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2nd Department of Medicine, J. W. Goethe University, 60590 Frankfurt/Main, Germany
2To whom correspondence should be addressed. E-mail: j.stein{at}em.uni-frankfurt.de.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • resveratrol • Caco-2 cells • cell cycle • colon cancer
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Red wine
[contains up to 13.4 mg resveratrol/L (2)
] and peanuts
[contain 0.02–1.79 µg resveratrol/g (3)
]
represent the main sources in the human diet. The compound has been
shown to exhibit cancer chemopreventive properties in different cell
culture and animal models. Resveratrol induces HL-60 promyelocytic
leukemia cell differentiation, suppresses prostanoid synthesis by
inhibition of cyclooxygenases and induces phase II carcinogen
detoxifying enzymes. In addition, it has been reported to inhibit the
growth of breast cancer cells in vitro and to reduce the number of
preneoplastic lesions in a mouse mammary gland culture model
(4)
. Moreover, resveratrol induces apoptosis by enhancing
the expression of Fas-ligand and activating caspases in HL-60 cells
(5)
, whereas it decreases the level of the antiapoptotic
protein Bcl-2 (6)
. Recent studies have demonstrated the
inhibitory effect of resveratrol on cell cycle progression
(7
8
9)
. Most studies report perturbation of the S/G2 phase
transition with a decrease of cells in G0/G1 phase of the cell cycle
and an increase of cells in S phase (10
11
12
13
14)
. Despite a
large amount of data on the antiproliferative and proapoptotic
properties, the exact mechanism by which resveratrol exerts its effects
on tumor cells is unknown. Even fewer data exist concerning inhibition
of tumorigenesis and cell growth in the colon. Schneider et al.
(15)
demonstrated reduced ornithine decarboxylase activity
and growth inhibition at the S/G2 phase transition of Caco-2 cells
after treatment with 25 µmol/L resveratrol. In addition,
it has been shown that resveratrol inhibits growth of colorectal
aberrant crypt foci and upregulates bax in azoxymethane-induced
carcinogenesis of the rat colon, whereas it reduces
p21WAF1/CIP1 protein level overall in the normal
surrounding mucosa (16)
.
Colonic epithelial cells undergo a sequential process of proliferation,
differentiation, apoptosis and exfoliation as they migrate along the
crypt-villus axis, which is deregulated in carcinogenesis. This
process is largely regulated by periodical activation and inactivation
of a highly conserved family of cyclin-dependent kinases
(cdk)3
(17)
. Cdk activity is modulated by the cyclins, which bind
to and activate the cdk (18)
. They are regulated primarily
by their expression levels. A critical event in the development of
malignant colorectal carcinomas from benign adenomas is mutation of the
tumor suppressor p53 (19)
. Loss of p53 function leads to
impaired control of cell cycle and apoptosis. Another common
characteristic among colon carcinomas is overexpression of
cyclooxygenase (COX)-2. The chemopreventive effects of nonsteroidal
anti-inflammatory drugs have been associated with their ability to
inhibit COX (20)
. Because resveratrol is a COX inhibitor,
it might exert its effects on colon cancer cells via this pathway.
The primary objective of the present study was to elucidate the
underlying molecular mechanisms of inhibition of cell cycle progression
by resveratrol. Because of the importance of cell cycle regulators in
carcinogenesis, we determined whether they can be affected by
resveratrol. Therefore, we assessed the influence of resveratrol on
positive and negative regulators of the cell cycle in Caco-2 cells and
HCT-116, which present well-established cell culture models and
differ in p53 and COX expression. Caco-2 cells express mutated p53 and
possess active COX-2, whereas HCT-116 cells express wild-type p53,
but have no detectable COX activity (21
22
23)
. Our interest
was in determining whether effects of resveratrol on the cell cycle
would differ in these two cell lines.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The human colon cancer cell lines Caco-2 and HCT-116 were obtained from
the American Type Culture Collection (ATCC, Rockville, MD). Caco-2
cells of passages 45–55 were cultured in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal calf serum (FCS), penicillin (1000
U/L) and streptomycin (1 mg/L). HCT-116 cells of passages
31–37 were cultured in McCoy’s 5A supplemented with 10% FCS,
penicillin (1000 U/L) and streptomycin (1 mg/L). Both cell
lines were maintained at 37°C under an atmosphere of 5%
CO2 in air. The medium was changed three times per week.
