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Carcinogenesis |
Department of Environmental Health Sciences [C. P. N., L. R. K., J. D. G., T. W. K.], Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205, and Departments of Oncology [J. S., W. B. I., W. G. N., J. D. G., T. W. K.], Urology [J. S., W. B. I., W. G. N.], Pathology [A. M. D.], Pharmacology and Molecular Sciences [W. G. N., T. W. K.], and Medicine [W. G. N.], Johns Hopkins University School of Medicine, Baltimore, Maryland 21287
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 
class isoforms of glutathione
S-transferases (GSTs) have been shown to inhibit
adduction of activated PhIP metabolites to DNA in cell-free systems. In
humans, silencing of GST (GSTP1) through CpG island
hypermethylation is found in nearly all prostate carcinomas and is
believed to be an early event in prostate carcinogenesis. We
hypothesized that suppressed GSTP1 expression in prostate cells would
increase their vulnerability to cytotoxicity and DNA adduct formation
mediated by activated PhIP metabolites. To test this hypothesis, the
human prostate adenocarcinoma cell line, LNCaP, which contains a
silenced GSTP1 gene, was genetically modified to
constitutively express high levels of GSTP1. Both LNCaP and LNCaP-GSTP1
cells exposed to N-OH-PhIP, but not parent PhIP, for
24 h showed a dose-dependent decrease in cell viability.
GSTP1-overexpressing cells had LC50s 30–40% higher than
cells transfected with the vector alone. PhIP-DNA adducts isolated from
LNCaP-derived cells and primary human prostate tissue cultures exposed
to N-OH-PhIP were analyzed by liquid
chromatography/electrospray ionization mass spectrometry. Primary
cultures of human prostate tissue and LNCaP-GSTP1 cells had 
50%
lower adduct levels than parental LNCaP and vector control cells.
Bioactivation assays using LNCaP cytosols showed that enzymatic
activation of N-OH-PhIP to a DNA binding species was
dependent on ATP and could be inhibited by recombinant human GSTP1 in
the presence of glutathione. This evidence confirms that
N-OH-PhIP can be bioactivated to a DNA binding species
in human prostate and human prostate-derived cells. These observations
provide the basis for using LNCaP and LNCaP-GSTP1 cells as a model
system for studying the role of this enzyme in protection against
N-OH-PhIP induced DNA damage in prostate carcinogenesis.
Loss of GSTP1 expression in human prostate may, therefore, enhance its
susceptibility to carcinogenic insult by compounds such as
N-OH-PhIP. Conversely, induction of GSTs in early-stage
prostate carcinogenesis may be a useful protective strategy. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Heterocyclic amines are pyrolysis products that have been isolated from cooked fish and meat and have been shown to be highly mutagenic in the Ames assay and are carcinogenic in rodents. PhIP,3 the most abundant of the mutagenic heterocyclic amines found in cooked meats, has been shown to have the highest carcinogenic potential of these compounds to humans and has also been detected in cigarette smoke condensate (3 , 4) . Initial long-term dietary studies involving PhIP revealed it to be carcinogenic to the colon, mammary gland, and lymphoid system in rodents (5 , 6) . Evaluation of rat prostate tissues from the original mammary and colon carcinogenesis study has also implicated PhIP in the development of prostate cancer (7) . Moreover, feeding of PhIP to lacI transgenic mice indicates that PhIP is a powerful prostate mutagen (8) . The collective findings of high incidences of lesions and carcinomas of the ventral prostate along with high levels of PhIP-DNA adduct formation and mutagenesis in the rat prostate suggest that PhIP could be an important factor in the development of human prostate cancer.
Bioactivation of PhIP to a DNA binding species in target tissues is believed to play a major role in its carcinogenicity. N-Hydroxylation of PhIP by cytochrome P-450s (primarily CYP1A2) in the liver is an obligatory step in the pathway leading to DNA adduct formation. Subsequent esterification by NATs, STs, or kinases has been shown to lead to the formation of a major adduct at the C8 of 2'dG (reviewed in Ref. 9 ). Cell-free studies have shown that GSTs can inhibit the binding of N-acetoxy-PhIP to calf thymus DNA by as much as 90% (10) . GSTP1, encoding the major GST isoform expressed in normal human prostate, is silenced by CpG island hypermethylation in nearly all prostate tumors and may be an early target for somatic alteration in multistep prostate carcinogenesis (11) . This study sought to assess the impact of GSTP1 in prostate-derived cells on protection against cytotoxicity and DNA adduct formation by the prostate carcinogen, PhIP.
