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INTRODUCTION |
Lipid signaling molecules markedly modulate prostate
tumorigenesis. Animal and epidemiological studies suggest a strong link between fat (in particular, polyunsaturated fatty acids) intake/content in the diet and prostate cancer development and progression (1-3). The
major polyunsaturated fatty acids in the human diet is linoleic acid,
which is the precursor to arachidonic acid
(AA).1 AA is metabolized via
three major biochemical pathways as follows: the cyclooxygenase pathway
leading to prostaglandins, prostacyclin, and thromboxane; the
lipoxygenase (LOX) pathway giving rise to various hydroperoxy and
hydroxy (HETE) fatty acids as well as leukotrienes; and the
P450-dependent epoxygenase pathway generating epoxyeicosatrienoic acids. AA itself and many of its metabolites (i.e. eicosanoids) are involved in growth-promoting
signaling, which may help drive cell proliferation and promote tumor
development (4-7). They may also contribute to tumorigenesis by
modulating cell death (7-9).
AA is metabolized to a variety of bioactive hydroperoxy and hydroxy
fatty acid molecules via LOX. To date at least 18 different LOX
sequences have been published, but the biological functions for most of
these LOXs remain unknown (10, 11). In human prostate, four LOX
molecules, i.e. 5-LOX, 12-LOX, 15-LOX1, and 15-LOX2, have
been reported at the mRNA, protein, or activity level (12-23). Different LOXs have been proposed to play different contributory roles
in prostate tumorigenesis, e.g. 5-LOX being a survival
factor (12, 13), 12-LOX being a proangiogenic factor (16), and 15-LOX1
somehow affecting p53 functions (18). These three LOXs have been
reported to be up-regulated in prostate cancer cells (14, 15, 17, 18),
although, in many cases, this correlation has not been corroborated by
simultaneous measurement of protein (or mRNA) expression and
enzymatic activities.
15-LOX2 is yet another LOX molecule implicated in prostate cancer
development. Cloned first by Brash and colleagues (19), 15-LOX2 is most
homologous (~80% amino acid identity) to murine 8-LOX and shows only
~40% identity to human 5-LOX, 12-LOX, or 15-LOX1 (19, 20). A splice
variant of 15-LOX2 with an in-frame 87-bp deletion has also been
reported (20). 15-LOX2 metabolizes exclusively AA to produce
15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) (19). 15-LOX2 shows an
interesting tissue expression pattern, i.e. only in
prostate, lung, skin, and cornea (19, 20). This tissue-restricted
expression pattern suggests that 15-LOX2 may play a role in the normal
development of prostate and the other three tissues, and its abnormal
expression/function could contribute to tumorigenesis in some of these
tissues. Indeed, work by Shappell and co-workers (21, 22) indicates
that both 15-LOX2 protein expression and its enzymatic activity are
decreased in prostate cancer tissues, and the expression levels of
15-LOX2 are inversely correlated with the pathological grade and
Gleason scores of the patients. These findings suggest that 15-LOX2 may
normally help maintain the differentiated phenotype of prostate
epithelial cells and that loss of 15-LOX2 expression may contribute to
prostate cancer development and progression. The major goal of our
study is to elucidate the biological functions of 15-LOX2 in normal prostate development as well as in prostate cancer development. In this
paper we provide evidence that 15-LOX2 is a negative cell cycle
regulator in normal prostate epithelial cells, which may explain why it
is advantageous for prostate cancer cells to lose its expression.
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MATERIALS AND METHODS |
Cells and Reagents--
Five normal human prostate (NHP)
epithelial cell strains, NHP1-NHP5, were primary cultures freshly
isolated from five different donors. TP1 were primary prostate
adenocarcinoma cells isolated from the same patient as NHP2 (24-26).
NHP1, NHP3, and NHP4 cells were obtained from Clonetics (Walkersville,
MD). All these primary strains were cultured in serum-free, PrEBM
medium (Clonetics) supplemented with insulin, epidermal growth factor,
hydrocortisone, bovine pituitary extract, and cholera toxin (24-26)
and used at passage 2-6. PPC-1 cells were isolated from primary
prostate carcinoma (27). MDA PCa 2a and 2b cell lines (abbreviated as
2a and 2b, respectively) (28) were derived from primary cultures of
bone marrow metastases from a single patient. LNCaP (29), an
androgen-responsive cell line and its androgen-independent sublines
(C4-2 and C5 (30), PC3 (31), Du145 (32), JCA-1 (33), and Tsu-Pr (34)
are all established metastatic cell lines. All cancer cell lines were cultured in RPMI 1640 supplemented with 5% heat-inactivated fetal bovine serum (FBS) and antibiotics. HEK 293 cells, which were used in
the transfection experiments, were purchased from ATCC and cultured in
Dulbecco's modified Eagle's medium supplemented with 5% FBS and antibiotics.
Rabbit polyclonal anti-5- and 
12-LOX and sheep polyclonal
anti-15-LOX1 antibodies were obtained from Cayman Chemicals (Ann Arbor,
MI). Rabbit polyclonal anti-15-LOX2 antibody (21-23) was purchased
from Oxford Biomedical (Rochester, MI). Monoclonal anti-actin and
anti-cytochrome c antibodies were purchased from ICN
(Indianapolis, IN) and BD PharMingen, respectively. A monoclonal
anti-BrdUrd (5-bromo-2'-deoxyuridine) antibody (hybridoma culture
supernatant) was kindly provided by Dr. Martin Raff. Anti-GFP (green
fluorescence protein) antibodies were obtained from
CLONTECH (Palo Alto, CA). All secondary antibodies
(goat anti-mouse or -rabbit IgG or rabbit anti-sheep IgG conjugated to
horseradish peroxidase, fluorescein isothiocyanate, or rhodamine) were
acquired from Amersham Biosciences. 15(S)-HETE and other eicosanoids
were bought from Cayman Chemicals. All other chemicals were purchased
from Sigma unless specified otherwise. Liposome FuGENE 6 was bought
from Roche Diagnostics.
Immunohistochemistry of 15-LOX2 Expression in Tissue
Sections--
Paraffin-embedded sections of normal prostate tissues
and prostate cancers were stained by immunohistochemistry using rabbit anti-15-LOX2 and goat anti-rabbit IgG conjugated to alkaline
phosphatase followed by substrate 3'3-diamino benzidine (DAB)
development. Sections of LNCaP cells grown in nude mice as well as
their metastatic variants in bone marrow and lymph node (30) were
similarly processed for 15-LOX2 immunostaining.
Immunofluorescence Detection of 15-LOX2 Expression in Cultured
Prostate (Cancer) Cells--
NHP cells or prostate cancer cells were
grown on 13-mm2 circular glass coverslips. To detect
endogenous 15-LOX2 expression in these cells, we used two different
permeabilization protocols. Specifically, following fixation in 4%
paraformaldehyde (PFA) at 4 °C for 1 h, cells were
permeabilized with either 1% Triton X-100 in PBS for 20 min or
20 °C acidic alcohol (95% ethanol, 5% glacial acetic acid) for
10 min. After washing, coverslips were blocked in 30% goat whole serum
followed by incubation in primary antibody (1:2000 dilution of
anti-15-LOX2) and secondary antibody (goat anti-rabbit IgG-fluorescein
isothiocyanate; 1:2000). Finally, cells were incubated with 1 µg/ml
propidium iodide to label all nuclei. After extensive washing,
coverslips were mounted on slides using Vectorshield mounting medium
(Vector Laboratories, Inc., Burlingame, CA) and observed under an
Olympus BX40 epifluorescence microscope. Images were captured with
MagnaFire software and processed in Adobe Photoshop.
Western Blotting--
Whole cell lysates were prepared in
complete RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.5%
Triton X-100, 10 mM EDTA) containing protease inhibitor
mixture. Protein concentrations were determined by MicroBCA kit
(Pierce). Various amounts of proteins were loaded on a 15% SDS-PAGE.
