Specific Structural Determinants Are Responsible for the
Antioxidant Activity and the Cell Cycle Effects of Resveratrol*
Lucia A.
Stivala
,
Monica
Savio,
Federico
Carafoli,
Paola
Perucca,
Livia
Bianchi,
Giovanni
Maga§,
Luca
Forti¶,
Ugo M.
Pagnoni¶,
Angelo
Albini
,
Ennio
Prosperi**, and
Vanio
Vannini
From the Dipartimento di Medicina Sperimentale, sez. Patologia
Generale, the
Dipartimento di Chimica Organica, Università
di Pavia, the ** Centro di Studio per l'Istochimica del CNR,
§ Istituto di Genetica Biochimica ed Evoluzionistica
IGBE-CNR, 27100 Pavia, and the ¶ Dipartimento di Chimica,
Università di Modena e Reggio Emilia, 41100 Modena, Italy
Received for publication, February 28, 2001, and in revised form, April 11, 2001
 |
ABSTRACT |
Resveratrol
(3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin
found in grapes and wine, which shows antioxidant and antiproliferative
activities. In this study we have investigated whether these properties
are dependent on similar or different structural determinants of the
molecule. To this purpose, resveratrol derivatives, in which all or
each single hydroxylic function were selectively substituted with
methyl groups, were synthesized. Analogues with the stilbenic double
bond reduced or with the stereoisomery modified were also investigated.
The antioxidant activity of these compounds was evaluated by measuring
the inhibition of citronellal thermo-oxidation, or the reduction of
2,2-diphenyl-1-picrylhydrazyl radical. In addition, the
protection against lipid peroxidation was determined in rat liver
microsomes, and in human primary cell cultures. The antiproliferative
activity was evaluated by a clonogenic assay, and by analysis of cell
cycle progression and DNA synthesis. The results showed that the
hydroxyl group in 4' position is not the sole determinant for
antioxidant activity. In contrast, the presence of 4'-OH together with
stereoisomery in the trans-conformation (4'-hydroxystyryl
moiety) was absolutely required for inhibition of cell proliferation.
Enzymatic assays in vitro demonstrated that inhibition of
DNA synthesis was induced by a direct interaction of resveratrol with
DNA polymerases
and
.
 |
INTRODUCTION |
Resveratrol (3,4',5-trihydroxystilbene) is synthesized by several
plants in response to adverse conditions such as environmental stress
or pathogenic attack. For this reason, it is classified as a
phytoalexin, a class of antibiotics of plant origin (1-3). Resveratrol
has been found in a multitude of dietary plants, such as peanuts,
mulberries, and in grape skin (4). Thus, relatively high concentrations
of this compound are present in grape juice and, especially, in red
wine (5-8). Growing evidence suggest that resveratrol plays a role in
the prevention of human pathological processes, such as inflammation
(9-11), atherosclerosis (12-14), and carcinogenesis (4, 15, 16). The
protective effect has been attributed to its antioxidant properties
(11, 17, 18), to an anticyclooxygenase activity (4, 20), and to
a modulating activity of lipid and lipoprotein metabolism (9, 21, 22). Resveratrol also inhibits platelet aggregation (13, 23) and exhibits
antiestrogenic activity (24, 25). However, these effects do not
exhaustively explain the antiproliferative and anticarcinogenic
properties of resveratrol. The proliferation of various human malignant
cell lines is slowed down by resveratrol (15, 16, 26). The inhibition
of cell growth, which has also been described in normal cells (27-29),
is accompanied by the accumulation of cells in S and G2
phases (16, 26, 30). Conflicting results have been reported on the
induction of apoptosis by resveratrol (26, 31, 32).
A number of antioxidants, such as vitamin E,
N-acetylcysteine, flavonoids, and carotenoids have been
reported to interfere with cell cycle progression by inducing the
expression of cdk inhibitors, like p21waf1-cip1,
p16ink4a, and p27kip1 (33-36). In the case of
resveratrol, such a mechanism is still controversial (26, 27). The
effects on cell cycle progression may be also explained by the direct
inhibition of ribonucleotide reductase (37) and DNA polymerase
(38).
The structural determinants of these diverse properties of the
resveratrol molecule are obscure, but the number and position of the
hydroxylic groups have been suggested to play an important role in the
antioxidant activity (14, 19, 39). The aim of this study was to extend
these studies on the structural determinants of the activity of
resveratrol, and in particular to establish whether the antioxidant and
antiproliferative activities are dependent on (i) the stereoisomery,
(ii) the position of the different phenolic hydroxyl groups, and (iii)
the stilbenic double bond of the molecule. For this purpose, the
cis form (II) was obtained by UV irradiation of
trans-resveratrol; three different derivatives were
synthesized in which the hydroxylic functions were selectively
protected by methyl groups: 3,5-dihydroxy-4'-methoxystilbene (III),
3,5-dimethoxy-4'-hydroxystilbene (IV), and 3,4',5-trimethoxystilbene
(V). Finally, the
,
-dihydro-3,4',5-trihydroxystilbene (VI) was
obtained by reduction of the stilbenic double bond.
The biological properties of trans-resveratrol were compared
with those of the above derivatives. In particular, the antioxidant activity was investigated in vitro by measuring the
inhibition of citronellal thermo-oxidation or the radical scavenging
ability using the free radical
DPPH.1 The protection against
lipid peroxidation induced by Fe/ascorbate and
tert-butylhydroperoxide TBHP was also assessed in rat liver microsomes, or in cultured human fibroblasts, respectively. The effects
on cell proliferation were studied by analyzing the cell clonogenic
efficiency and cell cycle progression. In addition, the recruitment of
proliferating cell nuclear antigen (PCNA) and replication protein A
(RPA) to the DNA replication sites were investigated. These proteins
are required for the initiation and elongation steps of DNA
replication, respectively. Finally, the ability of resveratrol and its
derivatives to inhibit replicative DNA polymerases was also assessed
with in vitro assays.
The results have shown that the hydroxyl group at the 4' position is
not the sole determinant for the antioxidant activity. Similarly, the
4'-hydroxyl group is necessary for the antiproliferative activity and
the DNA polymerase inhibition, but the trans conformation is
absolutely required for these effects.
 |
EXPERIMENTAL PROCEDURES |
Reagents
trans-Resveratrol (99% purity) and DPPH were
obtained from Sigma; aphidicolin was obtained from Roche Molecular
Biochemicals. Monoclonal antibodies anti-bromodeoxyuridine
(BrdUrd clone BU20) and anti-PCNA (clone PC10) were obtained
from Dako, while the anti-RPA (32 kDa subunit) 9H8 monoclonal antibody
was kindly provided by M. Wold (Iowa University). The fluorescein
isothiocyanate (FITC)-conjugated anti-mouse antibody was purchased from Sigma.
