Polyamines interact with DNA as molecular aggregatesLuciano D’Agostino1and Aldo Di Luccia2 1 Department of Clinical and Experimental Medicine, Federico II University, Naples, Italy; 2 In
Trang 1Polyamines interact with DNA as molecular aggregates
Luciano D’Agostino1and Aldo Di Luccia2
1 Department of Clinical and Experimental Medicine, Federico II University, Naples, Italy; 2 Institute of Food Science
and Technology – National Research Council, Avellino, Italy
New compounds, named nuclear aggregates of polyamines,
having a molecular mass of 8000, 4800 and < 1000 Da, were
found in the nuclear extracts of several replicating cells Their
molecular structure is based on the formation of ionic bonds
between polyamine ammonium and phosphate groups The
production of the 4800 Da compound, resulting from the
aggregation of five or more < 1000 Da units, was increased
in Caco-2 cells treated with the mitogen gastrin Dissolving
single polyamines in phosphate buffer resulted in the in vitro
aggregation of polyamines with the formation of
com-pounds with molecular masses identical to those of natural
aggregates After the interaction of the 4800 Da molecular
aggregate with the genomic DNA at 37C, both the absorbance of DNA in phosphate buffer and the DNA mobility in agarose gel increased greatly Furthermore, these compounds were able to protect the genomic DNA from digestion by DNase I, a phosphodiesterasic endonuclease Our data indicate that the nuclear aggregate of polyamines interacts with DNA phosphate groups and influence, more efficaciously than single polyamines, both the conformation and the protection of the DNA
Keywords: DNA conformation; DNA protection; apoptosis; molecular aggregates; polyamines
An increased intracellular concentration of polyamines is
necessary for the activation of DNA synthesis and cell
replication [1–4] The intestinal replicating cells are
partic-ularly capable of accumulating polyamines promoting both
their synthesis, through the activation of the enzyme
ornithine decarboxylase, and their uptake from the
extra-cellular space [5–9] Caco-2 cells, derived from a human
colon carcinoma, after confluence spontaneously
differen-tiate assuming morphological and functional features
sim-ilar to those of the small intestinal enterocytes This cell line
represents a useful in vitro model for studying the
mecha-nisms involved in polyamine-dependent cell replication [6,7]
Gastrin, a powerful mitogen for gastro-intestinal cells,
stimulates the growth of Caco-2 cells and increases the
intracellular concentration of polyamines promoting both
their endogenous synthesis and their uptake [7]
The interactions of the cationic polyamines with
negat-ively charged phosphate groups of nucleotidic
macromole-cules are considered to be of great biological importance In
particular, the interaction of polyamines with DNA
induces important conformational modifications in DNA
structure [10]
In a previous study, we aimed to investigate the fate of
putrescine when taken up from the medium of Caco-2 cells
and to analyse its binding to nuclear proteins We reported
the presence of compounds with molecular masses of about
8000, 4800 and < 1000 Da (actually, named 180 Da) in the
nuclear extracts of replicating cells In contrast, nuclear
extracts of differentiated Caco-2 cells lacked the 4800 Da
compound It was shown that these compounds, detected
by gel permeation chromatography (a separation technique that does not alter the molecular interactions) were able to establish noncovalent bonds with the exogenous radioactive polyamines We hypothesized that these compounds were oligopeptides [11]
Our aim in the present work was to: (a) better define the chemical structure of these nuclear compounds, herein named NAPs (nuclear aggregates of polyamines); (b) study their fluctuating concentrations during the various phases of Caco-2 cell replication induced by gastrin treatment; (c) ascertain their presence in other replicating cell lines; and (d) investigate the effects of NAP–DNA interaction on DNA conformation and DNA protection by means of spectro-photometric and electrophoretic analyses
M A T E R I A L S A N D M E T H O D S Cells
Pre-confluent (replicating) Caco-2 cells at day 6 of culture were used for the experiments [7] In order to favour cell synchronization, Caco-2 cells were left without changing the media for 60 h Cell replication was promoted by adding
10)10Mgastrin (ICN) to the dishes Nuclear extracts of cells treated with gastrin for 0, 2, 4, 8 and 12 h were fractionated
by gel permeation chromatography (GPC) The GPC peaks with the molecular masses of 8000, 4800 and < 1000 Da were collected and analysed for the detection of Fmoc derivative polyamines using reversed phase-HPLC The replication rate of these cells was evaluated by assessing bromodeoxyuridine (BrdU) incorporation, an S-phase marker [12]
NAP formation was also investigated in Caco-2 cells starved for 4 days
The following replicating cells, generously donated by other laboratories, were also used for NAP isolation: the
Correspondence to L D’Agostino, Facolta` di Medicina,
Via S Pansini 5, 80131, Napoli, Italy Fax/Tel.: +39 81 746 2707,
E-mail: luciano@unina.it.