Western blotting was performed as described previously (8, 26) using ECL.
Determination of 15-HETE Production in Prostate (Cancer) Cells by
LC/MS/MS--
The enzymatic activities
of 15-LOX2 in normal and cancerous prostate cells were analyzed with
LC/MS/MS using the modified procedure described by Kempen et
al. (35). Briefly, log-phase NHP1, NHP3, LNCaP, PC3, and DU145
were harvested with trypsinization, and cell pellets were washed in 5 ml of PBS and resuspended in 0.5 ml of PBS with 1 mM
calcium chloride solution. Cell suspension (1 × 107/ml) was incubated at 37 °C for 2 min. Then an
aliquot of 2.5 µl of calcium ionophore A23187 (1 mM) was
added, followed by the addition of 100 µM of AA. The
reaction mixture was incubated at 37 °C for 10 min at minimum light.
At the end of incubation, 40 µl of 1 N citric acid, 5 µl of 1% butylated hydroxytoluene, and 20 µl of
15-HETE-d8 (internal standard) were added to the samples. The lipids were extracted from the reaction mixture three times with 2 ml of hexane:ethyl acetate (1:1). Organic phases were
collected and dried under a stream of nitrogen. Samples were reconstituted in 200 µl of methanol, 10 mM ammonium
acetate, pH 8.5 (70:30), and analyzed using LC/MS/MS. Reverse-phase
high pressure liquid chromatography and mass spectrometry was performed
using a Quattro Ultima tandem mass spectrometer (Micromass, Beverly, MA) equipped with an Agilent HP1100 binary pump high pressure liquid
chromatography inlet. Eicosanoid metabolites were separated using a YMC
ODS-AQ 2.0 × 100-mm column (S3 µm, 120 Å, Waters Associates). Mobile phases consisted of 10 mM ammonium acetate, pH 8.5 (phase A) and methanol (phase B). Flow rate was 250 µl/min with
column maintained at 40 °C. Sample injection volume was 25 µl. The
mass spectrometer was operated in electrospray negative ion mode with a
cone voltage of 100, source temperature of 120 °C, and collision cell pressure of 2.10 × 10
3 torr using argon as
collision gas. The collision energy was 19 V. A minimum of duplicate
samples was measured in each experiment, and experiments were repeated
three times. The results were expressed as nanograms of
15(S)-HETE/5 × 106 cells pooled from three
independent experiments.
RT-PCR Analysis of 15-LOX2 mRNA Expression--
Total RNA
and mRNA were isolated using Tri-reagent (Invitrogen) and
Poly(A)Pure kit (Ambion, Austin, TX), respectively, according to the
manufacturers' instructions. Either 2 µg of total RNA or 0.5 µg of
mRNA from each cell type was used in RT (42 °C × 2 h) in a total of 20 µl reaction containing random hexamers and
Superscript II reverse transcriptase (Invitrogen). Two pairs of PCR
primers were designed based on the published 15-LOX2 cDNA sequence
(19; GenBankTM accession number U78294). Primers A
(sense, 5'-AACTCACCCCCACCACCATACACA-3') and B (antisense,
5'-TTCCCGCCTCCATCTCCCAAAGT-3') cover nucleotides 2234-2584 in the
3'-untranslated region (3'-untranslated region) of 15-LOX2. Primers C
(sense, 5'-ACTACCTCCCAAAGAACTTCCCC-3') and D (antisense,
5'-TTCAATGCCGATGCCTGTG-3') cover nucleotides 835-1379 in the
15-LOX2 coding region. For PCR, 2 µl of cDNA from each cell type
was used in a 25-µl reaction containing 0.5 µM primers, dNTPs, Taq, using the cycling profile 94 °C for 30 s, 60 °C for 45 s, and 72 °C for 1 min for 35 cycles. PCR
products were analyzed by agarose gel electrophoresis. RT-PCR of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control
(36).
Northern Blotting--
One µg of mRNA from each cell type
(i.e. NHP3, NHP4, PPC-1, JCA-1, LNCaP, C4-2, and PC3) was
fractionated on a 1.5% formaldehyde-agarose gel. Following alkaline
hydrolysis, RNA was transferred to Hybond+ membrane
(Amersham Biosciences) followed by UV cross-linking. After
prehybridization in ExpressHyb solution (CLONTECH)
for 1 h, the membrane was hybridized to a 32P-labled
15-LOX2 cDNA probe overnight at 55 °C. The probe used was a
759-bp EglI fragment (from nucleotide 43 to 802), and the probe was labeled using Ready-to-Go labeling kit (Amersham
Biosciences). Following hybridization, the membrane was washed twice
(30 min each) at 50 °C in 2× SSC buffer containing 0.1% SDS,
followed by washing twice (30 min each) in 0.1× SSC buffer containing
0.1% SDS. Then the membrane was stripped and rehybridized to a
32P-labeled 
-actin cDNA probe.
Treatment with Inhibitors of DNA Methylation and Histone
Deacetylation (HDAC)--
Log-phase LNCaP, PC3, and Du145 cells
(50-60% confluence) were treated with 5'-azadeoxycytidine (3 µM), which inhibits DNA methyltransferase (37), or
trichostatin A (0.5 µM), which inhibits HDAC (38, 39), or
both for 7 days with fresh media changed and drugs re-added on the 3.5 day. At the end of the treatment, cells were harvested either to
prepare proteins for Western blotting or to prepare total RNA for
RT-PCR using C-D primers.
Cloning of 15-LOX2 and Splice Variants--
We first cloned out
two large 15-LOX2 fragments from NHP2 and TP1 cells. cDNA was
synthesized from total RNA using the SMART cDNA synthesis kit
(CLONTECH) according to the manufacturer's instructions and PCR-amplified. 15-LOX2 fragments G-H (nucleotide 1880-2638) and I-J (nucleotides 131-2253) were amplified
using primers G (sense, 5'-CATCCTTGCTCTCTGGTTGC-3') and H (antisense, 5'-TGGAGTCTCGCTATGTCGTC-3'), and I (sense,
5'-CAAAGTGTCTGTCAGCATCGTGG-3') and J (antisense,
5'-TATGGTGGTGGGGGTGAGTTAC-3'), respectively. Subsequently, we cloned
the full-length 15-LOX2 and its splice variants using PCR-based
strategies. cDNA synthesis and PCR amplification were carried out
using a SuperScript One-step long distance PCR kit (Invitrogen), using
1 µg of NHP2 mRNA and several more pairs of cloning primers.
Primers CP-5' (sense, GCTAGCCTGGCAGCATGGCCGAGTTCAG-3') and CP-3'
(antisense, CTCGAGGATGGAGACGCTGTTCTCG-3') cover the entire coding
region (nucleotides 64-2099) from the translation-initiation codon/Kozak sequence to the sequence immediately in front of TAA stop
codon, with the NheI and XhoI restriction
enzyme sites incorporated to the 5'-ends of the primers, respectively.
Primers K (sense, TGCGCCGTAGAGAGCTGGACTT-3') and H (see above)
cover nearly the whole published 15-LOX2 sequence from nucleotides 36 to 2638. mRNA was denatured at 65 °C for 5 min and then mixed
with the buffer containing various enzymes. Then coupled cDNA
synthesis and PCR amplification were carried out at 50 °C for 30 min
followed by 94 °C for 3 min and then 94 °C for 15 s,
55 °C for 30 s, and 68 °C for 4 min for 40 cycles, using
either CP-5'/CP-3' or K/H primer pairs. Following PCR amplification,
all distinct bands were excised and cloned into pCRII-TOPO
(Invitrogen). Colonies were screened using a combination of PCR with C
and D primers and restriction digestion with EcoRI. Multiple
positive colonies of different sizes of inserts as well as various PCR
fragments were sequenced from both directions and characterized.
