Only the oligomers that did not form G-based structures or had a poor structure, under physiological conditions, were able to exert significant cellular growth inhibition effect.. Here, w
Trang 1associated eEF1A is correlated with their antiproliferative effect in haematopoietic human cancer cell lines
Bruna Scaggiante1, Barbara Dapas1, Gabriele Grassi2and Giorgio Manzini1
1 Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy
2 Department of Clinical, Morphological, and Technological Sciences, Division of Internal Medicine, University of Trieste, Italy
Single-stranded DNA may act as aptamer in
recogni-zing proteins with an affinity similar to or higher than
that of antibodies [1] Novel strategic applications of
aptameric single-stranded DNA encompass probes for
protein localization [1], therapeutic oligomers [2–4] and
microarrays of proteins [5]
Among oligomers able to adopt structures that are
recognized by specific proteins, there are those with a
high G content Within eukaryotic cells, G-rich
single-stranded structures appear to be involved in senescence
and aging by affecting telomere structure [6]
Chromo-somes end with a G-rich single-stranded overhang,
which is able to adopt a four-stranded G-quadruplex
structure that is a poor substrate for telomerase and
can be stabilized by ligands One of these, telomestatin,
stabilizes G-quadruplexes, thus inhibiting telomerase
activity [7] Moreover, a human protein named translin
was recently shown to stimulate telomerase activity by
specifically binding to the G-rich Tetrahymena and
human telomeric repeats [8] Furthermore, the
forma-tion of G-quadruplex structures is thought to contrib-ute to nonantisense effects by their ability to bind to cellular proteins [9,10] In particular, some protein tar-gets of these G-rich oligonucleotides have been identi-fied as nucleolin and a helicase [10,11]
Other proteins able to bind to G-quadruplex struc-tures have been recently discovered For example, it has been demonstrated that the human ribosomal pro-tein L7a interacts in vitro with a presumably G-rich RNA structure [12] G-quartet-forming oligodeoxy-nucleotides interacting with the SH2 domain of Stat3,
a protein encoded by a proto-oncogene that is activa-ted in many human cancer cells, represent a novel class
of aptameric therapeutic agents for the treatment of metastasis in cancer [13] Stat3 mediates upregulation
of bcl-x and mcl-1 gene expression and thus cell prolif-eration [14]
GT oligomers have been demonstrated to exert a specific, dose-dependent growth inhibition effect on
a variety of human cancer cell lines [15–17] The
Keywords
aptamers; CCRF-CEM; cell growth
inhibition; eEF1A; G-rich GT oligonucleotides
Correspondence
B Scaggiante, Molecular Biology Section,
Department of Biochemistry, Biophysics and
Macromolecular Chemistry, via Giorgeri, 1,
34127-Trieste, Italy
Fax: +39 040558 3691
Tel.: +39 040558 3678
E-mail: scaggiante@bbcm.units.it
(Received 30 August 2005, revised 12
Janu-ary 2006, accepted 18 JanuJanu-ary 2006)
doi:10.1111/j.1742-4658.2006.05143.x
G-rich GT oligonucleotides with a different content of G clusters have been evaluated for their ability to exert cytotoxicity and to bind to nuclear-associated proteins in T-lymphoblast CCRF-CEM cells Only the oligomers that did not form G-based structures or had a poor structure, under physiological conditions, were able to exert significant cellular growth inhibition effect The cytotoxicity of these oligomers was related
to their binding to the nuclear-associated eEF1A protein, but not to the recognition of nucleolin or other proteins In particular, GT oligomers adopting a conformation compatible with G-quadruplex, did not exert cytotoxicity and did not bind to eEF1A The overall results suggest that the ability of oligomers to adopt a G-quadruplex-type secondary structure
in a physiological buffer containing 150 mm NaCl is not a prerequisite for antiproliferative effect in haematopoietic cancer cells The cytotoxicity
of G-rich GT oligomers was shown to be tightly related to their binding affinity for eEF1A protein
Abbreviations
CRC, cytotoxicity-related complex; eEF1A, Elongation Factor 1 A.