[3H]dATP (40 Ci/mmol) and [3H]dTTP (40 Ci/mmol) were from Amersham Pharmacia Biotech. Activated calf thymus
DNA was prepared as described (40). Unlabeled dNTPs and poly(dA) and
oligo(dT)12-18 homopolymers were from Amersham Pharmacia
Biotech. Whatman was the supplier of the GF/C filters. All other
reagents were of analytical grade and purchased from Merck, Fluka, and Aldrich.
Poly(dA)/oligo(dT)12-18 primer-template was prepared
according to the manufacturer's protocol. Briefly, poly(dA) template oligonucleotide was mixed with the complementary
oligo(dT)12-18 oligonucleotide in a 10:1 molar ratio (w/w,
nucleotides) in 20 mM Tris-HCl (pH 8.0), containing 20 mM KCl and 1 mM EDTA, heated at 90 °C for 5 min then incubated at 65 °C for 2 h and slowly cooled at room temperature.
Calf thymus DNA polymerase
(pol
) and
(pol
) were
purified as described (40). The pol
used in this study was 2,200 units/ml (0.08 mg/ml). Pol
was 250 units/ml (0.2 mg/ml). 1 unit of
polymerase activity corresponds to the incorporation of 1 nmol of total
dTMP into acid-precipitable material in 60 min at 37 °C in a
standard assay containing 0.5 µg (nucleotides) of
poly(dA)/oligo(dT)10:1 and 20 µM dTTP.
Recombinant human wt PCNA was prepared as described (41).
Resveratrol Derivatives Synthesis
The cis form (II) was obtained by photoisomerization.
The trans-isomer (95 mg) was dissolved in 20 ml of
acetonitrile, flushed with nitrogen for 20 min, capped, and irradiated
by means of 4 external 15 W phosphor-coated lamps until the
photostationary state (about 1 to 1) was reached (40 min). Repeated
separation by column chromatography eluting with cyclohexane/ethyl
acetate (1:1) gave a sample (20% yield) of 98% pure (HPLC)
cis derivative. 1H NMR (CD3CN, 300 MHz):
6.15 (dd, 1H, J4,2 = J4,6 = 2.0 Hz, H-4); 6.25 (d, 2H,
J2,4 = J6,4 = 2.0 Hz, H-2
and H-6); 6.38 and 6.50 (AB system, 2H, J = 12.0 Hz,
CH=CH); 6.62 (d, 2H, J3',2' = J5',6' = 8.5 Hz, H-3' and H-5'); 6.75 (bs, 2H,
2xOH); 6.90 (bs, 1H, OH); 7.13 (d, 2H, J2',3' = J6',5' = 8.5 Hz, H-2' and H-6').
trans-3,5-Dihydroxy-4'-methoxystilbene
(4'-O-methylresveratrol) (compound III) was obtained as previously
described (44) by Wittig reaction between the phosphonium salt of the
commercially available 4-methoxybenzyl chloride and
3,5-bis-(tert-butyldimethylsilyloxy)benzaldehyde. Butyllithium (2.3 ml, 1.6 M in hexane, 3.6 mmol) was added
dropwise to a suspension of (4-methoxybenzyl)triphenylphosphonium
chloride (3.6 mmol) in tetrahydrofuran (50 ml) at
15 °C.
The resulting reddish solution was allowed to warm at room temperature
and stirred for 30 min.
3,5-Bis-(tert-butyldimethylsilyloxy)benzaldehyde (3.6 mmol)
was then added and the reaction mixture was stirred for 1 h,
diluted with ice-cold water (2 × 25 ml), and extracted with ethyl
acetate (3 × 30 ml). The organic extracts were washed with water
and the solvent was removed under reduced pressure to obtain a mixture
of trans- and
cis-3,5-di-(tert-butyldimethylsilyloxy)-4'-methoxystilbene, which were desilylated in tetrahydrofuran at room temperature with
tetrabutylammonium fluoride. After addition of ethyl ether the solution
was washed with water and the solvent was removed under reduced
pressure. The residue was filtered over silica gel to obtain
3,5-dihydroxy-4'-methoxystilbene as a 2:1 mixture of the
(trans/cis)-isomers in 75% yield. After crystallization
from CHCl3/pentane, the pure trans-isomer (III)
was obtained (33% yield). 1H NMR
(Me2SO-d6, 200 MHz):
3.78 (s, 3H,
OCH3); 6.14 (dd, 1H, J4,2 = J4, 6 = 2.05 Hz, H-4); 6.42 (d, 2H,
J2,4 = J6,4 = 2.05 Hz,
H-2 and H-6); 6.90 and 7.00 (AB system, 2H, J = 16.28 Hz, CH=CH); 6.94 (d, 2H, J3',2' = J5',6' = 8.77 Hz, H-3' and H-5'); 7.53 (d, 2H,
J2',3' = J6', 5' = 8.77 Hz, H-2' and H-6'); 9.23 (s, 2H, 2 × OH).
trans-3,5-Dimethoxy-4'-hydroxystilbene (IV) was obtained by
Perkin condensation between 4-hydroxybenzaldehyde and
3,5-dimethoxyphenylacetic acid (Aldrich), as reported by Pezet and Pont
(45). An alternative synthetic procedure was attempted, consisting in
the Wittig reaction between the phosphonium salt of
3,5-dimethoxybenzylbromide (synthesized from the commercially available
3, 5-dimethoxybenzyl alcohol) and 4-acetoxybenzaldehyde on 3.6 mmol
scale, as reported for the compound III. In this case, a 1:1 mixture of
the trans/cis-isomers was obtained in 48% yield. The
4'-hydroxyl function was deprotected by treatment with
K2CO3 in methanol at room temperature. After addition of ethyl acetate, the solution was washed with water and the
solvent removed under reduced pressure: the residue was then
chromatographed over silica gel using petroleum ether/diethyl ether
gradient, giving trans-3,5-dimethoxy-4'-hydroxystilbene in
20% yield. 1H NMR (Me2SO-d6, 200 MHz):
3.78 (s, 6H, 2 × OCH3); 6.38 (dd, 1H,
J4,2 = J4,6 = 2.22 Hz,
H-4); 6.73 (d, 2H, J2,4 = J6,4 = 2.22 Hz, H-2 and H-6); 6.78 (d, 2H,
J3',2' = J5',6' = 8.61 Hz, H-3' and H-5'); 6.94 and 7.17 (AB system, 2H, J = 16.42 Hz, CH=CH); 7.43 (d, 2H, J2',3' = J6',5' = 8.61 Hz, H-2' and H-6'); 9.56 (s, 1H, OH).