Abbreviations: GPC, Gel permeation chromatography; NAP, nuclear
aggregate of polyamines; BrdU, bromodeoxyuridine.
(Received 26 March 2002, revised 24 June 2002, accepted 19 July 2002)
Trang 2primary cultures chicken embryo chondrocyte, chicken
embryo fibroblast and quail embryo chondrocyte, and the
cell lines KB human epidermoid oropharingeal carcinoma,
PCCl3 rat thyroid and NA101 chicken embryo chondrocyte
transformed by RSV These cells were cultured in the
recommended standard conditions and used when
pre-confluent
Nuclei and nuclear extract preparations
Cells were solubilized in solution 1 (15 mM NaCl, 60 mM
KCl, 14 mM 2-mercaptoethanol, 2 mM EDTA, 15 mM
Hepes pH 7.9, 0.3Msucrose) containing 1% Triton-X100
and phenylmethanesulfonyl fluoride The crude nuclear
pellet was prepared by spinning the extracts at 3500 r.p.m
for 10 min at 4C on a 1-Msucrose cushion in solution 1
The purity of nuclei preparations was tested by light
microscopy after Crystal violet staining Nuclear extracts
were prepared as described [13] The nuclear pellet was
re-suspended in high-salt concentration (NaCl 400 mM)
solution 1 and centrifuged at 10 000 g for 10 min
GPC
The nuclear extracts were analysed by GPC–HPLC using a
Superose 12 prepacked column HR 10/30, which has a
separation range of 1000–300 000 Da (Pharmacia) The
column was equilibrated with 0.05M sodium phosphate
buffer (pH 7.2) containing 0.15M NaCl and calibrated
using compounds with varying molecular masses, as
indicated by the manufacturer Fifty lL of the nuclear
extracts were diluted in equal volume of equilibration buffer
and loaded onto the column The nuclear extracts were
eluted with the same buffer at 0.4 mLÆmin)1and detected at
280 nm The single GPC peaks with a molecular mass
< 10 000 Da were collected and stored at )20 C The
GPC analysis allowed the study of the nuclear extracts in
native conditions and in the absence of strong interactions
(electric field or denaturing and reducing conditions), which
disrupt noncovalent bonds Therefore, it was our sole
possible choice
RP-HPLC Fmoc-polyamine derivatives
The presence of polyamines in the GPC peaks was analysed
by RP-HPLC using a precolumn Fmoc derivatization [14]
The excitation wavelength was set at 265 nm and
fluores-cence emission was monitored at 305 nm to increase
sensibility in the Fmoc derivative analyses
Amino acid analysis of GPG peaks by RP-HPLC
of Fmoc derivatives
The presence of oligopeptides and/or free amino acids was
excluded by performing the amino acid analyses by
RP-HPLC of Fmoc derivatives before and after the
hydrolysis of GPC peaks Dried GPC peaks were dissolved
in 500 lL 6M HCl Each solution was put into vacuum
hydrolysis tubes (Pierce, Rockford, IL, USA), gassed with
nitrogen and sealed The tubes were incubated at 110C for
24 h in a Reacti-Therm for dry block heating apparatus
(Pierce) Derivatization of amino acids with Fmoc and their
RP-HPLC analysis were both performed as described [15]
The wavelength excitation was set at 265 nm and fluores-cence emission was monitored at 305 nm
Spectrophotometric scan of NAPs One ml of each NAP, obtained from GPC collection of Caco-2 cells nuclear extracts, was scanned at room temperature from 400 to 190 nm at 10 nmÆs)1by a Cary spectrophotometer 1E series (Varian Inc., Walnut Creek,
CA, USA)
In vitro aggregation of polyamines The in vitro aggregation of polyamines was studied dissol-ving putrescine, spermidine and spermine (Sigma) at equal molar concentrations in 0.05M sodium phosphate buffer (pH 7.2) containing 0.15MNaCl to obtain a mixture with a final concentration of 25 lM This polyamine solution was then analysed by GPC
In order to assess the role of spermine in NAP formation, the concentration of this 25 lM polyamine solution was brought to 50 lMby adding spermine and then performing
a new GPC run The GPC analyses were carried out as described above, using as mobile phase phosphate or Tris/HCl buffers
NMR analysis Putrescine, spermidine and spermine were dissolved in D2O
or in D2O phosphate buffer (0.05M pH 7.2, containing 0.15MNaCl) at a concentration of 10 mgÆmL)1
All spectra were recorded by a Bruker DRX-600 NMR spectrometer, operating at 599.19 MHz for1H, using the UXNMR software package; 1D-TOCSY experiments were carried out using the conventional pulse sequences, as described [16]
NAP–DNA spectrophotometry Spectrophotometric assays were performed by mixing
200 lL of the 8000, 4800 and < 1000 Da (the most retained) peaks with 100 lL of human genomic DNA in Tris/EDTA buffer (1.3 lgÆlL)1) This solution was brought
to a volume of 800 lL with 0.05Msodium phosphate buffer (pH 7.2) to obtain a concentration of 0.25 ng total polyamineÆlg)1DNA ratio The absorbance (A) of each NAP–DNA sample was measured with a thermostated Cary spectrophotometer 1E series (Varian Inc., Walnut Creek, CA, USA) at 260 nm after 6 min incubation at 15,
