Inhibition of Tumor Growth by the Antiangiogenic Placental Hormone, Proliferin-Related Protein
Nancy W. Bengtson and
Daniel I. H. Linzer
Department of Biochemistry, Molecular Biology, and Cell Biology
Northwestern University Evanston, Illinois 60208
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ABSTRACT
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Proliferin-related protein (PRP) is a potent
placental antiangiogenic hormone. To test the antiangiogenic potential
of PRP to block tumor growth, we engineered tumor cells to express this
hormone. Both SV40-transformed BALB/c mouse 3T3 fibroblasts and rat C6
glioma cells have markedly reduced growth rates as tumors in mice if
they express high levels of PRP. In both models, the small tumors that
form are largely avascular, whereas control tumors are rich in blood
vessels, consistent with PRP limiting tumor growth by preventing
neovascularization of the tumors. The antiangiogenic effects of PRP are
also detected on human endothelial cells, suggesting that the receptor
and signaling pathway of this mouse hormone are conserved
between mouse and human and may represent useful targets for the
development of antiangiogenic therapeutics. That signaling pathway
appears to involve an inhibition of arachidonic acid release, based on
the ability of arachidonic acid to overcome the antiangiogenic effects
of PRP.
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INTRODUCTION
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Angiogenesis, the growth of new blood vessels from existing
vessels, is an essential process in both physiology and
pathophysiology. During reproduction, remodeling of the maternal
vasculature at the implantation site and the growth of fetal vessels
into the placenta are required for the establishment of efficient gas,
nutrient, and waste exchange. Two placental hormones in the PRL family,
proliferin (PLF) and proliferin-related protein (PRP), contribute to
this process in mice as positive and negative regulators, respectively,
of angiogenesis (1). PLF appears to be important for attracting
maternal endothelial cells toward the trophoblasts (2), and PRP is
predicted to slow angiogenesis late in pregnancy, to prevent
endothelial cells from resealing open vessels that create the maternal
blood sinuses in which the trophoblasts bathe, and to limit the growth
of maternal and fetal vessels across the placenta (3).
Both PLF and PRP circulate in the maternal bloodstream at high levels,
PLF in early- to midgestation (4) and PRP in the latter part of
gestation (5). Thus, the activities of these hormones may be directed
both locally at endothelial cells in the uterus and placenta, and at
more distal maternal targets. PLF also enters the fetal compartment
(4), where it can bind to specific developing tissues (6). In contrast,
PRP appears to be excluded from the fetus (6, 7), which would allow
fetal vascularization to proceed in the absence of this antiangiogenic
hormone.
The accumulation of PRP in the maternal circulation may be expected to
have specific physiological consequences as part of normal mouse
pregnancy but may also contribute to limiting any
angiogenesis-dependent pathology. One such pathological condition is
tumor growth (8). Avascular tumors grow slowly and depend on
angiogenesis for a shift to a rapidly growing state (9, 10), whereas
tumors placed into vessel-rich regions may have immediate access to a
blood supply that supports rapid growth (11). Although tumors can grow
independently to a small size before requiring an outside source of
nutrients, more extensive growth depends upon the ability of tumors to
attract endothelial cells by secreting angiogenic factors or by
recruiting neighboring cells to secrete such factors. These endothelial
cells will form new blood vessels and vascularize the tumor, thereby
providing the tumor cells with nutrients and with access to the
bloodstream, potentially leading to metastasis (12).
That a placental hormone might be responsible for the inhibition of
tumor growth during pregnancy was suggested a few years ago by Gallo
and colleagues (13), who pointed out that human immunodeficiency virus
(HIV)-infected pregnant women were less prone to Kaposis sarcoma than
nonpregnant women or males (13). In that report, human CG (hCG) was put
forward as the placental factor responsible for the observed inhibition
of tumorigenesis during pregnancy. Subsequent reports have demonstrated
that an hCG-associated factor (HAF) and antineoplastic urinary protein
(ANUP), but not hCG itself, are two components responsible for the
observed effect on tumor cells in culture (14, 15, 16).
Despite the mistaken identification of hCG as the active factor
in inhibiting tumor growth, it is likely that placental hormones
provide a range of unusual activities as evidenced by the dramatic
physiological changes that occur specifically during pregnancy. The
activities of these placental hormones might then potentially be
exploited to regulate pathophysiology in the nonpregnant state. PRP
provides one such possibility. We therefore decided to test whether the
antiangiogenic activity of PRP is able to restrict tumor growth.
Furthermore, if any such activity is to prove useful in the design of
novel antitumor therapies, the effect of this hormone on mouse
endothelial cells must also be demonstrated to extend to human
endothelial cells, a prediction that we have also now examined.
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RESULTS
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Generation of SVT2 Tumor Cell Lines Expressing PRP
To test the effect of PRP on tumor growth, an
SV40-transformed BALB/c 3T3 fibroblast cell line (SVT2) was engineered
to secrete PRP. The SVT2 cell line (17) was selected in part due to its
rapid growth as a tumor when injected subcutaneously into BALB/c mice.
In addition, this would provide a homologous system in which effects of
a mouse hormone could be examined on mouse tumor cells, in an
appropriate location for the tumor cell type, and in immunocompetent
mice. A PRP expression construct containing the neomycin resistance
gene was transfected into SVT2 cells, and G418-resistant cell lines
were selected and then individually screened for the production of PRP.
Four stable transfectants were chosen for further analysis based on
their secretion of high levels of PRP (Fig. 1
). PRP produced by stably transfected
tumor cells is secreted, soluble, and of a higher apparent molecular
mass than the 24 kDa predicted from its amino acid sequence.
This higher apparent molecular mass is identical to that of PRP
produced by stably transfected Chinese hamster ovary cells; this larger
size has been demonstrated to result from N-linked glycosylation (5).
Three of these stably transfected cell lines, designated SVT2-PRP2,
SVT2-PRP3, and SVT2-PRP4, secrete quite high levels of PRP; by
comparison to the purified protein control, PRP represents
approximately 10% of the total secreted protein that accumulated in
the conditioned medium from each of these cell lines. No PRP is
detected in the conditioned medium of cultures of SVT2 cells stably
transfected with the empty expression vector, indicating that SVT2
cells do not express the endogenous PRP gene.