3,4',5-trans-Trimethoxystilbene (derivative V) was
synthesized by direct methylation, refluxing a mixture of
trans-resveratrol (0.44 mmol) and CH3I (9.24 mmol) in acetone (15 ml) in the presence of anhydrous potassium
carbonate (6.6 mmol). After addition of ethyl ether, the solution was
washed with water and the solvent was removed under reduced pressure:
the residue was then chromatographed over silica gel using petroleum
ether/diethyl ether gradient, giving
trans-3,4',5-trimethoxystilbene in quantitative yield. 1H NMR (CDCl3, 200 MHz):
3.87 (s, 9H,
3 × OCH3); 6.42 (dd, 1H, J4,2 = J4,6 = 2.26 Hz, H-4); 6.69 (d, 2H,
J2,4 = J6,4 = 2.26 Hz, H-2 and H-6); 6.93 and 7.08 (AB system, 2H, J = 16.26 Hz, CH=CH); 6.934 (d, 2H, J3',2' = J5',6' = 8.71 Hz, H-3' and H-5'); 7.48 (d, 2H,
J2',3' = J6',5' = 8.71 Hz, H-2' and H-6').
,
-Dihydro-3,4',5-trihydroxystilbene (
,
-dihydroresveratrol)
(derivative VI) was obtained by catalytic hydrogenation of trans-resveratrol (0.15 mmol) with 10% Pd/C catalyst in
methanol (5 ml) at room temperature and atmospheric pressure. The
catalyst was filtered off through Celite and washed with methanol; the solvent was then removed under reduced pressure and the residue chromatographed over silica gel (7:3, hexane/ethyl acetate) to obtain
,
-dihydro-3,4',5-trihydroxystilbene (87% yield). 1H
NMR (Me2SO-d6, 200 MHz):
2.55-2.77 (m, 4H,
CH2CH2); 6.03 (dd, 1H,
J4,2 = J4,6 = 2.11 Hz,
H-4); 6.07(d, 2H, J2,4 = J6,4 = 2.11 Hz, H-2 and H-6); 6.66 (d, 2H,
J3',2' = J5', 6' = 8.52 Hz, H-3' and H-5'); 7.00 (d, 2H, J2',3' = J6',5' = 8.52 Hz, H-2' and H-6'); 8.99 (s, 2H,
2 × OH); 9.08 (s, 1H, OH). The products synthesized were
further purified by crystallization to reach a final purity > 98%, as determined by high performance liquid chromatography.
Stock solutions of each substance were prepared in
N,N-dimethylformamide and final dilution was
performed in chlorobenzene for the in vitro oxidation test.
For cell culture experiments, stock solutions were prepared in
Me2SO and diluted directly in cell culture medium.
Cell Culture and Treatments
Normal human embryonic fibroblasts and HT1080 fibrosarcoma cells
(Istituto Zooprofilattico, Brescia, Italy) were cultured in Earle's
minimal essential medium (Life Technologies, Inc.) supplemented
with 10% fetal bovine serum (Life Technologies, Inc.), 100 IU/ml
penicillin, and 100 µg/ml streptomycin. Normal fibroblasts were used
between the 5th and 20th passages. Cell treatments were performed by
adding trans-resveratrol or its derivatives in culture medium at final concentrations ranging from 7 to 100 µM.
For resveratrol, these concentrations are comparable with doses found
in red wine and grapes (39). Untreated cultures received the same
amount of the solvent alone (Me2SO < 0.1%).
Aphidicolin (60 nM) was used as positive control for cell
cycle arrest in S phase. For the antioxidant activity determination,
normal fibroblasts were incubated for 30 min with 60 µM
trans-resveratrol or its derivatives, before the oxidative
treatment. For cell cycle analysis, normal fibroblasts and HT1080 cells
were incubated for 24, 48, and 72 h with
trans-resveratrol at the concentration of 15, 30, and 90 µM, then cells were washed twice with phosphate-buffered
saline (PBS), and detached with a standard trypsinization procedure.
Antioxidant Activity
The antioxidant activity of trans-resveratrol and its
derivatives was evaluated in vitro both by the citronellal
thermo-oxidation inhibition test (46), and the DPPH method (47, 48). In
addition, antioxidant activity was assessed in rat liver microsomes by
measuring lipid peroxidation inhibition after
Fe2+/ascorbate treatment, and in human fibroblast cultures
after TBHP treatment.
Citronellal Thermo-oxidation Method
In this test, the aldehyde (
)-citronellal is used as the
oxidation substrate: it is subjected to heating and intensive
oxygenation in chlorobenzene, and its disappearance with the consequent
formation of its degradation products are monitored by gas
chromatography. Chlorobenzene was selected as the reaction solvent
because of (i) its stability to oxidation; (ii) the ability to dissolve
both polar and nonpolar compounds, better than dimethylformamide; and (iii) the boiling point is higher then 80 °C (temperature test). Fifteen ml of a chlorobenzene solution, containing 150 µl of dodecane (Aldrich) and 150 µl of tridecane (Aldrich) as internal standards, were poured into a two-necked flask equipped with a condenser to
prevent evaporation. Resveratrol, or its derivatives, dissolved in
dimethylformamide, were added to the chlorobenzene solution to reach
final concentrations ranging from 60 to 120 µM. The
mixture was then heated at 80 °C and intensively oxygenated by
bubbling in O2 at a flow rate of 10 ml/min. At time 0, 300 µl of (
)-citronellal (Fluka) were added to the reaction medium.
Immediately and at periodic intervals, 0.1-µl samples were withdrawn
and analyzed by gas chromatography. The antioxidant power of
resveratrol and the derivatives was measured by determining the
efficient quantity (EQ), i.e. the concentration required for
each compound to double the half-life with respect to control reaction
(citronellal without antioxidant).
