Download Ganoderma lucidum Suppresses Growth of Breast Cancer Cells

NUTRITION AND CANCER, 49(2), 209–216
Copyright © 2004, Lawrence Erlbaum Associates, Inc.
Ganoderma lucidum Suppresses Growth of Breast Cancer Cells
Through the Inhibition of Akt/NF-κB Signaling
Jiahua Jiang, Veronika Slivova, Kevin Harvey, Tatiana Valachovicova, and Daniel Sliva
Abstract: Ganoderma lucidum (Reishi, Lingzhi) is a popular
Asian mushroom that has been used for more than 2 millennia for the general promotion of health and was therefore
called the “Mushroom of Immortality.” Ganoderma lucidum
was also used in traditional Chinese medicine to prevent or
treat a variety of diseases, including cancer. We previously
demonstrated that Ganoderma lucidum suppresses the invasive behavior of breast cancer cells by inhibiting the transcription factor NF-kB. However, the molecular mechanisms
responsible for the inhibitory effects of Ganoderma lucidum
on the growth of highly invasive and metastatic breast cancer
cells has not been fully elucidated. Here, we show that
Ganoderma lucidum inhibits proliferation of breast cancer
MDA-MB-231 cells by downregulating Akt/NF-kB signaling.
Ganoderma lucidum suppresses phosphorylation of Akt on
Ser473 and downregulates the expression of Akt, which results
in the inhibition of NF-kB activity in MDA-MB-231 cells.
The biological effect of Ganoderma lucidum was demonstrated by cell cycle arrest at G0/G1, which was the result of
the downregulation of expression of NF-kB-regulated cyclin
D1, followed by the inhibition of cdk4. Our results suggest
that Ganoderma lucidum inhibits the growth of
MDA-MB-231 breast cancer cells by modulating Akt/NF-kB
signaling and could have potential therapeutic use for the
treatment of breast cancer.
Introduction
One third of all newly diagnosed cancers among women
in the United States are breast cancers (1). Because breast
cancer often progresses from the therapy-responsive phenotype to the highly invasive and metastatic phenotype, breast
cancer is the second leading cause of cancer death in the U.S.
female population (2). A comprehensive review by the World
Cancer Research Fund and the American Institute of Cancer
Research clearly demonstrates the importance of nutrition in
the prevention of cancer, which could also contribute to the
low incidence of breast cancers among Asian women (3).
Therefore, it was suggested that some nutritional products
have chemopreventive and therapeutic effects against cancer.
These anticancer effects also significantly increased the popularity of dietary supplements in cancer patients (4).
The popular edible mushroom Ganoderma lucidum has
been widely used in eastern Asia to promote health and longevity. The regular consumption of Ganoderma lucidum in the
form of teas was believed to fortify the mind and the body, and
therefore Ganoderma lucidum was called the “Mushroom of
Immortality” (5). The dried powder of Ganoderma lucidum
has been used in traditional Chinese medicine for more than
2,000 years to prevent or treat different diseases, including
cancer (6). The anticancer properties of Ganoderma lucidum
have been attributed to either the isolated polysaccharides,
which are responsible for the stimulation of the immune system, or triterpenes, which demonstrate cytotoxic activity
against cancer cells (for review, see ref. 7). However,
Ganoderma lucidum is currently available as a dietary supplement in the form of extracts or capsules containing fruiting
bodies and/or spores of this mushroom. We recently reported
that Ganoderma lucidum suppresses constitutively active
transcription factors AP-1 and NF-κB, which resulted in the
downregulation of expression of urokinase-type plasminogen
activator (uPA) and its receptor uPAR in human breast and
prostate cancer cells (8). The anticancer effect of Ganoderma
lucidum was also demonstrated by the inhibition of adhesion,
migration, and invasion of the highly metastatic breast cancer
cells MDA-MB-231 (9).
The highly invasive potential of breast cancers was linked
to the overexpression of epidermal growth factor receptors
(EGFRs) (10), which control signaling through the activation
of the phosphatidylinositol 3-kinase (PI3K)/NF-κB pathway
responsible for the aberrant cell cycle progression of breast
cancer cells (11). In addition, PI3K also activates
serine/threonine protein kinase Akt (12), which can be specifically phosphorylated on Thr308 or Ser473 (13). Furthermore,
activated Akt can subsequently regulate the NF-κB pathway
(14,15), and NF-κB controls the expression of the cell cycle
regulator cyclin D1, which is responsible for the transition
from the G1 to the S phase during cell cycle progression (16).
