Identification of different isoforms of eEF1A in the nuclear fraction
of human T-lymphoblastic cancer cell line specifically binding
to aptameric cytotoxic GT oligomers
Barbara Dapas
1
, Gianluca Tell
2
, Andrea Scaloni
3
, Alex Pines
2
, Lino Ferrara
3
, Franco Quadrifoglio
1
and Bruna Scaggiante
1
1
Department of Biomedical Sciences and Technologies, University of Udine, Italy;
2
Department of Biochemistry, Biophysics
and Macromolecular Chemistry, University of Trieste, Italy;
3
Proteomics and Mass Spectrometry Laboratory, ISPAAM,
National Research Council, Naples, Italy
GT oligomers, showing a dose-dependent cytotoxic effect on
a variety of human cancer cell lines, but not on normal
human lymphocytes, recognize and form complexes with
nuclear proteins. By working with human T-lymphoblastic
CCRF-CEM cells and by using MS and SouthWestern
blotting, we identified eukaryotic elongation factor 1 alpha

stranded DNA oligomers are widely recognized to be
involved in important mechanisms associated with DNA
replication, repair and recombination [5–7]. Furthermore,
many reports evidenced that modulation of gene expression
[8,9], and stimulation or inhibition of cellular replication
[10,11], are influenced by single-stranded DNA sequences
specifically interacting with cellular proteins.
Oligonucleotides composed exclusively of G and T bases
have previously been shown to exert a specific, selective and
dose-dependent effect of cell growth inhibition on a variety
of human cancer cell lines [12]. The cytotoxic effect of these
GT oligomers was shown to be highly related to their ability
to form complexes with nuclear proteins, as measured by
UV cross-linking assays [12–15]. However, the nature of
these nuclear proteins behaving as single-stranded DNA-
binding proteins has not yet been identified [12–15]. A
protein isolated from fibroblasts with such an activity has
been already described [16], but it was able to tightly bind
either GA or GT oligomers. On the contrary, the nuclear
proteins binding to our GT oligomers did not specifically
recognize GA sequences [12]. More recently, it has been
shown that GT oligonucleotides, capable of forming
G-quartet structures, exerted a cytotoxic effect on human
cancer cell lines. By UV cross-linking assay, these oligomers
have been reported to interact with nucleolin, forming a
main complex of >100 kDa molecular mass [17]. This
complex was not formed when GT oligomers unable to
form a G-quartet structure were used [17,18]. The oligonu-
cleotide under our investigation (a 27-mer; see the Materials
and methods, below) did not present appreciable G-quartet

more basic isoforms of eEF1A in cancer cells, but not in
normal lymphocytes.
Materials and methods
Oligonucleotides
Oligonucleotides were purchased from MWG Biotech
(Ebersberg, Germany) as HPLC pure species and their
purity was confirmed by electrophoresis on an 18%
polyacrylamide/7
M
urea gel. For cell cultures, oligonucleo-
tides were resuspended in water and sterilized by centri-
fugation on a spin-X tube provided with a 0.22-l
M
filter
(Costar, Cambridge, MA, USA). The GT oligomer
sequence was: 5¢-TGT TTG TTT GTT TGT TTG TTT
GTT TGT-3¢; and the control CT sequence was: 5¢-TCT
TTC TTT CTT TCT TTC TTT CTT TCT-3¢. The
oligomers were 5¢ end-labelled by [c-
32
P]ATP with T4
polynucleotide kinase (MBI, Fermentas, MGMBH, St
Leon-Rot, Germany).
Cell culture and cytotoxic assay
The T-lymphoblastic leukaemic cell line (CCRF-CEM) and
normal human lymphocytes, obtained from peripheral
blood by separation on Ficoll–Isopaque (Gibco BRL, Life
Technologies, Milan, Italy), were cultured in RPMI-1640
supplemented with 10% fetal calf serum (FCS), 2 m
M