DPPH Reduction Method
Antioxidant solution in methanol (0.1 ml) was added to 3.9 ml of
a 6 × 10
5 M DPPH solution in methanol
(48). The exact initial DPPH concentration in the reaction medium was
calculated from a calibration curve. The decrease in absorbance was
determined at 515 nm at 0 min, every 5 min for 1 h, and every 60 min until the reaction reached a plateau (about 6 h). Antiradical
activity was expressed as the EC50, i.e. the
antioxidant concentration necessary to decrease the initial amount of
DPPH by 50%.
Lipid Peroxidation in Rat Liver Microsomes
Rat liver microsomes were prepared from Wistar rats by tissue
homogenization with 5 volumes of ice-cold 0.25 M sucrose
containing 5 mM Hepes, 0.5 mM EDTA (pH 7.5).
Briefly, microsomal fractions were isolated by removal of the nuclear
fraction at 8,000 × g for 10 min, removal of
mitochondrial fraction at 18,000 × g for 10 min, and
sedimentation at 105,000 × g for 60 min. Microsomal fractions were diluted in phosphate buffer, 0.1 M (pH 7.5),
at the final protein concentration of 1 mg/ml. The microsomes were preincubated in a shaking water bath at 37 °C for 10 min with varying concentrations (0.01-100 µM) of each compound
before starting lipid peroxidation with 100 µM
Fe2+, 500 µM ascorbate. After 60 min
incubation, the inhibitory effect on lipid peroxidation was assessed by
measuring thiobarbituric acid reactive substances (TBARS) by the method
of Yagi et al. (49). Briefly, 500 µl of microsomal
fraction were added to 500 µl of 20% trichloroacetic acid to stop
the lipid peroxidation reaction, and then 500 µl of 0.74%
thiobarbituric acid was added. The mixture was then heated in a boiling
water bath for 15 min. After centrifugation, 200 µl of supernatant
was transferred to the microtiter plate, the absorbance was measured at
535 nm and compared with standards prepared from the acid hydrolysis of
malonaldehyde tetraethylacetyl (Sigma). Inhibition of lipid
peroxidation was expressed as percentage, and the effective
concentration giving 50% inhibition (EC50) was calculated
from the inhibition curve.
Lipid Peroxidation in Cell Cultures
Lipid peroxidation was induced in cultured normal human
fibroblasts by TBHP (Sigma). Cells were preincubated for 30 min with 60 µM trans-resveratrol or its analogues in PBS,
and then 250 µM TBHP was added for 60 min. The production
of TBARS was assessed, as above described. In both experimental models,
microsomes and cells, TBHP-treated and untreated control samples
received the same amount of the solvent (Me2SO < 0.02%).
Clonogenic Efficiency Assay
The clonogenic efficiency was determined after incubation of
cells in culture medium containing trans-resveratrol or its
derivatives. Briefly, the cells were diluted in complete Earle's
minimal essential medium to ~4000 cells/ml, and volumes of this
suspension containing 200 cells were transferred to 30-mm dishes. After
24 h of treatment with trans-resveratrol and the
different derivatives, the cells were washed twice and 5 ml of fresh
medium was added. After 10 days, the colonies were stained with crystal
violet and counted, and the clonogenic efficiency was calculated as the
mean percentage with respect to control cells. Clonogenic efficiency of
untreated control cultures was about 35%.
Cell Cycle Analysis
Cell cycle distribution was assessed by determining BrdUrd
incorporation versus DNA content. Normal fibroblasts and
HT1080 cells were incubated with 30 µM BrdUrd (Sigma)
during the last hour of culture, harvested, and fixed in cold 70%
ethanol. Fixed cells were washed in PBS, resuspended in 2 N
HCl for 30 min at room temperature, pelletted, and then resuspended in
0.1 N sodium tetraborate for 15 min. The samples were then
washed in PBS, incubated for 15 min in PBS containing 1% bovine serum
albumin and 0.2% Tween 20 (PBT), and then for 60 min in 100 µl of
anti-BrdUrd monoclonal antibody diluted 1:20 in PBT. After two washes
with PBT, cells were incubated for 30 min with 100 µl of
FITC-conjugated anti-mouse antibody diluted 1:50 in PBT, then washed
twice, and resuspended in PBS containing 5 µg/ml propidium iodide and
2 mg/ml RNase A. Cells were analyzed with a Coulter Epics XL (Coulter
Corp.) flow cytometer. Ten thousand cells were measured for each
sample. Computer statistical analysis of mean fluorescence intensity
(MFI) and graphic representation were performed with the XL2 software (Coulter).
Immunofluorescence Determination of Nuclear-bound PCNA and RPA
(32 kDa)
In order to determine the nuclear-bound (detergent-insoluble)
fraction of protein involved in the DNA replication complex, a
hypotonic lysis was performed according to the following protocol (50).
Briefly, cells were resuspended in hypotonic lysis buffer 10 mM Tris-HCl (pH 7.4), containing 2.5 mM
MgCl2, 1 mM phenylmethylsulfonyl fluoride
(BDH), and 0.5% Nonidet P-40 (Sigma). After the lysis was completed,
cells were resuspended in saline, fixed in cold ethanol to a final 70%
concentration, and stored at
20 °C until analysis. Fixed cells
were pelleted by centrifugation, washed in PBT, and incubated for 60 min with 100 µl of monoclonal antibodies anti-PCNA or anti-RPA,
diluted 1:100 in PBT. At the end of incubation, cells were washed twice
with PBT and incubated for 30 min with 100 µl of FITC-conjugated
anti-mouse antibody, diluted 1:100. After the secondary antibody
incubation, cells were washed twice in PBT, and resuspended in PBS.
Negative controls consisted of cells incubated only with the secondary
FITC-conjugated antibody, or cells in which the primary antibody was
replaced by an isotypic irrelevant antibody (Sigma). For biparametric
analysis of immunofluorescence versus DNA content, cells
were stained with propidium iodide as described above.