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Figure 1. PRP Expression in SVT2-PRP Cell Lines
Conditioned media from stably transfected SVT2 cell lines were
harvested and analyzed for PRP expression. Equal amounts (15 µg)
of total protein from vector-transfected SVT2 cells (SVT2-V) or from
four different SVT2-derived cell lines stably transfected with the PRP
expression construct were loaded per lane. After transfer to a filter,
the samples were incubated with an antiserum against PRP followed by an
enzyme-linked secondary antibody. The leftmost lane contains
2 µg of purified PRP. The positions of the mol wt standards are
indicated on the left.
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Expression of PRP by the SVT2 tumor cells had no effect on cell growth
in culture. All four cell lines were readily recloned in soft agar,
indicating that SVT2 cells expressing PRP retain their fully
transformed phenotype. Furthermore, the rate of cell growth for each of
these cell lines was indistinguishable from the growth rate of the
control, vector-transfected SVT2 cell line (Fig. 2
). Thus, any effect that might be
observed on tumor growth in vivo would not be attributable
to direct effects of PRP on the growth of these cells.

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Figure 2. Cell Growth Curve of Stably Transfected SVT2 Cell
Lines
SVT2-V and each of the four SVT2-PRP cell lines were plated at 7,000
cells per well on day 0 and allowed to grow under standard culture
conditions. Cell number was determined in triplicate every day over a
period of 7 days, and the mean ± SD is shown.
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Inhibition of SVT2 Tumor Growth by PRP
Equivalent numbers of cells from each of the PRP-expressing SVT2
cell lines or the vector-transfected cell line were injected
subcutaneously into the flanks of BALB/c mice. After 3 weeks, tumors
were excised and analyzed; tumor growth was not maintained beyond this
point because the size of the control tumors had reached the maximum
allowed under the animal care guidelines. Each of the four SVT2 cell
lines producing PRP resulted in tumors that were significantly reduced
in volume and mass compared with the control (P <
0.01) (Fig. 3
). Notably, even the
SVT2-PRP1 cells, which express lower levels of PRP than the other cell
lines, grew poorly as a tumor.