All cell culture reagents were obtained from Gibco (Eggenstein,
Germany). A 1 mol/L stock solution of resveratrol (Sigma Chemical,
Deisenhofen, Germany) was prepared in dimethyl sulfoxide (DMSO) and
stored at -20°C. Control cells not receiving resveratrol routinely
had equal amounts of DMSO added to the culture media (
0.1%). For
treatment with resveratrol, cells were cultured until nearly confluent.
The medium was then removed and replaced by a medium containing either
solvent or 12.5–200 µmol/L resveratrol. Cytotoxicity
was excluded by lactate dehydrogenase (LDH) release assay (Boehringer
Mannheim, Mannheim, Germany). Stilbenemethanol and rhapontin were
obtained from Sigma Chemical.
Cell number.
Determination of cell numbers was carried out using a modification of
the method of Matsubara et al. (24)
. Briefly, cells were
plated at a densitiy of 7 x 103 cells per well in
96-well microtiter plates. Treatment with increasing concentrations of
resveratrol was carried out for 24, 48 and 72 h
(resveratrol-containing medium was changed after 48 h). At the end
of the incubation period, the medium was removed and any adherent cells
were fixed to the plate with 5% formaldehyde in PBS. The cells were
then stained with an aqueous solution of crystal violet (5 g/L)
followed by elution of the dye with 33% aqueous acetic acid.
Absorbance at 570 nm was determined with a Tecan Spectrafluor Plus
microplate reader (Tecan, Crailshaim, Germany) and the number of cells
was determined from a standard curve of absorbance against cell numbers
calculated from a mean of six experiments.
Western blot analysis.
Cells were plated on 80 cm2 flasks at a density of 2
x 106 cells per flask, allowed to attach overnight
and then exposed to resveratrol vs. DMSO for 24 h. After the cells
were washed three times with ice-cold PBS, they were incubated with
cell lysis buffer (New England Biolabs, Beverly, MA) containing
multiple protease inhibitors (Boehringer Mannheim, Germany) for 20 min
at 4°C. Cells were sonicated on ice and centrifuged at 10,000 x g for 5 min to sediment the particulate material.
Aliquots of the supernatant were assayed for total protein (BioRad
Laboratories, Muenchen, Germany) according to the method described by
Bradford (25)
. Protein (20 µg/lane) was
separated by SDS-PAGE along with prestained molecular weight
markers (BioRad Laboratories). The separated proteins were transferred
to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany)
with a semidry blotting device. Membranes were reversibly stained with
Ponceau-S red to verify homogeneity of protein blotting. The blots
were blocked overnight with Tris-buffered saline containing 0.05%
Tween-20 and 30 g/L nonfat milk at 4°C. The level of proteins was
assayed using the primary antibodies for 1 h with agitation at
room temperature. Immunoreactivity was demonstrated by enhanced
chemiluminescence system (Amersham Pharmacia Biotech, Buckinghamshire,
UK) using appropriate horseradish peroxidase conjugated secondary
antibodies (1:2000). Reprobing of blots for expression of actin was
done routinely. Antibodies against p27KIP1, cdc2, cdk2,
cdk4, cdk6, cyclin A and cyclin D1 were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-p21WAF1/CIP1 (Ab-1)
and anti-proliferating cell nuclear antigen (anti-PCNA) were
purchased from Oncogene (Cambridge, MA) and anti-pRb and
anti-cyclin E from Pharmingen (Becton-Dickinson, Heidelberg,
Germany). For quantification, the density of the bands was detected
with scanning densitometry, using a Desaga CabUVIS scanner and Desaga
ProViDoc software (Wiesloch, Germany). Results were expressed as a
percentage of the control.
Incorporation of [3H]thymidine and [14C]leucine.