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MATERIALS AND METHODS |
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Human Prostate Tissue and Cell Lines.
Grossly normal human prostatic tissues, opposite neoplastic prostate,
were dissected from resection specimens obtained from men (age range,
51–67 years) treated for prostatic carcinoma by radical prostatectomy
at The Johns Hopkins Hospital (Baltimore, MD). Human prostate tissues
used for enzyme activity assays and background adduct analysis were
frozen in liquid nitrogen and stored at -80°C, whereas tissues used
for carcinogen exposure were divided into three equivalent pieces of
200 mg each and minced into 2 x 2-mm sections
with a scalpel. Minced tissues were then placed in six-well plates with
RPMI 1640 supplemented with 10% fetal bovine serum (Life Technologies,
Gaithersburg, MD), 100 units/ml penicillin, 100 µg/ml streptomycin,
and 0.25µg/ml Fungizone. Tissues were preincubated for 1 h
(37°C, 5% CO2) before the addition of fresh
medium and carcinogen exposure.
The human prostatic carcinoma cell line LNCaP and GSTP1 cDNA were obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured (37°C, 5% CO2) in RPMI 1640 supplemented with 10% fetal bovine serum. LNCaP cell lines stably transfected with a constitutively expressing GSTP1 vector, designated GSTP1-C1 and GSTP1-C5, as will be described elsewhere,4 were cultured in LNCaP medium containing 200 µg/ml Geneticin to maintain selection. GSTP1 cDNA represented the most common GSTP1 allele type (Ile105 and Ala114).
Enzyme Assays.
LNCaP and LNCaP-GSTP1 cells were grown to confluency in
75-cm2 flasks, washed with PBS, trypsinized,
harvested, centrifuged, resuspended in 1 ml PBS, and frozen at
-80°C. The frozen cell suspensions were thawed and sonicated. The
resultant homogenates were placed in Eppendorf tubes and centrifuged at
10,000 x g. In the case of human prostate
samples, cytosols were prepared from fresh tissue, as described
previously (10)
. The supernatants were used for enzyme
activity and protein determinations. GST activity, expressed as
nmol/min/mg cytosolic protein, was measured using CDNB and ECA as
substrates to determine overall GST activity and GSTP1-specific
activity, respectively (12)
. Because of limited amounts of
human prostate tissues available and the known heterogeneity in GST
isozyme expression, only CDNB conjugation activity was measured in
those cytosols because this substrate gave a more comprehensive
approximation of total GST activity. Protein concentrations were
determined using BCA Protein Assay Reagent (Pierce, Rockford, IL) with
BSA as the standard.
Western Blot Analysis.
Cytosolic protein (50 µg), isolated and determined as above for
enzyme assays, was boiled for 15 min and resolved on a 12% SDS
polyacrylamide gel. After blotting, the membrane was blocked with 5%
nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20,
followed by a 1-h incubation with rabbit antihuman-GSTP1 serum (1:1000;
Calbiochem, San Diego, CA). The secondary horseradish
peroxidase-labeled goat antirabbit IgG (1:1000) was added for 1 h.
The GSTP1 protein was visualized using the supersignal chemiluminescent
substrate of Pierce (Rockford, IL). Recombinant human GSTP1
(Calbiochem, San Diego, CA) was boiled and loaded as a positive control
and comigrated with GSTP1 protein isolated from LNCaP cells stably
transfected with the GSTP1-expressing vector.
Cytotoxicity Assay.
The colorimetric MTT assay measures the activity of various
dehydrogenase enzymes in active mitochondria in living cells and has a
linear relationship with cell number (13)
. Approximately
1 x 104
cells/well were seeded in
96-well plates and treated 72 h later with graded concentrations
of PhIP or N-OH-PhIP using DMSO:ethanol (4:1) as the vehicle
at a final concentration of 0.1%. After 24 h, the carcinogen was
removed, and the plates of cells were washed with PBS. Medium was added
back to the plates, and 50 µl of MTT (2 mg/ml) in PBS were then
incubated with the cells for 4 h at 37°C. The MTT and medium
were removed, and DMSO was added to solubilize the cells. The
absorbance of the formazan product was detected using a UV max Kinetic
Microplate Reader (Molecular Devices, Menlo Park, CA) set at a
wavelength of 505 nm.
