2-ClATP exerts anti-tumoural actions not mediated by P2 receptors in neuronal and glial cell lines

Article abstract of DOI:10.1016/j.bcp.2003.09.015

We investigated the effects of the ATP analogue and P2 receptor agonist 2-ClATP on growth and survival of different neuronal (PC12, PC12nnr5 and SH-SY5Y) and glial (U87 and U373) cell lines, by the use of direct count of intact nuclei, fluorescence microscopy, fluorescence-activated cell sorter analysis (FACS) and high pressure liquid chromatography (HPLC). 2-ClATP lowered the number of cultured PC12nnr5, SH-SY5Y, U87 and U373 cells to almost 5%, and of PC12 cells to about 35% after 3-4 days of treatment. EC₅₀ was in the 5-25μM range, with 2-ClATP behaving as a cytotoxic or cytostatic agent. Analysis of the biological mechanisms demonstrated that pyridoxalphosphate-6- azophenyl-2′,4′-disulphonic acid (P2 receptor antagonist and nucleotidases inhibitor), but not Caffeine or CGS-15493 (P1 receptor antagonists) effectively prevented 2-ClATP-induced toxicity. 2-ClATP metabolic products (2-ClADP, 2-ClAMP, 2-Cladenosine) and new synthesis derivatives (2-CldAMP, 2-Cldadenosine-3′,5′-bisphosphate and 2-CldATP) exerted similar cytotoxic actions. Inhibition of both serum nucleotidases and purine nucleoside transporters strongly reduced 2-ClATP-induced cell death, which was conversely increased by the nucleotide hydrolyzing enzyme apyrase. The adenosine kinase inhibitor 5-iodotubericidin totally prevented 2-ClATP or 2-Cladenosine-induced toxicity. In summary, our findings indicate that 2-ClATP exerts either cell cycle arrest or cell death, acting neither on P2 nor on P1 receptors, but being extracellularly metabolized into 2-Cladenosine, intracellularly transported and re-phosphorylated.

Full text of DOI:10.1016/j.bcp.2003.09.015

Biochemical Pharmacology 67 (2004) 621–630
2-ClATP exerts anti-tumoural actions not mediated by
P2 receptors in neuronal and glial cell lines
Nadia D’Ambrosia,b, Stefano Costanzic, Daniela F. Angelinid, Rosaria Volpinic,
b
Giuseppe Sancesario , Gloria Cristalli , Cinzia Volonte
c
a,e,*
´
aFondazione Santa Lucia, Cellular Neurobiology Unit, Rome, Italy
bDepartment of Neuroscience, University of Rome Tor Vergata, Rome, Italy
cDepartment of Chemical Sciences, University of Camerino, Camerino, Italy
dFondazione Santa Lucia, Neuroimmunology Unit, Rome, Italy
eC.N.R. Institute of Neurobiology and Molecular Medicine, Rome, Italy
Received 14 May 2003; accepted 17 September 2003
Abstract
We investigated the effects of the ATP analogue and P2 receptor agonist 2-ClATP on growth and survival of different neuronal (PC12,
PC12nnr5 and SH-SY5Y) and glial (U87 and U373) cell lines, by the use of direct count of intact nuclei, fluorescence microscopy,
fluorescence-activated cell sorter analysis (FACS) and high pressure liquid chromatography (HPLC). 2-ClATP lowered the number of
cultured PC12nnr5, SH-SY5Y, U87 and U373 cells to almost 5%, and of PC12 cells to about 35% after 3–4 days of treatment. EC50 was in
the 5–25 mM range, with 2-ClATP behaving as a cytotoxic or cytostatic agent. Analysis of the biological mechanisms demonstrated
that pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid (P2 receptor antagonist and nucleotidases inhibitor), but not Caffeine or
CGS-15493 (P1 receptor antagonists) effectively prevented 2-ClATP-induced toxicity. 2-ClATP metabolic products (2-ClADP, 2-ClAMP,
2-Cladenosine) and new synthesis derivatives (2-CldAMP, 2-Cldadenosine-30,50-bisphosphate and 2-CldATP) exerted similar cytotoxic
actions. Inhibition of both serum nucleotidases and purine nucleoside transporters strongly reduced 2-ClATP-induced cell death, which
was conversely increased by the nucleotide hydrolyzing enzyme apyrase. The adenosine kinase inhibitor 5-iodotubericidin totally
prevented 2-ClATP or 2-Cladenosine-induced toxicity.
In summary, our findings indicate that 2-ClATP exerts either cell cycle arrest or cell death, acting neither on P2 nor on P1 receptors, but
being extracellularly metabolized into 2-Cladenosine, intracellularly transported and re-phosphorylated.
