Interaction of G-rich GT oligonucleotides with nuclear-associated eEF1A is correlated with their antiproliferative effect in haematopoietic human cancer cell lines

Interaction of G-rich GT oligonucleotides with nuclear-associated eEF1A is correlated with their antiproliferativeeffect in haematopoietic human cancer cell linesBruna Scaggiante1, Barbara Dapas1, Gabriele Grassi2 and 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: [email protected]

(Received 30 August 2005, revised 12 Janu-

ary 2006, accepted 18 January 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.

1350 FEBS Journal 273 (2006) 1350–1361 ª 2006 The Authors Journal compilation ª 2006 FEBS

resultant 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 electrophoretic 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 intermolecular

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.

Sequence Name

Length

(-mer)

5¢-TGTTTGTTTGTTTGTTTGTTTGTTTGT-3¢ GT 27

5¢-TGGTGTGTGTGGGGTGGTTGGTG-3¢ GT-G1 23

5¢-TGGGGTGTGTGGGGTGGTTGGTG-3¢ GT-G2 23

5¢-TGGGGTGTGTGGGGGGGTTGGTG-3¢ GT-G3 23

5¢-TGGTTGGGGTGGGGGGGGGGGTG-3¢ GT-G4 23

B. Scaggiante et al. GT oligonucleotides, eEF1A and antiproliferative effect

FEBS Journal 273 (2006) 1350–1361 ª 2006 The Authors Journal compilation ª 2006 FEBS 1351

of 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

GTnt

A

45

30

25

20

10

GT-G1

GT-G2

GT-G3

GT-G4

B

GTnt

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 [32P]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 mM NaCl, 10 mM K2HPO4 ⁄ KH2PO4, 1 mM 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).

GT oligonucleotides, eEF1A and antiproliferative effect B. Scaggiante et al.


formation 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-recognizing proteins confirmed

the results obtained with the total nuclear-enriched pro-

teins: a decreasing ability from GT-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-G420

00 5 10

ODN concentration(µM)

% o

f ce

llula

r gr

owth

15

Fig. 2. Cytotoxicity of G-rich GT oligomers. CCRF-CEM cells

(5 · 103) 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)1 of 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

kDaCompetitor

Labelled oligomer

119

76

47

29

1 2 3 4 5 6

–

GT GT-G1

GT-G2

GT-G3

GT-G4

1 2 3 4 5

kDa119

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¢-end32P-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 32P-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 32P-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.



nonlabelled 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-- -- kDa

66 kDa

45 kDa

WB:ααeEF1A

Competitor116

45

66

29

1 2 3 4 5 6 7

GT-G1

GT-G1

GT-G2

GT-G3

GT-G4

GT-G4

116 kDaαnucle

olin

αeEF1A

47

85

36

1 2

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 ⁄ Pi and incubated with eEF1A (lane 2) or nucleolin (lane 1) mAb

(1 lgÆmL)1) in NaCl ⁄ Pi, 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 ⁄ Pi 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 32P-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 32P-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 ⁄ Pi and incubated with eEF1A mAb (1 lgÆmL)1) in NaCl ⁄ Pi overnight, at 4 �C with constant

rocking. The blot was developed as described in (A).



complex. 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

37 �C. 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

37 �C, 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

CompetitoreEF1A protein

Labelled oligomer GT-G4 GT

eEF1A protein

+ + + + + + –116

4679

31

1 2 3 4 5 6 7

47 kDa

WB:ααeEF1A

GT-G1

GT-G2

GT-G3

GT-G4

A

B1 2 3 4 5 6 7 8

2.5 µL

-- --5.0 µL

10 µL

2.5 µL

5.0 µL

10 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¢-end32P-labelled GT-G4 (lanes 1–4) or 1 ng of 5¢-end 32P-labelled GT

(lanes 5–8) in 25 mM Tris ⁄HCl pH 8.0, containing 0.05% SDS,

0.05 mgÆmL)1 BSA, 0.1 mM 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 32P-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.



structure, 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

90 �C. They showed CD spectra indicative of absence

of structure only when they were resuspended in water

and heated at 65 �C (dashed green spectrum). On the

contrary, the structure of GT-G3 was not so stable

and it was disrupted by increasing the temperature to

65 �C (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

GTGT-G1GT-G2GT-G3GT-G4

GT-G4

GRO26AGRO29A

GRO26A

20ºC

20ºC, H2O65ºC, H2O

37ºC50ºC65ºC80ºC

100ºC

220

76

4

2

0

-2

240 260 280 300Wavelength [nm]

8

5

0

-3

CD[mdeg]

CD[mdeg]

CD[mdeg]

320

220 240 260 280 300Wavelength [nm]

320

220 240 260 280 300Wavelength [nm]

320

8

5

0

-4

A

B

Fig. 6. Circular dichroism of oligomers.

(A) A 10 lM solution of the indicated

oligomer was diluted in renaturation buffer

(150 mM NaCl, 10 mM K2HPO4 ⁄ KH2PO4,

1 mM EDTA pH 7.0) to a final concentration

of 0.5 lM recording the spectra at 37�C as

described in Experimental procedures. (B)

GRO26A and GT-G4 were diluted to 0.5 lM

final concentration in water and the spectra

were recorded at the indicated tempera-

tures.