Synthesis of Adduct Standards.
The 2'dG-C8 adduct of PhIP was synthesized by modifying a method
described previously (14)
. Briefly, 100 nmol of
N-OH-PhIP in DMSO:ethanol (4:1) was reacted with 1 µl of
acetic anhydride on an ice-salt bath for 15 min under argon, resulting
in the formation of N-acetoxy-PhIP. The reaction mixture was
then added to a 0.9-ml PBS solution containing 2.4 mg of 2'dG and 0.4
mg of BSA (added to enhance the stability of N-acetoxy-PhIP
in solution, thereby increasing the yield of the end product) and
incubated at 37°C for 30 min in a shaking Eppendorf warmer. The
protein was then precipitated out using 2 volumes of ethanol and
centrifugation at 12,000 x g for 10 min. The
synthesized 2'dG-C8-PhIP adduct was passed through a Sep pak and
purified by high-performance liquid chromatography as described
previously (14)
. The
[15N5]-adduct was
synthesized using the aforementioned methodology; however
[15N5]-2'dG was
substituted for 2'dG. Purified standards were redissolved in
DMSO:ethanol (4:1), quantitated spectrophotometrically, and stored at
-80°C.
DNA Adduction and Isolation.
LNCaP and LNCaP-GSTP1 cells (1 x 106) were plated in 75-cm2
flasks and grown to 75% confluence. Cells were then exposed to 20
µM N-OH-PhIP or DMSO:ethanol (4:1) vehicle in
fresh medium for 3 h. After the 3-h exposure, the cells were
washed with ice-cold PBS, trypsinized, harvested, centrifuged, washed
again with ice-cold PBS, and then frozen in liquid nitrogen and stored
at -80°C until DNA isolation. Human prostate used for
N-OH-PhIP incubations were prepared and cultured as
described above. After the 3-h N-OH-PhIP exposure, the
tissues were centrifuged, washed twice with ice-cold PBS, frozen in
liquid nitrogen, and stored at -80°C until DNA isolation. For DNA
isolation, cells or tissues were thawed and homogenized in an ice-cold
Teflon glass homogenizer, and DNA was isolated using an A.S.A.P.
Genomic DNA Isolation kit (Boehringer Mannheim, Indianapolis, IN). DNA
samples were washed twice with 70% ethanol, resuspended in 30
mM sodium acetate/1 mM zinc
sulfate buffer (pH 5.3), and quantitated by measuring the absorbance at
260 nm, assuming 1 absorbance unit = 50 µg/ml
double-stranded DNA
(A260/A280
ratios, 1.7–1.9). DNA (100 µg) from each sample was then digested
with 10 units of nuclease P1 at 70°C for 2 h, followed by 10
units of alkaline phosphatase at 70°C for 2 h. Complete
digestion to the resulting nucleoside bases was determined by
high-performance liquid chromatography with absorbance monitoring at
260 nm. The [15N5]-adduct
standard (500 pg in methanol) was added to each sample after the
addition of 2 volumes of ethanol and centrifugation to precipitate out
the protein. The supernatants were evaporated to dryness in a rotary
evaporator and redissolved in 50 µl of methanol prior to LC/ESI-MS
analysis.
LC/ESI-MS Analysis.
Mass spectral data were acquired on an LCQ (Thermoquest Corp., San
Jose, CA) quadrupole ion trap mass analyzer equipped with an
elecrospray probe coupled to a Thermal Systems Products liquid
chromatography system, which consisted of a quaternary pump, an
autosampler, and a variable wavelength UV detector. An ODS J-sphere
M-80 column (2 x 250 mm; YMC, Inc., Wilmington, NC)
heated to 45°C was used for sample separation. The mobile phase was
as follows: A, mass spectrometry grade H2O
containing 0.1% formic acid; B, mass spectrometry grade methanol
containing 0.1% formic acid. The LC conditions were as follows: flow
rate, 0.2 ml/min; a linear gradient from 0% B to 100% B in 10 min,
isocratic B to 13 min, and linear to 0% B by 13.5 min to reset at
starting conditions. Collision-induced dissociation and selected
reaction monitoring were optimized for analysis of the 2'dG-C8-PhIP
adduct, and a standard curve was generated based on the signal area of
known concentrations. Recovery of the
[15N5]-internal standard
was also monitored in the same chromatographic run for each sample and
was highly variable as a result of sample preparation and
reconstitution in methanol. 2'dG was analyzed by LC/ESI-MS under the
same chromatographic and mass spectral conditions. Data are represented
as pmol 2'dG-C8-PhIP (corrected for recovery of internal standard) per
µmol 2'dG, with a detection limit of 0.7 pmol adduct per µmol 2'dG
(equivalent to 2 adducts/107 nucleotides).