# 2003 Elsevier Inc. All rights reserved.
Keywords: PC12 cells; SH-SY5Y cells; U87, U373 cells; Cell death; Adenosine kinase
1. Introduction
to the molecular repertoire of the plasma membrane. In
particular, significant are the presence of P1-adenosine
receptors [1], P2-ATP receptors [2], nucleoside transpor-
ters [3] and degrading enzymes, such as ecto-nucleoti-
dases [4] or nucleoside-deaminases [5]. The variety and
interplay of these activities contribute to render the extra-
cellular milieu a very dynamic and heterogeneous source
of signaling inputs. As a matter of fact, ATP is a very
unstable compound, being metabolized by ecto-nucleoti-
dases to generate several intermediate metabolites to the
final product adenosine [6]. ATP and adenosine have been
in turn recognized as molecules with both trophic and
toxic actions in different cell types. It was actually
reported that extracellular ATP and its analogs can support
cell survival and neurite regeneration in PC12 cells [7],
Nucleotides and nucleosides represent a class of signal-
ing molecules in essentially all tissues. They can exert
different biological functions in various systems, accord-
ing to the extracellular environment of each target cell and
* Corresponding author. Tel.: þ39-06-5150-1557;
fax: þ39-06-5150-1556.
´
E-mail address: cinzia@in.rm.cnr.it (C. Volonte).
Abbreviations: 2-ClAdo, 2-Cladenosine; FCS, fetal calf serum; HS,
horse serum; 50IT, 5-iodotubericidin; NBTI, nitrobenzyl-6-thioinosine; NGF,
nerve growth factor; PBS, phosphate-buffered saline; PF1, 2-ClAMP; PF3,
2-CldAMP; PF4, 2-CldAdo-30,50-bisphosphate; PF5, 2-CldATP; PF10,
2-ClADP; PPADS, pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid;
PI, propidium iodide.
0006-2952/$ – see front matter # 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2003.09.015

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regulate the differentiation of skeletal muscle cells [8], and
induce proliferation of different cell types [9–14]. While
these trophic effects seem to be mediated by P2 receptors,
the toxic actions exerted by extracellular ATP can be due
to P2 receptor activation [15,16] and/or additional path-
ways [17–20]. For instance, P2X7 is mainly characterized
as a cytolytic pore in macrophages and lymphocytes [21],
while P2 receptor-mediated toxicity generally involves the
activation of multiple receptor subclasses. Cell death
induced by P2 receptors can also result from indirect
mechanisms, such as release of pro-inflammatory cyto-
kines in macrophages and microglia [22,23]. On the other
hand, adenosine can induce cell death both by binding to
P1 receptors [24–26] or being intracellularly transported
to produce ATP through the salvage pathway [27,28].
Although more stable than ATP itself, 2-ClATP can be
degraded by ATP-diphosphohydrolases to finally generate
the deaminase-resistant compound, 2-Cladenosine (2-
ClAdo) [29]. This last, in contrast to the trophic actions
exerted by 2-ClATP, is known to possess anti-tumoural and
cytotoxic effects in several cells [30,31]. In addition, the 20-
deoxyderivative of 2-ClAdo (cladribine) is clinically used
for the treatment of indolent lymphoid malignancies and,
in particular, against hairy cell leukemia [32]. The mole-
cular mechanisms responsible for the antiproliferative
effects induced by 2-chloroderivatives of adenosine are
generally mediated by intracellular pathways [33], albeit
the activation of extracellular adenosine receptors has also
been reported [34].
Here we describe the synthesis of PF3, PF5, and PF10.
Briefly, the nucleoside monophosphates were synthesized
from the corresponding nucleosides by means of the
Yoshikawa reaction [37], utilizing POCl3 as phosphorylat-
ing agent (Fig. 1). On the other hand, the synthesis of the
nucleosides polyphosphates was performed treating the
corresponding monophosphates, previously activated by
reaction with 1-10-carbonyldiimidazole, with ammonium
monophosphate or pyrophosphate (Fig. 1) [38].
2.2.1. General synthetic procedures
1H and 31P nuclear magnetic resonance (NMR) spectra
were obtained with Varian VXR 300 MHz spectrometer; d
in ppm, J in Hz. All exchangeable protons were confirmed
by addition of D2O. The reactions were monitored by thin
layer chromatography (TLC), using pre-coated TLC plates
with silica gel 60 F-254 (Merck) and iC3H7OH–H2O–
NH4OH (30%) (5.5/1/3.5) as mobile phase. The nucleo-
tides were purified by means of ionic exchange chromato-
graphy on a Sephadex DEAE A-25 column (HCO3À form)
equilibrated with H2O and eluted with a linear gradient of
H2O/0.5 M NH4HCO3.