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Figure 3. PRP Inhibition of SVT2 Tumor Growth
Tumors formed from injected SVT2-V cells or from each of the four
different SVT2-PRP cell lines were isolated after 3 weeks of growth,
and their weights (A) and volumes (B) were determined. Values are
presented as mean ± SEM with n = 7 to 12
mice per group; *, P < 0.01; **,
P < 0.001.
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Not all of the tumors from the SVT2-PRP lines were large enough for
further study. In each case, for those tumors that could be analyzed,
PRP mRNA was present in the tumor extracts (Fig. 4A
) and PRP was secreted by tumor tissue
placed in culture (Fig. 4B
). Thus, tumors generated from the
PRP-expressing SVT2 cell lines maintain PRP expression. The RNA and
protein expression analyses also revealed that distinct tumors arising
from a PRP-expressing SVT2 cell line display very similar levels of PRP
expression, demonstrating that this approach leads to consistent
hormone expression in vivo. In addition, compared with the
purified PRP standard, the glycoprotein hormone produced by the tumor
tissue was again similar in size and produced at high levels,
indicating that no significant change occurred in PRP expression upon
growth of the cell lines as tumors.

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Figure 4. PRP Expression in SVT2 Tumors
A, RNA (10 µg per lane) from an SVT2-V tumor and from two different
tumors derived from each of two SVT2-PRP cell lines was purified and
analyzed for PRP mRNA by filter hybridization. B, Tumor tissue samples
were also placed in culture, and conditioned media (15 µg protein per
lane) from these cultures were examined for PRP by immunoblotting.
Three independent tumors from one SVT2-PRP cell line are shown to
highlight the consistent levels of PRP expression. The leftmost
lane contains 2 µg of purified PRP.
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Tumors that developed from the three cell lines expressing high levels
of PRP (SVT2-PRP2, SVT2-PRP3, and SVT2-PRP4) were uniformly small,
solid masses. Closer inspection of these tumors by sectioning and
staining for the endothelial cell marker PECAM-1 revealed a marked
reduction in vascular density in the PRP-expressing tumors, in contrast
to the vessel-rich control tumors (Fig. 5
and Table 1
). SVT2-PRP1 cells, which
secrete lower levels of this antiangiogenic hormone, gave rise to
tumors that were reproducibly soft and fluid filled. Histologically,
these tumors have an outer vascularized region overlaying a region of
high tumor cell density but lacking vascular structures, and a core
that appears to have mostly cellular debris (Fig. 5
).

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Figure 5. PRP Inhibition of SVT2 Tumor Vascularization
Tumor tissue was isolated and sectioned for detection of endothelial
cells and vessels using an antibody against the endothelial antigen,
PECAM-1. Many cross-sectioned vessels are present in tumor tissue from
SVT2-V cells (A and B), several of which are indicated by the
arrows, but not in high expressing SVT2-PRP tumors (C
and D). SVT2-PRP1 tumor tissue (E) reveals a vascularized cortex (1 ),
an underlying avascular and dense cellular layer (2, with a higher
magnification of this region in panel F), and a core that appears to be
filled with fluid and cellular debris (3 ). Antibody binding to mark
endothelial cells is evident as a dark precipitate in
the higher magnification of SVT2-V tumor tissue (B). Tissue sections
were counterstained with Harriss hematoxylin. The bar
in the low magnification panels (A, C, and E) corresponds to 100 µm,
and in the high magnification panels (B, D, and F) to 10 µm.
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PRP Inhibition of C6 Glioma Tumor Growth
To verify that the effects of PRP on tumor growth were not
restricted to a single model system, we repeated the above experiments
with a second tumor cell line, C6 glioma cells (18). These cells also
grow rapidly as a tumor and secrete high levels of angiogenic activity
(19, 20, 21). Similar transfections were carried out with the PRP
expression construct, and the resultant stable transfectants were
screened for PRP expression by immunoblot analysis (Fig. 6
). Again, a range of PRP secretion was
observed among the isolated lines, and both high-expressing and
low-expressing lines were chosen for further study. As with the
SVT2-derived cell lines, all of the stably transfected C6 lines
displayed identical growth kinetics in cell culture (data not shown),
demonstrating that neither the transfection procedure nor the
production of PRP was detrimental to cell growth.