37 and 55C Controls were NAP solutions in the absence
of DNA or single polyamines at 1 lM concentration in water
DNA electrophoresis Electrophoresis of human genomic DNA or 1 kb DNA ladder (Sigma-Aldrich) was carried out in a HE 100 supersub (Amersham Pharmacia Biotech) at a constant temperature of 37C applying an electric field strength of 11.1 VÆcm)1in Tris/borate/EDTA
Ten lL of a mixture of genomic DNA and 8000 or 4800
or < 1000 NAP (0.25 ng total polyamineÆlg)1DNA) were loaded on a 1.5% ultrapure DNA grade agarose gel after an
Trang 3incubation period of 6 min at 37C The final
concentra-tion of DNA was 0.4 lgÆlL)1 Ethidium bromide buffer,
0.1 lgÆmL)1, was added to the gel and to the electrophoresis
buffer The duration of electrophoresis was 3.5 h
The influence of NAPs on the electrophoretic mobility of
small linear fragments of DNA was evaluated using a
241 base pair PCR product of the BRCA 1 gene The
electrophoretic conditions were the same as those used for
the genomic DNA
Two microliters of 1 kb DNA ladder (200 lgÆlL)1) were
mixed with 3 lL of the 8000, 4800 or < 1000 NAP These
solutions were incubated at 37C for 6 min, 5 lL
exonuclease III (65 UÆlL)1) were added and incubation
was continued for 30 min The samples were then separated
by electrophoresis for 1 h in the conditions described above
Genomic DNA (4 lg per 2.5 lL phosphate buffer) was
incubated for 6 min at 37C with 4.5 lL of 8000, 4800
or < 1000 NAPs (mean polyamine concentration:
0.25 ngÆlg)1DNA) or aqueous solutions of single
polyam-ines (0.25 ngÆlg)1DNA) The degradation of the genomic
DNA was examined by means of DNase I (RQ1RNase-free
DNase, Promega) at concentration of 0.025 UÆlg)1DNA
Briefly, 1 lL of the DNase I solution was added to 1 lL of
the reaction buffer solution (400 mM Tris/HCl at pH 8,
100 mMMgSO4, 10 mMCaCl2) and then mixed with NAP–
DNA or polyamine–DNA solutions Enzyme action was
stopped after 30 min at 37C adding 1 lL 20 mMEDTA
pH 8 Samples were then separated by electrophoresis for
1 h using the conditions described above
Each gel was photographed with a Polaroid MP-4 L
camera and migration distances were measured with a ruler
from photographs
Statistics
Differences in polyamine concentrations among NAPs were
tested for significance by one-wayANOVAwith Bonferroni
test for multiple means comparisons using SPSS software
package forWINDOWS, release 10.0.7 Values were
consid-ered significant at P < 0.05
R E S U L T S
GPC of nuclear extracts of replicating cells
A representative profile of GPC analysis of the nuclear
extracts of Caco-2 cells stimulated to replicate by gastrin is
shown in Fig 1 The chromatograms showed three peaks
The molecular masses of first two were estimated to be 8000
and 4800 Da The third peak, the most retained, fell out of
the column separation range and, for this reason was
marked as < 1000 Da Compelling variations in GPC
peaks were recorded after 2, 4, 8 and 12 h of gastrin
treatment The chromatograms at time 0 showed two minor
peaks corresponding to 8000 and 4800 Da (19.6 and 16.1%,
respectively) and a major one corresponding to < 1000 Da
(64.3%) Two hours after gastrin treatment, there was a
huge increase in the 4800 Da peak area value (62.2%) This
peak declined at 4 and 8 h and returned to the initial value
after 12 h of gastrin stimulation The 8000 Da peak
increased at 4 h (34.1%), remained the same at 8 h and
declined at 12 h The < 1000 Da peak strongly decreased at
2 and 4 h (18.9 and 19.8%, respectively) after which it
progressively increased, reaching the basal value in the final stage of observation The S phase entrance values indicated that gastrin promotes cell replication: before gastrin treatment, 31% of Caco-cells incorporated BrdU, whereas after 2 and 4 h 46% and 50% incorporated BrdU, respectively An increased BrdU incorporation (40%) was recorded as long as 8 and 12 h after gastrin treatment The GPC analysis performed on the nuclear extracts of Caco-2 cells starved for 4 days revealed a very low
< 1000 Da peak at the initial conditions (0 h), and the retarded and scarce formation of 4800 NAP at 4 and 8 h The 8000 Da NAP was essentially unaffected by prolonged starvation (data not shown)
The GPC analyses were also performed on the nuclear extracts of primary cultures, chicken embryo chondrocyte, chicken embryo fibroblast and quail embryo chondrocyte, and the cell lines KB human epidermoid oropharingeal carcinoma, PC Cl 3 rat thyroid and NA101 chicken embryo chondrocyte transformed by RSV In the chromatograms of these replicating cells, the 8000, 4800 and < 1000 Da peaks were always distinguishable (data not shown)
