernadakis A
(1992) Comparative biochemical, morphological, and
immunocytochemical studies between C-6 glial cells of
early and late passages and advanced passages of glial
cells derived from aged mouse cerebral hemispheres.
Glia 6, 245–257.
33 Deng W & Poretz RD (2003) Oligodendroglia in devel-
opmental neurotoxicity. Neurotoxicology 24, 161–178.
34 Krzeminski P, Misiewicz I, Pomorski P, Kasprzycka-
Guttman T & Baranska J (2007) Mitochondrial localiza-
tion of P2Y1, P2Y2 and P2Y12 receptors in rat astrocytes
and glioma C6 cells. Brain Res Bull 71, 587–592.
35 Lazarowski ER, Boucher RC & Harden TK (2000)
Constitutive release of ATP and evidence for major

contribution of ecto-nucleotide pyrophosphatase and
nucleoside diphosphokinase to extracellular nucleotide
concentrations. J Biol Chem 275, 31061–31068.
36 Hardy AR, Jones ML, Mundell SJ & Poole AW (2004)
Reciprocal cross-talk between P2Y1 and P2Y12 recep-
tors at the level of calcium signaling in human platelets.
Blood 104, 1745–1752.
37 Cantley LC & Neel BG (1999) New insights into tumor
suppression: PTEN suppresses tumor formation by
restraining the phosphoinositide 3-kinase ⁄ AKT path-
way. Proc Natl Acad Sci USA 96, 4240–4245.
38 Dugan LL, Kim JS, Zhang Y, Bart RD, Sun Y,
Holtzman DM & Gutmann DH (1999) Differential
effects of cAMP in neurons and astrocytes. Role of
B-raf. J Biol Chem 274, 25842–25848.
39 Kim S, Jee K, Kim D, Koh H & Chung J (2001) Cyclic
AMP inhibits Akt activity by blocking the membrane
localization of PDK1. J Biol Chem 276, 12864–12870.
P. Krzemin
´
ski et al. P2Y
1
and P2Y
12
in serum-deprived glioma C6
FEBS Journal 274 (2007) 1970–1982 ª 2007 The Authors Journal compilation ª 2007 FEBS 1981
40 Kurino M, Fukunaga K, Ushio Y & Miyamoto E
(1996) Cyclic AMP inhibits activation of mitogen-
activated protein kinase and cell proliferation in
response to growth factors in cultured rat cortical astro-

cytes. J Neurochem 67, 2246–2255.
41 Qiu W, Zhuang S, von Lintig FC, Boss GR & Pilz RB
(2000) Cell type-specific regulation of B-Raf kinase by
cAMP and 14-3-3 proteins. J Biol Chem 275, 31921–
31929.
42 Wang L, Liu F & Adamo ML (2001) Cyclic AMP inhi-
bits extracellular signal-regulated kinase and phosphati-
dylinositol 3-kinase ⁄ Akt pathways by inhibiting Rap1.
J Biol Chem 276, 37242–37249.
43 Van Kolen K & Slegers H (2004) P2Y12 receptor stimu-
lation inhibits beta-adrenergic receptor-induced differen-
tiation by reversing the cyclic AMP-dependent inhibition
of protein kinase B. J Neurochem 89, 442–453.
44 Coyle DE (1995) Adaptation of C6 glioma cells to
serum-free conditions leads to the expression of a mixed
astrocyte-oligodendrocyte phenotype and increased pro-
duction of neurite-promoting activity. J Neurosci Res
41, 374–385.
45 Prasad KN, Hovland AR, Nahreini P, Cole WC,
Hovland P, Kumar B & Prasad KC (2001) Differen-
tiation genes: are they primary targets for human
carcinogenesis? Exp Biol Med (Maywood) 226,
805–813.
46 Auguste P, Gursel DB, Lemiere S, Reimers D, Cuevas
P, Carceller F, Di Santo JP & Bikfalvi A (2001)
Inhibition of fibroblast growth factor ⁄ fibroblast
growth factor receptor activity in glioma cells
impedes tumor growth by both angiogenesis-dependent
and -independent mechanisms. Cancer Res 61, 1717–
1726.

47 Machein MR, Risau W & Plate KH (1999) Antiangio-
genic gene therapy in a rat glioma model using a domi-
nant-negative vascular endothelial growth factor
receptor 2. Hum Gene Ther 10, 1117–1128.
P2Y
1
and P2Y
12
in serum-deprived glioma C6 P. Krzemin
´
ski et al.
1982 FEBS Journal 274 (2007) 1970–1982 ª 2007 The Authors Journal compilation ª 2007 FEBS