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Biochemistry and Biophysics |
University of Nottingham Laboratory of Molecular Oncology, Cancer Research Campaign Department of Clinical Oncology, City Hospital, Nottingham NG5 1PB, United Kingdom
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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into murine
fibrosarcomas results in intravascular coagulation at the tumor site as
well as a major decrease in tumor blood flow (3)
. This led
to speculation that soluble factors produced by tumor cells prime
tumor-associated endothelial cells to respond to TNF-. EMAP-II
protein was subsequently identified and purified from supernatants of
cultured murine fibrosarcoma cells, based on its ability to potentiate
the endothelial procoagulant-inducing activity of TNF-
(4)
. Purified EMAP-II protein possesses a wide range of activities toward endothelial cells, neutrophils, and monocyte/macrophages in vitro. In addition to the induction of TF-dependent coagulation on endothelial cells and monocytes, EMAP-II up-regulates endothelial E- and P-selectin expression and release of von Willebrand factor (4) . It is also chemotactic for neutrophils and monocytes and induces release of myeloperoxidase activity from neutrophils (4) . In vivo, local injection of EMAP-II into the mouse footpad evokes an acute inflammatory response characterized by edema and a neutrophil-rich infiltrate (5) . Furthermore, direct injection into s.c. tumors in mice leads to hemorrhage and inflammatory infiltrates, followed by a decrease in tumor volume (4) , consistent with the activities of a pleiotropic, proinflammatory cytokine. EMAP-II is likely to be identical to the bladder carcinoma-derived cytokine BCDC and to the FO-1 and HS-1 proteins derived from the FO-1 and BLM human melanoma cell lines, respectively (6, 7, 8) .
Murine EMAP-II and human EMAP-II have been cloned and expressed in bacteria (4 , 9) . The cDNA sequence of EMAP-II is consistent with a Mr 34,000 precursor molecule, which is cleaved at a critical aspartate residue to produce the mature polypeptide (4) . This precursor lacks a classic hydrophobic signal peptide necessary for membrane translocation, and the mature molecule may be secreted via a novel pathway, in a manner similar to that of the leaderless precursor of interleukin-1ß, which undergoes proteolytic cleavage at the plasma membrane with subsequent release into the extracellular space (10) .
Given the potency of the mature EMAP-II molecule, it is important to understand how its biological availability and activity are regulated. A recent study of EMAP-II expression in the mouse embryo noted the coincidence of EMAP-II transcripts and the presence of macrophages in areas with high levels of apoptosis (11) . These findings led the authors to speculate that EMAP-II is released by apoptotic cells in tissues undergoing remodeling, thereby providing a chemoattractant signal for phagocytic cells required to remove cellular debris (11) . This hypothesis is consistent with a role for caspase-like enzymes in the cleavage of the precursor molecule, which was predicted earlier by Kao et al. (4) , based on the amino acid sequence of the putative cleavage site.
Because processing and release of EMAP-II may be associated with programmed cell death (4 , 11) , we decided to examine the expression of EMAP-II in two established prostate adenocarcinoma cell lines, LNCaP and DU-145. In general, prostate tumor cells proliferate slowly (12) , making them poor targets for conventional antimitotic chemotherapy (13) . In the absence of cell proliferation, programmed cell death can be activated in androgen-dependent prostatic cancer cells by androgen withdrawal (14) . On the other hand, androgen-independent tumor cells are resistant to androgen ablation because they have a defect in the initiating step of the apoptosis pathway (15) . However, these cells retain downstream components of the pathway (14) and can be induced to undergo apoptosis by agents that elevate intracellular calcium levels, such as ionomycin (16) and thapsigargin (17) , suggesting some potentially novel therapeutic approaches. We therefore examined the effects of chemical stress on LNCaP and DU-145 cells using known inducers of apoptosis to determine whether these might cause the release of biologically active EMAP-II. We compared these effects with those of necrosis-inducing agents. We also examined the effects of physiological stresses, in particular, hypoxia, which is often a constitutive component of the tumor microenvironment.