Measurement of Cellular AcCoA and ATP in LNCaP Cells.
LNCaP cells in log growth were assayed for AcCoA by reversed-phase
high-performance liquid chromatography as described previously
(15)
. LNCaP cells at 1.5 x 105
cells/ml were plated into 96-well plates with
each well containing 0.2 ml of cell suspension. Forty-eight h later,
cellular ATP content was determined using a luciferin-luciferase ATP
assay kit (bioluminescent somatic cell assay kit; Sigma) as described
previously (16)
.
Cytosolic Activation and Inhibition Assays.
Assays were conducted to determine relative contributions of NAT, ST,
and ATP-dependent kinase(s) in the bioactivation of
N-OH-PhIP to a DNA binding species by LNCaP cytosols using
enzyme-specific cofactors as described previously, with modifications
(17)
, and to assess the ability of rhGSTP1 (Calbiochem,
San Diego, CA), allele type unknown, to inhibit bioactivation. Covalent
binding of N-OH-PhIP (20 µM) to calf
thymus DNA (200 mg) in the presence or absence of PAPS (0.2
mM), AcCoA (1 mM), or ATP
(1 mM) was measured as described below. Assay
mixtures, containing 0.5 mg of either native or boiled cytosolic
protein, were incubated at 37°C for 30 min and stopped by the
addition of 2 ml of cold lysis buffer from the DNA isolation kit.
Experiments assessing the ability of GSTP1 to inhibit bioactivation of
N-OH-PhIP contained 60 µg of the recombinant human protein
in the presence or absence of GSH (3 mM). DNA
samples were isolated using the A.S.A.P. Genomic DNA Isolation kit and
processed for LC/ESI-MS analysis as described above.
Statistical Analyses.
Data were analyzed using either the Student’s t test or the
paired Student’s t test procedure in SigmaPlot statistical
computer software (Jandel Scientific, San Rafael, CA).
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RESULTS |
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. GSTP1-specific activity, determined using ECA as the
substrate, was significantly higher in both GSTP1-C1 and GSTP1-C5
cytosols, 7.5- and 20.5-fold, respectively, compared with LNCaP-Neo
(Fig. 1B)
. Assessment of GSTP1 content in LNCaP, LNCaP-Neo,
GSTP1-C1, and GSTP1-C5 cells by Western blot analysis indicates that
LNCaP and LNCaP-Neo cells have essentially no expression of GSTP1
protein, whereas GSTP1-C1 and GSTP1-C5 cells have progressively higher
levels of protein expression that coincides with the activity assays
(Fig. 1C)
. Western blot analysis for class GST was
weakly positive in LNCaP cells (data not shown) and may account for the
basal level of GST activity in these cells.
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, µ, and ) of GST were expressed at varied levels in
the epithelial cells of the peripheral zone (data not shown).
Cytotoxicity of N-OH-PhIP and PhIP in Parental LNCaP
Cells and LNCaP Cells with Elevated GSTP1 Expression.
The MTT assay revealed that exposure to N-OH-PhIP (0–50
µM) for 24 h was cytotoxic to all four
cell lines in a concentration-dependent manner (Fig. 2)
. Both clones of LNCaP-GSTP1 cells were more resistant to cytotoxicity
compared with LNCaP or LNCaP-Neo cells. The concentration of
N-OH-PhIP required to kill 50% of the cells
(LC50) increased significantly by 34 and 40% in
GSTP1-C1 and GSTP1-C5 cells, respectively, compared with LNCaP-Neo
cells (P < 0.05, paired Student’s
t test; Fig. 2
, inset). Exposure of LNCaP cells
to PhIP for 24 h at concentrations as high as 100
µM showed no concentration-dependent
cytotoxicity (Fig. 2)
.