Analysis of polyamine and amino acid by RP-HPLC
of purified GPC peaks The molar concentrations of polyamines forming NAPs are shown in Table 1: statistical differences were due to the lower total polyamines, spermine and spermidine concen-trations of < 1000 NAP with respect to those of 4800 NAP Polyamine molar concentrations allowed us to define the
Fig 1 Gel permeation chromatography of nuclear extracts from Caco-2 cells at 0, 2, 4, 8 and12 h following treatment with 10)10M gastrin The cells used for the experiment were preconfluent and starved for 60 h Each chromatographic run was performed using the fourth part of the entire nuclear extract of 10 6 cells The modifications in the 4800 and
< 1000 Da peaks showed an inverse trend after gastrin stimulation Minor modifications were observed in the 8000 Da peak.
Trang 4simplest formulae of NAPs The concentration of
phos-phates was calculated considering that they have, at
physiological pH, two negative charges (pKa1¼ 2.12;
pKa2¼ 7.21) Thus, we estimated there were two moles of
polyamines per mole of phosphate
NAPs extracted from the nuclei of the other cell types had
analogous polyamine composition (data not shown)
Acid hydrolysis ensured the absence of amino acids and
peptidic amino acid residues in NAP composition The
RP-HPLC profiles of Fmoc-derivatives before and after
acid hydrolysis were, in fact, identical and showed only the
typical polyamine peaks, and not any added and/or
increased peaks that could indicate the presence of peptides
(data not shown)
Polyamine aggregation studies
The absence of oligopeptides and free amino acids,
partic-ularly those exhibiting an absorbance at 280 nm, prompted
us to investigate the absorbance range We therefore
scanned NAPs between 400 and 190 nm The maximal
absorbance peak of NAPs obtained from Caco-2 cells was
at 200 nm Moreover, a lower peak, ranging from 240 to
290 nm, with the maximum at 265 nm, was observed The
height of this peak was different in each NAP, being lowest
in < 1000 NAP and highest in the 4800 NAP The absence
of a shoulder at 220 nm confirmed the absence of peptide
bonds (data not shown)
Because both results of acid hydrolysis and
spectropho-tometric scansions were inconsistent with a peptidic
struc-ture of NAPs, we supposed that these compounds could be
formed by the interaction and aggregation between
phos-phates and polyamines This hypothesis was tested by
performing GPC chromatography of a 25 lM polyamine
mixture in phosphate buffer pH 7.2, using
tris(hydroxy-methyl)aminomethane/HCl or phosphate buffer as the
mobile phase (Fig 2A) Whatever the buffer used, the
GPC profiles that resulted after the in vitro aggregation of
polyamines was very similar to those obtained by analysing
the nuclear extracts of replicating cells Furthermore, a huge
increase in the 4800 Da GPC fraction and the formation of
intermediate compounds with molecular mass ranging from
<1000 to 4800 Da occurred when polyamine solution was
brought to 50 lM by the addition of 125 lM spermine
(Fig 2B)
NMR was used for analysis of the effects of polyamine–
phosphate interaction on the molecular arrangement of
polyamines In Fig 3, the NMR-spectra of the single
polyamines (putrescine, spermidine and spermine) dissolved
in DO (A) or in DO phosphate buffer (B) are shown The
signals of polyamines in D2O phosphate buffer show chemical shifts of about 0.05 p.p.m higher than the signals
of the same polyamines dissolved in D2O The CH2proton resonances of putrescine dissolved both in D2O and in phosphate buffer were represented by two single peaks at 2.9 p.p.m (peaks 2) determined by the protons of methylene adjacent to the NH2terminus and at 1.7 p.p.m (peaks 1) due to the b CH2protons In addition to these two signals, spermidine and spermine gave signals that fall in the resonance field of 2.0–2.1 p.p.m determined by the methy-lene protons in position b included between nitrogen
Fig 2 In vitro aggregation of polyamines (A) GPC profile of equal molar concentrations of putrescine, spermidine and spermine (25 l M
final concentration) dissolved in phosphate buffer (pH 7.2) The GPC profile was the same when either the phosphate buffer or a tris (hydroxyl methyl)aminomethane/HCl buffer was used as the mobile phase (B) GPC was repeated after the addition of 125 l M spermine to this polyamine solution, resulting in a huge increase in the 4800 Da peak This result indicates that spermine concentration is a determin-ant of the formation of this compound This experiment demonstrates that it is possible to realize an in vitro aggregation of polyamines that gives rise to compounds with molecular masses identical to those of the natural aggregates extracted from nuclei of replicating cells.