Trang 2resultant cytotoxicity was tightly related to the
apta-meric behaviours of these GTs and in particular to
their ability to specifically bind to nuclear proteins
forming a major cytotoxicity-related complex (CRC)
of apparent molecular mass 45 ± 7 kDa [15–20]
Recently, a component of this complex has been
isola-ted from the nuclear enriched fraction of
haematopoi-etic cancer cell lines and identified as the eukaryotic
Elongation Factor 1 A (eEF1A) [21] Factors involved
in the translation of mRNA are known to contribute
to development of cancer [22,23]
It has been reported that the GT sequences with a
G-rich content can exert antiproliferative effects and
display aptameric properties by binding to nucleolin
[10] or to SV40 large T antigen helicase [11] Moreover
G-quartet-forming GTs have been shown to bind to
Stat3 and to induce tumour cell apoptosis [13] Here,
we wish to elucidate if GT oligomers with a G-rich
content can exert their antiproliferative activity in
human T-lymphoblast cancer cells and if they bind to
eEF1A protein
Results
The GT sequences are listed in Table 1 The 27-mer GT
was the reference oligomer able to exert cytotoxicity
and displaying the specific protein binding activities
[15–21] Starting from the GT and GT-G4 sequences,
the following oligomers were planned in order to
con-tain different clusters of G: GT-G1 has one cluster of
four guanines, GT-G2 has two clusters of four
gua-nines, GT-G3 has one cluster of seven guanines and
one of four guanines The human T-lymphoblast
CCRF-CEM cell line was used to perform the analysis,
being the reference cells extensively used in previous
works on GT oligomers and their protein interactors
[15–21]
The electrophoretic mobility of these oligomers
under native and denaturing conditions is illustrated in
Fig 1 To evidence different conformations the
oligo-mers were labelled at their 5¢-end by [32P]dATP[cP]
Figure 1A shows that under denaturing conditions, all
oligomers migrate according to their lengths It was previously demonstrated that GT does not fold into intra- or intermolecular structures and thus it migrates according to its length also in native conditions [18] Figure 1B shows the migrations in native conditions of the oligomers denatured and renatured overnight in a buffer with salt composition similar to that of the extracellular medium With respect to the unstructured
GT, GT-G1 does not appear to form significant
inter-or intramolecular structures, the electrophinter-oretic mobil-ity being in accord with its length GT-G2 shows a band migrating on the basis of its length, and a slightly slower nonresolved migrating band that might
be due to a dynamic interconversion with a bimole-cular structure GT-G3 can form an intermolebimole-cular structure of higher order demonstrated by the slowly migrating band, albeit a major band corresponding to its length was also present GT-G4 was shown to fold into an intramolecular structure (the faster migra-ting band), and to associate into an intermolecular one (the slower migrating band) Analogous results were obtained when the oligomers were renatured in a potassium phosphate buffer similar to the intracellular medium (data not shown)
To test the effect of these oligomers on cellular growth we performed a cytotoxicity assay The oligo-mers were applied to human lymphoblast CCRF-CEM cells in serum-containing medium and cell growth was evaluated after 72 h without changing the medium [15] As illustrated in Fig 2, GT-G1 and GT-G2 caused a reduction of cell growth at a level comparable to that of GT, showing almost complete inhibition at 15 lm This effect was cytotoxic, as pre-viously demonstrated for GT [15], as no recovery of cell growth was observed by prolonging cell culture for up to a further 4 days (data not shown) GT-G3 showed a moderate effect, giving not more than 50%
of cell growth inhibition at the highest dose (15 lm), probably due to a cytostatic effect as shown by the absence of cellular debris by microscope observation
On the contrary, no cell growth inhibition effect was observed for GT-G4
The ability of these sequences to bind to proteins forming the CRC of 45 ± 7 kDa was checked by UV cross-linking assays in competition experiments As illustrated in Fig 3A, the nonlabelled GT-G1, GT-G2 and GT-G3 were able to displace the labelled GT from binding to the CRC (white arrow) in the order GT-G1 > GT-G2 > GT-G3 (lanes 3, 4, 5) GT-G3 (lane 5) was the least efficient in acting as competitor
of GT, in agreement with the fact that it displayed a reduced cytotoxicity with respect to GT All competi-tors were able to displace GT from the minor complex
Table 1 Oligonucleotide sequences and names.
Length (-mer)
Trang 3of 100 kDa (black arrow) Self-competition of GT was
reported as reference (lane 2)
On the contrary, GT-G4 did not displace GT from
the CRC (lane 6) This evidence was in agreement with
the finding that GT-G4 did not exert any growth
inhi-bition effect It was able to displace the GT oligomer
only from the minor 100-kDa complex probably
formed with nucleolin [21] In fact, nucleolin was
pre-viously found to recognize the structured GT-G4
oligomer and to form with it the 100-kDa complex
[21] Figure 3B shows the binding of labelled oligomers
to the nuclear-enriched fraction of proteins The
effi-ciency of labelling of the single oligomers being not
homogenous, the ratios of the different binding signals
within each lane instead of their absolute values have
to be considered It can be seen that GT is present
mainly in the CRC and in a minor complex of about