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. Although the LC/ESI-MS method developed was highly specific for the
adducts measured, some minor peaks that did not correspond to the
elution of known standards were observed in chromatographic runs. These
minor peaks were also observed in some blank runs and in digested DNA
samples form LNCaP cells exposed to vehicle alone. 2'dG-C8-PhIP
formation, expressed as pmol per normal 2'dG (µmol) and corrected for
the percentage of recovery of the internal standard, in LNCaP cells was
concentration dependent over the range of 3–100
µM (data not shown). Shown in Fig. 4
, transfected cells that overexpressed GSTP1 had adduct levels nearly
one-half the amount seen in parental LNCaP and LNCaP-Neo cells.
2'dG-C8-PhIP adduct levels in primary cultures of human prostate were
on average 50% lower than levels seen in LNCaP or LNCaP-Neo cells. The
human prostate tissues used for GST activity measurements and
N-OH-PhIP exposure in culture were the same. No correlation
between levels of 2'dG-C8-PhIP formation and GST activity or expression
was observed. Human prostate tissues not exposed to
N-OH-PhIP in culture had no detectable background levels of
2'dG-C8-PhIP. Moreover, treatment of LNCaP cells or primary human
prostate tissues with 100 µM PhIP for up to
6 h also did not yield any detectable 2'dG-C8-PhIP.
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. Of the three major cofactors (PAPS, ATP, and AcCoA) used
in these determinations, the only one that enhanced binding to calf
thymus DNA, as determined by formation of 2'dG-C8-PhIP, compared with
controls was ATP. Slightly higher binding, compared with control
incubations with no cofactor, was seen in the presence of AcCoA, but
these levels were also seen in the heat-inactivated cytosol control.
The physiological levels of AcCoA and ATP measured in LNCaP cells were
6.1 ± 0.5 µM (mean ± SE; n = 3) and 1.8 ± 0.1 mM (mean ± SE;
n = 5), respectively. Assay conditions that
involved concentrations of AcCoA >100-fold over observed physiological
levels did not significantly elevate the formation of 2'dG-C8-PhIP,
whereas concentrations of ATP that were actually less than observed
physiological levels produced significant adduct formation with native
LNCaP cytosol. ATP-dependent 2'dG-C8-PhIP formation was significantly
inhibited by GSH alone, but this inhibition was extended to near
background levels by the addition of rhGSTP1 (Fig. 5B)
.
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DISCUSSION |
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We have developed a very sensitive and highly specific LC/ESI-MS
methodology for detecting and quantitating the major PhIP-DNA adduct,
2'dG-C8-PhIP, formed in in vivo and in vitro
systems. No other DNA adducts were detected by LC/ESI-MS; however, it
should be noted that the conditions were optimized for detecting the
2'dG-C8-PhIP adduct. This method was used to measure PhIP-DNA adduct
formation in prostate-derived cells and in a cell-free system through
bioactivation of N-OH-PHIP. Our data (Fig. 5A)
suggest that the pathway leading to DNA adduct formation in LNCaP cells
involves ATP-dependent enzymes. One ATP-dependent enzyme believed to be
involved in N-OH-PhIP activation is tRNA synthetase/kinase,
but the evidence is inconclusive as to whether activation is
attributable solely to the tRNA synthetase/kinase or to other kinases
as well (17
, 21) . The highly reactive derivatives
resulting from this ATP-dependent activation of N-OH-PhIP
are believed to be either the N-prolyloxy or the
N-phosphatyl esters at the exocyclic amino group. These
ester moieties serve as leaving groups, giving rise to putative
electrophilic arylnitrenium ion intermediates, which subsequently bind
to the C8 position of guanine (9)
. This ATP-dependent
activation of N-OH-PhIP has been observed in various tissues
of humans, rodents, and nonhuman primates (17
, 21
, 22)
and
may play a significant role in the susceptibility of extrahepatic
tissues to heterocyclic amine-induced DNA adduct formation,
particularly in tissues where NAT and ST activities are low. Activation
of heterocyclic amines by NATs and STs has been demonstrated in
cytosolic fractions isolated from human colon and mammary cells
(21
, 23, 24, 25)
. NAT-dependent O-acetylation of
heterocyclic amines is believed to be an important factor in the
susceptibility of the colon and mammary gland to heterocyclic
amine-induced carcinogenesis (21
, 23
, 25)
. There is recent
evidence showing the presence of NAT mRNA in human prostate epithelium
(26)
, which may account for some activation of
N-OH-PhIP in situ. However, Leff et
al. (27)
have shown that overexpression of
human NAT2, the isoform with the highest relative capacity to convert
N-OH-PhIP to a DNA-binding species (28)
,
specifically in the prostate of transgenic mice, does not enhance
PhIP-DNA adduct formation in that tissue. This evidence, along with our
findings, suggests that this ATP-dependent activation pathway may be
very important in determining prostate-related toxicities in contrast
to colonic and mammary tissues.