2.2.2. 2-Chloro-20-deoxyadenosine-50-monophosphate
(2-CldAMP, PF3)
To 86 mg of 2-Cl-20-deoxyadenosine (0.30 mmol) dis-
solved in 1.5 mL of trimethyl phosphate were added 4
equivalents of POCl3 (112 mL, 184 mg, 1.2 mmol). The
solution was stirred 45 min at 08, H2O was added (1.5 mL)
and the solution was neutralized by adding triethylamine
dropwise.
After ion exchange chromatography, 72 mg of PF3
(0.19 mmol, 63%) were obtained. The reaction gave also
34 mg of PF4 (0.07 mmol, 23%) as secondary product.
1H NMR (D2O) d 2.54 and 2.72 (1H each, m, H-20), 3.97
(2H, m, H-50), 4.22 (1H, m, H-40), 4.66 (1H, m, H-30), 6.35
(1H, t, H-10, J ¼ 6:96 Hz), 8.38 (1H, s, H-8).
31P NMR (D2O) d 1.83 (s).
In this work, we studied the actions of extracellular
nucleotides on the growth and survival of phaeochromo-
cytoma PC12 and variant PC12nnr5, on neuroblastoma
SH-SY5Y and on glioma U87 and U373 cell lines. In
particular, we analyzed the effects induced by the ATP
analogue 2-ClATP, previously shown to induce differentia-
tion and survival of PC12 cells [7] mainly acting on P2
receptors. We now demonstrate that 2-ClATP induces
cytostaticity or cytotoxicity not via P2 or P1 receptors,
but through the extracellular formation of 2-ClAdo, its
consequent intracellular transport and re-phosphorylation.
2.2.3. 2-Chloro-20-deoxyadenosine-50-trisphosphate
(2-CldATP, PF5)
To 56 mg of PF3 (0.15 mmol), dissolved in 1 mL of dry
dimethylformamide (DMF), were added 36 mL of tri-n-
butylamine (28 mg, 0.15 mmol). The solution was stirred
for 20 min at room temperature and then evaporated to
dryness under anhydrous conditions. After resuspension
in 1.4 mL of dry DMF, 1-10-carbonyldiimidazole (122 mg,
0.75 mmol) was added and the mixture was stirred for
3 hr at room temperature. Methanol (49 mL, 38.5 mg
1.2 mmol) was added and left for 30 min. Then 6 mL of
0.5 M bis(tri-n-butylammonium)pyrophosphate in DMF
(3 mmol) was added and the mixture was left for 14 hr at
room temperature. The solvent was removed in vacuo,
the mixture dissolved in H2O and purified by means of ion
exchange chromatography to obtain 44 mg of PF5
(0.07 mmol, 47%).
2. Materials and methods
2.1. Materials
All drugs used in this work were purchased from Sigma–
Aldrich, except XAMR 0721, which was acquired from
Calbiochem.
2.2. Synthesis of 2-chloroderivatives of adenosine
phosphates
PF1 [35] and PF4 [36] were synthesized following
procedures similar to those described in the literature.
The analytical data are consistent with those reported.

N. D’Ambrosi et al. / Biochemical Pharmacology 67 (2004) 621–630
623
Fig. 1. Schematic view of new-synthesis 2-chloroderivatives of adenosine phosphate. The compounds were obtained with synthesis procedures described in
Section 2.
1H NMR (D2O) d 2.76 (2H, m, H-20), 4.23 (2H, m, H-50),
4.53 (1H, m, H-40), 5.48 (1H, m, H-30), 6.40 (1H, t, H-10,
J ¼ 6:46 Hz), 8.47 (1H, s, H-8).
31P NMR (D2O) d À10.63 (m, Pb), À10.12 (m, Pa).
2.3. Cell cultures
31P NMR (D2O) d À22.42 (m, Pb), À10.81 (d, Pg),
À10.02 (d, Pa).
Rat phaeochromocytoma PC12 and PC12nnr5 cell lines
were cultured on collagen-coated dishes in RPMI 1640
medium supplemented with 10% heat-inactivated HS and
5% FCS; human neuroblastoma SH-SY5Y cells were grown
in Dulbecco’s modified Eagle’s medium (DMEM)/F12,
supplemented with 15% FCS; human glioma U87 and
U373 cell lines were grown in 10% FCS in DMEM. All
culture media were supplemented with glutamine (2 mM),
penicillin (50 U/mL), and streptomycin (50 mg/mL) and all
cell lines grown at 378 in 5% CO2.