Table 1 Concentration of polyamines (nmolÆmL)1) in the nuclear aggregates of polyamines (NAPs) extractedfrom nuclei of replicating Caco-2 cells The results are expressed as mean ± SD of four determinations carried out on the GPC eluted peaks The means marked by different letters differ for P < 0.05 by Bonferroni test Ph, Phosphate group.
8000 NAP 4800 NAP < 1000 NAP P (one-way ANOVA ) Putrescine (Put) 0.272 ± 0.141 0.307 ± 0.044 0.153 ± 0.053 N.S.
Spermidine (Spd) 0.256 ± 0.109 a,b 0.448 ± 0.166 a 0.187 ± 0.085 b 0.04
Spermine (Spm) 0.360 ± 0.153 a,b 0.618 ± 0.207 a 0.306 ± 0.082 b 0.04
Total 0.888 ± 0.299a,b 1.373 ± 0.379a 0.646 ± 0.182b 0.02
Simplest formula Put-Ph-Spd-Ph-Spm Put-Ph-Spd-(Ph-Spm) 2 Put-Ph-Spd-(Ph-Spm) 2
Trang 5(peaks 3) and of 2.9–3.1 p.p.m due to methylene protons
adjacent to NH (peaks 4 and 5) Furthermore, spermine
showed a singlet at 1.9 p.p.m (peaks 6)
Different profiles were recorded in the spectra of
spermine and spermidine, when dissolved in D2O or in
D2O phosphate buffer: peaks 3, 4 and 5 showed differences
in multiplicity of signals and in peak wideness
The observed variations in chemical shift and in the
width, shape and number of signals, due to the different
proton exchange and the spin–spin coupling, are
represen-tative of the interaction between phosphate groups and
polyammonium cations and, in the case of spermidine and
spermine, could be indicative of their different
conforma-tional arrangement in phosphate buffer
Effects of NAP–DNA interaction
To consider the likely relevance of NAPs and DNA
interaction to DNA conformation and, in particular, to
assess the effects of this interaction on the exposition of the
inner bases, NAP–DNA solutions were evaluated by
measuring A at 260 nm and at different temperatures The
Avalues of the different NAP–DNA solutions, monitored
for 6 min at 15, 37 and 55C, are shown in Fig 4A Each A
value was calculated by subtracting the A value of the DNA
from those of the NAP–DNA solutions Only the 4800
NAP–DNA solution showed an isolated huge A increase at
37C (0.7 absorbance units), while no absorbance variations
were recorded in the 8000 and < 1000 NAP–DNA solutions
at the three temperatures The highest A value of 4800 NAP–
DNA solution at 37C was reached in about 10 s
To exclude that the variation of absorbance was due to
NAPs and not to DNA conformational changes, NAP
solutions were evaluated in the absence of the genomic DNA: we did not observe any modification in the O.D values at the different temperatures of the experiment (data not shown) Furthermore, the absorbance of genomic DNA solutions did not change in presence of 1 lM single polyamines, a concentration similar to that of polyamines forming the NAPs (data not shown)
These spectrophotometric results motivated us to investi-gate the electrophoretical behaviour of NAP–genomic DNA solutions, in view of the fact that a different electrophoretic mobility of DNA on agarose gel can suggest
a modification of DNA conformation The electrophoretic pattern of the NAP–DNA solutions on a 1.5% agarose gel
is shown in Fig 4B: lane C, corresponding to the 4800 NAP–DNA, illustrates a faster migrating DNA band compared to lanes B and D corresponding to 8000 and
< 1000 NAP–DNA solutions A temperature of 37C was essential to the visualization of the fastest migration of 4800 NAP–DNA When this electrophoretic experiment was repeated using the 241 base pair DNA fragment, no significant difference in the migration of NAP–DNA oligomer solutions was found (data not shown)
To identify the sites of interaction of NAPs on the DNA strands, the 1 kb DNA ladder fragments preincubated with the single NAPs were exposed to the phosphodiesterasic activity of exonuclease III, assuming that the sparing of DNA fragments from degradation was due to the impedi-ment of the enzyme action on the DNA strand phospho-diester bridges that were occupied by NAPs (Fig 5) The degradation of the 1 kb DNA ladder by exonuc-lease III was strongly impeded by previous incubation with NAPs In fact, in the absence of these compounds, the enzymatic digestion was complete for the small fragments
Fig 3 NMR spectra of polyamines.1H
spec-tra of polyamines (putrescine, spermidine and
spermine) dissolved in (A) D 2 O or (B) D 2 O
phosphate buffer (50 m M , pH 7.2, containing
0.15 M NaCl) The concentration of
polyam-ines was 10 mgÆmL)1 Peaks 4 and 5 of
sper-mine and 2, 4 and 5 of spermidine changed in
chemical shift and signal multiplicity when
these polyamines were dissolved in phosphate
buffer Only differences in chemical shifts were
recorded for putrescine (data not shown).