DNA Polymerase Assays
Enzymatic Assays--
Pol
activity on activated DNA was
assayed in a final volume of 25 µl containing: 50 mM
Tris-HCl (pH 7.6), 0.25 mg/ml bovine serum albumin, 1 mM
dithiothreitol, 6 mM MgCl2, 10 µM
each [3H]dATP (5 Ci/mmol), dGTP, dCTP, and
[3H]dTTP (5 Ci/mmol), 0.05 units of pol
and 3 µg of
activated DNA. All reactions were incubated for 15 min at 37 °C
unless otherwise stated and the DNA precipitated with 10%
trichloroacetic acid. Insoluble radioactive material was determined as
described (51). Enzymes and proteins were added as indicated in the
figure legends. Pol
activity was assayed on poly(dA)/oligo(dT) in a
final volume of 25 µl containing: 50 mM BisTris-HCl (pH
6.6), 0.25 mg/ml bovine serum albumin, 1 mM dithiothreitol,
6 mM MgCl2, 10 µM
[3H]dTTP (5 Ci/mmol), 50 ng of PCNA, 0.035 units of pol
, and 200 nM (3'-OH ends) of poly(dA)/oligo(dT). All
reactions were incubated for 15 min at 37 °C unless otherwise stated
and the DNA precipitated with 10% trichloroacetic acid. Insoluble
radioactive material was determined as described (51).
Inhibition Assays--
Assays were performed under the
conditions described above. Different concentrations of the inhibitors
to be tested were added as indicated in figure legends to the reaction
mixture in the absence of DNA template and nucleotides. After 5 min of
incubation at room temperature, the reaction was started by addition of
the missing reagents and incubation was as described above.
Ki values were calculated by Dixon plot of the
experimental data.
Electronic Structure and Thermodynamic Stability of the Phenoxyl
Radicals from Resveratrol
The electronic structure and the formation enthalpy of the three
different phenoxyl radicals arising from the loss of hydrogens at the
3-, 5-, and 4'-OH groups in resveratrol were determined by
semiempirical Molecular Orbital calculations using to the MNDO-PM3 semiempirical method. Full optimization of molecular geometries were
achieved using a gradient best fit procedure with an energy convergence
criterium of 10
4 kcal/mol. The imput geometries were
determined by best fit using a Molecular Mechanics method (all methods
implemented in the Hyperchem package, release 5.1).
Statistics
All experiments were performed at least three times.
Experimental data were analyzed with one-way analysis of variance
(ANOVA) followed by Tukey's multiple range test for significant differences.
 |
RESULTS |
Fig. 1 shows the chemical structures
of trans (I) and cis-resveratrol (II), of three
trans-form derivatives in which each hydroxylic group is
protected by methyl groups (III, IV, and V), and a derivative with
reduced double bond (VI).

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|
Fig. 1.
Chemical structures of trans
(I), cis-resveratrol (II), and derivatives
III-VI. trans-Resveratrol derivatives were synthesized,
as described under "Experimental Procedures," in order to change
the stereoisomery in the cis conformation (II), to
selectively substitute the hydroxylic functions with methoxy
(OCH3) groups (III, IV, and V), or to reduce the stilbenic
double bond (VI).
|
|
Antioxidant Activity of trans-Resveratrol and Derivatives
II-VI--
The comparison of antioxidant activity of
trans-resveratrol and the derivatives, as estimated in
in vitro tests, is reported in Table
I. The results are expressed as the EQ
(citronellal test) or the EC50 (microsomes and DPPH assays)
of each compound used. The stronger the antioxidant, the smaller the EQ
or EC50 value. In all three tests,
trans-resveratrol (I) showed the highest antioxidant
activity, whereas compound V did not exert any significant effect.
Increasing values of EQ and EC50 were observed for
derivatives II, IV, and VI, and compound III reaching values about 5 and 3 times higher (p < 0.01) than
trans-resveratrol, in the citronellal and microsome test,
respectively. With the DPPH assay, the trans (I) and the
cis (II) forms showed a similar EC50, and
derivative IV gave a comparable value. Derivatives III and VI were less
efficient, showing EC50 values about 2 (p < 0.05) and 4 times (p < 0.01) higher than
trans-resveratrol, respectively. Fig.
2 shows the effects of
trans-resveratrol (I) and the derivatives on TBARS production induced by TBHP in normal human fibroblasts. Incubation of
the cells for 1 h in the presence of 250 µM TBHP
significantly increased membrane lipid peroxidation, raising the TBARS
production to 4.04 nmol/5 × 105 cells, from the level
of 0.92 nmol/5 × 105 cells measured in untreated
control samples. trans-Resveratrol (I) inhibited the TBARS
production by about 67% (p < 0.01). Among the
derivatives, compounds II, IV, and VI exhibited significant antioxidant
activity, by reducing TBARS production by about 42% (p < 0.01), 61% (p < 0.01), and 33% (p < 0.01), respectively. Derivatives III and V did not exert any
statistically significant activity in this assay. The concentration of
Me2SO used (<0.02%) did not induce any significant
protective effect against lipid peroxidation (data not shown). Bybenzil
(compound without double bond and OH groups) and
trans-stilbene (compound with double bond, without OH
groups) did not show any detectable antioxidant activity, with any of
the three in vitro methods (results not shown).
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Table I
Antioxidant activities of trans-resveratrol and derivatives II-VI
The antioxidant power is expressed by EQ and EC50, as described
under "Experimental Procedures." Each value is the mean of at least
three independent experiments ± S.D.
|
|

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Fig. 2.
Antioxidant activity of
trans-resveratrol (I) and derivatives II-VI.
Antioxidant activity of 60 µM
trans-resveratrol (I) or derivatives II-VI, expressed as the
percentage of inhibition of TBARS production in normal human
fibroblasts treated with TBHP. Mean values ± S.D. of at least
five independent experiments are shown. *, = p < 0.05;
**, = p < 0.01 significantly different as compared
with control by one way ANOVA-Tukey's test.
|
|
Effect of trans-Resveratrol and Derivatives II-VI on the Clonogenic
Efficiency of Normal Fibroblasts--
The clonogenic efficiency was
studied in normal fibroblasts treated for 24 h with
trans-resveratrol or the derivatives at concentrations
ranging from 0 to 90 µM. Fig.
3 shows that only trans-resveratrol and derivative IV induced a
dose-dependent reduction in clonogenic efficiency, with an
estimated IC50 of about 60 µM for both. All
other derivatives failed to induce a significant inhibition in cell
growth.

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Fig. 3.