Our data indicate that LNCaP and DU-145 cells express EMAP-II in vitro, but the protein is normally retained intracellularly as a Mr 34,000 precursor. Treatment of these cells with hypoxia or chemical agents known to induce apoptotic or necrotic cell death leads to the release and partial processing of EMAP-II. Importantly, EMAP-II release can occur in the absence of apoptosis. We suggest that the release of EMAP-II and its subsequent interaction with other host cells may contribute to the overall response of tumors to cytotoxic therapy.
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MATERIALS AND METHODS |
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All experiments were performed with cells at 80–90%
confluence, unless otherwise indicated. For chemical treatment of
cells, medium was replaced with fresh medium supplemented with freshly
dissolved drug at a concentration either derived from the literature or
based on initial dose-ranging experiments (data not shown). Incubations
with drugs were carried out for up to 72 h, depending on the agent
(see Table 1
). For hormone withdrawal experiments, cells were grown in medium
containing 5% charcoal-stripped calf serum or charcoal-stripped calf
serum supplemented with 10 nM dihydrotestosterone.
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RT-PCR.
RNA from cultures of LNCaP and DU-145 cells was purified using
a Purescript RNA Isolation Kit (Gentra Systems Inc.). cDNA was prepared
using the Promega Reverse Transcription System (Promega, Southampton,
United Kingdom). Specific primers were used to amplify EMAP-II cDNA by
PCR. One µl of cDNA was mixed with 500 ng of primers specific for
EMAP-II or control gene (human porphobilinogen deaminase) and 0.2
mM deoxynucleotide triphosphates in a total volume of 50
µl of Dynazyme reaction buffer (Flowgen, Lichfield, United Kingdom).
After a hot start (96°C for 5 min and then 54°C for 3 min), 1 unit
of Dynazyme II DNA polymerase (Flowgen) was added to each tube. Samples
were then incubated at 72°C for 2 min. Thirty-five cycles were
completed under the following conditions: (a) 94°C
(denaturation), 45 s; (b) 55°C (primer annealing),
45 s; and (c) 72°C (primer extension), 60 s.
Primer sequences were as follows; (a) EMAP-II forward primer
EM7, 5'-CGTCTGGATCTTCGAATTGG-3'; (b) EMAP-II reverse primer
EM8, 5'-GCATCAAAAGTAATTCTGTC-3'; (c) control forward primer
PB5', 5'-ATGTCTGGTAACGGCAATGCGG-3'; and (d) control reverse
primer PB3', 5'-TGGTTCCCACCACACTCT TCTCTG-3'. PCR controls were
also run with cDNA omitted.
Recombinant Mr 22,000 and
Mr 34,000 EMAP-II.
These forms of EMAP-II were prepared as described previously
(9)
.
Antibodies.
Preparation and characterization of polyclonal antibodies against
recombinant human EMAP-II (R2B2) have been described in detail
elsewhere (9)
. Briefly, rabbits were immunized with
recombinant human EMAP-II expressed in Escherichia coli as a
fusion protein with GST. Serum was tested for reactivity with
recombinant EMAP-II and recombinant GST by ELISA
(9)
, and animals were exsanguinated. Reactivity with
recombinant GST and E. coli antigens was removed by
cross-absorption on a column of immobilized extract of E.
coli BL21 transformed with the expression plasmid pGEX-2T coupled
to CNBr-activated Sepharose 4B (Pharmacia Biotech). Polyclonal
antibodies were tested for reactivity with recombinant GST and
recombinant EMAP-II by ELISA and showed no reactivity for GST.
Fluorescence-activated Cell-sorting Analysis.
Immunofluorescent detection of EMAP-II was performed on LNCaP and
DU-145 cells with R2B2 antibodies. Staining was carried out on fixed,
permeabilized cell suspensions with R2B2 antibodies diluted 1:1000 in
TBS. The secondary antibody was goat antirabbit FITC conjugate (Sigma).
Samples were analyzed on a Becton Dickinson FACScan using the LYSIS
program.
Western Blotting.