100 kDa, but that GT-G1, GT-G2 and GT-G3 can form in addition to the CRC (white arrow) and the 100-kDa complex (black arrow), analogously to GT, a complex of about 70 kDa also Moreover, the G-rich oligomers produced a band of about 26 kDa, due to a nonspecific protein binding previously described, prob-ably derived from a cytoplasmic contaminant [15] The ratios of oligomer bound in the CRC to that bound
in the 70-kDa complex estimated for each lane by phosphoimager was shown to be 0.38, 0.37, 0.19, for GT-G1, GT-G2, GT-G3, respectively This may explain why GT-G3 exerted a reduced antiproliferative activity, i.e by its preferential binding to other pro-teins On the contrary, GT-G4 did not form the CRC, whereas it was found to produce the 70-kDa and the 100-kDa complexes, that therefore cannot be involved
in the cell growth inhibition effect Thus, the lack of
GT
nt
A
45
30
25
20
10
GT
-G1 GT
-G2 GT
-G3 GT -G4
B
GT
nt
45
30
25
20
10
GT
-G1 GT
-G2 GT
-G3 GT -G4
Fig 1 Denaturing and native electrophoresis of G-rich GT oligomers (A) Denaturing electrophoresis Five micrograms of oligomers were denatured by heating at 95 C for 10 min in 7 M urea and then cooled on ice The samples were then loaded onto a 20% polyacrylamide gel
in 0.1 M sodium acetate ⁄ acetic acid buffer pH 5.0, containing 7 M urea The gel was run in 0.1 M sodium acetate ⁄ acetic acid buffer pH 5.0,
at 42 C at 15 VÆcm)1 The gel was stained by 0.01% Stainsall dye in 50% formamide (v ⁄ v) The nucleotide length markers are noted on the left (nt) (B) Native electrophoresis The oligomers were 5¢-end labelled by using [ 32 P]dATP[cP] polynucleotide kinase as described in Experi-mental procedures One microgram of unlabelled oligomers and about 1 ng of corresponding labelled ones adjusted to a specific activity of
15 000 cpm, were added together in a total volume of 10 lL in 150 m M NaCl, 10 m M K2HPO4⁄ KH 2 PO4, 1 m M EDTA pH 7.0 The samples were denatured by heating at 95 C for 5 min and then slowly cooled overnight at room temperature After adding 3 lL of 50% glycerol in TBE buffer, the samples were loaded onto 20% polyacrylamide gel in TBE buffer and run at 10 VÆcm)1, at 4 C The gel was fixed in 10% acetic acid, dried and then exposed to X-LS Kodak film The nucleotide length markers are noted on the left (nt).
Trang 4formation of the CRC agrees with the absence of cell
growth inhibition by GT-G4
By affinity chromatography, using a GT biotinylated
51-mer, the proteins interacting with GT were isolated
from the pool of nuclear-enriched fraction and used to
perform competition experiments by UV crosslinking
assays Figure 4 shows a western blot of the total
nuc-lear extracts used for the affinity chromatography with
antinucleolin and anti-eEF1A antibodies It is evident
that at the level of eEF1A recognition no nucleolin
fragments are present This excludes interference from
possible proteolytic fragments of nucleolin to the GT
oligomer binding at the level of the eEF1A band As
illustrated in Fig 4B, the isolated proteins formed two
complexes with GT (lane 1): the most abundant,
com-patible with the binding of eEF1A in the CRC (white
arrow), the other of apparent molecular mass of about
100 kDa compatible with binding to nucleolin (black
arrow) The competition experiments in UV
cross-link-ing assays with the GT-recognizcross-link-ing proteins confirmed
the results obtained with the total nuclear-enriched
pro-teins: a decreasing ability from G1 (lane 3) to
GT-G3 (lane 5) to displace GT from the CRC and a lack
of competition by GT-G4 (lane 6) Competition by
120
100
80
60
40
GT GT-G1 GT-G2 GT-G3 GT-G4 20
0
ODN concentration
(µ M )
15
Fig 2 Cytotoxicity of G-rich GT oligomers CCRF-CEM cells
(5 · 10 3 ) in exponential growth phase were seeded in triplicate in
200 lL of serum-containing medium in a 96-well microtiter plate.
After overnight incubation, the oligomers were directly added to
the cell medium at the indicated concentrations Cell growth was
evaluated 72 h after oligonucleotide addition by incorporation of
0.5 mgÆmL)1of MTT, as described in Experimental procedures The
percentage cell growth was calculated by taking growth of an
internal nontreated control as 100% The results are mean ± SD of
5–10 independent experiments.
GT GT -G1 GT -G2 GT -G3 GT -G4 kDa Competitor
Labelled oligomer
119
76
47
29
–
GT GT -G1 GT -G2 GT -G3 GT -G4
kDa 119 76
47
29
A
B
Fig 3 Binding of the G-rich GT oligomers to total nuclear proteins (A) Competition of binding to GT Three micrograms of total nuclear CCRF-CEM cell extract were incubated with 2 ng of 5¢-end
32 P-labelled GT in buffer C in the presence of the nonspecific com-petitors (1 lg salmon sperm DNA, 1 lg of CT oligomer) and in the absence or in the presence of the indicated specific competitors added at 1000-fold molar excess After 30 min incubation at room temperature, the samples were exposed to UV light for 10 min and then denatured by adding SDS ⁄ PAGE loading buffer and boiling the samples The samples were loaded onto an SDS ⁄ PAGE gel (10% acrylamide) and run at 15 VÆcm)1 The gel was dried and exposed
to X-AR Kodak film The black arrow indicates the 100-kDa com-plex; the white arrow indicates the 45 ± 7-kDa complex (CRC) (B)
UV crosslinking of G-rich GT oligomers Three micrograms of total nuclear extract of CCRF-CEM cells were incubated in buffer C in the presence of 2 ng of the indicated 5¢-end 32 P-labelled G-rich GT oligomers and with 1 lg salmon sperm DNA and 1 lg of CT oligo-mer as nonspecific competitors After 30 min incubation at room temperature, the samples were exposed to UV light for 10 min and then denatured by adding SDS ⁄ PAGE loading buffer and boiling the samples The samples were loaded onto an SDS ⁄ PAGE gel (10% acrylamide) and run at 15 VÆcm)1 2 ng of 5¢-end 32 P-labelled GT sample were included as reference (lane 1) The gel was dried and exposed to X-LS Kodak film The black arrow indicates the 100-kDa complex; the white arrow indicates the CRC.