Caco-2 cells cultured in 24-well plates (5 x 104/well) were treated with resveratrol for 24 h. During treatment, the cells were pulsed with 1.8 x 107 Bq/well [3H]thymidine and 9.2 x 108 Bq/well [14C]leucine (Amersham Pharmacia Biotech, Freiburg, Germany). The medium was discarded, the monolayers were washed three times with PBS and the cellular macromolecules were precipitated using 5% trichloroacetic acid. The acid was aspirated, cells were washed with absolute methanol and formic acid (2.5 mol/L) was used to solubilize the precipitated macromolecules. Probes were transferred to scintillation vials; 3.0 mL scintillation fluid (Packard Biosciences, Groningen, Netherlands) was added and measurements were carried out with a liquid scintillation counter (Packard Instruments, Meridien, CT). Cellular protein concentrations were determined as described in the Western blot analysis section.
Determination of alkaline phosphatase (AP) activity.
Alkaline phosphatase activity, a marker of differentiation was measured using p-nitrophenylphosphate as substrate according to the manufacturer’s instructions (Merck, Darmstadt, Germany). Cell lysates of Caco-2 cells treated for 1, 4, 8 or 12 d with 12.5 µmol/L resveratrol were analyzed in the assay. Cellular protein concentrations were determined as described in the Western blot analysis section. AP activity was calculated in units per milligram protein (U/mg protein).
Cell cycle analysis.
Cells were seeded 24 h before treatment in 6-well plates at a density of 15 x 104/well. Cells were washed 24 h after treatment with PBS and harvested by trypsinization (2.5% trypsin/EDTA solution, Gibco). DNA contents of cells were measured using a DNA staining kit (CycleTEST PLUS DNA Reagent Kit, Becton Dickinson). Propidium iodide–stained nuclear fractions were obtained by following the kit protocol. Data were acquired using CellQuest Software (Becton Dickinson) with a FACScalibur (Becton Dickinson) flow cytometry system using 10,000 cells per analysis. Cell cycle distributions were calculated using ModFit LT 2.0 software (Verity Software House, Topsham, ME).
Apoptosis assay.
The EnzCheck caspase-3 assay kit #2 (Molecular Probes, Leiden, Netherlands) was used according to the manufacturers suggestions. Briefly, cells grown in 80 cm2 cell culture flasks were incubated with 200 µmol/L resveratrol, vehicle or 50 µmol/L camptothecin for 8, 24 and 48 h. Floating cells were collected with the medium, and attached cells were collected by trypsinization. Both fractions were counted together with a hemocytometer. Cells (1 x 106/sample) were collected in PBS and centrifuged at 200 x g for 10 min. The supernatant was discarded and the cell pellets were frozen at -80°C until all samples were collected. The pellets were thawed on ice and resuspended in lysis buffer. After complete lysis of the cells, the particulate material was sedimented by centrifugation at 2000 x g for 5 min. The supernatant was incubated with the Z- DEVD-R110 substrate for 30 min. Fluorescence was measured (excitation/emission, 496/520 nm) with the fluorescence microplate reader Tecan SpectraFluor PLUS.
Statistical analysis.
Data are expressed as means ± SD Differences were tested for statistical significance using two-way ANOVA. Individual differences between groups were assessed using the least significant differences (LSD) test (Microsoft Excel, Microsoft, Roselle, IL). A P-value < 0.05 was considered to indicate a significant difference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). When Caco-2 cells were treated with 100 µmol/L
resveratrol, cell number did not increase after 72 h of
incubation, whereas 200 µmol/L treatment reduced cell
counts at this time (P < 0.05). To discriminate
between an effect of resveratrol on the rate of cell growth as opposed
to cell death, thymidine and leucine uptake were used to estimate any
net change in biomass. Figure 2
shows the effect of increasing concentrations of resveratrol on the
proliferation of Caco-2 cells over 24 h when both thymidine and
leucine uptake were measured and related to protein content of the
cells. Treatment resulted in a dose-dependent inhibition of
[3H]thymidine and
[14C]leucine incorporation. A significant
inhibitory effect was observed with resveratrol concentrations 
12.5
µmol/L, and a concentration of 100 µmol/L
completely abolished proliferation of Caco-2 cells. The effects were
not associated with unspecific toxicity of the compound because LDH
activity in the cell culture medium was unaffected by resveratrol
treatment (data not shown). AP activity, a marker of differentiation,
was assessed in Caco-2 cells, which differentiate spontaneously after 1
wk in culture (increase of AP activity from 10.35 ± 2.23 at d 1
to 117.4 ± 14.06 U/mg protein at d 12).