PAGE was used to separate proteins from LNCaP and DU-145 cell extracts
and supernatants, and EMAP-II was detected by Western blotting with the
R2B2 polyclonal antibodies as described previously (9)
.
Antibody binding was revealed by enhanced chemiluminescence. An
unexpected band corresponding to a molecular weight of 
66,000 was
frequently seen on Western blots. We have established that this is an
artifact of the technique and should be disregarded.
Immunohistochemistry.
All tissues were fixed in 4% paraformaldehyde before embedding in
paraffin. Four-µm sections were cut onto glass slides and incubated
at 60°C for 30 min. Before antibody staining, sections were dewaxed
with Histolene clearing agent (Cell Path Plc, Hemel Hempstead, United
Kingdom) and rehydrated by passing through a graded series of alcohols
(100–30%), followed by PBS (pH 7.4). Endogenous peroxidase
activity was quenched by incubation of all slides in 0.3% (v/v)
hydrogen peroxide in methanol. Sections were microwaved in an 800 W
oven for 10 min in 0.1 M citrate buffer (2.1 g/liter citric
acid and 1.0 g/liter sodium hydroxide). For all immunohistochemical
reactions, a Vectastain Elite ABC Kit (Vector Laboratories,
Peterborough, United Kingdom) was used, and all incubations were
carried out at room temperature in a humidified chamber. Nonspecific
binding of antibodies was blocked by incubating sections in 20% normal
goat serum for 20 min. After shaking off excess blocking solution, R2B2
rabbit polyclonal antibodies against EMAP-II were added at a 1:500
dilution. Preimmune rabbit serum at the same dilution was used as a
control. Sections were incubated for an additional 60 min and then
washed three times with PBS. Sections were incubated for an additional
30 min with a biotinylated goat antirabbit secondary antibody and then
washed three times with PBS. Slides were then incubated with the
avidin-biotin complex reagent, followed by diaminobenzidine substrate.
After washing with distilled water, slides were counterstained with
Mayer’s hematoxylin. Finally, the slides were dehydrated with graded
alcohols and mounted with DPX. Sections were viewed with a Nikon
Optiphot microscope and photographed on Kodak Elite 100 ASA film.
Detection of Apoptotic and Necrotic Cells with H342/PI.
This method (18)
allows distinction of the nuclei of
living and dying cells by fluorescence microscopy after staining with
the fluorochromes H342 and PI. H342 stains all cell nuclei, whereas PI
stains only the nuclei of cells with disrupted plasma membranes.
Therefore, viable and necrotic cells will have blue round nuclei and
pink round nuclei, respectively. Apoptotic cells will have
condensed/fragmented blue or pink nuclei, depending on whether they are
in the "early" or "late" stages of apoptosis.
Cells were harvested by trypsinization, centrifuged in 10 ml of cell culture medium at 1500 rpm for 5 min, and then resuspended at a density of about 1 x 106 cells/ml in medium. Cells were stained with H342 (10 µM) and PI (10 µM) on ice for 5 min in the dark. A 50-µl aliquot was then dropped onto a glass microscope slide, a coverslip was applied, and the cells were examined under a Nikon fluorescence microscope with UV (UV-1A) and green (G-2A) filters to detect H342 and PI staining, respectively. A minimum number of 100 cells/sample were counted, and the percentage of viable, necrotic, and apoptotic cells was calculated.
Coagulation Assay.
Conditioned media from control and treated LNCaP cells were tested for
procoagulant activity using a two-stage coagulation assay
(19)
. HUVECs were grown to confluence on 0.2%
gelatin-coated, 24-well tissue culture plates in HUVEC medium (M199
medium; Life Technologies, Inc., Paisley, United Kingdom) supplemented
with 14 mM HEPES, 0.15% sodium hydrogen carbonate, 2
mM L-glutamine, 100 units/ml penicillin, 100
µg/ml streptomycin, 90 µg/ml heparin, 20 µg/ml endothelial cell
growth supplement (Boehringer Mannheim), and 20% FCS. Tumor CM was
diluted 1:1 in HUVEC medium, and 50 units/ml polymixin B was added to
all test solutions to quench the effects of the contaminating
lipopolysaccharide. As a positive control, TNF- (2.5 pM)
or recombinant EMAP-II (10 pg/ml) was added to selected test solutions.