Trang 6up to 1018 bp and partial for those of 1636 and 2036 bp
(lane B) In contrast, when exonoclease III incubation was
preceded by the interaction of each NAP with the DNA,
degradation of these bands was strongly reduced (lanes C,
D and E) Single polyamines, used as controls at a
concentration (1 lM) similar to that of polyamines forming
the NAPs, did not show any protective effect
Degradation of genomic DNA by DNase I, a
phospho-diesterasic endonuclease, was strongly reduced by previous
incubation with each NAP (Fig 6A) In contrast, the
incubation of genomic DNA with 1 lMspermine,
spermi-dine or putrescine solutions did not provide any relevant
protective effect (Fig 6B)
D I S C U S S I O N
In the present study we have demonstrated that in the nuclei
of replicating Caco-2 cells, and of all the other replicating cells tested) i.e epithelial or mesenchimal, mutated or nonmutated) polyamines aggregate with phosphate ani-ons by ionic bonds to form three molecular structures with estimated molecular masses of 8000, 4800 and < 1000 Da, uncharacterized so far Owing to their positive charge, these NAPs interact with the phosphate groups of the DNA strands Compelling modifications in the concentration of NAPs paralleled the mitogenic effects produced in Caco-2 cells by gastrin In particular, the 4800 NAP was apparently closely linked to the process of cell replication, as this NAP reached maximal concentration in the few hours following gastrin stimulation The importance of 4800 NAP in cell replication is also strongly supported by its absence in the differentiated Caco-2 cells, which lost their replicating activity when confluence was reached [11] The 8000 NAP was well represented in both replicating and differentiated Caco-2 cells [11], and its concentration did not vary much during Caco-2 cell replication
The biological role of 8000 NAP, which is presumably not played in the cell replication, is vaguely definable from our study However, other important interactive functions such as single-strand DNA stabilization and/or DNA repair and protection can be postulated for this NAP
Fig 4 Absorbance (A) values andelectrophoretic patterns of NAP–
DNA solutions (r, 8000 NAP-DNA; j, 4800 NAP-DNA; m, < 1000
NAP-DNA) (A) The 4800 NAP–DNA solution showed the highest A
values, which reached the maximum at 37 C Intermediate A values,
unchanged by temperature variations, were recorded for the 8000
NAP–DNA solution The < 1000 NAP–DNA solution did not show
any absorbance value A values were monitored for 6 min at 260 nm at
different temperatures and calculated by subtracting the A value of the
DNA from those of NAP–DNA solutions Human genomic DNA
was used NAP solutions without DNA did not show any variation in
A at the different temperatures used Furthermore, single polyamines,
used as control at a concentration equivalent (1 l M ) to that of
poly-amines composing the NAPs, did not cause any variation in A (data
not shown) (B) Lane A corresponds to the migration of human
genomic DNA Lanes B, C and D show the same DNA preincubated
for 6 min at 37 C with 8000 and 4800 and < 1000 NAPs, respectively.
A faster migration of the DNA in presence of 4800 NAP was shown.
Electrophoresis was performed in 1.5% agarose gel in Tris/borate/
EDTA and at a constant temperature of 37 C.
Fig 5 Effect of exonuclease III on the electrophoretic migration of NAPs )1 kb DNA ladder Lane A, 1 kb DNA ladder; lane B, the same DNA fragments incubated for 6 min at 37 C with exonuclease III Migration of the 8000, 4800 and < 1000 NAP )1kb DNA ladder solutions incubated with exonuclease III are shown in the lanes C, D and E, respectively In the absence of NAPs (lane B), enzymatic degradation was partial for the DNA fragments of 2036 and 1636 bp and complete for the smaller ones NAPs conferred huge protection against degradation by exonuclease III to the DNA fragments: bands
< 506 bp are faintly visible, while those of 1018 and 506 bp remain evident In the same lanes, only a negligible diminution in the intensity
of the bands corresponding to DNA fragments of > 1018 bp can be appraised Electrophoresis was for 1 h in a 1.5% agarose gel The sparing of DNA fragments from degradation, due to inhibition of the enzyme action on the DNA strand phosphodiester bridges occupied by NAPs, indicates that this is the site of NAP–DNA interaction Single polyamines, used as control at a concentration equivalent (1 l M ) to that of polyamines composing the NAPs, did not show any protective effect (data not shown).