mined by the Bradford method [19] using BSA as standard.
EMSA, UV crosslinking and supershift assays
IntheEMSA,1ngof[c-
32
P]ATP-labelled oligonucleotide
was incubated with 2 lg of total nuclear or cytoplasmic
extracts supplemented with protease inhibitors (2 lgÆmL
)1
apoprotinin, 1 lgÆmL
)1
pepstatin, 1 m
M
dithiothreitol)
(Sigma Chemical Co.) in 20 m
M
Hepes, 0.42
M
NaCl,
1.5 m
M
MgCl
2
,0.2m
M
EDTA, 25% glycerol, pH 7.0,
containing nonspecific competitors [1 lg of salmon-sperm
DNA or 1 lg of poly(dIdC)] (Pharmacia, Uppsala, Sweden)
and the indicated amounts of unlabelled specific CT
oligomer competitor. When indicated, the protein excised
from the Coomassie-stained gel was recovered in 50 m

buffer (TBE) and electrophoresed at 10 V cm
)1
,ata
temperature of 4 °C.
In the supershift gel-mobility assay, samples of total
nuclear extracts were diluted 1 : 5 (v/v) in water and 0.5 lg
of protein was incubated for 2.5 h at room temperature with
the indicated amounts of specific rabbit polyclonal anti-
eEF1A serum or with corresponding amounts of control
total serum obtained from unimmunized rabbits. Then,
2 ng of specific [c-
32
P]-labelled GT oligonucleotide was
added to 30 lLof20m
M
Tris/HCl buffer, pH 7.5,
containing 75 m
M
KCl, 5 m
M
dithiotreitol, 6 lgBSA,
0.1% Tween 20, 0.025 m
M
Escherichia coli DNA, and 15%
glycerol. After 30 min of incubation at room temperature,
the samples were loaded onto a 7% native polyacrylamide
gel in 20 m
M
TBE buffer and electrophoresed for 90 min
at 10 V cm

M
guanidine hydrochloride solution in water. The membrane
was incubated overnight on a rocking shaker, at 4 °C, in
50 m
M
Hepes, pH 7.2, containing 0.1
M
KCl, 1 m
M
MgCl
2
,
5m
M
dithiothreitol, 1 m
M
EDTA and 10% glycerol.
Membranes derived from bidimensional PAGE blotting
were processed without the denaturation/renaturation pro-
cedure. Membranes were blocked by washing with the same
buffer, containing 5% nonfat dried milk and 5 m
M
dithiothreitol, for 1 h. Protein–DNA interaction was per-
formed overnight, at 4 °C, with 10 pmol of [c-
32
P]-labelled
oligonucleotide. Membranes were then washed between two
and four times for 10 min at room temperature, until the
background radioactivity started to decline, and were then
exposed to autoradiography.

EDTA, 0.25% Tween-20, 0.3 lgÆlL
)1
lysozyme) per gram of bacterial pellet and disrupted by
sonication. The lysate was centrifuged (10 000 g,20min,
4 °C) and the recombinant eEF1A protein collected in the
supernatant as a soluble protein.
Western blotting analysis
The blotted membrane was blocked with 3% nonfat dried
milk in PBS (NaCl/P
i
) and incubated with eEF1A mono-
clonal antibody (mAb) (1 lg/mL) (Upstate Biotechnology,
Lake Placid, NY, USA) in NaCl/P
i
,overnight,at4°Cwith
constant rocking. Then, it was washed twice with deionized
water and incubated for 1.5 h with an anti-mouse IgG-
conjugated horseradish peroxidase secondary antibody
(Promega, Madison, WI, USA). After washing once with
NaCl/P
i
containing 0.05% Tween-20 and four times with
deionized water, the nitrocellulose blot was developed using
enhanced chemiluminescence detection (Pierce, Rockford,
IL, USA) according to the manufacturer’s protocols, and
thenexposedtoX-rayfilm.
The same filter was stripped by a 10-min incubation in
4
M
guanidine hydrochloride, rinsed with 10 volumes of