HUVEC medium was carefully removed from plates by aspiration and
replaced with test medium (500 µl/well). The samples were incubated
for 4.5 h at 37°C and then assayed for procoagulant activity.
Test medium was removed by careful inversion of the plate onto tissue paper, and each well was washed twice with 1 ml of PBS. Owren’s (barbital) buffer (100 µl) and 100 µl of citrated human plasma from a normal donor were then added to each well. The plate was placed in a 37°C water bath for 5 min to equilibrate. Coagulation was then initiated by the addition of 100 µl of 30 mM calcium chloride. Time to formation of visible fibrin strands/gel was measured with a stopwatch. Clotting times were converted to TF equivalents (pg/106 cells) by reference to a bilogarithmic calibration curve constructed using purified reconstituted human TF (19) .
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RESULTS |
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A, Lanes 3 and 5).
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). To determine the size distribution of the
immunoreactive polypeptides, we carried out Western blotting on
extracts and CM from both cell lines with R2B2. We have previously
shown (9)
that these antibodies detect a major band of
Mr 34,000 corresponding to the
precursor of EMAP-II in lysates of U937 transformed human monocytic
cells and a weaker band of Mr
22,000 in concentrated growth medium from the same cells. Lysates of
LNCaP and DU-145 cells showed a major band on Western blots of
Mr 34,000, indicating the presence
of a precursor of EMAP-II within both cell types (Fig. 2
A, Lanes 1 and 7). Concentrated media in which the
same cells had been grown also contained detectable levels of
Mr 34,000 EMAP-II antigen, more
in the case of LNCaP cells (Fig. 2A
, Lanes 2 and
8). We concluded from these experiments that both prostate
cancer cell lines constitutively express EMAP-II protein as the
Mr 34,000 form, releasing minimal
levels under normal conditions of culture. We therefore examined the
effects of a variety of cellular stresses known to cause apoptotic or
necrotic death or both to determine whether these might stimulate the
release of EMAP-II.
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. In brief, the most
potent inducers of apoptosis were ionomycin, camptothecin, and
thapsigargin. Etoposide induced approximately equal levels of necrosis
and apoptosis by 48 h. Overall, DU-145 cells appeared to be
somewhat more resistant to the effects of apoptosis-inducing agents
than LNCaP cells. Hydrogen peroxide, saponin, and antimycin rapidly
induced necrosis in both cell lines. Both cell lines appear resistant
to 8 Gy of X-rays given as a single dose. Hypoxia (2.5%
O2 for 48 h), on the other hand, is clearly
a potent physiological stress, because approximately 49% of LNCaP
cells and 11% of DU-145 cells were necrotic by 48 h. Hypoxia
appears to primarily induce necrosis and not apoptosis in these cell
lines. In response to hormone withdrawal, androgen-sensitive LNCaP
cells ceased proliferating and showed morphological alterations, but
there was no evidence of apoptosis or necrosis after 7 days of
observation (data not shown). Hormone withdrawal had no effect on
DU-145 cells. Heat shock produced extremely variable and inconsistent
results (data not shown).
Effect of Stress on Processing and Release of EMAP-II.
We examined the effect of different stresses on the release and
processing of EMAP-II by Western blotting. Fig. 2A
shows a
Western blot of lysates and concentrated media from LNCaP and DU-145
cells after treatment for 24 h with ionomycin, which we found to
be a potent inducer of apoptosis. The blots show a relative increase in
Mr 34,000 EMAP-II in the supernatants
of LNCaP and DU-145 cells after treatment (Fig. 2A
, compare
Lane 2 with Lanes 4 and 6 and compare
Lane 8 with Lanes 10 and 12).