Construction and Characterization of Expression Vectors of
15-LOX2 and Its Splice Variants--
Full-length 15-LOX2 and its
splice variants were re-amplified from pCRII-TOPO vectors using
Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) and
primers CP-5' and CP-3' with TTA stop codon added in the CP-3' primer.
Individual products were recloned into pCRII-TOPO vector, and the
EcoRI fragments containing the full-length molecules were
subcloned into pCMS-EGFP vector (CLONTECH), in which EGFP and target genes are driven by separate promoters. Alternatively, the K-H fragments were directly released from pCRII-TOPO with EcoRI and then subcloned into pCMS-EGFP. The resultant
expression vectors were subjected to restriction mapping as well as
sequencing to determine the orientation of expression.
To characterize these vectors, which we have designated pEGFP-15LOX2,
pEGFP-15LOX2sv-a, pEGFP-15LOX2sv-b, and pEGFP-15LOX2sv-c, respectively,
we first transfected them into HEK 293 cells using FuGENE 6. Seventy
two h later, cells were harvested for either Western blotting or
LC/MS/MS determination of 15(S)-HETE production.
Effect of Exogenous 15(S)-HETE on Cell Proliferation and
Survival--
NHP2, TP1, LNCaP, PC3, and Du145 cells were plated in
24-well culture plates at 1 × 104 cells/well. The
next day, cells were treated with 15(S)-HETE at 0.1, 1, 10, 25, 50, and
75 µM. Seventy two h later, cells were harvested, and the
number of live and dead cells was determined using trypan blue dye
exclusion assays (8). Each condition was run in quadruplicate, and the
results are expressed as the mean percentage cell number (relative to
vehicle (ethanol) control) ± S.E. obtained from three repeat experiments.
To determine whether inhibition of cell proliferation was caused by
inhibition of cell cycle progression, we performed BrdUrd incorporation
experiments. Various cells were plated on glass coverslips and treated
with 15(S)-HETE for 72 h, similarly as described above. During the
last 6 h, BrdUrd (10 µM final concentration) was
added, and cells were then processed for BrdUrd immunostaining, using
the protocol we established previously (36). Briefly, at the end of
BrdUrd pulse, cells were fixed in PFA and permeabilized in acidic
alcohol, and DNA was denatured in acidic HCl. Then cells were incubated
with the monoclonal anti-BrdUrd antibody followed by goat anti-mouse
IgG conjugated to fluorescein isothiocyanate. Cells were counterstained
with bisbenzimide (Hoechst 33342) or DAPI. An average of 500-1000
cells were counted for each cell type, and the results were expressed
as % BrdUrd+ cells (36, 40).
Studies on 15-LOX2 Expression and Cell Cycle in NHP
Cells--
We first used immunofluorescent staining to assess the
relationship between 15-LOX2 expression and cell cycle. Log-phase NHP1 or NHP2 cells grown on glass coverslips (1 × 104
cells/13 mm2) were pulsed with BrdUrd (10 µM)
for 4 h followed by double labeling of 15-LOX2 and BrdUrd,
basically using the protocol described above for 15-LOX2 and BrdUrd
staining alone. An average of 200-1000 cells was counted, and the
results are expressed as percentage of BrdUrd+ cells in the
total number of 15-LOX2+ and 15-LOX2 cells.
To study further the relationship between 15-LOX2 expression and cell
cycle progression, cell cycle arrest was induced in NHP2 cells by
subjecting them to growth factor deprivation for 1 day. In another
experiment, starved NHP2 cells were re-cultured in factor-containing
medium for 1 day (i.e. release 1 day). At the end, cells
were fixed and used for 15-LOX2-BrdUrd double labeling.
Restoration of 15-LOX2 Expression in Prostate Cancer
Cells--
PC3 or PPC-1 cells grown on glass coverslips (3 × 103 cells/13 mm2) were either untransfected or
transiently transfected with empty vector (i.e. pCMS-EGFP),
pEGFP-15LOX2, or its splice variants (1 µg of plasmid/coverslip) with
FuGENE 6. Seventy two h later, cells were pulsed with 10 µM BrdUrd for the final 4 h and then processed for
BrdUrd immunostaining using a modified protocol. Briefly, cells were
first fixed in 4% PFA at 4 °C for 1 h to preserve GFP,
followed by permeabilization in 1% Triton X-100 for 20 min at room
temperature. To denature DNA, we incubated cells with 100 µg/ml DNase
in PBS containing 1% Triton for 90 min at room temperature instead of
the regular HCl step, which destroys the GFP signal. Then cells were
stained for BrdUrd, as described above. The results were expressed as
% BrdUrd+ cells of the total number of GFP+
and GFP cells. Two individuals independently counted an
average of 600-1500 cells for each condition, and experiments were
repeated three times.
To confirm the 15-LOX2 expression in transfected cells, immunostaining
with anti-15-LOX2 antibody was performed. We also developed a double
labeling, tri-color fluorescence protocol that allowed us to monitor
simultaneously 15-LOX2 expression and BrdUrd incorporation in
GFP+ cells. Briefly, 72 h after transfection with
control vector or various 15-LOX2 expression vectors, cells were pulsed
with BrdUrd (10 µM; 4 h) and then fixed cells in 4%
PFA followed by permeabilization in 1% Triton. Cells were then stained
for 15-LOX2 using the rabbit polyclonal anti-15-LOX2 followed by goat
anti-rabbit IgG conjugated to Texas Red. Finally, cells were processed
for BrdUrd using monoclonal anti-BrdUrd followed by goat anti-mouse IgG
conjugated to Cascade Blue (Molecular Probes, Eugene, OR), which was
observed using the DAPI bandpass.
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RESULTS |
15-LOX2 Protein Expression Is Lost in all Prostate Cancer Cells
Examined--
15-LOX2 was recently shown to be down-regulated/lost in
prostate cancer tissues (21, 22). We first sought to confirm this independently. An immunohistochemical analysis with a small sample size
(1 normal, 1 prostate intraepithelial neoplasia (PIN), and 4 prostate
cancer sections), using the same rabbit polyclonal anti-15-LOX2
antibody (21, 22), revealed that 15-LOX2 was indeed abundantly
expressed in normal prostate epithelia, greatly reduced in PIN, and
completely lost in prostate cancer tissues (see Supplemental Material,
Fig. 1). Our data, therefore, support the previous observations by
Shappell and co-workers (21, 22). A similar immunohistochemical
staining for 15-LOX2 on sections of LNCaP and its metastatic
variants (30) grown in nude mice also did not reveal any positive
staining (not shown).
To circumvent certain inherent pitfalls associated with studies using
tissues or tissue sections (e.g. correlative nature, mixed
tumor, and host cells and difficulties in performing
biochemical/molecular characterizations and establishing the
cause-and-effect relationship), we chose to study early passage
(i.e. 2-6) primary prostate epithelial cells in an attempt
to understand the biological functions of 15-LOX2. We first performed
Western blotting analysis of 15-LOX2 expression using 5 primary strains
of normal human prostate (NHP1-5) epithelial cells and 9 established
prostate cancer cell lines. As shown in Fig.
1, all 5 NHP strains expressed abundant
~76-kDa 15-LOX2 protein, whereas none of the cancer cells expressed
the protein. In addition to 15-LOX2, we also detected two lower bands migrating at ~70 and ~67 kDa, respectively, which were
significantly reduced in all prostate cancer cells (Fig. 1).
Furthermore, at least one upper band (~83 kDa) and two lower bands
(~52 and 26 kDa, respectively) were observed specifically in NHP but
not in cancer cells (Fig. 1, arrowheads).
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Fig. 1.