rated and washed with water. Proteins were in-gel reduced,
S-alkylated and digested with trypsin, as previously des-
cribed [22]. Digest aliquots were removed and used directly
or subjected to a desalting/concentration step on lZip-
TipC
18
(Millipore Corp., Bedford, MA, USA) before
analysis by MALDI-MS. Peptide mixtures were loaded
onto the MALDI target, using the dried droplet technique
and a-cyano-4-hydroxycinnamic as matrix, and analysed by
using a Voyager-DE PRO mass spectrometer (Applied
Biosystems, Framingham, MA, USA). Internal-mass calib-
ration was performed with peptides deriving from trypsin
autoproteolysis. The mass spectra were acquired in either
reflectron or linear mode with delayed extraction. Post-
source decay (PSD) fragment ion spectra were acquired for
intense signals after isolation of the appropriate precursor
by using timed ion selection. Fragment ions were refocused
onto the detector by stepping the voltage applied to the
reflectron in the following ratios: 1.000 (precursor ion
segment), 0.960, 0.750, 0.563, 0.422, 0.316, 0.237, 0.178,
0.133, 0.100, 0.075, 0.056 and 0.042 (fragment segments).
Individual segments were superimposed by using the
DATA
EXPLORER
4.0 software (Applied Biosystems). All precursor
ion segments were acquired at low laser power (variable
attenuator ¼ 1950), for less than 200 laser pulses, to avoid
saturating the detector. The laser power was increased to
200 units for all the remaining segments of the PSD

Equilibration buffer containing 50 m
M
Tris/HCl (pH 8.8),
6
M
urea, 30% glycerol, 2% SDS, 2% dithiothreitol and
2.5% iodoacetamide. Gels were then used for Western or
SouthWestern blot analysis, as described above. As internal
normalizer, the presence of the nuclear protein, Ran-GTP,
was detected by Western blot on the same filters used to
analyse eEF1A protein, by using a specific mAb (BD
Pharmingen, CA, USA).
Results
Identification of eEF1A as a nuclear protein specifically
related to the cytotoxicity of GT oligomer in cancer cells
In order to identify the nuclear proteins that specifically
recognize cytotoxic GT oligomers, a 27-mer GT sequence
was used [12]. This oligomer forms a specific CRC with an
apparent molecular weight of 45 ± 7 kDa [12–15]. The
T-lymphoblastic CCRF-CEM cancer cell line was chosen
for this investigation as it was previously used to
demonstrate the specific cytotoxic action of the GT
oligomers [12–15]. Figure 1A shows that in SouthWestern
blots, the labelled GT oligomer bound (in a specific
manner) two main proteins, named P1 and P2, compared
with the binding of a labelled nontoxic CT oligomer, used
as a control. The latter showed a weak interaction with P1
and P2 proteins, whereas it preferentially bound to a
nuclear protein with a mass of 70 kDa (marked by an
asterisk), recognized to the same extent also by the GT

32
P-labelled GT probe (GT) and the other
half with a c
32
P-labelled CT oligomer (CT) as a control, at 4 °C(each
probe counted 650 000 c.p.m.). After incubation overnight, the filters
were rinsed and then exposed to Omat XAR Kodak film. (B) Immu-
noblotting of subcellular fractions. Twenty micrograms of cytoplasmic
(lane 1) or nuclear extract (lane 2) fractions of CCRF-CEM cells was
separated by SDS/PAGE (8% gel). After blotting onto nitrocellulose
membrane, the filter was probed with the nuclear-specific antibodies
anti-TBP or anti-Egr1, as described in the Materials and methods. As a
loading control, the presence of b-actin protein was also confirmed by
using specific antibody, as described in the Materials and methods.
3254 B. Dapas et al. (Eur. J. Biochem. 270) Ó FEBS 2003
The comparison of this peptide-mass fingerprint with the
theoretical ones, calculated by an in silico digestion of all
human sequences occurring in the databases, identified P1
as eEF1A. Furthermore, PSD experiments performed on
selected peptide precursor ions (i.e. m/z 1405.4 and 1780.9)
generated internal sequence tags that unambiguously con-
firmed the nature of this protein (data not shown).
Moreover, the spectrum reported in the figure showed the
occurrence of a series of signals that were not interpreted
simply on the basis of the eEF1A sequence, but according to
the post-translational modifications already described
for this protein [25]. It demonstrated the occurrence of
Ne-dimethyllysine (Lys55, Lys165) and Ne-trimethyllysine
(Lys79, Lys318) in the eEF1A sample purified from
T-lymphoblastic CCRF-CEM cancer cells. No data on