Trang 5nonlabelled GT is shown as reference (lane 2)
More-over, full ability to compete in the binding to the higher
molecular weight protein (i.e nucleolin; black arrow)
was observed for all G-rich oligomers In panel B
at right it can be observed that GT-G1 formed both complexes, whereas GT-G4 formed only the 100 kDa
GT
66 kDa
45 kDa
WB:ααeEF1A
Competitor
116
45 66
29
1 2 3 4 5 6 7
GT
-G1
GT
-G1 GT
-G2 GT
-G3 GT
-G4
GT -G4
116 kDa
47 85
36
1 2 A
B
C
Fig 4 (A) Western blotting of nuclear extract with anti-eEF1A and anti-nucleolin IgG Twenty micrograms of nuclear extract were separated
by SDS ⁄ PAGE (12% acrylamide) and then blotted onto a 0.22-lm nitrocellulose membrane as described in Experimental procedures The blotted membrane was blocked with 3% nonfat dried milk in NaCl ⁄ P i and incubated with eEF1A (lane 2) or nucleolin (lane 1) mAb (1 lgÆmL)1) in NaCl ⁄ P i , overnight, at 4 C with constant rocking After washing, the membrane was incubated for 1.5 h with a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody, then rinsed once with NaCl ⁄ P i containing 0.05% Tween-20 and four times with deionized water The blot was developed as described in Experimental procedures (B) Binding of G-rich GT to affinity-purified proteins: (left) 3.5 lL of proteins purified by affinity chromatography as described in Experimental procedures were incubated in 10 lL buffer C containing
2 ng of 5¢-end 32 P-labelled GT and in the presence or in the absence of the indicated specific competitors added at 500-fold molar excess,
or (right) in 10 lL buffer C containing 2 ng of the indicated 5¢-end 32
P-labelled oligomers without specific competitors After 30 min incuba-tion at room temperature, the samples were crosslinked by exposure to UV light for 10 min, denatured by adding SDS ⁄ PAGE loading buffer and boiling The samples were loaded onto 10% SDS ⁄ PAGE and run at 15 VÆcm)1 The gel was dried and exposed to X-AR Kodak film The black arrow indicates the 100-kDa complex; the white arrow indicates the CRC (C) Western blotting of the affinity-purified proteins with anti-eEF1A Thirty micrograms of affinity purified proteins (lane 1) or 7.5 lg total nuclear proteins (lane 2) were separated by SDS ⁄ PAGE (12% acrylamide) and then blotted onto a 0.22 lm nitrocellulose membrane as described in Experimental procedures The blotted membrane was blocked with 3% nonfat dried milk in NaCl ⁄ P i and incubated with eEF1A mAb (1 lgÆmL)1) in NaCl ⁄ P i overnight, at 4 C with constant rocking The blot was developed as described in (A).
Trang 6complex The presence of eEF1A in the purified protein
mix was confirmed by western blotting with the specific
antibody (Fig 4C, lane 1) The eEF1A protein from
total nuclear extract is shown as control in lane 2
To test the binding abilities of the oligomers toward
the isolated protein, eEF1A was excised from
Coomassie-stained gel and recovered as previously
demonstrated [21] Figure 5 illustrates EMSA and UV
cross-linking assays with purified eEF1A Figure 5A
shows that in the absence of competition the
noncyto-toxic oligomer GT-G4 was found to bind very faintly
to eEF1A, also with addition of increasing amounts of
protein (lanes 2–4) In contrast, labelled GT was found
to bind with stronger affinity to eEF1A in a manner
directly proportional to protein quantities (lanes 5–7)
The presence of a minor slower migrating band in lanes
5–7 might be due to a complex of higher molecularity
The slightly faster mobility of the complex between
GT-G4 and eEF1A (lanes 2–4) is probably accounted
for by the difference in length and thus in migration of
the free oligomer (lane 1 vs lane 8) Moreover, it seems
conceivable that GT-G4, forming the G-quartet
struc-ture, gave a more compact (i.e faster) complex than
that generated by the nonstructured GT
Figure 5B shows competition experiments performed
with the isolated eEF1A in UV cross-linking assays
GT-G1 (lane 3) and, to a lesser extent, GT-G2 (lane 4)
were able to displace GT from eEF1A GT-G3 (lane
5) and GT-G4 (lane 6) resulted inefficient in producing
competition On the left the western blot with the
anti-eEF1A antibody of the protein recovered from the gel
band is shown
To completely elucidate the relationship between the
structure of the GTs and their ability to inhibit cell
growth by forming the CRC, we performed CD at
37C As a control we used two oligomers, GRO29A
and GRO26A, whose structures were related to