Effect of trans-resveratrol
(I) and derivatives II-VI on the clonogenic efficiency of normal human
fibroblasts. Cells were incubated 24 h after plating with the
different compounds, and grown for 10 days before counting colonies, as
described under "Experimental Procedures." Each point was assayed
in triplicate dishes, and each experiment was repeated at least three
times. Values are expressed in percentage and referred to untreated
control cultures. Clonogenic efficiency of untreated control cultures
was about 35%.
|
|
Effects of trans-Resveratrol and Derivatives II-VI on Cell Cycle
Progression of Normal and Tumor Cell Lines--
To further investigate
the effect of the various derivatives on cell proliferation, the
distribution in each phase of the cell cycle was analyzed by
determining the DNA content with flow cytometry. Fig.
4 compares the effects in normal
fibroblasts treated for 24 h with trans-resveratrol or
derivatives II-VI, at the 30 µM concentration, because
significant effects could be already observed in these conditions. A
significant accumulation (p < 0.01) in S phase, and a
consequent reduction in the number of cells in G1 phase,
was observed with trans-resveratrol (G1 = 44.5%, S = 42.5%, G2 + M = 13.0%), and
with derivative IV (G1 = 48.0%, S = 40.1%,
G2 + M = 11.9%), with respect to the cell cycle
distribution of control cells (G1 = 61.3%, S = 22.3%, G2 + M = 16.4%). The cell cycle
distribution of samples treated with the other derivatives (II, III, V,
and VI) was comparable to that observed in the controls. Aphidicolin, a
well known inhibitor of DNA polymerases
and
, used as a positive
control, induced a significant accumulation of cells in S phase
(G1 = 34.5%, S = 58%, G2 + M = 7.5%), comparable to that induced by compounds I and IV. Cells
incubated with the solvent alone (Me2SO < 0.1%) did
not show any alteration of cell cycle progression. The inhibitory
effect on cell growth induced by trans-resveratrol and
derivative IV was reversible, since 48 h after removal of the
compounds, the percentage of cells in S phase returned to the control
value (not shown).

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Fig. 4.
Cell cycle analysis of normal human
fibroblasts treated with trans-resveratrol (I) and the
derivatives II-VI. Determination of cell cycle distribution was
performed in samples incubated for 24 h with 30 µM
of the above compounds, stained with propidium iodide, and measured by
flow cytometry, as described under "Experimental Procedures."
Aphidicolin (60 nM) was used as positive control for cell
cycle arrest in S phase. Mean values of the percentage of cells in each
phase of cell cycle were obtained from three independent experiments.
**, p < 0.01 significantly different from to control
by one way ANOVA-Tukey's test.
|
|
To study whether the observed cell cycle imbalance induced by
trans-resveratrol and derivative IV was consequent upon a
change in DNA synthesis, DNA replication was assessed by BrdUrd
incorporation and determined with immunofluorescence and flow
cytometric analysis. Fig. 5 shows the dot
plots of BrdUrd immunofluorescence versus DNA content in
control cells and in fibroblasts treated with 30 µM
trans-resveratrol for 24, 48, and 72 h. The results
showed that cells progressed through S phase at a slower rate than
control cells and incorporated significantly lower amounts of BrdUrd
incorporation. Quantitative analysis of BrdUrd immunofluorescence in
the region corresponding to S-phase cells indicated that
trans-resveratrol inhibited BrdUrd incorporation by about
55% at 24 h (see Figs. 5B and 7A), and by
about 70 and 80% at 48 and 72 h (Fig. 5, C and
D), respectively. Similar results were obtained in HT1080 fibrosarcoma cells (not shown).

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Fig. 5.
Effect of trans-resveratrol
on DNA synthesis in normal human fibroblasts. Two-parameter dot
plots of BrdUrd incorporation versus DNA content in control
cells and in samples treated with 30 µM
trans-resveratrol for 24, 48, and 72 h. During the last
hour of trans-resveratrol treatment, 30 µM
BrdUrd was added to each sample. Immunostaining and flow cytometric
analysis of BrdUrd incorporation were performed as described under
"Experimental Procedures." Results shown are from one out of three
independent experiments. Control cells (A) and fibroblasts
treated with 30 µM trans-resveratrol for
24 h (B), 48 h (C), and 72 h
(D).
|
|
Effects of trans-Resveratrol on the Recruitment of PCNA and RPA
Proteins to DNA Replication Sites--
To explore the basis underlying
the inhibition of DNA synthesis, the recruitment of PCNA and RPA
(32-kDa subunit) proteins to DNA replication sites was investigated
next. To this aim, cells were lysed in hypotonic buffer to separate the
detergent-insoluble forms of the two proteins. Fig.
6A shows the dot plots of PCNA immunofluorescence versus DNA content in control cells
(a) and in fibroblasts treated for 24 h with 15 (b), 30 (c), or 90 µM (d) trans-resveratrol. Accumulation of cells in S
phase was evident at the lowest concentrations, while with 90 µM trans-resveratrol, cells were blocked at
the G1/S phase transition, as indicated by the high PCNA
immunofluorescence levels typical of cells entering S phase (52).
Quantitative analysis of immunofluorescence intensity in S phase
indicated that the amount of PCNA assembled in replication foci in
treated samples, was about 12% higher (not significant) than that of
control cells. The amount of RPA protein (32 kDa) assembled at the
replication foci was also not significantly modified after treatment
with trans-resveratrol (Fig. 6B).

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Fig. 6.
Effect of trans-resveratrol
on the recruitment of PCNA and RPA to DNA replication sites in normal
human fibroblasts. A, two-parameter dot plots of PCNA
immunofluorescence versus DNA content in control cells (a)
and in fibroblasts treated for 24 h with
trans-resveratrol 15 (b), 30 (c), and
90 (d) µM. B, two parameter dot
plots of RPA (32 kDa) immunofluorescence versus DNA content
in control cells (a) and in fibroblasts treated for 24 h with trans-resveratrol 30 µM (b).
In both experiments (A and B), determination of
detergent-insoluble form of PCNA or RPA was performed by immunostaining
and flow cytometric analysis, as described under "Experimental
Procedures." Results shown are from one out of three independent
experiments. IFS, immunofluorescence value of
S-phase.
|
|
Effects of trans-Resveratrol and Derivatives II-VI on DNA
Synthesis--
The above results indicated that BrdUrd incorporation
was inhibited at a step following to the recruitment of RPA and PCNA to
the replication foci, thus suggesting that the inhibition of DNA
synthesis occurred at the level of DNA polymerase activity, as also
suggested by the reduction in BrdUrd incorporation. For this purpose,
the ability of trans-resveratrol to inhibit DNA synthesis
was compared with the other derivatives, both in cells and in in
vitro assays testing the activity of replicative DNA pol
and
pol
. The results reported in Fig.