specifically generate a 100-kDa complex with GT oligomers
that form a G-quartet structure, thus exerting a cytotoxic
effect on human cancer cell lines [10,17]. PSD experiments
performed on selected precursor ions (i.e. m/z 2201.3 and
1649.7) allowed internal sequence tags to be obtained,
definitively demonstrating the nature of this species
(Fig. 2B).
The identity of P1 was also assayed by Western-blotting
experiments with a mAb for eEF1A. As illustrated in
Fig. 3A, the protein excised from the Coomassie-stained gel
was recognized by the specific eEF1A antibody (Fig. 3A,
lane 3). As controls, recombinant eEF1A protein (Fig. 3A,
lane 1) and a sample obtained from total nuclear extracts
(Fig.3A,lane2)weretested.
The EMSA with the protein eluted from the P1 band
excised from the Coomassie-stained gel of CCRF-CEM cell
nuclear extracts showed that this protein selectively recog-
nized the GT oligomer with respect to control CT sequence,
similarly to results obtained with the total nuclear extracts
[12–15]. It is noteworthy that all the EMSA and UV cross-
linking assays were performed using a buffer containing
25% glycerol to preserve the activity of eEF1A. As
illustrated in Fig. 3B, the eEF1A recovered from the
P1-excised band showed a stronger interaction when
incubated with the labelled GT oligomer (Fig. 3B, lane 1)
than when incubated with the labelled control CT oligomer
(Fig. 3B, lane 7). Moreover, the presence of a fivefold molar
excess of CT-unlabelled competitor (Fig. 3B, lane 5), did
not completely displace the GT oligonucleotide from the
protein interaction. On the contrary, only a fivefold molar

More interestingly, as illustrated in Fig. 5A, with respect
to the protein of the nuclear extracts, the soluble eEF1A
recovered from the cytoplamic fraction did not bind to a GT
oligomer in SouthWestern blotting. Although comparable
quantities of the protein were loaded onto the gel, as
evidenced by Western blotting, in the cytoplasmic extract,
only the nucleolin band was evident. Similarly to this and to
the previous results [12], in the UV cross-linking assay the
specific CRC displayed by the nuclear extract was not
present in the cytoplasmic sample (Fig. 5B). On the
contrary, the cytoplasmic extract showed a band of about
28 kDa, previously demonstrated to bind to GT in a
nonspecific manner [12].
Characterization of eEF1A in normal and cancer cells
In order to compare the binding properties of the nuclear
eEF1A in normal cells compared with those in cancer
CCRF-CEM cells, human lymphocytes were isolated
from the peripheral blood of normal donors. These cells
were not sensitive to the cytotoxic effect of GT oligomers
Fig. 3. P1 Western blotting analysis and affinity measurements for GT
oligomer. (A) Western blotting analysis. Protein samples were separ-
ated by SDS/PAGE (12% gel) and then transferred onto a nitrocel-
lulose filter and incubated with mAb for eEF1A, as described in the
Materials and methods. Lane 1, bacterial recombinant eEF1A protein
(R eEF1A); lane 2, eEF1A protein from total nuclear extracts (NE
eEF1A); lane 3, P1 band excised from an SDS/PAGE gel (P1). (B) P1
affinity for GT oligomer. P1 protein, excised from an SDS/PAGE gel
loaded with 50 lg of total nuclear extract, was renatured as described
in the Materials and methods. Five microlitres of sample was then
incubated with 2 ng of [c-