anti-proliferative activity in tumour cells [10] As illustrated
in Fig 6A, the weak CD bands of GT and GT-G1
indicate absence of appreciable secondary structure at
37C, under conditions similar to those of the
extra-cellular medium The spectra of GT-G2 and GRO29A
showed a small band at 263 nm, suggesting the
forma-tion of a limited structure GT-G3 was found to give a
peak at 263 nm compatible with G-quartet structure
However, no full structure in the G-quadruplex was
detected as shown by the low intensity of the 263-nm
band A clear structure formation was found for
GRO26A and GT-G4: a positive peak at 263 nm and
a negative one at 242 nm These CD spectra were
compatible with parallel G-quadruplex Moreover,
GRO26A showed a slight signal at 295 nm that might
be related to a minor amount of antiparallel
G-quad-ruplex The CD spectra of GRO29A and GRO26A agree with their electrophoretic mobilities under non-denaturing conditions: GRO29A, forming a poor
GT
kDa
Competitor eEF1A protein
Labelled oligomer GT-G4 GT eEF1A protein
116 46 79
31
1 2 3 4 5 6 7
47 kDa
WB: ααeEF1A
GT
-G1 GT
-G2 GT
-G3 GT -G4
A
B
1 2 3 4 5 6 7 8
2.5 µL
Fig 5 Binding of G-rich GTs to eEF1A protein The eEF1A protein was purified from a Coomassie blue-stained gel, as described in Experimental procedures (A) Band-shift assay From 2.5 to 10 lL
of the isolated eEF1A protein were incubated with 1 ng of 5¢-end
32 P-labelled GT-G4 (lanes 1–4) or 1 ng of 5¢-end 32 P-labelled GT (lanes 5–8) in 25 m M Tris ⁄ HCl pH 8.0, containing 0.05% SDS, 0.05 mgÆmL)1BSA, 0.1 m M EDTA, 1.25% glycerol and 0.1 M NaCl, for 30 min at room temperature The samples were then loaded onto 8% polyacrylamide in TBE buffer and run at 20 VÆcm)1 at
4 C The gel was then dried and exposed to X-AR Kodak film (B)
UV cross-linking assay Ten microlitres of the isolated eEF1A pro-tein were incubated in buffer C with 1 ng of 5¢-end 32 P-labelled GT
in the presence or absence of the indicated specific nonlabelled competitors added at 10-fold molar excess After 30 min incubation
at room temperature, the samples were exposed to UV light for
10 min, denatured by adding SDS ⁄ PAGE loading buffer and boiling The samples were separated by SDS ⁄ PAGE (12% acrylamide) and run at 10 VÆcm)1 The gel was then dried and exposed to X-AR Ko-dak film On the left is shown western blotting of 50 lL of the recovered protein performed after SDS ⁄ PAGE (12% acrylamide) with an anti-eEF1A mAb as described in Experimental procedures.
Trang 7structure, was found to run on the basis of its length,
whereas GRO26A, demonstrating a full G-based
struc-ture, showed a fast and a slow migrating band
simi-larly to GT-G4 (data not shown) Figure 6B illustrates
the stability of GRO26A and GT-G4: these structures
were not disrupted by increasing the temperature to
90C They showed CD spectra indicative of absence
of structure only when they were resuspended in water
and heated at 65C (dashed green spectrum) On the
contrary, the structure of GT-G3 was not so stable
and it was disrupted by increasing the temperature to
65C (data not shown) The effect of GRO29A and
GRO26A on CCRF-CEM cell growth is shown in
Fig 7A: in accordance with previous data, we found
that GRO29A exerted a significant growth inhibition effect, similar to GT, whereas the G-quartet forming GRO26A did not alter cellular growth, similar to GT-G4 Moreover, as illustrated in Fig 7B, GRO29A (lane 4) was able to compete in the binding to specific nuclear proteins (CRC) as did GT (lane 3) On the contrary, no competition was observed using GRO26A (lane 5)
Discussion
A series of guanosine-rich phosphodiester oligodeoxy-nucleotides strongly inhibits proliferation in a number
of human tumour cell lines and the presence of
GT GT-G1 GT-G2 GT-G3 GT-G4
GT-G4
GRO26A GRO29A
GRO26A
20ºC
20ºC, H 2 O 65ºC, H 2 O
37ºC 50ºC 65ºC 80ºC 100ºC
220
7
6
4
2
0
-2
240 260 280 300 Wavelength [nm]
8
5
0
-3
CD[mdeg]
CD[mdeg]
CD[mdeg]
320
220 240 260 280 300
Wavelength [nm]
320
220 240 260 280 300
Wavelength [nm]
320
8
5
0
-4
A
B
Fig 6 Circular dichroism of oligomers (A) A 10 l M solution of the indicated oligomer was diluted in renaturation buffer (150 m M NaCl, 10 m M K2HPO4⁄ KH 2 PO4,
1 m M EDTA pH 7.0) to a final concentration
of 0.5 l M recording the spectra at 37C as described in Experimental procedures (B) GRO26A and GT-G4 were diluted to 0.5 l M final concentration in water and the spectra were recorded at the indicated tempera-tures.