Finally, PI3K, Akt, and NF-κB are constitutively active in
All authors are affiliated with the Cancer Research Laboratory, Methodist Research Institute, Indianapolis, IN 46202. D. Sliva is also affiliated with the Department of Medicine, School of Medicine, Indiana University, Indianapolis, IN 46202.
highly invasive human MDA-MB-231 breast cancer cells
(17,18) and therefore they are suitable targets for cancer treatment (19). In the present study, we show that Ganoderma
lucidum inhibits the growth of breast cancer cells by inducing
cell cycle arrest at G0/G1 by suppressing NF-κB activity
through the inhibition of Akt phosphorylation in
MDA-MB-231 cells. We also report that Ganoderma lucidum
suppresses the expression of cyclin D1 followed by the inhibition of cyclin-dependent protein kinase cdk4.
Materials and Methods
Materials
Ganoderma lucidum (Reishimax) was purchased from
Pharmanex (Provo, UT). According to the manufacturer, this
sample contains powdered extract (20:1) with spores and is
standardized to 13.5% polysaccharides and 6% triterpenes.
Stock solution was prepared by dissolving Ganoderma
lucidum in sterile water at a concentration of 50 mg/ml and
stored at 4°C.
Cell Culture
The human breast cells MCF-10A and breast cancer cells
MDA-MB-231 were obtained from ATCC (Manassas, VA).
MCF-10A cells were maintained in DMEM/F12 medium
containing 5% horse serum (HS), insulin (10 µg/ml), epidermal growth factor (EGF; 20 ng/ml), cholera toxin (100
µg/ml), hydrocortisone (0.5 µg/ml), penicillin (50 U/ml), and
streptomycin (50 U/ml). MDA-MB-231 cells were maintained in DMEM medium containing penicillin (50 U/ml),
streptomycin (50 U/ml), and 10% fetal bovine serum (FBS).
Media and supplements came from GIBCO BRL (Grand Island, NY). HS and FBS were obtained from Hyclone (Logan,
UT).
resuspended in propidium iodine (50 µg/ml). Cell cycle analysis was performed on a FACStarPLUS flow cytometer
(Becton-Dickinson, San Jose, CA), as previously described
(20). Data are the mean ± standard deviation from 3 independent experiments.
DNA Transfection and Chloramphenicol
Acetyltransferase (CAT) Assay
MDA-MB-231 cells were transfected with NF-κB-CAT
reporter constructs and β-galactosidase expression vector
pCH110, as previously described (8). Twenty-four hours after transfection, cells were treated with Ganoderma lucidum
for an additional 24 h at 37°C, as indicated in the text. Cell
lysates were prepared and CAT assays performed, as described (8). Data points represent the mean ± standard deviation of three independent transfection experiments.
Western Blot Analysis
MDA-MB-231 cells (1 × 107) were treated with Ganoderma lucidum (1.0 mg/ml) for 24, 48, 72, and 96 h. After incubation, cells were washed twice with ice-cold DPBS, lysed
with 1 ml of ice-cold lysis buffer (50 mM Tris-HCl pH 7.4,
150 mM NaCl, 1% NP-40, 1 mM EGTA, 1 mM EDTA, and
protease inhibitor cocktail Complete™ [Boehringer Mannheim, Indianapolis, IN]) at 4°C for 30 min. The lysates were
collected and cleared of nuclei by centrifugation for 10 min
at 14,000 g. The equal amounts of proteins (20 µg/lane) were
separated on 15% SDS-PAGE and transferred to a PVDF
membrane (Millipore, Bedford, MA). The protein expression was detected with the corresponding primary antibodies: anti-Akt, anti-phospho-Akt (Thr308), anti-phospho-Akt
(Ser473; Cell Signaling, Beverly, MA), anti-cyclin D1,
anti-Cdk4, and anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Protein expression was visualized
using the ECL Western Blotting Detection System
(Amersham Biosciences, Buckinghamshire, UK).