total serum (CRS), for 2.5 h at room temperature. Then, 2 ng of c
32
P-
labelled GT oligomer was added to the samples, as reported in the
Materials and methods. After a further 30 min of incubation at room
temperature, the samples were loaded onto a 7% polyacrylamide gel in
0.5 · Tris/borate/EDTA (TBE) buffer and electrophoresed at 4 °C.
The gel was dried and exposed to Omat XAR Kodak film. c
32
P-
Labelled GT oligomer was incubated with buffer (lane 1), with 9 lgof
polyclonal anti-eEF1A (lane 2), with 9 lg of total CRS (lane 3),
with nuclear proteins and 9 lg of polyclonal anti-eEF1A (lane 4), with
nuclearproteinsand0.9lg of polyclonal anti-eEF1A (lane 5), with
nuclearproteinsand9lg of total CRS (lane 6), with nuclear proteins
and 0.9 lg of total CRS (lane 7) or with nuclear proteins only (lane 8).
The arrow indicates the supershift.
3256 B. Dapas et al. (Eur. J. Biochem. 270) Ó FEBS 2003
(Fig. 6A), and their nuclear proteins did not form the
CRC with the GT sequence (marked by arrow), as shown
by EMSA or UV cross-linking assays (Fig. 6B,C). To
investigate the binding properties of the lymphocyte
eEF1A protein, SouthWestern blots were performed. It
was found that the CCRF-CEM nuclear extracts con-
tained higher amounts of eEF1A than those of human
lymphocytes. In fact, Western blotting experiments dem-
onstrated that the relative amount of eEF1A recovered
from lymphocyte nuclear extracts was 2.7 ± 0.8-fold less
than that obtained from cancer CCRF-CEM T lympho-
blasts (mean of three independent experiments). For this

4
CCRF-CEM cells or peripheral
normal human lymphocytes were seeded in 100 lL of complete
medium on 96-well microtiter plates. After 4 h of incubation, 7.5 l
M
of
GT oligomer or control CT sequence were added to the cells. The
percentage of viable cells was assayed after 72 h of incubation by
determining the incorporation of 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl-tetrazolium bromide, as described in Materials and methods.
(B) EMSA assay. Two micrograms of total nuclear proteins derived
from CCRF-CEM cells or from human lymphocytes were incubated
with 1.5 lg of poly(dIdC), 1 lgofCTand1ngofc
32
P-labelled GT
oligomer in a buffer containing 25% glycerol, as described in the
Materials and methods. After incubation at room temperature for
30 min, the samples were loaded onto a 7% polyacrylamide gel in Tris/
borate/EDTA (TBE) buffer and run at 4 °C. (C) UV cross-linking
assay. Two micrograms of total nuclear proteins derived from CCRF-
CEM cells or from human lymphocytes were incubated with 1.5 lgof
poly(dIdC), 1 lgofCTand1ngofc
32
P-labelled GT oligomer, as
described in the Materials and methods. The samples were then
exposed for 10 min to a 302 nm UV light, added to SDS/PAGE
loading buffer and separated by SDS/PAGE (12% gel). The arrows
indicated the specific cytotoxicity-related complex.
Ó FEBS 2003 eEF1A binding to aptameric cytotoxic GT oligomers (Eur. J. Biochem. 270) 3257
normalizing the quantities of loaded proteins on a

nuclear protein from normal human lymphocytes were separated by
SDS/PAGE (8% gel) and transferred onto a nitrocellulose filter, as
described in the Materials and methods. The proteins were denatured
and renatured as described above, and the filter was hybridized with
c
32
P-labelled GT probe at 4 °C. After overnight incubation with the
probe, the filters were rinsed, as described in the Materials and
methods, and then exposed to Omat XAR Kodak film. (B) Western-
blotting analysis. Protein samples reported in (A) were analysed for
eEF1A and b-actin content.
Fig. 8. Bidimensional PAGE analysis of nuclear elongation factor 1
alpha (eEF1A). (A) Thirty micrograms of nuclear extracts from CCRF
CEM cells and normal human lymphocytes, calculated from evalua-
tion of the protein content in a Coomassie-stained gel, were analysed
by bidimensional PAGE, as described in the Materials and methods.
The presence of eEF1A was tested by Western blot analysis by using
the specific antibody anti-eEF1A. As an internal normalizer of loading
amount and focusing position, the presence of the constitutive nuclear
transporter, Ran-GTP, was also tested by using a specific monoclonal
antibody. (B) Bidimensional PAGE analysis of other samples, con-
firming the reproducibility of data obtained. Thirty micrograms of
nuclear extracts from normal human lymphocytes were similarly
analysed by bidimensional PAGE and compared with CCRF-CEM
nuclear extracts. The presence of eEF1A was confirmed by Western
blot analysis using the specific antibody, anti-eEF1A. Only the higher
magnification of IEF of the eEF1A region is reported.
3258 B. Dapas et al. (Eur. J. Biochem. 270) Ó FEBS 2003
cells, the presence of two different clusters of eEF1A
isoforms (cluster 1 and cluster 2). The apparent pI of cluster