LNCaP cells, a well-characterized prostate adenocarcinoma cell line,
serve as a model for understanding many aspects of prostatic epithelial
cell biochemistry and molecular biology. These cells have an
experimentally useful feature in that they possess a GSTP1
gene that has been silenced because of CpG island hypermethylation of
the promoter region. This silencing, which occurs in >90% of
prostatic carcinomas and is increased in prostatic intraepithelial
neoplasia, may be an early genetic lesion that predisposes select cells
to carcinogenic insult and may be useful as a biomarker of early-stage
prostate carcinoma (11
, 29)
. Restored expression of GSTP1
in LNCaP cells has allowed us to explore the ability of GSTP1 to
protect these prostate-derived cells from toxicities associated with
exposure to a potential human prostate carcinogen. Our results show
that overexpression of GSTP1 in LNCaP cells does partially protect
against cytotoxicity and DNA adduct formation caused by exposure to
N-OH-PhIP. The observed cytotoxicity suggests, but does not
definitively prove, that these prostate-derived cells express enzymes
that can bioactivate N-OH-PhIP to an esterified form, which
could in turn bind to cellular macromolecules and cause cell death. The
relevance of this model system is further supported by the observation
that 2'dG-C8-PhIP levels seen in human prostate surgical samples
exposed to N-OH-PhIP at equivalent concentrations and time
points are comparable with levels seen in GSTP1-overexpressing cells
(Fig. 4)
. This feature not withstanding, there remain obvious
difficulties in comparing data from prostate cancer cells grown in
culture to prostate tissue that contains a heterogeneous population of
cells. Differences in activation enzymes as well as detoxification
enzymes (e.g., the varied expression of GST isoforms)
between LNCaP cells and human prostate tissue could potentially affect
the cytotoxicity and adduct formation caused by N-OH-PhIP.
In fact, no correlation between levels of 2'dG-C8-PhIP formation and
GST activity or GST expression in human prostate cultures was observed.
Because of the heterogeneity of GST isozyme expression in any given
human prostate tissue section, along with the possibility for
differences in N-OH-PhIP activation, associations between
DNA adduct formation and qualitative enzyme expression in individual
tissues are difficult. Immunohistochemical staining of
GSTP1-C1-overexpressing cells showed that there is very heterogeneous
expression of the protein, with some cells expressing very high levels
whereas others express either very low or intermediate
levels,5
somewhat mimicking the in vivo situation. Although derived
from clonal expansion of a single colony, heterogeneous expression of
GSTP1 may be caused by differential regulation in individual cells.
A previous study has shown that several GST isoforms were able to
inhibit DNA binding of N-acetoxy-PhIP (an ultimate DNA
binding species) to calf thymus DNA by as much as 90%
(10)
. Specifically, GSTP1–1 was able to significantly
inhibit binding by 30%. Our results confirm that GSTP1 can inhibit
binding of activated PhIP metabolites to DNA in cellular and cell-free
systems and that there may be biological relevance in the prevention of
toxicities to the prostate. GSH, a highly abundant cellular
nucleophile, alone was able to significantly inhibit ATP-dependent
adduct formation in a cell-free system (Fig. 5B)
.
This result could be attributable to nonenzymatic binding of GSH to the
reactive intermediate(s) or to low levels of other GST isoforms (low
levels of class GST were detected by Western blot analysis) present
in LNCaP cytosol binding to and inactivating the reactive
intermediate(s). The mechanism of inactivation of
N-esterified-PhIP intermediates has not yet been determined,
but Lin et al. (10)
demonstrated that the end
products of a reaction mixture that contained
N-acetoxy-PhIP, GST, and GSH were PhIP and oxidized
glutathione. The stoichiometry of the end products, PhIP and oxidized
glutathione, suggested that the reaction was not a simple redox
reaction. There is evidence that a glutathione conjugate is formed, but
that it is highly labile and degrades to form a 5-hydroxy-PhIP
metabolite (30)
. Our observations of nearly complete
inhibition of ATP-dependent N-OH-PhIP-induced DNA adduct
formation in a cell-free system by GSTP1 suggests that this GST isoform
may have a higher substrate specificity for the ATP-dependent
metabolite(s) of N-OH-PhIP versus
N-acetoxy-PhIP, thus enhancing its ability to inhibit
binding to DNA. Further studies should focus on determining the
enzyme(s) responsible for producing the ultimate DNA binding species in
prostate-derived cells as well as characterizing the structural
properties of the activated metabolite(s). The effectiveness of other
GST isoforms should also be assessed in their ability to inhibit the
cytotoxic and genotoxic effects of activated PhIP metabolites in the
prostate.