Cell Proliferation Assay
Cell proliferation was determined by the tetrazolium salt
method, according to the manufacturer’s instructions
(Promega, Madison, WI). Briefly, MCF-10A and MDAMB-231 cells (5 × 103/well) were cultured in a 96-well plate
and treated at indicated times with Ganoderma lucidum
(0–1.0 mg/ml). At the end of the incubation period, the cells
were harvested and absorption was determined with an
ELISA plate reader at 570 nm. Data points represent mean ±
standard deviation in one experiment repeated at least twice.
Cell Cycle Analysis
MDA-MB-231 cells (1 × 106) were seeded and after 24 h
treated with Ganoderma lucidum (0.5 mg/ml) for the indicated period of time (0–48 h). After incubation, the cells
were harvested by trypsinization, washed with Dulbecco’s
phosphate buffered saline (DPBS) containing 2% FBS, and
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Results
Ganoderma lucidum Inhibits Proliferation of
Highly Invasive Human Breast Cancer Cells
by Cell Cycle Arrest at G0/G1
We recently reported that Ganoderma lucidum inhibits
the invasiveness of breast cancer cells by suppressing cell adhesion, cell migration, cell invasion, and colony formation
(9). To demonstrate the effect of Ganoderma lucidum on cell
growth, we treated MDA-MB-231 breast cancer cells with
increasing concentrations of Ganoderma lucidum (0–1.0
mg/ml) for 24, 48, and 72 h, and cell proliferation was determined. As expected, Ganoderma lucidum suppressed the
proliferation of MDA-MB-231 cells in a dose- and time-dependent manner (Fig. 1). To investigate the mechanism by
which Ganoderma lucidum inhibits cell growth, we analyzed
Nutrition and Cancer 2004
Figure 1. Ganoderma lucidum inhibits proliferation of MDA-MB-231
cells. MDA-MB-3 cells were treated with 0, 0.25, 0.5, and 1.0 mg/ml of
Ganoderma lucidum. Proliferation was assessed after 24, 48, and 72 h, as described in Materials and Methods. Each bar represents the mean ± standard
deviation of 3 experiments.
tivity at the DNA-binding and at the transactivation levels in
breast cancer cells (8). However, the amounts of biologically
active components in our original samples of Ganoderma
lucidum were not determined, which could result in a wide
range of their potency to inhibit cancer cells (8,21). Thus, in
the present study, we used Ganoderma lucidum in the form of
powdered extract (20:1) with spores, which contains 13.5%
polysaccharides and 6% triterpenes. In accordance with our
previous data, Ganoderma lucidum inhibited constitutively
active NF-κB in the reporter gene assay in MDA-MB-231
cells in a dose-response manner (Fig. 3).
Because Akt serine-threonine kinase controls the activity
of NF-κB (14,15), we investigated whether the inhibitory effect of Ganoderma lucidum on NF-κB is mediated through the
suppression of Akt in breast cancer cells. MDA-MB-231 cells
were treated with Ganoderma lucidum (1.0 mg/ml) for 0, 24,
48, 72, and 96 h, and the expression of Akt was determined in
whole cell extracts by Western blot analysis. As seen in Fig.
4A, Ganoderma lucidum inhibits the expression of Akt kinase
in a time-response manner. However, the activity of Akt requires phosphorylation at Thr308 and Ser473 (22). Therefore,
we investigated whether Ganoderma lucidum affects the
phosphorylation of Akt. Although we did not observe significant inhibition of p-Akt- Thr308 (not shown), we found that
Ganoderma lucidum markedly decreased phosphorylation of
Akt at Ser473 (Fig. 4B). To determine whether the effect of
Ganoderma lucidum on the activity of Akt is reversible, we
treated MDA-MB-231 cells with Ganoderma lucidum (1.0
mg/ml), and after 24 and 48 h of incubation, culture media
were replaced with media without Ganoderma lucidum, and
the incubation continued for an additional 24 and 48 h. As
shown in Fig. 4C, removal of Ganoderma lucidum restored the
activity of Akt in MDA-MB-231 cells, as demonstrated by the
Figure 2. Effect of Ganoderma lucidum on cell cycle distribution.
MDA-MB-231 cells were treated with Ganoderma lucidum (0.5 mg/ml) for
0, 24, and 48 h, and the amount of cells at G0/G1, S, and G2/M phases were
determined by flow cytometry, as described in Materials and Methods.