more basic form of eEF1A, whereas a very weak interaction
was found for the isoform of eEF1A focusing at a pH of
9.0. The recognition seemed highly specific because, under
these experimental conditions, no other interactions were
detected on the filter. Moreover, no significant interaction in
bidimensional PAGE SouthWestern blots was found on
human lymphocytes at the position corresponding to
eEF1A (see Fig. 10), once more indicating that normal
eEF1A did not react with the GT oligomer.
Fig. 9. Comparative analysis of bidimensional PAGE analysis of
SouthWestern and Western blots for CCRF-CEM cells. Two samples of
50 lg of total nuclear protein from CCRF-CEM cells were separated,
in parallel, by bidimensional PAGE and blotted onto nitrocellulose
filters, as described in the Materials and methods. (A) Western blot-
ting. The filter was assayed using anti-eEF1A mAb, as described in the
Materials and methods. The position of the eEF1A protein was con-
firmed by revealing the presence of the Ran–GTP protein. (B)
SouthWestern blotting. The filter was assayed for SouthWestern
blotting, as described in the experimental section using, as probe, c
32
P-
labelled GT oligomer and then exposed to Omat XAR Kodak film.
Fig. 10. Comparative analysis of bidimensional PAGE, SouthWestern
and Western blotting for human lymphocytes. Two samples of 50 lgof
total nuclear protein of normal human lymphocytes, previously nor-
malized with respect to CCRF-CEM cell protein by a Coomassie-
stained gel, were separated, in parallel, by bidimensional PAGE and
blotted onto nitrocellulose filters, as described in the Materials and
methods. (A) Western blotting. The filter was assayed for Western blot
using anti-eEF1A mAb, as described in the Materials and methods.

complex with vigilin, for exporting tRNA [37], and with
ZPR1, for inducing cell proliferation upon mitogen stimu-
lation [38]. Thus, the elucidation of a possible role for the
nuclear fraction of eEF1A in modulating nuclear func-
tion and gene expression could gain new insights in
tumorigenesis.
In this manuscript, we demonstrate that eEF1A, isolated
from nuclear extracts of CCRF-CEM cancer cells, is
specifically recognized by a cytotoxic GT sequence. This
protein was found to be the polypeptide component of the
CRC,basedonMALDI-MSanalysis,Westernblotting
experiments and supershift assays. In contrast, the GT
oligomer did not bind to the eEF1A of normal human
lymphocytes and these cells were not sensitive to the
cytotoxic action of the GT. It should be noted that nucleolin
was also recognized by the GT oligomer [10,17]. However,
under native conditions, the more abundant CRC observed,
migrated with an apparent mass not associated with the
nucleolin–oligomer complex [12–15], probably because the
GT sequence used does not form, in appreciable quantity,
the G-quartet structure specifically recognized by this
protein, as revealed by gel electrophoresis and circular
dichroism studies [12,14]. Moreover, nucleolin, and not
eEF1A, was bound by the GT oligomer in lymphocyte
sample on one-dimensional SouthWestern assay; however,
lymphocyte viability was not affected by GT. In cytoplasmic
extracts, the nucleolin was found to bind to GT in a
SouthWestern assay, but not in EMSA or UV crosslinking
assays. Furthermore, we analysed a G-rich GT sequence
able to form the G-quartet structure and thus to bind to