15-LOX2 protein expression is lost in
prostate cancer cells. Whole cell lysates (50 µg) from each cell
type were separated by 15% SDS-PAGE. The membrane was first probed
with the polyclonal anti-15-LOX2 and then stripped and reprobed with
the antibodies against the molecules indicated. Cytochrome c
(cyt. c) was used as the loading control. 15-LOX2 and two
lower bands are indicated on the right. The molecular
mass markers are indicated on the left. Arrowheads,
additional bands detected only in NHP cells but not in prostate cancer
cells. Note that no 12-LOX band was detected.
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In contrast to 15-LOX2, 15-LOX1 was expressed at low levels in all
cells examined (Fig. 1). 5-LOX was expressed at variable levels in
these cells, and it was, in general, reduced in prostate cancer cells
(Fig. 1). We could not detect 12-LOX in any of these cells, even at 150 µg/lane protein loading (Fig. 1 and not shown).
Loss of Expression of 15-LOX2 in Prostate Cancer Cells Leads to
Significantly Reduced 15(S)-HETE Production--
The above Western
blotting data are consistent with our immunohistochemical results
(Supplemental Material, Fig. 1) and the results of others (21, 22). To
determine whether loss of 15-LOX2 expression would result in reduced
enzymatic activity in prostate cancer cells, we measured 15(S)-HETE
production in several NHP and prostate cancer cells using LC/MS/MS. As
shown in Table I, the 15(S)-HETE
production in prostate cancer cells (LNCaP, PC3, and Du145) was indeed
much lower (~10-fold decrease) than in NHP (i.e. NHP1 and
NHP3) cells.
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Table I
15(S)-HETE production in primary prostate epithelial cells and prostate
cancer cells
15(S)-HETE production was measured in log phase cells using LC/MS/MS
analysis as detailed in the text. Data were obtained from three
independent experiments, and the values are mean ± S.D. derived
from six samples with each cell type (n = 6).
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Loss of 15-LOX2 Expression in Prostate Cancer Cells Occurs at the
mRNA Level--
To explore the mechanism(s) underlying the loss of
15-LOX2 protein expression in prostate cancer cells, we carried out
RT-PCR analyses and Northern blotting (Fig.
2). RT-PCR using primers A and B in the
3'-untranslated region detected abundant 15-LOX2 mRNA in all 5 NHP
strains but detected little or no message in prostate cancer cells
(Fig. 2a, upper panel). Similarly, RT-PCR using
primers C and D in the 15-LOX2 coding region detected the expected
545-bp 15-LOX2 band in all NHP cells, which was not detected or only
faintly seen in prostate cancer cells (Fig. 2a, middle panels, upper band). Both the A-B band and the C-D
upper band were confirmed to be 15-LOX2 by sequencing the PCR
fragments. RT-PCR using primers C-D also detected a lower band of 459 bp in NHP cells (Fig. 2a, middle panel,
lower band). This band represents both 15-LOX2 splice variants a
and b (15-LOX2sv-a and 15-LOX2sv-b, respectively), which
could not be distinguished from each other because they have the same
splicing structure in the region covered by primers C and D (see
below).
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Fig. 2.
15-LOX2 mRNA expression is reduced or
lost in prostate cancer cells. a, RT-PCR analysis of 15-LOX2
mRNA expression in NHP and prostate cancer cells. Upper
panel, PCR using A-B primer pair (see "Materials and
Methods"). The expected 15-LOX2 product is 351 bp. Middle
panel, PCR using C-D primer pair. The expected 15-LOX2 product is
545 bp (the upper band), and the 15-LOX2sv-a/15-LOX2sv-b
band is ~460 bp (the lower band). Note that the
middle panel was underexposed to clearly show the two bands.
Lower panel, PCR of GAPDH. b, Northern blotting.
Upper panel, 15-LOX2. Lower panel,
-actin.
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Northern blotting similarly detected the ~2.6-kb 15-LOX2 mRNA in
NHP cells but not in prostate cancer cells (Fig. 2b). We could not detect the alternatively spliced 15-LOX2 isoforms, even in
NHP cells (Fig. 2b), probably because their mRNA levels
are too low.
The above results together suggest that loss of 15-LOX2 protein
expression in prostate cancer cells results from transcriptional repression or mRNA instability.
Loss of 15-LOX2 Expression in Prostate Cancer Cells Is Caused by
Mechanisms Other Than Promoter (Gene) Hypermethylation or Histone
Deacetylation-induced Chromatin Over-compaction--
Transcriptional
repression or silencing of genes in many cases results from epigenetic
changes, especially promoter hypermethylation or chromatin compaction
due to histone deacetylation or both, for example (37, 39). To test
whether these mechanisms may explain, at least partially, the loss of
15-LOX2 expression in prostate cancer cells, we treated LNCaP, PC3, and
Du145 cells with 5'-azadeoxycytidine, which inhibits DNA
methyltransferase (37), or trichostatin A, which inhibits HDAC (38,
39), either individually or in combination and then analyzed 15-LOX2
mRNA and protein expression. As shown in Fig.
3, these treatments did not lead to the
re-expression of 15-LOX2 mRNA (Fig. 3a, upper panel) or protein (Fig. 3b, upper panel). By
contrast, both 5'-azadeoxycytidine and TSA were effective in
up-regulating the maternally inherited mitochondrial gene, cytochrome
c oxidase subunit II (41), especially in LNCaP cells (Fig.
3b, middle panel). Treatment of NHP2 cells with
these two inhibitors did not affect the endogenous 15-LOX2 expression
(not shown). Together, these data suggest that mechanisms other than
hypermethylation and histone deacetylation are responsible for the
silencing of 15-LOX2 (ALOX15B) gene in prostate
cancer cells.
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Fig. 3.
Inhibitors of DNA methylation or histone
deacetylation could not induce the re-expression of 15-LOX2 mRNA
(a) or protein (b) in prostate cancer
cells. LNCaP, PC3, and Du145 cells were treated with either
vehicle ethanol (control), 5'-azadeoxycytidine
(Aza), or trichostatin A (TSA) either
individually or in combination (Both) as detailed under
"Materials and Methods." Untreated NHP cells were used as positive
control. a, RT was performed with 2 µg of total RNA from
each condition using random hexamers, and PCR was performed using 2 µl of cDNA and primers A and B. RT-PCR of GAPDH was used as
control. b, 60 µg/lane protein was separated by 10%
SDS-PAGE and then used in Western blotting for 15-LOX2. The membrane
was stripped and re-probed with a monoclonal anti-cytochrome
c oxidase subunit II (COX II) (middle
panel) or anti-actin (lower panel).
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NHP Cells Express Multiple 15-LOX2 Splice Variants--
To
understand better the biological functions of 15-LOX2, we decided to
clone out the full-length 15-LOX2 and any apparent splice variants. We
adopted a one-step RT-PCR cloning strategy for this purpose. As shown
in Fig. 4a, using primers
CP-5' and CP-3' that cover the full coding sequence of 15-LOX2, we
detected the expected ~2.0-kb 15-LOX2 in NHP3 cells. In addition, one
distinct upper band (~2.1 kb) and 2 lower bands (1.94 and ~1.8 kb,
respectively), which may represent 15-LOX2 splice variants, were also
detected in NHP3 cells (Fig. 4a). The same expression
pattern was also observed in NHP2 cells (not shown). All these bands
were greatly reduced in primary carcinoma TP1 cells and undetectable in
PPC-1, PC3, and LNCaP C4-2 cells (Fig. 4a). As expected,
TP1 cells expressed barely detectable 15-LOX2 protein (not shown). When
we used the K and H primers that cover nearly the full length of
15-LOX2 cDNA (see "Materials and Methods") to perform the PCR
cloning, we observed more upper and lower bands in addition to the
expected 15-LOX2 band in NHP2 but not in cancer cells (not shown).
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Fig. 4.