Each bar represents the mean ± standard deviation of 3 experiments.
cell cycle distribution by flow cytometry. As shown in Fig. 2,
treatment with Ganoderma lucidum (0.5 mg/ml) caused cell
cycle arrest at the G0/G1 phase, where the amount of
MDA-MB-231 cells at G0/G1 increased from 38.7% (0 h) to
66.5% (24 h) and 63.9% (48 h).
Ganoderma lucidum Suppresses NF-κB
Activity by Inhibiting Akt Kinase
We have previously demonstrated that purified spores or
fruiting bodies of Ganoderma lucidum decrease NF-κB acVol. 49, No. 2
Figure 3. Ganoderma lucidum inhibits NF-κB in MDA-MB-231 cells.
MDA-MB-231 cells were transfected with NF-κB-CAT reporter construct
and β-galactosidase expression vector. Twenty-four hours after transfection,
the cells were treated with Ganoderma lucidum and NF-κB was measured,
as described in Materials and Methods. The results are expressed as the
percentage of relative NF-κB activity. Each bar represents the mean ± standard deviation of 3 experiments.
211
Figure 4. Ganoderma lucidum inhibits Akt in MDA-MB-231 cells. MDA-MB-231 cells were treated with Ganoderma lucidum (Gl, 1.0 mg/ml) for the indicated times. Whole cell extracts were prepared and subjected to Western blot analysis with A: anti-Akt and B: anti-p-Akt-Ser473 antibodies, as described in Materials and Methods. C: MDA-MB-231 cells were untreated for 24 h (1), and 48 h (2), or treated with Gl for 24 h (3), and 48 h (4), or treated with Gl for 24 h followed by the incubation with fresh media for an additional 24 h (5) and 48 h (6), or treated with Gl for 48 h followed by the incubation with fresh media for an
additional 24 h (7) and 48 h (8). Western blot analysis with anti-p-Akt-Ser473 antibodies was performed as described previously. The equivalent amount of protein was verified by reprobing the blots with anti-β-actin antibody. The results are representative of 3 separate experiments.
phosphorylation of Akt at Ser473. Thus, Ganoderma lucidum
inhibits constitutively active NF-κB by suppressing Akt
kinase activity, and the effect of Ganoderma lucidum on Akt
activity is reversible.
Ganoderma lucidum Downregulates the
Expression of Cyclin D1 and cdk4
Because Akt kinase controls the activation of NF-κB (23)
and because NF-κB regulates the expression of cyclin D1
(16), we hypothesized that cell cycle arrest at G0/G1 by
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Ganoderma lucidum is the result of downregulation of the
expression of cyclin D1. The cell extracts from
MDA-MB-231 cells treated with Ganoderma lucidum were
subjected to Western blot analysis with anti-cyclin D1 antibody. As shown in Fig. 5A, Ganoderma lucidum markedly
decreased the expression of cyclin D1 in a time-response
manner. Because cyclin D1 regulates the activity of
cyclin-dependent protein kinase cdk4, which controls the G1
cell cycle checkpoint (24), we investigated the effect of
Ganoderma lucidum on cdk4 kinase. As expected,
Ganoderma lucidum also inhibits expression of cdk4 (Fig.
Nutrition and Cancer 2004
Vol. 49, No. 2
Figure 5. Ganoderma lucidum downregulates the expression of cyclin D1 and cdk4 in MDA-MB-231 cells. MDA-MB-231 cells were treated with Ganoderma lucidum (1.0 mg/ml) for the indicated times. Whole cell
extracts were subjected to Western blot analysis with A: anti-cyclin D1 and B: anti-cdk4 antibodies. The equivalent amount of protein was verified by reprobing the blots with anti-β-actin antibody. The results are representative of 3 separate experiments.
213
5B), suggesting that cell cycle arrest at G0/G1 by
Ganoderma lucidum is a result of the inhibition of the cell cycle regulatory proteins cyclin D1 and cdk4.