Accordingly, GT oligomers did not elicit cytotoxic action on
these cells, and did not form the CRC with the nuclear
proteins when a fourfold increase in protein content was
loaded onto the gel (data not shown). These results
underline that eEF1A from CCRF-CEM cell nuclear
extracts displays specificity in recognizing GT oligomers,
and the selective cytotoxic action on CCRF-CEM cells
suggests a possible role for eEF1A in maintaining the
viability and proliferative activity of cancer cells. One
hypothesis may be that these oligomers exert their action by
blocking the binding of eEF1A to its ligand in cancer cells,
perhaps to zinc finger proteins involved in the modulation
of cell proliferation, as proposed by Gangwani et al. [38].
Bidimensional PAGE analysis of eEF1A combined with
a specific Western blotting assay showed the occurrence of
two distinct clusters of spots in T-lymphoblastic CCRF-
CEM cells, whereas normal lymphocytes presented only one
cluster. In particular, the newly occurring components in
cancer cells (cluster 2) focused at a more basic pH. This
result could hypothetically explain the higher affinity of the
protein towards oligonucleotides simply on the basis of a
charge increase at specific amino acids in its nucleotide-
binding site, but not its binding selectivity for the GT
sequences.
Different post-translational modifications have been
reported to occur in the eEF1A polypeptide chain, such as
phosphorylation, methylation and glyceryl-phosphoryl-
ethanolamine addition [25,27,29,39,40] but, to date, their
functional significance has not been totally solved. Differ-
ences in the level of phosphorylation of eEF1A have already

above.
By using SouthWestern assays on bidimensional PAGE
of CCRF-CEM cell nuclear extracts, we observed that the
more basic isoform of eEF1A binds to the GT oligomer,
whereas only a weak interaction was found for the eEF1A
migrating like the normal constitutive molecule. Accord-
ingly, eEF1A from normal human lymphocytes did not
recognize GT oligomer at all. On this basis, it could be
tempting to speculate that different isoforms of nuclear
eEF1A, in particular a more basic molecule, should
account for the different sensitivities to cytotoxic effect
exerted by GT oligomers. These differences might be
related to a variable degree of post-translational process-
ing, although the high shift in the apparent pI of the more
basic molecules cannot alone be simply explained by
modifications such as methylation of the lysine residues.
Even if theoretically possible, this phenomenon might
account for a low probable high number of methylated
lysine residues in a protein with a total low-density
charge. Thus, in these high basic proteins it might be that
either methylation of the lysine or specific substitutions in
the amino acid sequence, increasing the number of basic
residues of lysine, arginine and histidine, could occur.
However it seemed probable that the eEF1A isoforms
could act as a controlling event in maintaining the
viability and/or promoting the growth of T-lymphoblastic
tumour cells. Supporting evidence for the specific role of
this nuclear-associated eEF1A species come from the
observation that the soluble cytoplasmic eEF1A from
cancer cells did not bind cytotoxic GT oligomers in

single-stranded DNA binding protein detected in mammalian cell
extracts by gel retardation assays and UV cross-linking of long
and short single-stranded DNA molecules. J. Biol. Chem. 268,
7147–7154.
6. Coverley, D., Kenny, M.K., Lane, D.P. & Wood, R.D. (1992) A
role for the human single-stranded DNA binding protein HSSB/
RPA in an early stage of nucleotide excision repair. Nucleic Acids
Res. 20, 3873–3880.
7. Yee, H.A., Wong, A.K.C., van de Sande, J.H. & Rattner, J.B.
(1991) Identification of novel single-stranded d[TC]n binding
proteins in several mammalian species. Nucleic Acids Res. 19,
949–953.
8. Michelotti,E.F.,Tomonaga,T.,Krutzsch,H.&Levens,D.(1995)
Cellular nucleic acid binding protein regulates the CT element of
the human c-myc protooncogene. J. Biol. Chem. 270, 9494–9499.
9. Yano-Yanagisawa, H., Li, Y., Wang, H. & Kowi, Y. (1995) Sin-
gle-stranded DNA binding proteins isolated from mouse brain
recognize specific trinucleotide repeat sequences in vitro. Nucleic
Acids Res. 23, 2654–2660.
10. Xu, X., Hamhouyia, F., Thomas, S.D., Burke, T.J., Girvan, A.C.,
McGregor,W.G.,Trent,J.O.,Miller,D.M.&Bates,P.J.(2001)
Inhibition of DNA replication and induction of S phase cell
cycle arrest by G-rich oligonucleotides. J. Biol. Chem. 276,
43221–43230.
11. Lang,R.,Hultner,L.,Lipford,G.B.,Wagner,H.&Heeg,K.
(1999) Guanosine-rich oligodeoxynucleotides induce proliferation
of macrophage progenitors in cultures of murine bone marrow
cells. Eur. J. Immunol. 29, 3496–3506.
12. Scaggiante,B.,Morassutti,C.,Dapas,B.,Tolazzi,G.,Ustulin,F.
& Quadrifoglio, F. (1998) Human cancer cell lines growth