Trang 8G-quartets in the active oligonucleotides was found to
determine cell growth inhibition activity [10,13,14]
The G-rich oligonucleotides bind to specific cellular
proteins in both nuclear and cytoplasmic extracts and
to proteins derived from the plasma membrane, and
their biological activity correlates with binding to these
proteins Strong evidence showed that one of these
proteins is nucleolin, a multifunctional phosphoprotein
whose levels are related to the rate of cell proliferation
in a variety of solid tumour cell lines [10] The biologi-cal activity of the G-rich oligomers was found to be associated with their ability to form stable G-quartet-containing structures and with their binding to specific cellular proteins, most likely nucleolin [10] More recently, the antiproliferative activity of G-rich oligo-nucleotides has been directly related to their inhibition effect on DNA replication, resulting from negative modulation of a helicase activity [11] Independently, other authors found that G-quartet-forming oligomers bind to Stat3, a protein involved in tumour cell pro-gression The oligomers inhibited Stat3 binding to DNA, thus blocking the transcription of Stat3-regula-ted genes and the progression of prostate and breast cancers in mice [13]
Here we demonstrate that G-rich GT oligomers can exert cytotoxicity on haematopoietic T-lymphoblast CCRF-CEM cells only if the oligomers bind to nuclear proteins forming the CRC, derived from eEF1A recog-nition Similar results were confirmed in other cell lines
of haematopoietic tumour origin, such as Jurkat, CEM-VLB, Raji, HL60, K562 (data not shown) The cytotoxicity and the formation of the CRC with nuc-lear proteins seem related to the presence of oligomers migrating according to their length, as demonstrated
by electrophoresis for GT-G1, GT-G2 and GT-G3 GT-G4, demonstrating the formation of a full struc-ture, did not form the CRC and did not exert any appreciable growth inhibition effect on the tumour cells The nontoxic GT-G4 has 78% G-content, two clusters of four and 11 consecutive Gs, and it appears
to form structure in native electrophoresis, probably as
a G-quadruplex Our CD spectrum clearly indicates a very stable G-quartet structure at physiological condi-tions A melting curve, recorded at 295 nm, compatible with the disruption of a G-quartet structure [10] was also found for GT-G4 (data not shown) A NMR study has shown that a DNA oligonucleotide contain-ing different G clusters adopts an asymmetric bimole-cular G-quadruplex structure in solution [24], and the topology of this structure is distinct from the folds of the closely related and well-characterized sequences d(G4T4G4) and d(G3T4G3) [25] Recently, the ability of the G-rich oligomers to exert an antiproliferative effect has been related to their binding to specific cellular proteins, rather than to G-quadruplex formation [26] The absence of cytotoxicity of GT-G4 appears not to
be related to a reduced intracellular accumulation of this oligomer In fact, the incorporation of 32P-labelled oligomers into viable cells showed similar uptakes with the only exception of GT-G4, whose internalization rate was even higher (data not shown)
100
80
60
40
20
0
116 kDa
70 kDa
46 kDa
32 kDa
23 kDa
1 2 3 4 5
Competitor
+ + + +
Proteins
GT
GT
GR O29A
GR
O29A GR O26A
GR
A
B
Fig 7 Cytotoxic assay of GRO26A and GRO29A and their binding
to nuclear proteins (A) CCRF-CEM cells (5 · 10 3 ) in exponential
growth phase were seeded in triplicate in 200 lL of
serum-contain-ing medium in 96-well microtiter plates After overnight incubation,
the oligomers were directly added to the cell medium at 10 l M
concentration Cell growth was evaluated 72 h after oligonucleotide
addition by incorporation of 0.5 mgÆmL)1of MTT, as described in
Experimental procedures As reference oligomers GT and GT-G4
were used in the same experiment (B) Two micrograms of total
nuclear proteins (lanes 2–5) were incubated with 2 ng of 5¢-end
32 P-labelled GT in buffer C in the presence of the nonspecific
com-petitors (1 lg poly(dIdC) and 1 lg of CT oligomer) and with the
indicated specific competitors added at 500-fold molar excess
(lanes 3–5) After 30 min incubation at room temperature, the
sam-ples were cross-linked by UV exposure and then separated by
SDS ⁄ PAGE (12% acrylamide) as described in Experimental
proce-dures Reference lane 1 shows the migration of the free oligomer;
the open arrow shows the CRC.