Cloning and characterization of 15-LOX2 and
its splice variants. a, RT-PCR analysis of 15-LOX2
expression. mRNA (0.5 µg) from each cell type indicated was used
in a one-step RT-PCR protocol using primers CP-5' and CP-3' (see
"Materials and Methods") that cover the full coding sequence of
15-LOX2. For comparative purposes, primary carcinoma cells TP1 and
three cancer cell lines, PPC-1, PC3, and C4-2, were also included. The
molecular weight markers are indicated at the left. Note
that NHP3 cells showed four bands, whose intensities were greatly
reduced in TP1 cells and lost in the three prostate cancer cell lines.
b, the cloned products of 15-LOX2 and its splice variants.
c, Western blotting of 15-LOX2 and its splice variants in
HEK 293 cells transiently transfected (72 h) with respective expression
constructs. d, amino acid sequence alignment using MegAlign
program Clustal algorithm with PAM250 residue weight table. Only the
divergent sequences corresponding to amino acids 361-676 of 15-LOX2
are shown. The divergent parts are highlighted by dark
boxes. Several amino acids conserved in all mammalian LOXs are in
bold. The three His residues (i.e.
His373, His378, and His553 that
correspond to His361, His366, and
His553 of the rabbit reticulocyte 15-LOX; see Ref. 10) and
the C-terminal Ile residue important in coordinating iron are marked by
asterisks. The Asp602-Val603
sequences important for determining 15-LOX2 substrate specificity as
well as the R486H are boxed. See text for detailed
discussions.
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We subsequently cut out several bands and cloned out, from NHP2
cells, the full-length 15-LOX2 and three of its splice variants. Eight
sequence variants that were different from the published 15-LOX2
cDNA sequence (19) were identified. Six of these sequence variants,
i.e. 561(A/G) (numbering from ATG), 921(G/T), 1440(T/C), 1584insA, 1587delT, and 1650(A/C), were silent nucleic acid changes. The other two, 811(T/G) and 1457(G/A), resulted in conserved amino acid
changes, i.e. L270V and R486H, respectively. Interestingly, another sequence variant, L271V, was described previously (20). The
sequence variants reported here were found in 100% of our clones
(>20) sequenced. They might represent rare sequence differences in our
sample (i.e. prostate-specific 15-LOX2) or true
polymorphisms. In NHP cells, there are at least three 15-LOX2 splice
variants, which we named 15-LOX2sv-a, 15-LOX2sv-b, and 15-LOX2sv-c,
respectively (Fig. 4 and Table II). In
15-LOX2sv-a, exon 9 (87 bp) was spliced out, generating a protein of
647 amino acids with an estimated molecular mass of ~73 kDa (Table II
and Fig. 4d). This splice variant is identical to the
15-LOX2 splice variant reported previously (20;
GenBankTM accession number AF149095). In
15-LOX2sv-b, in addition to exon 9 being spliced out, another splicing
event took place between nucleotides 1516 and 1650, removing part of
exon 10 and the entire exon 11 (Fig. 4d and Table II). This
splice variant is predicted to encode a 602-amino acid protein with an
estimated molecular mass of ~67 kDa. When performing RT-PCR using
primers C and D that covers nucleotides 835-1379, both 15-LOX2sv-a and
15-LOX2sv-b would be detected as an ~459-bp fragment (see Fig.
2a, middle panel, lower band), because both variants have
exon 9 spliced out. In 15-LOX2sv-c, intron 12 (80 bp) was retained,
generating a molecule of ~2.1 kb in coding region, which was the
biggest in the four 15-LOX2 molecules we cloned (Fig. 4b).
However, the retention of intron 12 resulted in a frameshift and
premature stop codon and led to a protein of 617 amino acids with an
estimated molecular mass of ~70 kDa (Fig. 4d and Table
II).
To characterize these splice variants, we subcloned 15-LOX2 and its
splice variants into a binary expression vector pCMS-EGFP, in which
EGFP and target genes are driven by different promoters. When we
transiently transfected 15-LOX2 and its splice variants into HEK 293 cells, we detected the protein bands of the expected molecular weight
in the transfected cells (Fig. 4c). In subsequent biological
studies, we focused on 15-LOX2 and 15-LOX2sv-a, both of which had been
well characterized before (19, 20). We are currently performing further
biochemical characterizations of 15-LOX2sv-b and 15-LOX2sv-c, and the
results will be reported elsewhere.
15-LOX2 Product, 15(S)-HETE, Demonstrates More Pronounced
Inhibitory Effects on the Proliferation/Survival of Prostate Cancer
Cells Than NHP Cells--
To understand the biological functions of
15-LOX2, we first analyzed the effect of its product, 15(S)-HETE, on
the proliferation and survival of normal (NHP2), primary carcinoma
(TP1), and metastatic (LNCaP, PC3, and Du145) prostate cancer cells. As
shown in Table III and Fig.
5a, exogenous 15(S)-HETE, in a
concentration-dependent manner, reduced cell number,
especially in prostate cancer cells. At concentrations <25
µM, there was no obvious cell death in any cell type,
but, at 
25 µM, 15(S)-HETE induced concentration-related apoptosis, as judged by morphology and DNA fragmentation assays (not
shown). Interestingly, the 15(S)-HETE effect was more pronounced in
prostate cancer cells than in NHP cells (Table III). For example, at 10 µM, 15(S)-HETE inhibited prostate cancer cell
proliferation by 30-50%; however, it had only a marginal effect on
NHP2 cells (Table III). At 75 µM 15(S)-HETE, all cancer
cells died but ~30% of NHP2 cells survived. We determined the
IC50 values of 15(S)-HETE inhibition of cell
proliferation/survival to be ~50, 25, 25, 25, and 10 µM
for NHP2, TP1, LNCaP, PC3, and Du145 cells, respectively (Table
III).
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Table III
Effect of 15(S)-HETE on prostate (cancer) cell proliferation and
survival
NHP2, TP1, LNCaP, PC3, and Du145 cells were plated in 24-well culture
plates at 1 × 104 cells/well. Next day cells were treated
with 15(S)-HETE. NHP2 and TP1 cells were treated in their normal
culture medium (see "Materials and Methods") and LNCaP, PC3, and
Du145 cells were treated in RPMI 1640 medium supplemented with 2% FBS
(instead of 5% FBS in their normal culture medium to reduce 15(S)-HETE
binding to serum proteins). Seventy two h later, cells were harvested,
and the number of live cells was determined using trypan blue dye
exclusion assays (8). Each condition was run in quadruplicate, and the
results are expressed as the mean % cell number (relative to vehicle
(ethanol) control) ± S.E.
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Fig. 5.
15(S)-HETE inhibits proliferation/survival of
prostate cancer cells. a, TP1 cells treated with 0 (left), 15 (middle), and 30 (right)
µM 15(S)-HETE for 72 h. Cells were then stained with
Giemsa, fixed, and photographed. b, 15(S)-HETE inhibits
BrdUrd incorporation in prostate cancer cells. Various cells were
treated with 15(S)-HETE at the doses indicated for 72 h. At the
last 6 h, cells were pulsed with BrdUrd and then processed for
BrdUrd staining. An average of 800-1000 cells were counted for each
condition. The results are expressed as % BrdUrd+ cells,
and bars represent means ± S.E. derived from three independent
experiments.
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15(S)-HETE at <25 µM exerted a
concentration-dependent inhibitory effect on prostate
cancer cells without inducing apoptosis, suggesting that, at the
lower doses, 15(S)-HETE probably inhibits cell cycle progression. To
test this possibility, we treated NHP2, TP1, LNCaP, PC3, and Du145
cells with 5, 10, and 25 µM 15(S)-HETE for 72 h.