757–766.
19. Bradford, M.M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72, 248–254.
20. Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680–685.
21. Tell, G., Pines, A., Paron, I., D’Elia, A., Bisca, A., Kelley, M.R.,
Manzini, G. & Damante, G. (2002) Mitochondrial localization of
APE/Ref-1 in thyroid cells. J. Biol. Chem. 277, 14564–14574.
22. Allegrini, S., Scaloni, A., Ferrara, L., Pesi, R., Pinna, P., Sgarrella,
F., Camici, M., Eriksson, F. & Tozzi, M.G. (2001) Bovine cyto-
solic 5¢-nucleotidase acts through the formation of an aspartate
52-phosphoenzyme intermediate. J. Biol. Chem. 276, 33526–33532.
23. Clauser, K.R., Baker, P.R. & Burlingame, A.L. (1999) Role of
accurate mass measurement [+/- 10 ppm] in protein identification
strategies employing MS or MS/MS and database searching. Anal.
Chem. 71, 2871–2882.
24. Zhang, W. & Chait, B.T. (2000) ProFound: an expert system for
protein identification using mass spectrometric peptide mapping
information. Anal. Chem. 72, 2482–2489.
25.Dever,T.E.,Costello,C.E.,Owens,C.L.,Rosenberry,T.L.&
Merrick, W.C. (1989) Location of seven post-translational
modifications in rabbit elongation factor 1 alpha including
dimethyllysine, trimethyllysine, and glycerylphosphorylethanol-
amine. J. Biol. Chem. 264, 20518–20525.
26. Gopalkrishnan, R.V., Su, Z.Z., Goldstein, N.I. & Fisher, P.B.
(1999) Translational infidelity and human cancer: role of the PTI-1
oncogene. Int. J. Biochem. Cell. Biol. 31, 151–162.
27. Sun, Y., Lin, J., Katz, A.E. & Fisher, P.B. (1997) Human prostatic
carcinoma oncogene PTI-1 is expressed in human tumor cell lines

35. Sanders, J., Brandsma, M., Janssen, G.M., Dijk, J. & Moller, W.
(1996) Immunofluorescence studies of human fibroblasts demon-
strate the presence of the complex of elongation factor-1
beta gamma delta in the endoplasmic reticulum. J. Cell Sci. 109,
1113–1117.
36. Janssen, G.M., van Damme, H.T., Kriek, J., Amons, R. & Moller,
W. (1994) The subunit structure of elongation factor 1 from
Artemia. Why two alpha-chains in this complex? J. Biol. Chem.
269, 31410–31417.
37. Kruse, C., Grunweller, A., Willkomm, D.K., Pfeiffer, T., Hart-
mann, R.K. & Muller, P.K. (1998) tRNA is entrapped in similar,
but distinct, nuclear and cytoplasmic ribonucleoprotein com-
plexes, both of which contain vigilin and elongation factor 1 alpha.
Biochem. J. 329, 615–621.
38. Gangwani, L., Mikrut, M., Galcheva-Gargova, Z. & Davis, R.J.
(1998) Interaction of ZPR1 with translation elongation factor-
1alpha in proliferating cells. J. Cell Biol. 143, 1471–1484.
39. Das, T., Mathur, M., Gupta, A.K., Janssen, G.M. & Banerjie,
A.Y. (1998) RNA polymerase of vesicular stomatitis virus spe-
cifically associates with translation elongation factor-1 alpha-
betagamma for its activity. Proc. Natl Acad. Sci. USA 95, 1449–
1454.
40. Ransom, W.D., Lao, P.C., Gage, D.A. & Boss, W.F. (1998)
Phosphoglycerylethanolamine posttranslational modification of
plant eukaryotic elongation factor 1alpha. Plant Physiol. 117,
949–960.
41. Blackwell, J.L. & Brinton, M.A. (1997) Translation elongation
factor-1 alpha interacts with the 3¢ stem–loop region of West Nile
virus genomic RNA. J. Virol. 71, 6433–6444.
42. Coppard, N.J., Clark, B.F. & Cramer, F. (1983) Methylation of