Trang 9GT-G4, showing a G-quartet-based structure, did
not exert cytotoxic effects on haematopoietic cancer cell
lines, whereas the G-rich GT oligomer GRO29A was
successfully used by Bates to significantly inhibit the
growth of a variety of human cancer cells derived from
solid tumours [10] The same author used as a control
GRO26A, that did not significantly alter cellular
growth Accordingly with these authors, we found that
GRO29A exerted on CCRF-CEM cells a growth
inhibi-tion effect similarly to GT, whereas GRO26A did not
significantly alter cellular proliferation On the
contrary, in our experimental conditions, which were
similar to those of the extracellular medium, the
CD spectra of GRO26A show that it formed a stable
G-quadruplex structure as GT-G4 did, whereas
GRO29A exhibited a CD indicative of a poor structure
In experimental conditions similar to that of the
intra-cellular medium (in 140 mm KCl containing buffer),
GRO29A did not show a CD spectrum diagnostic of
G-quartet-based structure (data not shown) In fact, the
spectra clearly show only minor differences with that
obtained in NaCl containing buffer, and this is
indicat-ive of a rather weak secondary structure The apparent
discrepancy with literature results [10,26] can be
explained by the fact that the formation of G-quartet
based structures from a rather various repertoire often
implies rather long kinetic processes, depending on
molecularity, oligomer concentration, salt, temperature
of annealing, and frequently different coexisting
com-peting forms In particular GRO29A needs 56 h
anneal-ing at 60C in 140 mm KCl to assume a G-quartet
containing structure [26] In agreement with our results,
competition experiments demonstrated that GRO29A
was able to displace the labelled oligomer from eEF1A,
similarly to GT, whereas GRO26 was not
Further-more, in nondenaturing electrophoresis under our
experimental conditions GRO29A migrated mostly on
the basis of its length in accordance with other findings
[26], whereas GRO26A demonstrated the formation of
a full structure Thus it seems likely that GRO29A can
exert a growth inhibition effect on human
haematopoi-etic cancer cells because in physiological conditions it
does not significantly form G-quartets and can bind to
eEF1A The binding of GRO29A to eEF1A was not
observed by Bates et al [10] in solid tumour, but this
might be related to the absence in these cells of the
eEF1A isoforms that we identified in the
haematopoiet-ic cell line [21], or to the buffer conditions used for the
binding Thus the G-quartet structure is clearly not a
prerequisite for the antiproliferative activity of G-rich
oligomers in haematopoietic cancer cells
The toxic GT-G1 has 60% G content and only one
cluster of four guanines It migrates on the basis of its
length in electrophoresis, it was as cytotoxic as GT and showed the formation of the CRC GT-G2, with 65% G-content, demonstrated a very faint structure and inhibited the cellular growth similarly to GT-G1 GT-G3, which, with 69% content, assumed) in part) intermolecular structures clearly related to the increase in the number of Gs in the cluster (from four
in GT-G2 to seven in GT-G3), gave a reduced cellular growth inhibition; accordingly, it showed a reduced capacity to form CRC The lower ability of GT-G2 to compete for the binding of GT to eEF1A with respect
to GT-G1 both in total nuclear extract and in affinity-chromatography-purified proteins does not agree with cytotoxicity data, the two oligomers showing irrelevant differences in growth inhibition The overall results suggest that the kinetics of binding of GT-G2 to eEF1A might be slower with respect to that of GT-G1 explaining its reduced ability to displace GT from the CRC and this might be related to the mild grade of structure formation observed in its CD spec-trum However, it cannot be completely excluded that GT-G1 and GT-G2 have a different in vivo intracellu-lar localization, i.e GT-G2 being predominately nuc-lear in localization with respect to GT-G1, thus taking into account the different binding ability vs the same cytotoxicity Although a different intracellular localiza-tion could explain differences in antiproliferative effect, the protein binding ability suggests that the biological activity of the G-rich GT oligomers is related to their recognition of nuclear-associated eEF1A Furthermore, all of the oligomers were able to displace GT from nucleolin (the complex of highest molecular weight), both in assays with total nuclear extract and with affinity chromatography purified proteins, but not all were able to exert cell growth inhibition Thus it seems unlikely that nucleolin is related to the antiproliferative effect exerted by G-rich GTs Moreover, the oligomers that did not bind to eEF1A, such as GT-G4, did not exert growth inhibition
It is interesting to note that GT-G1 recognizes also another nuclear protein, forming a complex of 70 kDa, just as GT-G2, GT-G3 and GT-G4 This complex is unlikely to involve already described proteins such as Stat3 [14] or a helicase [11], whose molecular masses are 80 and 124 kDa, respectively The formation of this complex clearly suggests that these G-rich oligomers target other proteins that GT does not engage, but this fact is not related to the cytotoxic effect Moreover, the binding of the G-rich oligomers GT-G1, GT-G2, GT-G3 and GT-G4 to proteins form-ing the 70-kDa complex might be due to the interac-tion with a proteolytic fragment of nucleolin observed