2.2.4. 2-Chloroadenosine-50-diphosphate (2-ClADP,
PF10)
To 60 mg of 2-ClAMP (0.15 mmol), dissolved in 1 mL
of dry DMF, were added 36 mL of tri-n-butylamine (28 mg,
0.15 mmol). The solution was stirred for 20 min at room
temperature and then evaporated to dryness under anhy-
drous conditions. After resuspension in 1.4 mL of dry
DMF, 1-10-carbonyldiimidazole (122 mg, 0.75 mmol)
was added and the mixture was stirred for 3 hr at room
temperature. Methanol (49 mL, 38.5 mg, 1.2 mmol) was
added and left for 30 min. Then 6 mL of 0.5 M (tri-n-
butylammonium)phosphate in DMF (3 mmol) was added
and the mixture was left for 14 hr at room temperature. The
solvent was removed in vacuo, the mixture dissolved in
H2O and purified by means of ion exchange chromato-
graphy to obtain 30 mg of 2-ClADP (PF10, 0.058 mmol,
39%).
2.4. Cell survival assay
Cells were grown in 24-well flat bottom plates at a
density of 3 ꢀ 105 per well, in the presence of the appro-
priate medium (15% sera), as cited above, or 1.5% sera
(1% heat-inactivated HS, 0.5% FCS) or inactivated 15%
sera (568 for 90 min). Drugs were added 4 hr after seeding
and the cell number was evaluated after various lengths of
times by direct count of intact nuclei [39]. Briefly, the
method is based on the solubilization of cellular mem-
branes by a mild detergent, followed by direct count of all
1H NMR (D2O) d 4.07 (2H, m, H-50), 4.24 (1H, m, H-40),
4.40 (1H, m, H-30), 4.59 (1H, t, H-20, J ¼ 4:58 Hz), 6.00
(1H, m, H-10, J ¼ 5:60 Hz), 8.34 (1H, s, H-8).

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N. D’Ambrosi et al. / Biochemical Pharmacology 67 (2004) 621–630
intact nuclei. In all cases, triplicate wells were scored and
counts represent means ꢁ SEM.
2.9. Statistical analysis
Statistical differences were verified by one-way analysis
of variance and P < 0:05 was considered significant:
ꢂP < 0:05, ꢂꢂP < 0:01, ꢂꢂꢂP < 0:001.
¨
2.5. Chromatin staining by Hoechst 33258
PC12 and PC12nnr5 cells seeded on 35 mm plates were
kept with or without 100 mM 2-ClATP for 24 hr. Cells were
then fixed for 15 min with 4% paraformaldehyde in PBS,
washed three times with PBS and incubated for 10 min
3. Results
¨
with 1 mg/mL Hoechst 33258. After three final washes in
PBS, nuclear condensation was visualized under UV light,
using fluorescence microscopy.
3.1. Extracellular 2-ClATP reduces phaeochromocytoma,
neuroblastoma and glioma cell number in culture
To study the biological effects of the slowly hydroly-
sable ATP analogue 2-ClATP on different models of
neuronal and glial tumoural cells, a single dose of
100 mM was added to the phaeochromocytoma PC12
and PC12nnr5 cells, to the neuroblastoma SH-SY5Y and
to the glioma U87 and U373 cells, in the presence of
optimal sera concentrations. As shown in Fig. 2A, 2-ClATP
always inhibited cell growth, as detected by count of
viable, intact nuclei after 4 days of treatment. Maximal
effect (90–100% inhibition) was obtained with PC12nnr5,
SH-SY5Y, U87 and U373 cells, while about 65% inhibition
was observed with PC12 cells.
2.6. Propidium iodide staining and FACS analysis
PC12 and PC12nnr5 cells, cultured on 24-well plates,
were treated with or without 100 mM 2-ClATP for different
times and were then collected and centrifuged for 10 min at
200 g. The pellet was resuspended in 1 mL PBS/5 mM
EDTA, dissociated thoroughly and centrifuged. The cells
were fixed in ice-cold 70% ethanol in PBS, kept at À208 for
more than 30 min, spun for 10 min at 200 g and incubated
for 30 min at room temperature with 1 mg/mL PI in PBS,
also containing 0.1% (v/v) Triton X-100 and 2 mg/mL
DNase-free RNase A (Sigma–Aldrich). Flow cytometry
was then performed with FACSCalibur (BD Biosciences)
flow cytometer. The data were analyzed using CellQuest
3.3 software. For the purpose of analysis, acquired events
were gated to eliminate cell aggregates.