7A show that among the
derivatives, only compound IV inhibited BrdUrd incorporation to a
similar extent of trans-resveratrol. Comparable results were obtained in the in vitro assays on DNA polymerase activity
(Fig. 7B). trans-Resveratrol inhibited both pol
and pol
with similar potencies, whereas the derivative IV
showed a 2-fold preference for pol
(p < 0.01) with
respect to pol
(p < 0.05). It must be noted that
this inhibition was enhanced by the preincubation with the enzyme,
suggesting a slow-binding mode of inhibition. All the other compounds
were 3-10-fold less active than the trans-resveratrol. The
cis-form (II) of resveratrol was also tested and found to be
inactive, confirming that for DNA synthesis inhibition, the active
configuration of the compound was in the trans-form. The corresponding Ki values for the active compounds are listed in Table II. None of the
derivatives inhibited Escherichia coli pol I (Klenow
fragment), HIV-1 reverse transcriptase, and HSV-1 DNA polymerase (data
not shown), confirming the specificity of the above inhibition
observed.

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Fig. 7.
Effect of trans-resveratrol
(I) and derivatives II-VI on DNA synthesis in cell cultures and
in vitro. A, quantitative evaluation
of BrdUrd incorporation. Data are BrdUrd immunofluorescence values
expressed as the percentage of samples treated with 30 µM
of each derivative versus untreated control cells.
B, DNA synthesis performed in vitro by purified
pol and pol in the absence (control) and presence of
trans-resveratrol and derivatives II-VI. Assays were
performed as described under "Experimental Procedures" in the
presence of 5 µM of each inhibitor to be tested. DNA
polymerase activity was expressed as percentage of the control reaction
without inhibitor which was 0.8 pmol × min 1 for pol
and 0.6 pmol × min 1 for pol and taken as
100%. *, p < 0.05; **, p < 0.01 significantly different from to control by one way ANOVA-Tukey's
test.
|
|
Electronic Structure and Thermodynamic Stability of the Phenoxyl
Radicals from trans/cis-Resveratrol--
Fig.
8 shows the limit surface diagrams for
the singly occupied molecular orbitals in the three phenoxyl-type
radicals of both the trans and cis conformations,
as were obtained from PM3. In all cases a delocalization of the
unpaired spin is observed. However, in the trans
configuration, the 4'-phenoxyl (picture A) is extended also to the
adjacent ring through the stilbene double bond, whereas in the 3- and
5-phenoxyl species (picture B) the delocalization is confined to the
aromatic ring bound to the oxygen radical center. This different
electronic structure leads to a greater resonance stabilization energy
for the 4'-phenoxyl radical, the formation of which is accordingly
predicted to be more exothermic, as indicated by
Hf° reported in Fig.
8.

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Fig. 8.
Electronic structure and thermodynamic
stability of the phenoxyl radicals from resveratrol. Picture of
the limit surface diagrams for the singly occupied molecular orbitals
in the 4'-phenoxyl radical (A), and 5- or 3-phenoxyl
radicals (B), from trans-resveratrol and from
cis-resveratrol (C and D),
respectively. Values of formation enthalpy
( Hf°) for each
phenoxyl radical are also shown.
|
|
The rationale for these results is obtained by considering that in
mononuclear phenoxyl radicals the maximum unpaired spin densities are
at the ring meta and para positions. In the
resveratrol 4'-phenoxyl radical the spin density can flow to the
adjacent ring since the stilbene double bond acts as a bridge being
bound to a maximum spin density center. This is not the case for the other two radical species where the double bond is bound at positions of minimum spin density. As a consequence of the greater resonance stabilization energy, the hydrogen abstraction from the 4'-OH bond is
expected to be favored being more exothermic with respect to the
analogue reactions at the 3-OH and 5-OH positions. According to the
above arguments, the absence of the olefinic double bond in
,
-dihydro-3,4',5-trihydroxystilbene is expected to cause a
decrease in the 4'-phenoxyl radical resonance stabilization energy and,
consequently, a decrease in the overall inhibition efficiency.
Based on molecular orbitals calculations, all the phenoxyl radicals of
resveratrol in the cis conformation (Fig. 8C and D) show an intrinsic thermodynamic stability lower than that of
corresponding radicals in the trans-form (Fig. 8,
A and B), since the formation enthalpies are less
exothermic by 7.1 kcal/mol for the 4'-phenoxyl radical
(
Hf°
trans (
55.8 kcal/mol)
Hf° cis
form (
48.7 kcal/mol)), and by 6.4 kcal/mol, for the 5- and 3-phenoxyl
radicals. Furthermore, within the cis configuration framework, the 3- and 5-phenoxyl radicals (Fig. 8D) are also
found to be less stable
(
Hf° =
46.77
kcal/mol) than the 4'-phenoxyl analogue (Fig. 8C,
Hf° =
48.71
kcal/mol). In fact, in the cis 4'-phenoxyl radical, the spin
delocalization to the phenyl ring via the double bond is partially
hindered by the lack of coplanarity of the
system. This
effect is witnessed by the higher resonance stabilization energy of the
trans, with respect to the cis analogue, which is obtained by this calculation
(
Hf°A

Hf°B =
2.7
kcal/mol;
Hf°C
Hf°D =
1.94 kcal/mol). The difference between these values leads to 0.76 kcal/mol, which represents a 30% decrease in the resonance stability.
For the sake of comparing the radical reactivities of cis-
and trans-resveratrol, the reaction enthalpies for the
hydrogen abstraction reaction have been calculated using the molecular orbitals method (Table III). The hydrogen
abstraction reactions from trans-resveratrol are predicted
to be more exothermic by 2-3 kcal/mol.