Trang 7In particular, a protective role is strongly suggested by its
inhibitory effect on the action of both exonuclease III and
DNase I, an effect exerted by the other NAPs also
Variations in the < 1000 NAP by GPC analyses indicate
that this compound functions as a store of material to be
used in the formation of 4800 NAP: the diminution of
< 1000 NAP occurs simultaneously with an increase in
4800 NAP Its highly concentrated presence in the nuclei
appears to be determinant of 4800 NAP production In fact,
prolongation of starvation for up to 4 days) conditions
that determine a strong reduction in the concentration of
polyamines in the cells [17]) caused a strong decrease in
< 1000 NAP and a reduced and delayed production of the
4800 NAP in the 8 h following gastrin treatment These
data indicate that the rapid formation of 4800 NAP occurs
only in conditions of efficient polyamine metabolism assuring the maintenance of adequate amounts of < 1000 NAP monomers in the nuclei
The hypothesis that < 1000 NAP is a precursor of 4800 NAP is also supported by in vitro aggregation studies that clearly demonstrated that the adjunct of spermine to the polyamines dissolved in phosphate buffer determines the increase in the 4800 Da GPC peak, paralleled by the disappearance of the < 1000 Da peak Therefore, it is possible to hypothesize that whenever the concentration of spermine, and probably also that of spermidine, increase in the cells, they interact with the < 1000 Da compounds to form new 4800 Da compounds with the consequent diminution of those of < 1000 Da
In spite of the wide biological differences of the cell types
we tested, the NAPs were detected in all nuclear extracts, thus indicating that nuclear polyamine aggregation could be regarded as a general biological phenomenon
The NAPs are rich in spermine and spermidine The appearance of these two polyamines in the nucleus is crucially important for the induction of cell mitosis [1–4] Because of their high positive charge, spermine and spermidine interact with the DNA phosphates by means of nonspecific electro-static binding [18] The induction of B-Z DNA conforma-tion, an important structural modification that prepares DNA for replication, was also accounted for by spermine and spermidine in various studies [19–21] Vajayanathan
et al.recently investigated DNA condensation by polyam-ines using static and dynamic laser light scattering tech-niques In particular, the structural modifications determined
by spermine and a series of its homologues on k-DNA were assessed These authors found that the variation of the number of methylene spacings in the bridging region between the two secondary amino groups of spermine had a profound effect on the molecule’s ability to provoke structural changes
in DNA and concluded that the regio-chemical distribution
of positive charge along polyamines plays a major role in the condensation of DNA [22]
Our data are consistent with the findings cited above, but further suggest that the interaction of polyamines with the genomic DNA is more complex, as these polycationic compounds have an extraordinary intrinsic tendency to form molecular aggregates, generating ionic bonds with the phosphate anions present both in buffer solutions or biological media and in DNA strands
The interaction of 4800 NAP with DNA determined interesting changes in both absorbance and electrophoretic migration of whole DNA, probably as a consequence of induced DNA conformational modifications The UV data indicate that the interaction of the whole DNA with this NAP is able to determine, at physiological temperature, the increased exposition of the bases, which, in usual experi-mental conditions, can be obtained only by denaturing the DNA at high temperatures or exposing it to a very high pH [23] Furthermore, the rapid increase in A values of the 4800 NAP–DNA solution at 37C is in accordance with a model
of gradual and oriented B-Z transition starting at (CG)5 motif: as the superhelical stress increases, the Z-structure propagates along the remaining part of the repeat by successive transitions until the full-length sequence is converted [24] Similarly, the faster electrophoretic mobility
of the 4800 NAP–genomic DNA solution at 37C, depending on the enhanced permeation into the gel showed
Fig 6 Electrophoresis of human genomic DNA incubatedwith NAPs
or single polyamines anddigestedby DNase I (A) m: 1 kb DNA ladder
DNA; lanes A and B, DNA and DNA + DNase I, respectively; lane
C, DNA + 8000 NAP + Dnase I; lane D, DNA + 4800 NAP +
Dnase I; lane E, DNA + < 1000 NAP + DNase I (B) Lanes A
and B, DNA and DNA + DNase I, respectively; lane C, DNA +
spermine + Dnase I; lane D, DNA + spermidine + DNase I; lane
E, DNA + putrescine + DNase I Electrophoresis of the DNA was
carried out for 1 h in a 1.5% agarose gel at 37 C The protective effect
of NAPs or single polyamines on genomic DNA was assayed after
their incubation with DNA and the successive exposition of these
solutions to DNase I NAPs were shown to be more efficient than
1 l M single polyamines in protecting genomic DNA from DNase I
degradation This polyamine concentration was equivalent to that of
polyamines composing NAPs.