3.2. 2-ClATP exerts a cytotoxic effect in PC12nnr5 and
a cytostatic action in PC12 cells
We further analyzed the inhibition of cell growth
induced by 2-ClATP and compared PC12 to PC12nnr5
cells. The two cell lines are apparently similar, being
PC12nnr5 a PC12 variant mostly lacking the Nerve
Growth Factor (NGF)-high affinity TrkA receptor
[40,41]. In the presence of sera, 2-ClATP inhibited cell
growth in 3 days, with EC50 at about 5 mM (for PC12 cells)
or 25 mM (for PC12nnr5 cells) and with maximal effects at
100 mM (data not shown). Time-course experiments
showed that 2-ClATP reduced PC12nnr5 cells of about
20% in 24 hr, 60% in 48 hr and 100% in 3 days (Fig. 2B).
Conversely, the number of PC12 cells was maintained
constant up to at least 7 days (Fig. 2C). These results were
confirmed by morphological observations: while 2-
ClATP-treated PC12nnr5 cultures showed cellular debris,
shrunken, phase-dark and floating cells, PC12 cells
remained instead round and phase-bright (data not shown).
In order to assess whether the effect of 2-ClATP was to
induce cell death, to arrest cell cycle or both, we performed
the following experiments. PC12nnr5 and PC12 cells were
2.7. [3H]thymidine incorporation
PC12 cells in complete medium were seeded on 96-well
plate (5 ꢀ 104 cells/well) in the presence or absence of
100 mM 2-ClATP. [3H]thymidine (25 mCi/mL, final con-
centration 10 mM) was added to the cultures 8 hr after
plating. At different times thereafter, the cells were
washed, harvested and DNA isolated on fiberglass filters
(Wallac) using a Tomtek 96-well harvester. The filters were
then air-dried and assessed for specifically bound radio-
activity, by liquid scintillation counting.
2.8. HPLC analysis
For the measurement of 2-ClATP breakdown, PC12nnr5
cells cultured on 90 mm dishes in the presence of 15% sera,
were exposed to 1 mM 2-ClATP. At different times, sam-
ples from the culture medium were collected, filtered on
0.2 mm NY membranes and analyzed by HPLC with a HP
1090 Liquid Chromatograph series II. Separations were
carried out on a 150 mm ꢀ 4:60 mm Phenomenex Sinergy
¨
incubated with 100 mM 2-ClATP and stained with Hoechst
33258 to analyze nuclear DNA condensation (Figs. 3A
and 4A). Typical apoptotic nuclei (indicated by arrows)
with DNA at different levels of condensation were
observed in PC12nnr5 (Fig. 3A) but not in PC12 cells
(Fig. 4A). We further assessed DNA fragmentation and
ploidy, by staining with PI and performing subsequent
cytofluorimetric analysis. As indicated in Fig. 3B, control
˚
4 mm Polar-RP 80 A column. The elution solvent was
CH3COOH 2% in H2O:CH3OH 75:25, the injection volume
was 20 mL, the flow rate was 700 mL/min and the detector
was DAD l1 ¼ 265 nm, l2 ¼ 254 nm, l3 ¼ 275 nm,
lr ¼ 550 nm.

N. D’Ambrosi et al. / Biochemical Pharmacology 67 (2004) 621–630
625
Table 1
Action of P2X (ꢂ) and P2Y (8) antagonists on 100 mM 2-ClATP-induced
toxicity in PC12nnr5 cells
Antagonist
Percent of intact nuclei
(ꢂ) oATP 50 mM
3 ꢁ 1
5 ꢁ 2
(ꢂ) DIDS 200 mM
(ꢂ) Brilliant Blue G 30 mM
(ꢂ) Calmidazolium 1 mM
(ꢂ) TNP-ATP 120 mM
(ꢂ) MRS 2159 100 mM
(ꢂ) NF 023 100 mM
(ꢂ) PPNDS 300 mM
(8) PIT 10 mM
7 ꢁ 3
1 ꢁ 0.5
8 ꢁ 0.5
9 ꢁ 3
4 ꢁ 3
47 ꢁ 1***
4 ꢁ 2
(8) MRS 2179 10 mM
(8) XAMR 0721 100 mM
(ꢂ,8) Suramine 300 mM
(ꢂ,8) P5P 200 mM
8 ꢁ 0.5
7 ꢁ 1
10 ꢁ 2
9 ꢁ 1
(ꢂ,8) PPADS 300 mM
(ꢂ,8) RB-2 300 mM
74 ꢁ 7.5***
9 ꢁ 3
PC12nnr5 cells were incubated in the simultaneous presence of
100 mM 2-ClATP and the above indicated P2 receptor antagonists. Cell
survival was assessed 4 days later, by direct count of intact nuclei. Counts
represent means ꢁ SEM (N ¼ 3).
Abbreviations: oATP, ATP-20,30-dialdehyde; DIDS, 4,40-diisothiocya-
natostilbene-2,20disulphonic acid; TNP-ATP, trinitrophenyl-ATP; PIT, 2,20-
pyridylsatogen; P5P, pyridoxal-50-phosphate; RB-2, reactive blue-2.