During the last 6 h, we pulsed the cells with 10 µM BrdUrd and analyzed cells in the S-phase by staining cells for BrdUrd
incorporation (36, 42). As shown in Fig. 5b, 15(S)-HETE at 5 and 10 µM inhibited, in a
concentration-dependent manner, BrdUrd incorporation in
prostate cancer cells including TP1 cells, whereas NHP2 cells were
completely resistant at these doses. At 25 µM, 15(S)-HETE
inhibits cell cycle progression in all cells examined with TP1 cells
being most sensitive (Fig. 5b).
We also analyzed the effects of several other related LOX products,
namely 5(S)-HETE (a major 5-LOX product), 12(S)-HETE (the major 12-LOX
product), and 13(S)-hydroxyoctadecadienoic acid (the major 15-LOX1
metabolite), on the same panel of cells. 5(S)-HETE induced significant
cell death at as low as 1 µM and killed all cells at
10-25 µM, and 12(S)-HETE promoted proliferation of all cells, whereas 13(S)-hydroxyoctadecadienoic acid showed minimal effect
up to 50 µM (not shown)
15-LOX2 Expression in NHP Cells Is Heterogeneous and Inversely
Correlated with Cell Cycle Progression--
To understand the
biological function(s) of 15-LOX2, its expression was analyzed in NHP
cells. To our surprise, 15-LOX2 expression in NHP2 (Fig.
6a), and NHP1 and NHP3 (not
shown) cells was heterogeneous; some cells were strongly positive for
15-LOX2 expression and some weakly positive, whereas the
majority of cells were negative. Overall, 12-20% of log-phase
(passage 2-6) NHP (i.e. NHP1, NHP2, and NHP3) cells were
positive for 15-LOX2 expression (not shown; also see Fig.
7d). The heterogeneous 15-LOX2
expression was seen even within clones; in a three-cell clone, for
example, one cell was positive for 15-LOX2, whereas the other two were
negative (Fig. 6b). In general, 15-LOX2-positive cells were
larger than 15-LOX2-negative cells (Fig. 6, a, b,
and d; also see Fig. 7). 15-LOX2 in positive NHP cells was
homogeneously distributed as granules in the cytosol (Fig. 6,
b and d). However, when a more harsh
permeabilization/extraction method (i.e. acidic alcohol) was
used, we found that a significant amount of 15-LOX2 was also localized
to cell-cell borders and perinuclear regions (Fig. 6c). When
we used the same protocols to stain PC3 and PPC-1 prostate cancer
cells, we found no specific 15-LOX2 staining (not shown).
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Fig. 6.
Heterogeneous 15-LOX2 expression and
inverse correlation with cell cycle. a, 15-LOX2 staining in
log-phase NHP2 (passage 4) cells. Cell nuclei were counterstained with
propidium iodide. Note that 15-LOX2 expression is heterogeneous in that
some cells were strongly positive, some cells were weakly positive, and
the majority of cells were negative for 15-LOX2 (green).
b, intraclonal heterogeneity of 15-LOX2 expression. NHP
cells (passage 5) were plated at clonal density (100 cells/13-mm2 coverslip) and cultured for 3 days. Then the
cells were used for 15-LOX2 immunostaining. Note that one cell is
strongly positive for 15-LOX2, whereas the other two cells in the same
clone were negative for 15-LOX2. c, localization of 15-LOX2
in cell-cell borders when cells were permeabilized/extracted with
acidic alcohol. d and e, inverse correlation
between 15-LOX2 expression and BrdUrd incorporation. The total number
of cells counted was indicated in e. Magnifications:
a, ×100; b-d, ×400.
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Fig. 7.
15-LOX2 expression is inversely correlated
with cell cycle progression (i.e. BrdUrd incorporation) in NHP2
cells. a, log-phase NHP2 (passage 4) cells double-labeled
with 15-LOX2 (green) and BrdUrd (red).
b, NHP2 cells deprived of growth factor for 1 day (starved 1 day) were double-labeled with 15-LOX2 (green) and BrdUrd
(red). Note that none of the cells shown was incorporating
BrdUrd. c, NHP2 cells starved for 1 day were released
(i.e. re-cultured in factor-containing medium) for 1 day and
then double-labeled with 15-LOX2 (green) and BrdUrd
(red). Magnifications in a-c, ×400.
d, quantification of 15-LOX2 expression and BrdUrd
positivity (following a 4-h pulse) in NHP2 cells under various culture
conditions. The results were means ± S.E. derived from two
independent experiments. starv 1d, starved for 1 day.
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The heterogeneous nature of 15-LOX2 expression as well as the
inhibitory effect of 15(S)-HETE on cell cycle (Fig. 5b) led us to think that 15-LOX2 expression in NHP cells may be normally coupled to cell cycle progression. To test this possibility, 15-LOX2 and BrdUrd double labeling studies were done. The results indeed revealed that although ~30% 15-LOX2 NHP2 cells were
BrdUrd+, <2% of 15-LOX2+ cells were
BrdUrd+ (Fig. 6, d and e).
To explore further the relationship between 15-LOX2 expression and cell
cycle, we induced cell cycle arrest in NHP2 cells by culturing them in
factor-free medium. In log-phase cultures (passages 2-6), ~13% NHP2
cells were 15-LOX2+ (Fig. 7d), and 99% of these
15-LOX2+ cells were BrdUrd (Fig.
7a and data not shown). Among 15-LOX2 cells,
~32% were incorporating BrdUrd (Fig. 7d). Upon
deprivation for 1 day, ~35% NHP2 cells became 15-LOX2+
and, correspondingly, the % BrdUrd+ cells dropped to <2%
(Fig. 7, b and d). When starved NHP2 cells were
re-cultured in normal factor-containing medium (i.e.
released) for 1 day, the 15-LOX2+ cells decreased to
~15%, and the BrdUrd+ cells increased to ~35% (Fig.
7, c and d). Together, these data reinforce our
earlier observations that 15-LOX2 expression is inversely correlated
with cell cycle progression.
Restoration of 15-LOX2 in Prostate Cancer Cells Inhibits Cell Cycle
Progression and Proliferation--
To establish a cause-and-effect
relationship between 15-LOX2 expression and cell cycle progression, we
attempted to restore its expression in prostate cancer cells that have
lost its expression (Fig. 1). To that end, we transiently transfected
GFP-tagged expression vectors of 15-LOX2 and its splice variants into
PPC-1 cells (because PPC-1 cells are most susceptible to
liposome-mediated transfections) and, in some cases, PC3 cells and
asked how this restoration of expression affects cell proliferation
and/or death. Restoration of 15-LOX2 expression significantly reduced
PPC-1 cell number, compared with transfection with vector alone (not
shown). 15-LOX2sv-a also showed some although reduced inhibitory effect
whereas 15-LOX2sv-b was much less inhibitory (not shown). The reduced
cell number did not result from increased cell death, as we did not
observe any obvious differences in apoptosis (ranging from 8 to 11%)
between untransfected cells and cells transfected with control vector or 15-LOX2 expression vectors. Similar inhibitory effects on cell number were also observed in transfected PC-3 prostate cancer cells and
293 cells (not shown).
These results suggest that the inhibitory effect of 15-LOX2 likely
resulted from an inhibition of cell cycle progression. We thus
developed a modified BrdUrd labeling protocol that allowed us to detect
cells in S-phase without jeopardizing the GFP signals (see "Materials
and Methods"). This protocol also allowed us to directly compare the
impact of restoring 15-LOX2 expression on cell cycle progression in
isogenic populations of cells by comparing BrdUrd incorporation in
GFP versus GFP+ cells. As shown in
Fig. 8, GFP and
GFP+-PPC-1 cells transfected with the control vector showed
no difference in the % BrdUrd incorporation (~37%), which was
slightly lower than that observed in untransfected cells (~42%).