Furthermore, in the treated samples, a strong immunoreactive band of
Mr 26,000–28,000 appears in
supernatants. Using radiolabeled Mr
34,000 precursor, we have shown that a
Mr 26,000–28,000 band is a common
intermediate in the processing of
EMAP-II.4
In the DU-145 cells, ionomycin treatment induces essentially the
complete disappearance of Mr 34,000
EMAP-II from the cells and the appearance of the
Mr 26,000–28,000 form in the
supernatants (Fig. 2A
, Lanes 10 and
12). On the other hand, thapsigargin seems to have less
effect on EMAP-II processing or release from DU-145 cells, with some
release into the medium and conversion after 48 h (compare Fig. 2
B, Lanes 10 and 14). Interestingly, DU-145 cells
do not undergo significant apoptosis or necrosis in response to
thapsigargin (see Table 1
). A Mr
22,000 band corresponding to the fully processed protein and migrating
with the same mobility as recombinant EMAP-II (Fig. 2
A, Lane
R) is just detectable in supernatant samples from LNCaP cells
treated with 10 µm ionomycin (Fig. 2
A, Lane 6). Etoposide
treatment gave rise to a similar profile; camptothecin also produced a
Mr 26,000–28,000 band, but not a
Mr 20,000–22,000 band (see Table 2
).
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. The data indicate changes similar to those seen with ionomycin,
thapsigargin, and etoposide, i.e., increased
Mr 34,000 EMAP-II in the cell
supernatants combined with the appearance of a
Mr 26,000–28,000 band. Excluding
glucose from the medium, which increases the levels of necrosis,
enhances this effect, and the appearance of the fully processed
Mr 20,000–22,000 form of EMAP-II
can be noted (Fig. 3
B, Lanes 23 and 24). Partial
processing to the Mr 26,000–28,000
forms was also observed in DU-145 cells (data not shown).
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shows the results of exposure of LNCaP and DU-145 cells to hypoxia,
which induced high levels of necrosis and very little apoptosis.
EMAP-II Western blot profiles were similar to those seen with
antimycin, which mimics some effects of hypoxia, and other inducers of
apoptosis. Hypoxia induced the appearance of a strong
Mr 22,000 band that was absent from
control cultures. Although treatment with hypoxia appeared to induce
increased immunoreactive material, there was no significant change in
total EMAP-II antigen in hypoxic cultures by competitive ELISA (data
not shown).
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. The first four bars of the graph demonstrate the well-documented
response of endothelial cells to EMAP-II, whereby recombinant EMAP-II
and TNF act synergistically to increase procoagulant activity. The next
two bars show that LNCaP CM has very low levels of
procoagulant-inducing activity; the addition of TNF produces a
supra-additive effect (Control CM + TNF).
Medium from cells subjected to hypoxia for 48 h does not have
significantly elevated coagulation-inducing activity. However, when TNF
is added to this medium, a strong coagulation response is seen, which
is approximately equal to the level obtained with recombinant
EMAP-II and TNF combined. Similar data were obtained with DU-145 CM
(data not shown).
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indicate that the Mr 34,000 form of
EMAP-II is only slightly but not significantly less potent than the
Mr 22,000 form in terms of activity in
the endothelial coagulation assay.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). Adenocarcinomas of the prostate show stronger staining of
the malignant epithelial component, with occasional weak staining of
the stromal component (Fig. 7B
). This pattern, which
occurred in all prostatic adenocarcinomas we studied, is consistent
with the intracellular expression of EMAP-II by the prostate
adenocarcinoma cell lines LNCaP and DU-145 in vitro.
Although the present study is limited to two cell lines, we believe
they accurately reflect EMAP-II expression in the human disease.