GSTs have been proposed to play a critical role in defending normal
cells against electrophilic carcinogens. Inactivation of
GSTP1 in prostate cells by promoter hypermethylation may
lead to increased vulnerability to electrophilic carcinogens. We have
demonstrated that restored GSTP1 expression in the prostate-derived
cell line LNCaP can inhibit cytotoxicity and DNA adduct formation
caused by a potential dietary carcinogen. The evidence presented leads
to the possibility of strategies for the prevention of initial and
cumulative DNA-damaging events caused by N-OH-PhIP and other
similar compounds that may lead to multistage prostate carcinogenesis.
A recent study by Montironi et al. (31)
suggests that finasteride, a 5--reductase inhibitor currently used
in a human prostate cancer prevention trial, may act as a GST
inducer in human prostate. Induction of GSTs in the prostate by
chemopreventive agents may therefore be a viable preventive strategy,
either alone or in concert with other mechanisms. This line of
reasoning may also help to explain the negative correlation of prostate
cancer with high vegetable intake seen in epidemiological studies
(2
, 32) , because these vegetables contain potent inducers
of GSTs such as isothiocyanates (33)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants CA44530, ES06052,
ES03819, ES07141, and CA70126 and the Alexander and Margaret Stewart
Trust. 
2 To whom requests for reprints should be
addressed, at Department of Environmental Health Sciences, Johns
Hopkins University School of Public Health, 615 North Wolfe Street,
Baltimore, MD 21205. Phone: (410) 955-4712. Fax: (410) 955-0116;
E-mail: tkensler{at}jhsph.edu
3 The abbreviations used are: PhIP,
2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; NAT,
N-acetyltransferase; ST, sulfotransferase; 2'dG,
2'deoxyguanosine; GST, glutathione S-transferase;
N-OH-PhIP, 2-hydroxyamino-PhIP; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; CDNB,
1-chloro-2,4-dinitrobenzene; ECA, ethacrynic acid; GSH, reduced
glutathione; AcCoA, acetyl CoA; PAPS, 3'-phosphoadenosine
5'-phosphosulfate; rhGSTP1, recombinant human GSTP1; LC/ESI-MS, liquid
chromatography/electrospray ionization mass spectrometry; 2'dG-C8-PhIP,
N-(deoxyguanosin-8-yl)-PhIP.
Received 3/ 2/00. Accepted 10/26/00.
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REFERENCES |
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-carboline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and a copper phthalocyanine cellulose extract of cigarette smoke condensate by cytochrome P-450 enzymes in rat and human liver microsomes. Cancer Res., 51: 5284-5291, 1991.-class glutathione S-transferase gene accompanies human prostate carcinogenesis. Proc. Natl. Acad. Sci. USA, 91: 11733-11737, 1994.This article has been cited by other articles:
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X. Lin, M. Tascilar, W.-H. Lee, W. J. Vles, B. H. Lee, R. Veeraswamy, K. Asgari, D. Freije, B. van Rees, W. R. Gage, et al. GSTP1 CpG Island Hypermethylation Is Responsible for the Absence of GSTP1 Expression in Human Prostate Cancer Cells Am. J. Pathol., November 1, 2001; 159(5): 1815 - 1826. [Abstract] [Full Text] [PDF]
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, LNCaP-Neo;
, GSTP1-C1; 
, GSTP1-C5 cells exposed to N-OH-PhIP;
, LNCaP cells exposed to PhIP. Inset, mean
LC50s (n = 3) for all four
cell lines exposed to N-OH-PhIP; bars,
SE. *, P < 0.05 compared with
LNCaP-Neo cells calculated by paired Student’s t
test.
m/z 374.3 transition in
selected reaction monitoring mode. B, mass chromatogram
of digested DNA from LNCaP cells exposed to 20 µM
N-OH-PhIP for 3 h monitoring the m/z
490.1 