Trang 8by the condensed molecules [25,26] is also indicative of a
strong rearrangement of the DNA double helix, probably
caused by the closing of phosphate groups in GC rich
regions and the DNA condensation [27,28] In the case of
the 241 base pair DNA fragment, we did not observe
significant differences in its electroforetic mobility in the
presence of NAPs This result confirms that the ratio
between the molecular dimension and gel matrix sieving is
crucial for the detection of the DNA conformational
changes, as already demonstrated by others [26] Thus, we
believe that these evidences are not in contradiction with our
conclusion that, namely, the electrophoretic pattern
deter-mined by the interaction of 4800 NAP with the genomic
DNA is due to a remarkable DNA conformational
modification
Unlike 4800 NAP, 8000 and < 1000 NAPs did not
change either absorbance or electrophoretic migration
properties of DNA However, as all of the NAPs are able
to interact with the DNA, as inferred by the fact that
incubation of DNA with these compounds hindered its
degradation by exonuclease III and DNase I, other
inter-active functions can be proposed, such as DNA protection
and/or stabilization and packaging
The role of NAPs in DNA protection seems to be
relevant, as they were able to protect the genomic DNA
from DNase I digestion with an efficacy hugely superior to
that shown by single polyamines These data indicate that
NAPs are probably crucial in the defence of DNA in the
case of inappropriate activation of the apoptotic process
[29,30]
As an ulterior aim of our study, we intended to shed light
on the NAP molecular structure The GPC analysis of
standard polyamines dissolved in phosphate buffer revealed
the formation of compounds with a molecular mass
identical to those of natural NAPs These results suggest
that NAP supramolecular arrangement derives from the
simple aggregation between the negatively charged
phos-phates and the positively charged polyamines NMR studies
performed on the polyamines dissolved in phosphate buffer
clearly indicate that the ionic interaction between phosphate
groups and polyammonium cations of spermidine and
spermine induce their molecular rearrangement We
sup-pose that this new molecular conformation can favour
aggregation among the polyamines and the phosphates,
with the consequent generation of NAPs Our belief is
supported by evidence from several NMR studies that
demonstrated the formation of complexes among
polyam-ines and phosphorylated molecules [31–33] The formation
of a polymeric aggregation of polyamines and phosphates
can be also inferred by our spectrophotometric analyses
showing that these compounds gave two absorbance peaks
at 200 and 265 nm These absorbance values can be
attributed to an electron delocalization similar to that of
the pfip* transition appearing in the spectra of molecules
with pfip bond conjugated structure such as polyene
systems or aromatic structures Therefore, it can be argued
that the NAP monomers (Table 1) tend to assume a sort of
cyclic structure by phosphate bridges, and form a polymeric
aggregation of a reticular appearance in which electrons are
delocalized in a p-like status, determining different e for
each NAP This phenomenon explains their detection at
280 nm in GPC analyses
The aggregation of polyamines and phosphates ascribes
to NAPs a positive charge due to a 2 : 1 polyamine : phos-phate ratio Thus, these compounds have a considerable number of free positive charges available for interaction with the negatively charged molecules Our results are all in favour of the interaction of NAPs with the DNA Theor-etically, this phenomenon should be consequent to the exposition of NAP positive charges to the DNA phosphate groups The hindrance to the phosphodiesterasic DNA degradation exerted by these compounds indicates that the DNA phosphate groups are really the negative ions involved in this kind of interaction Furthermore, as DNA integrity has been shown to be assured by NAPs with an efficiency much higher than that exerted by single polyam-ines, it is possible to suppose that, as already suggested for polyamines [34–36], the size and probably also the shape of each NAP are important for its appropriate arrangement in the DNA groove
In conclusion, we have demonstrated that in the nuclei of several replicating cell types there exist molecular aggregates that are able to interact with DNA phosphate groups, i.e NAPs, whose quasi-stable structure is based on ionic bonds between polyamines and phosphate groups
In our opinion, the identification of these compounds, which are able to induce DNA conformational changes and DNA protection, does not contradict the generally accepted functions of polyamines, but instead provides a better understanding of several aspects of the interaction of polyamines with DNA
A C K N O W L E D G E M E N T S
We are grateful to the following for their generous gift of cells: Drs
E Gionta, G Pontarelli, M Santoro and P.S Tagliaferri We wish also
to thank Drs A Menchise, C Verde, C Salzano, M.V Barone, M Di Pietro, N De Tommasi and A Contegiacomo, and Mr P Mastranzo for their help in performing parts of the experiments Our work was supported by a research grant from the Campania Region.
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