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Figure 6. PRP Expression in C6-PRP Cell Lines
Conditioned media from stably transfected C6 cell lines were harvested
and analyzed for PRP expression. Equal amounts (15 µg) of total
protein from parental (C6) and vector-transfected (C6-V) cells or from
two different C6-derived cell lines stably transfected with the PRP
expression construct (one high expressing and one low expressing line)
were loaded onto each lane, and after transfer to a filter the samples
were incubated with an antiserum against PRP followed by an
enzyme-linked secondary antibody. A sample of serum-free medium from
placental cultures generated from tissue isolated at day 12 of
gestation (day 12 PCM) was included as a positive control. The
positions of the mol wt standards are indicated on the
left; the arrow points to PRP.
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Because C6 cells are of rat origin, immunodeficient ("nude") mice
were used as hosts for the in vivo tumor growth analysis.
The absence of hair on these mice also made it possible to monitor
tumor size throughout the 3-week period after tumor cell injection. (In
contrast, SVT2 tumor size in BALB/c mice could only be measured
accurately at the end of the experiment.) Consistent with the results
in the SVT2 system, two C6-PRP cell lines expressing high levels of the
antiangiogenic hormone grew at significantly slower rates than the
control C6 tumors (P < 0.05) (results are shown for
one tumor line in Fig. 7
); C6-derived
cell lines expressing lower levels of PRP, though, grew at a rate
indistinguishable from that of the parental and vector-transfected C6
controls (Fig. 7
). The smaller tumors resulting from the C6-PRP
high-expressing lines were largely avascular, as revealed by the
histology of tumor tissue sections (Fig. 8
) and immunostaining with the antiserum
against the endothelial cell marker PECAM-1 to count vessels (Table 1
).
High PRP expression resulted in a greatly reduced density of larger
vessels, but only a modest reduction in small vessels (Table 1
); the
latter category, though, includes small groupings of PECAM-1-positive
cells even if a lumen is not evident and may therefore underestimate
the effectiveness of PRP.

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Figure 7. PRP Inhibition of C6 Tumor Growth
Equal numbers of C6-V (filled circles and solid line),
low expressing C6-PRP (open diamonds and small dashed
line) or high expressing C6-PRP (open squares and large
dashed line) cells were injected subcutaneously into nude mice.
The slopes of the lines are 0.26 ± 0.04 for C6-V, 0.23 ±
0.04 for low expressing C6-PRP, and 0.14 ± 0.01 for high
expressing C6-PRP (P < 0.05 vs.
control). Tumor size (mean ± SEM with n = 4
mice per group) was measured until 3 weeks after injection.
P < 0.05 for every individual time point comparing
high expressing C6-PRP tumor size to C6-V control.
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Figure 8. PRP Inhibition of C6 Tumor Vascularization
Tumor tissue from parental C6 (A) and vector-transfected C6 cells (B)
display numerous vessels. High-PRP-expressing tumors (C and D), but
much less so low-PRP-expressing tumors (E and F), showed a marked
diminution in vessel density. Tissue sections were counterstained with
Harriss hematoxylin. Bar = 100 µm.
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Effect and Mechanism of Action of PRP on Human Endothelial
Cells
We have previously demonstrated that PRP is able to inhibit the
migration of bovine endothelial cells in culture and angiogenesis in
the rat cornea (1). If the currently unidentified PRP receptor and
downstream signaling pathways are to provide potential targets for the
design of novel antitumor therapeutics, then it is also essential to
determine whether this conservation of PRP action on endothelial cells
from other species extends to human endothelial cells. We therefore
examined the effect of PRP on the migration of human dermal
microvascular endothelial cells in response to basic fibroblast growth
factor (bFGF) in a Boyden chamber assay. Cells on a collagen-coated
filter in upper wells of the chamber were exposed to medium alone or
medium supplemented with bFGF, PRP, or the combination of bFGF and PRP
in the lower chambers. Six hours later, the number of endothelial cells
that had migrated from the upper to the lower surface of the filter was
determined. Treatment with bFGF resulted in a 7-fold increase in
migration (Fig. 9
). PRP alone had no
effect on cell migration, but this hormone did reverse the effect of
bFGF. A monoclonal antibody against PRP (22), but not irrelevant
antibodies, completely reversed the inhibitory effect of the PRP
preparation, demonstrating that the antiangiogenic activity is fully
attributable to PRP (Fig. 9
). Thus, PRP is able to act directly on
human endothelial cells to block an angiogenic response.