Treatment with 12.5 µmol/L resveratrol increased AP
activity from 9.73 ± 4.25 at d 1 to 128.66 ± 4.34
U/mg protein at d 12 (values are means ± SD, n = 6, P < 0.05 vs.
control at d 12).
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). Untreated subconfluent Caco-2 cells revealed a constant cell cycle
phase distribution of cells in G0/G1 (42.27 ± 0.27%), in S
(44.57 ± 0.38%) and G2/M (13.17 ± 0.60%) phases (Fig. 3A
). At 50 µmol/L, resveratrol led to a 54%
decline (P < 0.05) of cells in G2/M phase. This effect
was associated with a 14% increase (P < 0.05) in
proportion of cells in S phase. At higher concentrations (100 and 200
µmol/L), the S phase arrest of the cell cycle was
reversed, with values of 44.13 ± 0.13 and 43.93 ± 0.47% in
the S phase population, respectively. Similar results were obtained for
HCT-116 cells, which possess wild-type p53, and the effects were
more dramatic (Fig. 3B
). Under control conditions, the
distribution of cells in G0/G1 (37.00 ± 0.84%), S (44.66 ± 0.46%) and G2/M (18.35 ± 0.38%) phases of the cell cycle
remained constant during a 24-h incubation. The distribution profile
for cells incubated with 50 µmol/L for 24 h recorded
13.23 ± 0.35% of cells in G0/G1, 83.60 ± 2.02% in S and
3.19 ± 2.37% in G2/M. This effect was reversible at higher
concentrations. The cell cycle distribution of HCT-116 cells treated
with 200 µmol/L resveratrol (32.45 ± 0.39% G0/G1,
48.36 ± 2.81% S, 19.20 ± 3.20% G2/M) differed little from
values obtained for controls. To test the specificity of the cell cycle
inhibitory effect of resveratrol, we treated Caco-2 cells with the
glycosylated stilbene analogs rhapontin (100 µmol/L; 41.37
± 0.23% G0/G1, 43.60 ± 0.70% S, 15.04 ± 0.30%
G2/M) and stilbenemethanol (25 µmol/L; 41.73 ± 0.69% G0/G1, 44.58 ± 0.24% S, 13.70 ± 0.93% G2/M). No
significant change in cell cycle distribution was observed
(n = 2).
|
). A dose-dependent reduction in cdk4 and cyclin D1 protein levels
was apparent. At concentrations 100 µmol/L, no cyclin D1
protein was detected. Cyclin A protein level was enhanced at
concentrations up to 100 µmol/L. The maximal effect was
observed after the addition of 25 µmol/L resveratrol. The
amount of cyclin E protein was elevated to a concentration
of 200 µmol/L. We further examined the expression of the
cell cycle inhibitors p21WAF1/CIP1 and
p27KIP1 by immunoblot (Fig. 4B
). A
dose-dependent decrease of p27KIP1 was
observed. The p21WAF1/CIP1 protein was
constitutively present in resveratrol-treated Caco-2 cells. As an
indication that resveratrol inhibits cdk activity, we determined the
phosphorylation state of pRb by immunoblot. The retinoblastoma protein
(pRb), as observed by a shift of the band in Western blot analysis, was
changed from the hyperphosphorylated to the hypophosphorylated form at
the highest concentration of resveratrol and total pRb protein was
decreased. To exclude the possibility that the observed effects of
resveratrol on the cell cycle–regulating proteins were cell line
specific we incubated HCT-116, a human colorectal cancer cell line with
200 µmol/L resveratrol vs. vehicle for 24 h and
performed Western blots with the cellular lysates. As observed in
Caco-2 cells, resveratrol treatment decreased cyclin D1, cdk4 and
p27KIP1 levels, whereas cdk2 and cdk6 levels were
unmodified. The increase in cyclin E protein expression was also
apparent in HCT-116 cells (Fig. 5
).