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The nature of the putative precursor of EMAP-II has recently become less clear because Quevillon et al. (23) noted the high degree of amino acid identity between EMAP-II and the p43 auxiliary component of the mammalian multisynthase complex. The hamster p43 protein is composed of 359 amino acids with a predicted molecular weight of Mr 40,000. This protein shares 86% and 85% amino acid identity with human and murine Mr 34,000 EMAP-II, respectively, whereas the human p43 and EMAP-II homologues appear to share 100% identity (23) . Quevillon et al. (23) have suggested that Mr 20,000 EMAP-II and, by implication, its Mr 34,000 precursor are truncated forms of the p43 auxiliary protein generated by partial proteolysis of the multisynthase complex, which may occur as a consequence of the disruption of protein synthesis in tumor cells. Our data demonstrate that LNCaP and DU-145 cells constitutively express the Mr 34,000 form of EMAP-II in vitro, with little evidence of further significant processing under normal culture conditions. Furthermore, our antibodies do not detect any proteins consistent with a human p43 auxiliary protein in any extracts, suggesting that Mr 34,000 EMAP-II is expressed as a unique protein and does not represent the partially processed form of a larger protein.
Under conditions of stress, however, EMAP-II is further processed and released by both cell lines into the extracellular environment. EMAP-II could play a role in two possible scenarios in vivo: regions of low oxygen tension are frequently present in rapidly growing experimental and human tumors, usually as a consequence of vascular insufficiency (24) . Hypoxia contributes directly to resistance to treatment with ionizing radiation but may also indirectly induce resistance to antimitotic chemotherapeutic agents through the initiation of a cell cycle block. On the basis of our data, rapidly growing, hypoxic tumors should release higher levels of the mature protein and should be more sensitive to TNF. Prostate cancer cells proliferate slowly in vivo (12) ; therefore, prostate tumors might be considered less likely to contain significant regions of hypoxia. However, recent evidence, including that derived from oxygen electrode measurements in situ (25) , supports the existence of hypoxic regions within prostate tumors. It might therefore be predicted that human prostate tumors do release some processed EMAP-II in these regions. Subjecting such tumors to added stress such as chemotherapeutic agents or other agents that induce cell death should enhance this release.
Treatment of tumors with chemotherapeutic agents is frequently associated with vascular pathologies, particularly disorders of coagulation (26) . Intravascular coagulation and hemorrhage are seen histologically in experimental tumors after treatment with conventional chemotherapy agents (27) . Damage to the vasculature may be due to direct effects on endothelial cells (28) or may occur indirectly through the action of molecules such as EMAP-II (29) released by damaged tumor cells. Our data show that several chemical agents, including camptothecin and etoposide (both used clinically), stimulate EMAP-II release, which could then contribute to the damage described above.
In summary, treatment of prostate tumor cells with stress-inducing agents promotes the release of biologically active EMAP-II. Furthermore, release and processing are not restricted to apoptotic cells but may also occur in cells that will ultimately die through necrosis. We suggest that released EMAP-II may act directly on tumor-associated endothelial cells, promoting a procoagulant phenotype, or may be involved in recruiting immune effector cells into the tumor milieu. These findings suggest a potential role for EMAP-II in tumors and, furthermore, mechanisms whereby its potent biological activity is expressed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Cancer Research
Campaign of the United Kingdom and the Association for International
Cancer Research. 
2 To whom requests for reprints should be
addressed, at University of Nottingham Laboratory of Molecular
Oncology, Cancer Research Campaign Department of Clinical Oncology,
City Hospital, Hucknall Road, Nottingham NG5 1PB, United Kingdom.
Phone: 44-1159-628014; Fax: 44-1159-627923; E-mail: cliff.murray{at}nott.ac.uk
3 The abbreviations used are: EMAP-II, endothelial
monocyte-activating polypeptide II; RT-PCR, reverse transcription-PCR;
TNF, tumor necrosis factor; GST, glutathione
S-transferase; PI, propidium iodide; H342, Hoechst
33342; HUVEC, human umbilical vein endothelial cell; TF, tissue factor;
CM, conditioned medium.
4 J. C. Murray, G. Barnett, M. P. R. Tas, J.
Brown, D. Powe, and C. Clelland. Immunohistochemical analysis of the
expression of the proinflammatory polypeptide EMAP-II in
vivo, submitted for publication.
5 A-M. Jakobsen, M. P. R. Tas, and J. C.
Murray. Processing of EMAP-II: cleavage by serine proteases and
caspases, manuscript in preparation.
Received 9/29/99. Accepted 3/30/00.
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REFERENCES |
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