This low level of GFP cytotoxicity has been reported previously in
other cell systems (43). In contrast, fewer GFP+-PPC-1
cells transfected with 15-LOX2 (~15%) or 15-LOX2sv-a (~28%) were
BrdUrd+ compared with GFP cells (~40%)
(Figs. 8 and 9). By contrast, 15-LOX2sv-b
demonstrated a much reduced inhibitory effect on PPC-1 cell BrdUrd
incorporation (Fig. 8). The 15-LOX2 expression in the transfected cells
was confirmed by immunostaining (Fig.
10). Experiments with a tri-color, double-labeling protocol (see "Materials and Methods") to
simultaneously label 15-LOX2 and BrdUrd in GFP+-cells
revealed similar results (not shown).
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Fig. 8.
Restoration of 15-LOX2 expression in PPC-1
prostate cancer cells inhibits cell cycle progression. PPC-1 cells
were either untransfected or transfected with the control vector,
pCMS-EGFP (control), or with various pEGFP-15-LOX2 expression vectors,
i.e. 15-LOX2, 15-LOX2sv-a, and 15-LOX2sv-b. Seventy two h
later, cells were pulsed with BrdUrd (for the last 4 h) and then
processed for BrdUrd immunostaining. BrdUrd positivity was then scored
in both GFP and GFP+ cell populations under
each condition. An average of 600-1500 cells was counted for each
condition by two individuals, and the results are expressed as
means ± S.E. derived from three to five independent experiments.
**, p < 0.05; *, p < 0.01 compared
with the vector-transfected cells.
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Fig. 9.
Restoration of 15-LOX2 expression in PPC-1
prostate cancer cells inhibits BrdUrd incorporation. PPC-1 cells
were either untransfected or transfected with the control vector,
pCMS-EGFP (Vector), or with various pEGFP-15-LOX2 expression
vectors, i.e. 15-LOX2, 15-LOX2sv-a, and 15-LOX2sv-b (not
shown). Seventy two h later, cells were pulsed for the final 4 h
with BrdUrd (10 µM) and then processed for BrdUrd
immunostaining. All cell nuclei were counterstained with DAPI. Shown
are representative micrographs showing that although some control
vector-transfected cells were still incorporating BrdUrd most PPC-1
cells transfected with 15-LOX2 or 15-LOX2sv-a were not. See Fig. 8 for
quantitative results. Original magnifications, ×200.
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Fig. 10.
Micrographs showing that PPC-1 cells
transfected with pEGFP-15-LOX2 or pEGFP-15-LOX2sv-a were stained
positive for 15-LOX2 (red), and cells transfected with
the control vector, pCMS-EGFP, were negative for 15-LOX2. Similar
positive staining was also observed in cells transfected with
pEGFP-15-LOX2sv-b and pEGFP-15-LOX2sv-c (not shown). Original
magnification, ×400.
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DISCUSSION |
The major findings of the present study are as follows: 1) 15-LOX2
is the major LOX expressed in NHP cells, which also express at least
three 15-LOX2 splice variants; 2) 15-LOX2 protein expression is lost
and its enzymatic activity greatly reduced in all prostate cancer cells
examined; 3) loss of 15-LOX2 expression results from transcriptional
repression via mechanisms other than promoter hypermethylation or
histone deacetylation-induced chromatin compaction; 4) 15-LOX2 product,
15(S)-HETE, preferentially inhibits cell cycle progression in prostate
cancer cells; and 5) most importantly, 15-LOX2 expression in NHP cells
is inversely correlated with cell cycle progression. Restoration of
15-LOX2 expression in prostate cancer cells induces cell cycle arrest.
Together, these data provide strong evidence that 15-LOX2 may be an
endogenous negative cell cycle regulator in NHP cells, which explains,
in part, why it is advantageous for prostate cancer cells to suppress
its expression.
NHP Cells Express Abundant 15-LOX2 and at Least Three Splice
Variants--
Several arachidonate LOXs, including 5-LOX, 12-LOX, and
15-LOX-1, have been reported to be up-regulated in prostate cancer cells (see Introduction). 15-LOX2, by contrast, is the first LOX found
to be expressed abundantly in normal prostate but decreased or lost in
prostate cancers in vivo (see Refs. 21 and 22 and see
Supplemental Fig. 1). 15-LOX2 gene is localized on
chromosome 17p13.1, a region where 12-LOX and 15-LOX1 genes also
reside (44). 15-LOX2 gene consists of 14 exons (Table II; see
Ref. 44), a structural organization similar to those of 5-LOX, 12-LOX,
and 15-LOX1 (10, 11). Interestingly, 15-LOX2 has at least three splice
variants. 15-LOX2sv-a, which is identical to the splice variant
previously identified (20), is missing exon 9. Three-dimensional modeling of 15-LOX2sv-a structure, based on the crystallography data of
15-LOX1, suggests that a complete 
-helix made of exon 9 is removed
from the substrate-binding pocket (20). Therefore, 15-LOX2sv-a
possesses reduced specific activity in metabolizing AA compared with
15-LOX2 (20). Consistent with its reduced enzymatic activity, we found
that 15-LOX2sv-a also has a reduced biological (i.e.
inhibiting BrdUrd incorporation) activity compared with 15-LOX2.
Whether 15-LOX2sv-b and 15-LOX2sv-c have reduced enzymatic activities
is currently under further investigation, although we did observe that
the transfected 15-LOX2sv-b did not significantly inhibit the BrdUrd
incorporation or cell proliferation (Fig. 8 and data not shown).
On Western blotting, we could variably detect three bands that migrate
immediately below the 15-LOX2 band in NHP cells (e.g. Fig. 1
and Fig. 3b). Based on their relative molecular weight, these three bands may represent 15-LOX2sv-a, 15-LOX2sv-c, and 15-LOX2sv-b, respectively. These splice variants are generally expressed at much lower levels than 15-LOX2; with 1 µg of mRNA we
could not detect their message (Fig. 2b), and their protein levels are also lower and variable (Fig. 1 and Fig. 3b).
However, unlike 15-LOX2, these splice variants are still expressed in
prostate cancer cells at variable levels. Interestingly, at least one
more upper band and two other lower bands are also detected only in NHP
but not in cancer cells (Fig. 1, arrowheads). Whether they also represent 15-LOX2 splice variants remains to be determined. When
we performed PCR cloning with K-H primers, we detected several additional bands in addition to the three splice
variants,2 suggesting that
there are probably more 15-LOX2 splice variants.
The biological functions of these splice variants are unclear. Like all
other alternatively spliced products (45), they may help regulate and
fine-tune the biological functions of the parental 15-LOX2. Because
these splice variants have reduced enzymatic activities (20) but still
can bind substrate (i.e. AA), it is conceivable that, under
certain circumstances, they may function to "sink" AA so as to
adjust the availability of AA to 15-LOX2.
15-LOX2 Expression Is Lost in All Prostate Cancer Cells
Examined--
That 15-LOX2 plays an important role in maintaining
normal prostate homeostasis is also supported by the fact that prostate cancer cells lose its expression in vivo (21, 22) and
in vitro (this study). The decrease or loss of 15-LOX2
expression in prostate cancer tissues occurs in the precursor lesion,
high grade PIN (see Refs. 21 and 22 and Supplemental Fig. 1),
suggesting that this may represent an early event in prostate tumorigenesis.
It is remarkable that all prostate cancer cells examined lost the
expression of 15-LOX2. Even SV40-immortalized but non-transformed prostate epithelial cells lose 15-LOX2
expression.3 Accompanying the
loss of 15-LOX2 expression, prostate cancer cells also show reduced
expression of 15-LOX2 splice variants. The decreased 15-LOX2 message
(Fig. 4a) and protein (not shown) in TP1 primary carcinoma
cells again suggest that suppression of 15-LOX2 expression may
represent an early event in prostate tumorigenesis. The loss of 15-LOX2
protein expression leads to much less 15(S)-HETE production in prostate
cancer cells compared with NHP cells (Table I). The low amount of
15(S)-HETE in prostate cancer cells may come from residual 15-LOX2
splice variants and/or 15-LOX1.