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Table III
Radical reactivities of cis- and trans-resveratrol, as determined by
reaction enthalpies for the hydrogen abstraction reaction, calculated
using the molecular orbital method
|
|
 |
DISCUSSION |
In agreement with abundant evidence obtained on other systems
in vitro (14, 17, 39, 53, 54) the work presented here has
documented a significant antioxidant activity of resveratrol. The
results have shown that the hydroxyl group in the 4' position is
required for the antioxidant activity, but acts synergistically with
the 3- and 5-OH groups. In fact, the derivative IV, which has a free
hydroxyl group in the 4' position, exert a significant antioxidant
activity in all the tests used. The trans-isomery and the
double bond in the stilbenic skeleton also play a role, at least as
indicated by the citronellal and microsome tests, since
cis-resveratrol and compound VI, in which the double bond is
reduced, are significantly less effective than the
trans-resveratrol. A similar conclusion is suggested by the
cell culture assay, based on the inhibition of TBHP-induced lipid
peroxidation. The major role played by the 4'-OH group in the radical
scavenging and antioxidant activity of trans-resveratrol
could be related to the electronic structure and the formation enthalpy
of the three different phenoxyl radicals arising from the loss of
hydrogens at the 3-, 5-, and 4'-OH groups in resveratrol (Fig. 8). The
hydrogen abstraction from 4'-OH bond is expected to be favored, being
more exothermic than the analogue reactions at the 3-OH and 5-OH
positions, as a consequence of the greater resonance stabilization
energy. In addition, the absence of the double bond is expected to
cause a decrease in the 4'-phenoxyl radical resonance stabilization energy. This would induce a decrease in the antioxidant activity of
compound VI, as observed in our experimental models. Finally, on
comparing the radical reactivity of trans- and
cis-resveratrol (Table III), the hydrogen abstraction
reactions from the trans-form are predicted to be more
exothermic. This provides a thermodynamic rationale for the
experimental observation in two in vitro methods and in cell
culture that the antioxidant power of cis-resveratrol is
lower than that of the trans-analogue. DPPH measurements
differ from the above results in that trans- and
cis-form have the same effect, while again both methylation
and, more markedly, reduction of the olefinic double bond, decrease the
antioxidant activity. Thus, in this assay, hydrogen transfer to the
radical is equally efficient from both isomers. It may be inferred from
these results that the calculated decrease in the stabilization energy
for the phenoxyl radical in the cis configuration does not
significantly influence the reaction kinetic activation parameters.
This in turn might be considered diagnostic of the activated complex
along the reaction path being closer to reactants than the products.
In agreement with studies by others (15, 16, 25-28, 30),
trans-resveratrol has been found to inhibit in a dose- and
time-dependent manner cell growth both in normal
fibroblasts and in fibrosarcoma cells. An antiproliferative activity
comparable to that of trans-resveratrol has been observed
only for derivative IV. Importantly, their effect was cytostatic and
reversible since no evidence of cell death was obtained by a number of
tests such as visual inspection for detached cells, trypan blue
exclusion, and annexin V staining (data not shown).
The cytostatic effect may be attributed to decreased DNA synthesis
given that a significant inhibition of BrdUrd incorporation was
previously found in other cell lines (19, 29, 30, 37), and also
observed in our study on normal fibroblasts and in fibrosarcoma cells.
The results described here suggest that inhibition of DNA synthesis
occurred at the level of DNA polymerase activity, since the recruitment
of PCNA and RPA (32 kDa) proteins to DNA replication sites was not
affected by trans-resveratrol.
The in vitro assays have demonstrated that only
trans-resveratrol and derivative IV inhibited significantly
DNA polymerases
and
. Interestingly, there was an increase in
specificity for the inhibition of pol
with respect to pol
, from
trans-resveratrol to compound IV suggesting a possible role
of the -OH groups in positions 3 and 5 in the binding to the different
DNA polymerases. The inhibition by resveratrol was found to be strictly
specific for the B-type DNA polymerases, since neither pol I (member of the A-type family of DNA polymerases), nor HIV-1 RT (belonging to the
reverse transcriptases/RNA-dependent RNA
polymerases/telomerases family) were inhibited. Moreover, within the
B-type family, trans-resveratrol discriminated between
eukaryotic and viral enzymes, i.e. HSV-1 pol was not
inhibited. These data suggest that the interaction of resveratrol with
the eukaryotic replicative DNA polymerases
and
is highly specific.
In conclusion, the results of this study indicate that (i) 4'-hydroxyl
group in trans-conformation (hydroxystyryl moiety) is not
the sole determinant for antioxidant properties, while it is absolutely
required for antiproliferative activity. (ii) There is a direct
correlation, from a structural point of view, between the
antiproliferative effect and the ability to inhibit DNA pol
and
. Thus, a mechanism underlying the inhibition of cell cycle
progression is the interaction between the 4'-hydroxystyryl moiety of
trans-resveratrol and DNA polymerases.
The structure-activity relationship revealed by this study should be
taken in account in studies aimed at synthesizing resveratrol derivatives with more selective antioxidant and/or antiproliferative activity. To this respect, the observation that the cis
conformation of resveratrol still showed antioxidant activity but was
totally inactive against DNA polymerases, is very interesting and
deserves further investigation.
 |
ACKNOWLEDGEMENTS |
We thank R. Pezet (Station
Fèdèrale de Recherches Agronomiques de Changins, Nyon,
Switzerland) for providing a chemically pure standard of
trans-3,5-dimethoxy-4'-hydroxystilbene (IV), and A. Faucitano (Dipartimento di Chimica generale, Università di Pavia)
for the helpful discussion on thermodynamic stability of resveratrol.
We also thank R. Melli for technical assistance, R. Pizzala for
statistical analysis, and E. Gherardi for critical review of the manuscript.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dipartimento di
Medicina Sperimentale Sez. Patologia generale "C. Golgi,"
Università di Pavia Piazza Botta, 10-27100 Pavia, Italy. Tel.:
39-0382-506333; Fax: 39-0382-303673; E-mail:
l.stivala@botta.unipv.it.
Published, JBC Papers in Press, April 20, 2001, DOI 10.1074/jbc.M101846200
 |
ABBREVIATIONS |
The abbreviations used are:
DPPH, 2,2-diphenyl-1-picrylhydrazyl radical;
TBHP, tert-butylhydroperoxide;
BrdUrd, bromodeoxyuridine;
Me2SO, dimethyl sulfoxide;
FITC, fluorescein
isothiocyanate;
PBS, phosphate-buffered saline;
PCNA, proliferating
cell nuclear antigen;
PMSF, phenylmethylsulfonyl fluoride;
pol
, DNA
polymerase
;
pol
, DNA polymerase
;
RPA, replication
protein A;
TBARS, thiobarbituric acid reactive substances;
BisTris, 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)propane-1,3-diol.
 |
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