***P < 0:001 with respect to 2-ClATP, one-way variance analysis.
3.3. Cytotoxicity induced by 2-ClATP is mediated by
neither P2 nor P1 receptors
The toxic effect induced by 2-ClATP in PC12nnr5 cells
was tested in the presence of several P2 antagonists with
different specificities towards P2X and P2Y receptors
(Table 1). Only pyridoxal-50-phosphate-6-(20-naphthy-
lazo-60-nitro-40,80-disulfonate) (PPNDS) and PPADS (both
used at 300 mM) sustained, respectively, 47 and 74%
survival in 3 days of 2-ClATP exposure (Table 1). We
then tested 2-ClATP metabolites and derivatives. As shown
in Fig. 5A, extracellular PF10 (Fig. 1), PF1 (Fig. 1) and 2-
ClAdo (100 mM) were able to induce complete cell death in
3 days. Similarly effective (Fig. 5A) were also the newly
synthesized derivatives PF3, PF4 and PF5 (Fig. 1). In order
to investigate the potential involvement of P1 receptors, the
non-selective adenosine antagonists Caffeine and CGS-
15493 were tested, respectively at 100 mM and 250 nM.
None reverted the toxic action exerted by 2-ClATP or 2-
ClAdo (Fig. 5B).
Fig. 2. 2-ClATP reduces the number of neuronal and glial cells in culture.
All cells maintained under proliferating conditions were treated with a
single dose of 100 mM 2-ClATP. Cell survival was assessed 4 days later (A)
or as indicated (B, C) by direct count of intact nuclei. Counts represent
means ꢁ SEM (N ¼ 3) and 100% survival is referred to untreated cultures.
In (A), ꢂꢂꢂP < 0:001 with respect to corresponding control, one-way
variance analysis.
PC12nnr5 cells displayed about 21% of cells in S þ G2
phases and only 0.93% in the apoptotic sub G1/G0 fraction.
The treatment with 2-ClATP for 30 hr increased the sub
G1/G0 fragmented DNA peak to 44% of the entire DNA
population, while the S þ G2 populations were reduced to
about 7%. The cytofluorimetric analysis revealed no sig-
nificant difference between controls and 2-ClATP-treated
PC12 cells up to 3 days (data not shown). [3H]Thymidine
incorporation into PC12 cells exposed to 2-ClATP for 36
and 60 hr showed instead that DNA duplication was
reduced of 40 and 50%, with respect to untreated cultures
(Fig. 4B).
3.4. Extracellular 2-ClATP is toxic only when
metabolized into 2-ClAdo mainly by serum ATPases
Having assessed that 2-ClAdo can exactly mimic the
toxic effect of 2-ClATP, we investigated the possible
extracellular degradation of the nucleotide. It was reported
that PPADS can block ecto-ATPases in various cell types,
acting therefore not only as a P2 receptor antagonist, but
also as an inhibitor of adenosine poly-phosphates hydro-
lyzing enzymes [42]. PC12nnr5 cells treated, respectively
Once established that 2-ClATP was cytostatic with PC12
cells (Figs. 2A–C and 4), but cytotoxic with PC12nnr5
(Figs. 2A–B and 3), we further pursued the analysis of the
toxic effect of 2-ClATP mainly in PC12nnr5 cells.

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¨
Fig. 3. 2-ClATP induces apoptosis in PC12nnr5 cells. PC12nnr5 cells were exposed to 100 mM 2-ClATP for 24 hr (A) or 30 hr (B) and stained with Hoechst
¨
33258 (1 mg/mL) (A) or PI (1 mg/mL) (B). Condensed nuclei stained with Hoechst 33258 (indicated by arrows) were visualized using Zeiss fluorescent
microscope (A), while DNA ploidy was measured by PI incorporation and cytofluorimetric analysis (B). In both cases, Ctrl represents untreated cultures.
Results are representative of three independent experiments.
with 2-ClATP or PF10 in the presence of 300 mM PPADS
showed a remarkable (80%) or partial (50%) reduction of
cell death (Fig. 6A). On the other hand, 300 mM PPADS
was only slightly (35%) or no effective on PF1- or 2-
ClAdo-induced toxicity (Fig. 6A). This indicates selec-
tivity of PPADS towards ATPases and ADPases, but not 50-
nucleotidases. In order to identify if mainly sera or cellular
ecto-enzymes were responsible for the potential 2-ClATP
hydrolysis, we either inactivated or lowered the sera in the
culture media. Cell death caused by 2-ClATP in PC12nnr5
was completely prevented by heat-inactivating the sera
(Fig. 6B). Moreover, 2-ClATP in 1.5% sera (instead of
15%) reduced its toxic effect from 95 to 60% (data not
shown). Consistently, the simultaneous addition of the
ATP-hydrolyzing enzyme apyrase (5 unit/mL), increased
cell death by 2-ClATP of nearly 50 and 80%, respectively
in the presence of active or inactivated sera (Fig. 6B).