Discussion
The wide consumption of mushroom Ganoderma
lucidum and its medicinal benefits have been reported since
ancient times, and references to its use were described in the
earliest written Chinese “Classic of Materia Medica” around
25–220 AD (25). Different biologically active compounds,
such as polysaccharides, triterpenes, proteins, and lipids,
were isolated from Ganoderma lucidum (7). However,
Ganoderma lucidum is usually consumed in the form of dietary supplements, powder, and tea (26). We have previously
reported that fruiting body or spores of Ganoderma lucidum
suppressed constitutively active transcription factors AP-1
and NF-κB, which resulted in the inhibition of secretion of
uPA and inhibition of cell motility of highly invasive cancer
cells (8). In the present study, we used Ganoderma lucidum
in the form of a dietary supplement containing powdered extract (20:1) and spores with 13.5% polysaccharides and 6%
triterpenes. It has been suggested that isolated polysaccharides and triterpenes have anticancer effects through different
mechanisms, including by stimulation of the immune response (polysaccharides) or by the cytotoxic effects against
cancer cells (triterpenes; for review see ref. 27). The use of
Ganoderma lucidum containing both polysaccharides and
triterpenes is in agreement with the practice of herbal medicine, and the use of the whole product can also decrease possible toxic effects of the isolated components. Although
Ganoderma lucidum inhibits cell proliferation, this effect is
not caused by cytotoxicity because the same dose (0–1.0
mg/ml) did not suppress the viability of breast cancer cells
(data not shown).
Here, we show that Ganoderma lucidum inhibits the
growth of highly invasive MDA-MB-231 breast cancer cells
by cell cycle arrest at the G0/G1 phase. Our data suggest that
the arrest at the G1 cell cycle checkpoint by Ganoderma
lucidum is a result of the inhibition of Akt kinase activity,
with subsequent inhibition of transcription factor NF-κB,
followed by downregulation of the expression of cyclin D1
and cdk4 kinase.
Serine-threonine kinase Akt, also known as protein kinase
B (PKB), consists of a family of highly conserved kinases
Akt1, Akt2, and Akt3 (28,29). Akt3 expression was significantly upregulated in estrogen receptor-negative (ERα-negative) primary breast cancer tumors, suggesting that Akt3 may
contribute to the more aggressive clinical phenotype (30).
The enzymatic activity of Akt3 was also significantly elevated in ERα-negative, highly invasive MDA-MB-231 breast
cancer cells, further confirming the role of Akt as an oncogene responsible for the survival and growth of cancer cells
(30). Therefore, Akt is a suitable target for inhibiting highly
invasive and metastatic cancers. Our data demonstrate that
Ganoderma lucidum downregulates the expression of Akt
214
and suppresses the activity of Akt by inhibiting
phosphorylation of Akt at Ser473, which is the regulatory
phosphorylation site responsible for the activity of Akt. Although Akt activity can be suppressed by specific synthetic
inhibitors of PI3K such as wortmannin and LY294002, here
we show the inhibition of Akt with the mushroom extract
from Ganoderma lucidum. Interestingly, other natural products, such as epigallocatechin-3-galate (EGCG), a major biologically active component of green tea, and genistein, an
isoflavonoid from soy beans, inhibited Akt in MDA-MB-231
breast cancer cells (31, 32). However, EGCG inhibited TGFα-dependent phosphorylation of Akt at Ser473, whereas the
same treatment did not affect the levels of Akt expression
(31). On the other hand, genistein inhibited both the kinase
activity of Akt as well as the expression of total Akt and
phosphorylated Akt at Ser473 (32). Considering all of these
findings, we can conclude that Ganoderma lucidum
downregulates the expression of constitutively active Akt
and inhibits phosphorylation of Akt at Ser473.
In the present study, we also demonstrated that Ganoderma lucidum suppresses the activity of NF-κB in MDAMB-231 cells. The suppression of NF-κB activity by Ganoderma lucidum is probably modulated through the inhibition
of Akt kinase, which controls the activity of NF-κB by
phosphorylation of IκB kinase (IKK) and liberation of
NF-κB by degradation of IκB (14, 15). Our data are consistent with studies by Masuda et al., which demonstrate that the
suppression of Akt by EGCG results in the inhibition of constitutively active and TNF-α-induced NF-κB in a reporter
gene assay (31). Furthermore, Gong et al. show that
genistein, which inhibits Akt, also inhibits the DNA-binding
activity of NF-κB in unstimulated or EGF-stimulated
MDA-MB-231 cells (32). Alternatively, the effect of Ganoderma lucidum on the activity of NF-κB can be caused by the
inhibition of PKC, and we have also recently demonstrated
that the PKC inhibitor bisindolylamelimide I suppresses
NF-κB activity in MDA-MB-231 cells (33). Finally, recent
studies demonstrate that polysaccharides isolated from
Ganoderma lucidum inhibit alloxan-induced activation of
NF-κB in pancreas (34) and that the herbal mixture PCSPES, which also contains Ganoderma lucidum, suppresses
lipopolysaccharide-induced NF-κB in macrophages (35).