G-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 kDa70 kDa

46 kDa32 kDa23 kDa

1 2 3 4 5

Competitor -- --

-- + + + +Proteins

% o

f ce

llula

r gr

owth

GT

GT

GRO29A

GRO

29A

GRO

26A

GRO26A

GT-G4

A

B

Fig. 7. Cytotoxic assay of GRO26A and GRO29A and their binding

to nuclear proteins. (A) CCRF-CEM cells (5 · 103) 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 lM

concentration. Cell growth was evaluated 72 h after oligonucleotide

addition by incorporation of 0.5 mgÆmL)1 of 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¢-end32P-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.



GT-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 60 �C 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



with 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 95 �Cfor 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)1 for 2.5 h

at 42 �C. In nondenaturing conditions, the oligomers (5 lg)were denatured by heating at 95 �C 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 32P-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)1 penicillin, 100 lgÆmL)1 streptomycin.

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)1 MTT 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 · 106 CCRF-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)1 beads. 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 4 �C 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 Coomassieblue-stained gel

We have previously shown that the protein eEF1A can be

isolated from a Coomassie blue stained gel [21]. Briefly, the



band corresponding to eEF1A was excised from the gel and

recovered in 50 mm Tris ⁄HCl pH 8.0, containing 0.1%

SDS, 0.1 mgÆmL)1 BSA, 0.2 mm EDTA, 2.5% glycerol.

After two steps of freeze ⁄ thawing, followed by precipitation

with cold acetone, the protein was rinsed with methanol,

denatured with 8 m urea and then renatured by overnight

incubation in a fixed volume of 50 mm Tris ⁄HCl pH 7.6,

100 mm KCl, 5 mm DTT, 0.1 mm PMSF. It was not poss-

ible to quantify the amount of recovered protein owing to

the presence of a high molar excess of BSA remaining in

the recovery buffer. Therefore, a fixed aliquot of the protein

was incubated with 1 ng of the indicated probes. After

30 min incubation at room temperature, the samples were

loaded onto a native 8% polyacrylamide gel in TBE and

run at 10 VÆcm)1, at a temperature of 4 �C.

EMSA and UV cross-linking assay

Samples containing 2 lg total nuclear proteins or 2.5–

10 lL of proteins purified by affinity chromatography or

by PAGE, were incubated with 2 ng [c-32P]ATP-labelled

probe and with 1 lg of poly(dIdC) and 1 lg of CT oligo-

mer (5¢-TCTTTCTTTCTTTCTTTCTTTCTTTCT-3¢), as

nonspecific competitors, in the absence or in the presence

of 500-fold molar excess of the indicated nonlabelled speci-

fic competitors. For EMSA, after 30 min incubation at

room temperature, the samples were loaded onto a 12%

polyacrylamide gel (acrylamide:bisacrylamide, 29 : 1, w ⁄w)in TBE and run at 10 VÆcm)1 for 2 h at 4 �C. The gels were

fixed in 10% acetic acid, then dried and exposed to KodaK

XAR-OMAT film. For the UV crosslinking, after 30 min

incubation at room temperature, samples were exposed to

UV light at 302 nm for 10 min using a transilluminator,

denatured and loaded onto 12% SDS ⁄PAGE gel. The gels

were dried and exposed to KodaK XAR-OMAT film.

Western blotting analysis

Five micrograms total nuclear proteins and 30 lL affinity

purified proteins, separated by 12% SDS ⁄PAGE were elect-

rophoretically transferred onto a 0.22-lm nitrocellulose

membrane (Schleicher & Schuell, Keene, NH) in 50 mm

Tris ⁄HCl, 40 mm glycine, 0.4% SDS, 20% methanol buffer,

using a transblot semidry apparatus system (Amersham

Pharmacia Biotech, Uppsala, Sweden). The membrane was

stained with Ponceau S (Sigma Chemical Co.) and destained

with deionized water. The blotted membrane was blocked

with 3% nonfat dried milk in NaCl ⁄Pi and incubated with

eEF1A mAb (1 lgÆmL)1) (Upstate Biotechnology, Lake Pla-

cid, NY) or with nucleolin mAb (Santa Cruz Biotechnology

Inc., CA) in NaCl ⁄Pi, overnight, at 4 �C with constant rock-

ing. Then, it was washed twice with deionized water and

incubated for 1.5 h with a horseradish peroxidase-conju-

gated antimouse IgG secondary antibody (Promega, Madi-

son, WI). After washing once with NaCl ⁄Pi containing

0.05% Tween-20 and four times with deionized water, the

nitrocellulose blot was developed using enhanced chemilumi-

nescence detection (Pierce, Rockford, IL) according to the

manufacturer’s protocols, and then exposed to X-ray film.

Circular dichroism

The oligomers were resuspended in 150 mm NaCl, 10 mm

K2HPO4 ⁄KH2PO4, 1 mm EDTA, pH 7.0 at 10 lm and

denatured at 100 �C and then renatured by slowly cooling

overnight. The oligomers were diluted in 150 mm NaCl,

10 mm K2HPO4 ⁄KH2PO4, 1 mm EDTA pH 7.0 buffer or in

water at a final concentration of 0.5 lm and then analysed

with a Jasco JT-710 CD spectrophotometer equipped with a

thermostatic bath. The spectra were recorded at different

temperatures by a Spectra manager analyser software.

Acknowledgments

This work was supported in part by FIRB number

RBNE0155LB and in part by the program ‘Rientro

cervelli’ art. 1 DM n.13 of Italian Ministry for Univer-

sity and Research, MIUR.

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Interaction of G-rich GT oligonucleotides with nuclear-associated eEF1A is correlated with their antiproliferative effect in haematopoietic human cancer cell lines

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