To further confirm the extracellular degradation of
2-ClATP into 2-ClAdo, we performed HPLC analysis
on the culture media from PC12nnr5 cells treated with
1 mM 2-ClATP. The extracellular pool of halogenated
nucleotides (tri-, di- and mono-phosphates) decreased
as a function of time in culture, while 2-ClAdo progres-
sively augmented (Fig. 6C). The calculated 2-ClAdo peak
areas expressed in mAbsorbance Units ꢀ second were
122.48, 183.43, 658.6, respectively, in 3, 6 and 24 hr, also
confirming the extracellular formation and accumulation
of 2-ClAdo.
3.5. Cytotoxicity is dependent on 2-ClAdo intracellular
phosphorylation
We finally investigated the effects of 2-ClATP/2-ClAdo
in the presence of inhibitors of nucleoside transport and
intracellular metabolism. When 2-ClATP or 2-ClAdo were
challenged with the purine transport inhibitor NBTI
(20 mM), survival increased from 20 to about 50%
(Fig. 7). Moreover, the adenosine kinase inhibitor 50IT,
used at 20 nM, completely reverted the toxic action of 2-
ClATP and 2-ClAdo (Fig. 7). Conversely and as expected,
the adenosine deaminase inhibitor erythro-9-(2-hydroxy-
3-nonyl)adenine hydrochloride (EHNA) didn’t influence
cell survival (Fig. 7), being 2-ClAdo a deaminase-resistant
molecule [30].

N. D’Ambrosi et al. / Biochemical Pharmacology 67 (2004) 621–630
627
Fig. 4. 2-ClATP reduces proliferation but doesn’t induce apoptosis in PC12 cells. PC12 cells were exposed to a single dose of 100 mM 2-ClATP. After 48 hr,
¨
untreated and drug-exposed cells were stained with Hoechst 33258 (1 mg/mL). Nuclear chromatin condensation was visualized using Zeiss fluorescent
microscope (A). Images are representative of three independent experiments. In (B) untreated and 2-ClATP-treated PC12 cells were incubated with
[3H]thymidine (25 mCi/mL) and after DNA isolation the incorporation of [3H]thymidine was measured by liquid scintillation counting. Counts represent
means ꢁ SEM (N ¼ 3). ꢂP < 0:05 with respect to corresponding 36 hr value, one-way variance analysis.
4. Discussion
[42]. Nevertheless, the lack of effect by additional ATPases
inhibitors such as Suramin and NF-023 could be explained
by their restricted efficacy with all cell lines [20]. More-
over, inactivation or reduction of sera respectively pre-
vented or attenuated cell death, while increased toxicity
was instead obtained with apyrase. Finally, HPLC analysis
proved the extracellular breakdown of 2-ClATP and the
consequent accumulation of 2-ClAdo. Consistently with a
P2 receptor-independent mechanism, several 2-ClATP
derivatives and metabolites (PF5, PF10, PF4, PF1, PF3
and 2-ClAdo) also induced cell death. Moreover, toxicity
of 2-ClATP was due to its hydrolysis into 2-ClAdo mainly
mediated by serum ATPases, while cellular ecto-nucleo-
tidases seemed to play only a minor role, although well
expressed and characterized in PC12 cells [47].
Once generated, 2-ClAdo is known to exert toxic actions
via two different mechanisms: acting on P1 receptors [25],
or being intracellularly transported and metabolized by the
nucleotide salvage pathway enzymes. In this last case, the
overall effect is inhibition of DNA synthesis and thus cell
cycle arrest and death [48]. All our data strongly support
the intracellular pathway: (1) the P1 antagonists Caffeine
and CGS-15493 did not prevent 2-ClATP- or 2-ClAdo-
induced cell death. (2) NBTI, inhibiting the intracellular
adenosine transport, was instead partially protective. This
Many recent findings reported new roles for extracel-
lular purines and pyrimidines, acting either as growth
factors to induce cell proliferation [9,10], survival [7,43]
and differentiation [7,13,44,45], or as toxic agents to
mediate cell death [46]. This variety of effects can be
sustained by several different means. It often requires
intense signaling, sometimes is dependent on multiple
reinforcing ligands and it can very often be triggered by
the coordination of partially dependent, overlapping or
even contrasting processes, rather then by all-or-none
responses. Given the ability of 2-ClATP to sustain PC12
cell differentiation in the complete absence of sera or NGF
[7], we analyzed the effect of 2-ClATP on growth and
survival of additional neuronal and glial cells. We have
shown here that 2-ClATP reduces the number of PC12,
PC12nnr5, SH-SY5Y, U87 and U373 cells in the presence
of sera and this occurs according to multiple features and
through several independent mechanisms. Differently from
the pro-survival and neuritogenic action [7], cell death
induced by 2-ClATP is P2 receptor independent. This was
established by the lack of effect exerted by various P2
receptor antagonists, once established that PPADS was
indirectly protective by inhibiting ecto-ATPase activities

628
N. D’Ambrosi et al. / Biochemical Pharmacology 67 (2004) 621–630
Fig. 5. 2-ClATP metabolites exert toxicity not mediated by P1 receptors.