by Bates [10] as well as by us (Fig 4A) This agrees
Trang 10with the observation that the nontoxic GT-G4 forms
the two complexes of 100 kDa and of 70 kDa, both
compatible with nucleolin recognition The possibility
that a 48-kDa fragment of nucleolin [27] could be a
major contaminant of eEF1A protein can be excluded
by MALDI TOF analysis of the Coomassie blue band
extract [21] and by the absence of a corresponding
nucleolin signal in western-blotting of our nuclear
extracts (Fig 4A)
Thus these results indicate that in haematopoietic
cancer cells G-rich GT oligomers exert a growth
inhi-bition effect by binding to nuclear-associated eEF1A
protein and this effect is inversely related to the ability
of oligomers to adopt G-quartet structures in
physiolo-gical conditions
Experimental procedures
Oligonucleotide sequences
HPLC-purified phosphodiester oligomers were from
MWG-Biotech AG (Ebersberg, Germany) The oligomers were
resuspended in physiological solution at 1000 lm stock
solution and sterilized by centrifugation in 0.2 lm filter
spin-X tubes
Native and denaturing electrophoresis
The oligomers (5 lg) were denatured by heating at 95C
for 10 min and supplemented with 7 m urea The samples
were loaded onto 20% polyacrylamide gel
(acrylamide:bis-acrylamide, 29 : 1 w⁄ w) in 0.1 m acetic acid, pH 5.0,
10 mm NaCl, 10 mm MgCl2 containing 7 m urea The gel
was run in 0.1 m acetic acid, pH 5.0, at 10 VÆcm)1for 2.5 h
at 42C In nondenaturing conditions, the oligomers (5 lg)
were denatured by heating at 95C for 10 min and
rena-tured in 150 mm NaCl, 10 mm K2HPO4⁄ KH2PO4, 1 mm
EDTA, pH 7.0, by slowly cooling at room temperature
overnight The samples were then electrophoresed through
20% polyacrylamide gel (acrylamide:bisacrylamide, 29 : 1
w⁄ w) in TBE buffer (0.09 m Tris ⁄ borate, pH 8.0, 2 mm
EDTA) at 5 VÆcm)1 for 4 h at room temperature The
gels were stained by using 0.01% Stainsall dye (Sigma
Chemical Co., St Louis, MO, USA) in 50% formamide
(v⁄ v) Alternatively, using 32
P-labelled oligomers, the gels were fixed in 10% acetic acid, dried and then exposed to
autoradiography on X-AR Omat Kodak film
Cell cultures and cytotoxicity assay
The human T-lymphoblastic leukaemic CCRF-CEM cell
line was cultured in RPMI 1640 medium supplemented with
10% foetal serum (Euroclone, Celbio, Devon, UK), 2 mm
L-Gln, 100 UÆmL)1penicillin, 100 lgÆmL)1streptomycin
CCRF-CEM cells (5· 103) in exponential growth phase were seeded in 200 lL foetal clone serum (Euroclone, Cel-bio, Devon, UK) containing medium, in 96-well microtiter plate in triplicate After overnight incubation, the oligomers were directly added to the cell medium at the indicated final concentrations Cell growth was evaluated 3 days of culture after oligonucleotide administration by incorporation of 0.5 mgÆmL)1MTT into viable cells [28] The percentage of cellular growth was estimated by considering 100% cell growth that of the internal-control nontreated cells
Total nuclear extracts preparation Total nuclear extracts were obtained from approximately
20· 106CCRF-CEM cells by a small modification of Dig-nam’s method [15] The protein content was determined by the Bradford method [29] using BSA (Sigma Chemical Co.)
as standard
Affinity chromatography The 5¢-biotin labelled oligomer 5¢-T(GTTT)9GT-3¢ (MWG-Biotech AG) was immobilized on streptavidin magnetic particles (Boehringer, Mannhein) in 10 mm Tris⁄ HCl, 1 mm EDTA, 100 mm NaCl, pH 7.5 (TEN 100) at a concentra-tion of 1 lg oligomerÆmg)1beads After 30 min incubation
at room temperature, the beads were washed twice with
10 mm Tris⁄ HCl, 1 mm EDTA, 1 m NaCl, pH 7.5 (TEN 1000) The beads were equilibrated in TEN 1000 For pro-tein binding, the beads were preincubated with 0.5 mgÆmL)1 BSA in TEN 100 for 10 min The beads were washed thrice with TEN 100 and then equilibrated in 20 mm Hepes, 1.5 mm MgCl2, 0.2 mm EDTA, 0.42 m NaCl, containing 10% glycerol and 0.05% NP40 (buffer C) The beads were incubated with 3 mg proteins from the nuclear-enriched fraction supplemented with 1 mm phenylmethanesulfonyl fluoride (PMSF), 0.01% NP40 and 0.05 mgÆmL)1 salmon sperm DNA, for 1 h with gentle stirring The beads were washed twice with buffer C and twice with buffer C with-out NP40 The elution was made with three volumes of buffer C without NP40 adjusted to 1.5 m NaCl, incubating for 10 min with gentle stirring The beads were then washed with TEN 100 and stored at 4C with 0.01% NaN3 The eluted proteins were dialysed with a 3000-Da cutoff mem-brane (Centricon) in 1 mm Hepes, 0.2 mm EDTA, 5% gly-cerol, 5 mm dithiothreitol (DTT), 5 mm PMSF and then lyophilized The protein was resuspended in 50 lL water containing 10% glycerol and 5 mm DTT
Purification of eEF1A from Coomassie blue-stained gel
We have previously shown that the protein eEF1A can be isolated from a Coomassie blue stained gel [21] Briefly, the