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). The topoisomerase inhibitor camptothecin, which induces
capase-3–dependent apoptosis, was used as a positive control at 50
µmol/L. After 24 h, a significant increase in
caspase-3 cleaving activity was found in resveratrol-treated cells,
which was even more apparent after 48 h. Camptothecin induced
capase-3 activity significantly after 8 h and led to higher values
of capase-3 activity after 24 and 48 h compared with resveratrol.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in which an inhibitory effect
has been shown in different stages of tumorigenesis.
This study demonstrates that resveratrol significantly inhibits the
growth and proliferation of the human colonic adenocarcinoma cell line
Caco-2 in vitro without influencing differentiation. Even though the
rise in AP activity, a hallmark of differentiation, was significant
after 12 d of treatment with resveratrol, these values cannot be
considered physiologically relevant. Comparison with the
differentiation-inducing agent butyrate showed that the
short-chain fatty acid enhanced Caco-2 cell AP activity at 2 mmol/L
six- to sevenfold (26)
.
Flow cytometry results indicated a significant reduction of cells in
the G2/M phase of the cell division cycle, whereas the S phase
population increased, which is consistent with the results obtained by
Schneider et al. (15)
. These effects are specific for
resveratrol because addition of the stilbene analogs stilbenemethanol
and rhapontin did not change cell cycle distribution. This prompted us
to investigate in further detail the mechanism of action of resveratrol
on the cell cycle machinery. Western blot analysis of positive cell
cycle regulators (cdc2, cdk2, cdk4, cdk6, cyclin A, cyclin D1 and
cyclin E) showed a dose-dependent increase in cyclin E levels and
an increase in cyclin A levels only at concentrations up to 100
µmol/L, suggesting the presence of an S to G2 block.
Resveratrol treatment significantly reduced cyclin D1 levels and its
related serine/threonine kinase cdk4. As a positive regulator of cdk4
and cdk6, cyclin D1 has been implicated in controlling the G1 phase of
the cell cycle and is frequently overexpressed in human colon
adenocarcinomas (27
,28)
. Overexpression of an antisense
cyclin D1 cDNA construct in a human colon carcinoma cell
line leads to impaired cell growth and tumorigenicity, implying
that cyclin D1 presents an oncogene (29)
. The
cyclin D1/cdk4 complex is responsible for cell cycle progression in
early G1 phase and for phosphorylation and thus inactivation of the
tumorsuppressor pRb (30
,31)
. Hypophosphorylated pRb is
able to sequester the transcription factor E2F in the cytosol, which
suppresses protein expression of the cell cycle machinery, thus causing
a blockade in G1 phase. As revealed by immunoblot, dephosphorylation of
pRb was observed in response to resveratrol treatment at a
concentration of 200 µmol/L. Hypophosphorylation of pRb
has been associated with inhibition of growth and arrest of cells in G1
phase (32)
. This might be a possible explanation for the
increase in G1/S ratio, a marker of G1 arrest, observed at
concentrations exceeding 50 µmol/L. It is tempting to
speculate that the reduction of cdk4, cyclin D1 and cyclin A levels is
responsible for this effect. Previous studies indicate that primary
human tumors and tumor-derived cell lines often display a
correlation between the levels of expression of cyclin D1 and the
pRb tumor suppressor protein (33)
. This could contribute
to the observed reduction of total pRb protein expression. The kinase
activity of the cdk is negatively regulated by binding of the
cyclin-dependent kinase inhibitors (cki)
p21WAF1/CIP1 and p27KIP1
(34)
, which are positive regulators of differentiation
(35)
. Subsequently, we determined protein
expression of cki p21WAF1/CIP1 and
p27KIP1. The p21WAF1/CIP1
level was unmodified by resveratrol addition. In contrast,
p27KIP1 expression was inhibited in treated
cells, consistent with the results obtained by Doki et al.
(33)
that expressions of cyclin D1 and
p27KIP1 are closely correlated. The level of
p27KIP1 in cyclin D1 overexpressing
cells is largely dependent on levels of cyclin D1 protein
(36)
, possibly contributing to the observed effect.