PC12nnr5 cells were exposed to the indicated purinergic compounds, all
used at 100 mM, in the absence (A) or in the simultaneous presence
of Caffeine (100 mM) or CGS-15493 (250 nM) (B). After 3 days of
treatment, cell survival was measured by direct count of intact nuclei.
Counts represent means ꢁ SEM (N ¼ 4) and 100% survival is referred to
untreated cultures.
incomplete effect is probably due to the presence on PC12
cells of transporters both sensitive (es-type or ENT1) and
insensitive (is-type or ENT2) to NBTI [49] and to the fact
that nucleosides can be simultaneously internalized by
these molecules [50]. Nevertheless, mechanisms conjoint
or additional to nucleoside transport may still be involved
in cell death. (3) The adenosine kinase inhibitor 50IT
prevented cell death, further confirming the intracellular
mechanism of 2-ClATP and 2-ClAdo. Antiproliferative
drugs are generally known to elicit toxic actions by inter-
fering with DNA synthesis directly through the poly-
merases or the enzyme ribonucleotide reductase [33],
causing in turn a nucleotide disequilibrium called ‘‘pyr-
imidine starvation’’ [48]. This last mechanism, however,
seemed to play a minor role in 2-ClATP/2-ClAdo-induced
toxicity, since the direct addition of uridine as an extra-
cellular pyrimidine source didn’t rescue PC12nnr5 cells
from death (data not shown).
Fig. 6. 2-ClATP-toxicity is dependent on the activity of extracellular
ATPases. PC12nnr5 cells were exposed to the indicated purinergic
compounds, all used at 100 mM, in the presence of 15% sera (A, B), or
heat-inactivated sera (HI 15% sera) (B). In (A and B), the cells were
simultaneously incubated with or without 300 mM PPADS or with 5 U/mL
apyrase and survival was assessed 60 hr (A) or 48 hr (B) later, by direct
count of intact nuclei. Counts represent means ꢁ SEM (N ¼ 3). In (A),
ꢂꢂꢂP < 0:001,ꢂ ꢂP < 0:05 with respect to corresponding PPADS; in (B),
ꢂꢂP < 0:005, P < 0:05 with respect to corresponding 15% sera, one-way
variance analysis. In (C), PC12nnr5 cells were exposed to 1 mM 2-ClATP
and the culture media was collected after 3, 6 and 24 hr and analyzed for
nucleotide breakdown by HPLC, all as described in Section 2. Values are
expressed as mAbsorbance Units (mAU).
A peculiarity emerging from our data is the cytostatic
effect of 2-ClATP/2-ClAdo in PC12 cells, compared to the
cytotoxicity induced in PC12nnr5 cells (demonstrated by
the presence of chromatin condensation and DNA frag-
mentation). Although highly speculative, this might rely on
the main difference between the two cell variants, that is
the absence of TrkA from PC12nnr5 cells. Indeed, in the
absence of TrkA, the low affinity p75 NGF receptor often
behaves as a pro-cell death effector [51] and its activation
(by growth factors present in sera and/or by P2 receptors
[52]) could therefore sustain apoptotic signals and rein-
force 2-ClATP/2-ClAdo-induced toxicity. In Trk-contain-
ing PC12 cells, 2-ClATP might instead exert trophic roles
through either P2 receptors [7] or TrkA trans-activation by
its adenosine-metabolites [53]. This would occur simulta-
neously to the toxicity induced by intracellular 2-ClAdo
and the overall effect would be cell cycle arrest, but not cell
death.

N. D’Ambrosi et al. / Biochemical Pharmacology 67 (2004) 621–630
629
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Acknowledgments
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The research presented was supported by Progetto
Coordinato CNR Agenzia 2000 and by Cofinanziamento
MURST 2001/2002. The useful suggestions and critical
reading of the manuscript by Prof. Sauro Vittori and the
professional style editing by Mrs. Catherine Wrenn are
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