The loss of 15-LOX2 protein expression in cultured prostate cancer
cells could result from transcriptional repression; Northern blotting
fails to reveal and RT-PCR reveals only low levels or no 15-LOX2
mRNA expression in these cells (Fig. 2). Epigenetic gene silencing
mechanisms, as a consequence of promoter hypermethylation and histone
deacetylation-induced chromatin over-compaction, account for the
transcriptional suppression of numerous mammalian (including some LOX)
genes (37, 39, 46). For example, 15-LOX-1 expression in human
colorectal carcinoma cells appears to be regulated by histone
acetylation (46). However, these mechanisms do not seem to be
responsible for the transcriptional silencing of 15-LOX2 in prostate
cancer cells, as inhibitors of DNA methylation and/or HDAC could not
relieve this suppression. Another potential mechanism for the loss of
15-LOX2 expression in prostate cancer cells is due to gene mutation,
especially when considering that 15-LOX2 locus (17p13.1) is very close
to p53 gene locus (17p13.2), which is frequently mutated in advanced
prostate cancers (47). However, that suppression of 15-LOX2 protein
expression occurs in precursor lesions (i.e. high grade PIN)
and in primary carcinoma cells (TP1) casts some doubts on this
possibility. 15-LOX1 mRNA expression is silenced during erythroid
differentiation as a result of regulation at the 3'-untranslated region
by heterogeneous nuclear ribonucleoproteins (48). Whether similar
mechanisms involving RNA-binding proteins or transcription factors
exist to silence the 15-LOX2 gene expression in prostate cancer
cells is one of our ongoing research projects.
What Is the Biological Function of 15-LOX2?--
Because 15-LOX2
is abundantly expressed in NHP cells but is lost in prostate cancer
cells, the molecule may normally function by helping maintain the
differentiated phenotype of prostate epithelial cells, restrict cell
cycle progression, induce apoptosis of damaged or worn-out cells, or
limit the migratory (or invasive) cellular behavior. These functions
may not be mutually exclusive. One clue to 15-LOX2 function(s) comes
from an analysis of the effect of its product, 15(S)-HETE, on prostate
(cancer) cell proliferation and survival. At 25 µM,
15(S)-HETE induces apoptosis, and, at <25 µM, 15(S)-HETE
reduces the number of cells in S-phase. 15(S)-HETE was recently shown
to have a concentration-dependent inhibitory effect on
colony formation of PC3 cells in soft agar with an IC50 at
~30 µM (23). This value is very similar to our data. It
is unlikely that the 15(S)-HETE effect on prostate cancer cells is caused by nonspecific fatty acid cytotoxicity (9), because several
other related eicosanoids tested show very different or even opposite
effects. It is worth pointing out that the effective inhibitory dose(s)
of 15(S)-HETE on prostate cancer cells are high (Table I) (23). This is
not very surprising because, very possibly, only a small fraction of
15(S)-HETE administered actually gets inside cells. Moreover,
eicosanoids including 15(S)-HETE have been proposed to exert some of
their biological functions through activating nuclear hormone
receptors, peroxisome proliferator-activating receptors (PPAR), and the
effective doses for eicosanoid activation of PPAR are at the 20-50
µM range (23, 49, 50). Therefore, if 15(S)-HETE is
exerting its inhibitory effects on prostate cancer cells via PPAR
(23, 50), one would have to use high doses of this agonist. Cultured
NHP cells produce ~30 ng of 15(S)-HETE/5 million cells when given
exogenous AA (Table I), which corresponds to ~20 pmol of
intracellular 15(S)-HETE/106 cells. Whether this
concentration is high enough to produce any biological effect and how
much endogenous 15(S)-HETE is produced by cultured NHP cells or NHP
cells in vivo remain unknown. The biological function(s) of
15-LOX2, if mediated through its enzymatic activity, will be influenced
by the availability of its substrate, AA. It is conceivable that under
stimulated conditions both the concentration of free AA and the
enzymatic activity of 15-LOX2 (20) may be up-regulated, leading to an
increased 15(S)-HETE production and resultant cell cycle arrest and/or
cell death.
Interestingly, NHP cells are more resistant to 15(S)-HETE-induced cell
cycle arrest and apoptosis (Table III and Fig. 5b). This
makes sense as these cells normally express abundant 15-LOX2 and
produce endogenous 15(S)-HETE. This differential response may partially
explain why prostate cancer cells lose 15-LOX2, because 15(S)-HETE is
inhibitory to their clonal expansion. That TP1 primary carcinoma cells
show more sensitive response than NHP2 cells, which are isolated from
the same patient as TP1 cells (24), to 15(S)-HETE again supports the
notion that decreased/lost 15-LOX2 expression and activity may
represent an early molecular event during prostate tumorigenesis.
But what is the biological function(s) of 15-LOX2? Immunofluorescent
staining surprisingly reveals that log-phase NHP cells heterogeneously
express 15-LOX2, and this heterogeneity is even reflected among cells
within one clone. Because most 15-LOX2+ cells are larger
than 15-LOX2 cells and also because 15(S)-HETE induces
cell cycle arrest, we suspect that 15-LOX2 expression may be inversely
correlated with cell cycle progression. Two lines of experiments
subsequently confirmed this hypothesis. First, in log-phase NHP
cell cultures, <2% 15-LOX2+ cells are
BrdUrd+, whereas ~35% 15-LOX2 cells are
BrdUrd+. The very few 15-LOX2+ cells that are
BrdUrd+ show low levels of 15-LOX2 expression, suggesting
that perhaps 15-LOX2 protein expression has to reach a certain
threshold to arrest cell cycle. Second, when NHP2 cells are subjected
to starvation, there is an up-regulation in 15-LOX2 expression, from
~13 to 35%, and there is a corresponding decrease in the number of
cells in S-phase, from ~35 to <2%. Although both sets of
experiments do not establish whether it is the 15-LOX2 accumulation
that arrests cell cycle or it is the cell cycle arrest that accumulates
15-LOX2, the observations do suggest that 15-LOX2 expression
is correlated with cell cycle arrest. Because ~65% of
NHP2 cells are not in cell cycle and only 13-20% of this population
of cells express 15-LOX2, these results suggest that either 15-LOX2
expression is only one of the contributing factors in arresting cell
cycle or, perhaps, 15-LOX2 protein is degraded before the cells are about to exit G1.
Subsequent expression studies provide convincing evidence that 15-LOX2
expression does cause cell cycle arrest; restoration of 15-LOX2
expression in prostate cancer cells that have lost 15-LOX2 expression
inhibits cell cycle progression and proliferation, leading to reduced
cell number. 15-LOX2 overexpression in HEK 293, PPC-1, PC3, and Du145
cells for 3-5 days does not result in increased apoptosis. Even
further overexpression of 15-LOX2 in NHP2 cells does not lead to
apoptosis.3 These observations suggest that, under
unstimulated conditions, 15-LOX2 expression is not pro-apoptotic. This
conclusion is consistent with the fact that NHP cells in
vivo and in vitro express abundant 15-LOX2. This
conclusion is also consistent with the effect of 15(S)-HETE, which only
causes apoptosis when used at non-physiologically high concentrations.
Together, these results suggest that, under basal conditions, perhaps
only certain amounts of 15(S)-HETE can be produced, due to limited
availability of AA, from either endogenous (in NHP cells) or exogenous
(in transfected cancer cells) 15-LOX2 to help induce cell cycle arrest.
These results also predict that, under stimulated conditions when
increased AA becomes available, 15-LOX2 might cause cell apoptosis.
In summary, in this study we provide evidence that 15-LOX2 is a
negative cell cycle regulator in normal prostat
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ABSTRACT
INTRODUCTION