Expression of PCNA, a protein involved in cell cycle regulation, DNA
synthesis and DNA repair, was not affected by resveratrol addition. As
revealed by flow cytometry, the cells accumulate in S phase at a
concentration of 50 µmol/L. This cell cycle arrest was
reversed when higher concentrations of resveratrol were used. The cell
cycle distribution corresponds well with expression of cyclin A, which
is synthesized during S phase (37)
. Disruption of cyclin A
function can inhibit chromosomal DNA replication (38)
. The
growth inhibitory effect of resveratrol seems to be more pronounced at
concentrations 50 µmol/L, which correlates with a
reduction of cyclin A levels. This effect, together with accumulation
of cyclin E, was also observed by Park et al. (10)
in U937
human leukemia cells. This might lead to the conclusion that the
effects of resveratrol on the cell division cycle are mainly a result
of hampered DNA synthesis. The mechanism of action of resveratrol on
tumor cells proposed to date includes inhibition of ribonucleotide
reductase (39)
, DNA polymerase (40)
and COX-1
(4)
and inhibition of COX-2 transcription
(41
,42)
. Because Caco-2 cells express little or no COX-1
(22)
and HCT-116 cells lack COX-1 or COX-2 activity
(23)
, we can conclude that the observed effects are not
mediated by COX inhibition.
It has been assumed that the cell cycle inhibition in S and G2 phase is
mediated by upregulation of p53 and induction of
p21WAF1/CIP1 (43)
. The
resveratrol-mediated cell cycle inhibition we observed was clearly
independent of p53 because Caco-2 cells possess mutated p53
(21)
. Our finding that resveratrol exerts the same effects
on HCT-116 cells, which express wild-type p53, confirms earlier
work showing that inhibition of cell cycle progression by resveratrol
can function independently of p53 (10
,44)
. In addition, no
changes in the p21WAF1/CIP1 protein
levels were observed in Caco-2 cells.
Another possible mechanism by which the antiproliferative activity of
resveratrol on Caco-2 cells may be exerted is induction of apoptosis.
Indeed it has been demonstrated that resveratrol induces apoptosis in
promyelocytic leukemia cells (HL-60) (6)
via upregulation
of CD95 ligand (5)
through a strictly p53-dependent
pathway in a mouse epidermal cell line (45)
, and
independent of the CD95 pathway in CEM-C7H2 acute leukemia cells
(44)
. We therefore determined whether resveratrol could
induce apoptosis in Caco-2 cells. This was done by measuring the
activity of caspase-3, a key protease in apoptosis. The increase of
caspase-3 activity in Caco-2 cells was significant after 24 h.
However, compared with camptothecin, this increase is very small.
Because caspase-3 cleaving activity represents an early event in
apoptosis, this might explain the reduction of cell counts, which was
observed in the crystal violet assay after 72 h with 200
µmol/L resveratrol. Elimination of transformed cells via
apoptosis is considered to be crucial for restoration of normal
epithelial growth in the colon (46)
.
Epidemiologic studies reveal a strong inverse association between
frequency of intake of plant-derived foods and cancer incidence.
Resveratrol, widely distributed in red wine, peanuts and other sources
of the human diet, exerts broad and potent anticarcinogenic and
antitumor activities when applied at a pharmacologic level. The fact
that the intestinal epithelium might be confronted with much higher
concentrations than cells in other tissues, because the absorption rate
of resveratrol in the perfused small intestine of the rat was estimated
to be only 20.5% (47)
, implies that resveratrol could
have an important role in the prevention of colon cancer by blocking
hyperproliferation of the epithelium and by promoting apoptosis.
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FOOTNOTES |
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3 Abbreviations used: AP, alkaline phosphatase; cdk, cyclin-dependent kinase; cki, cyclin-dependent kinase inhibitor; COX, cyclooxygenase; DMSO, dimethyl sulfoxide; FCS, fetal calf serum; LDH, lactate dehydrogenase; PCNA, proliferating cell nuclear antigen; pRb, retinoblastoma protein.
Manuscript received March 8, 2001. Initial review completed April 12, 2001. Revision accepted May 17, 2001.
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