As we demonstrated previously, Ganoderma lucidum inhibits cell proliferation and induces cell cycle arrest at
G0/G1. As we expected, this effect is a result of the inhibition
of cyclin D1, the expression of which is controlled by NF-κB
(16). Subsequently, inhibition of cyclin D1 suppressed cdk4
kinase, which resulted in cell cycle arrest at G0/G1 and the
inhibition of proliferation. In addition to inducing cell cycle
arrest, extended exposure of Ganoderma lucidum also induced apoptosis in MDA-MB-231 cells (not shown). Experiments are in progress to characterize the mechanism(s) by
which Ganoderma lucidum induces apoptosis in breast cancer cells. A recent study suggests that EGF-induced NF-κB
activation is a major pathway for the proliferation of
MDA-MB-231 cells, and the inhibition of NF-κB suppressed
regulatory proteins of G1/S cell cycle progression, such as
Nutrition and Cancer 2004
cyclin D1 and pRb (11). In addition to controlling cell proliferation, NF-κB also controls the expression of proteins responsible for metastasis and angiogenesis of cancers (19);
we have recently demonstrated that Ganoderma lucidum inhibits NF-κB-dependent expression of uPA and uPAR, which
resulted in the inhibition of the metastatic behavior of highly
invasive breast cancer cells (8,9).
Cell cycle arrest at G0/G1 phase and inhibition of cyclin
D1 by the alcohol extract of Ganoderma lucidum was also reported by Hu et al. (36). However, this effect was observed in
breast cancer MCF-7 cells (36), cancer cells that do not contain constitutively active NF-κB and are not as invasive as the
highly metastatic MDA-MB-231 cells (17). We have also
found that Ganoderma lucidum inhibits proliferation of the
normal breast cells MCF10A (not shown), cells that also do
not have constitutively active NF-κB, nor do they
overexpress cyclin D1 (37,38), suggesting that Ganoderma
lucidum inhibits cell proliferation by multiple mechanisms.
Furthermore, ethanol extracts of Ganoderma lucidum inhibited growth and induced cell cycle arrest at the G0/G1 phase
of human cervical cancer HeLa cells (39). Alternatively, Lin
et al. (40) recently described the inhibition of growth and G2
cell cycle arrest by Ganoderma lucidum in human hepatoma
cells, which were linked to the downregulation of expression
of cyclin B, and we found that Ganoderma lucidum arrested
PC-3 prostate cancer cells at the G2/M phase independently
of the inhibition of NF-κB (41). All of these findings together
suggest that Ganoderma lucidum can inhibit the proliferation
of normal and cancer cells by specific mechanism(s), which
are different in various cell lines.
In summary, our data demonstrate that Ganoderma
lucidum inhibited the growth of human breast cancer cells by
cell cycle arrest at the G0/G1 phase. The biological effects of
Ganoderma lucidum on MDA-MB-231 cells are mediated by
the inhibition of phosphorylation and downregulation of expression of Akt kinase, which results in the suppression of
NF-κB activity following the downregulation of cyclin D1
and cdk4 expression. The study presented demonstrates how
the edible mushroom Ganoderma lucidum inhibits the
growth of highly invasive and metastatic breast cancer cells
on the molecular level and could validate its possible use for
the prevention and/or treatment of breast cancer.
Acknowledgments and Notes
We thank Dr. Karen Spear for editing the manuscript. This work was
supported by a grant from the Showalter Foundation to D. Sliva. Address
correspondence to D. Sliva, Cancer Research Laboratory, Methodist Research Institute, 1800 N Capitol Ave, E504, Indianapolis, IN 46202. Phone:
(317) 962–5731. FAX: (317) 962–9369. E-mail: [email protected].
Submitted 11 February 2004; accepted in final form 15 June 2004.
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