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Figure 9. PRP Inhibition of bFGF-Induced Human Endothelial
Cell Migration
Total migration of human dermal microvascular endothelial cells in a
Boyden chamber assay was measured in response to 10 ng/ml bFGF, 1
µg/ml PRP, 10 ng/ml bFGF + 1 µg/ml PRP, and the combination of
these two factors in the presence of a 20 µg/ml monoclonal antibody
(mAb) against PRP or an equivalent amount of irrelevant immunoglobulin
(IgG). Data are the mean ± SEM for the number of
cells migrated in 10 high-power microscope fields per well, with n
= 6 wells; *, P < 0.01 relative to the untreated
control.
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PRP blocks endothelial cell migration in culture and neovascularization
in vivo in response to both bFGF and PLF. Stimulation of
endothelial cell migration by these angiogenic factors is inhibited by
pertussis toxin, implicating G protein signaling (23, 24). At least for
bFGF, the G-protein signaling pathway leads to arachidonic acid
production as an essential step (23), suggesting that the arachidonic
acid pathway may be an important target for PRP. Indeed, addition of 10
µM arachidonic acid reversed PRP inhibition of
bFGF-stimulated human endothelial cell migration (Fig. 10
).

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Figure 10. Arachidonic Acid Reversal of PRP Inhibition of
bFGF-Induced Endothelial Cell Migration
Total migration of human dermal microvascular endothelial cells in a
Boyden chamber assay was measured in response to 10 ng/ml bFGF, 1
µg/ml PRP, 1 or 10 µM arachidonic acid, or to
combinations of these factors. Data are the mean ±
SEM for the number of cells migrated in 10 high-power
microscope fields per well, with n = 6 wells; *,
P < 0.001 relative to the untreated control. Note
that PRP blocks the response to bFGF (compare columns 2 and 6), and
that 10 µM arachidonic acid reverses this effect of
PRP (column 8).
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DISCUSSION
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The experiments presented here demonstrate that PRP is able to
restrict both tumor angiogenesis and tumor growth in mice. These
results are consistent with the antiangiogenic effects of PRP being the
cause of the reduced tumor growth, but we cannot exclude at this time
other possible biological effects of PRP that may be relevant to tumor
growth. PRP thus joins several other antiangiogenic factors, including
angiostatin (25) and endostatin (26), as agents which can be used to
inhibit tumor growth, at least in model systems. While it may be
tempting to assume that any antiangiogenic factor will inhibit tumor
growth, experimental evidence is required to establish the
effectiveness of a factor for several reasons. An antiangiogenic factor
may act on only some classes of endothelial cells, or only prevent
angiogenesis induced by a limited set of angiogenic factors, or not be
sufficiently robust in vivo because of posttranslational
modification (such as proteolytic cleavage) or rapid clearance. The
establishment of PRP as an effective agent in interfering with tumor
angiogenesis in mice suggests that its receptor and downstream
signaling pathways may be useful targets for antitumor drug design. The
finding that the antiangiogenic effects of PRP can be overcome by
addition of exogenous arachidonic acid represents the first clue to the
PRP signaling mechanism. Predictions to be tested based on this result
are that PRP inhibits the bFGF- or PLF-induced activation of
phospholipase A2, and that a specific metabolic
product of arachidonic acid (for example, a particular prostaglandin)
may be an essential signal for endothelial cell migration in response
to both bFGF and PLF.
The ability of PRP to act on tumors at sites outside of the uterus
suggests that the high levels of PRP that circulate during pregnancy
may have systemic effects. One possible contribution of PRP outside of
the implantation site might be to restrict angiogenesis-dependent cell
proliferation during pregnancy. The brief period of PRP expression in
gestation (
1 week) makes it difficult to examine the effect of
endogenous PRP on tumor growth. It should become possible, though, to
identify both implantation site and distal effects of PRP by disruption
of this gene in mice.
The tumor cells and hosts that we chose to examine provide a
combination of homologous mouse components in the case of the SVT2
system, and a potent and widely used angiogenic tumor model in the C6
system. In both cases, by engineering tumor cells to secrete PRP,
rather than injecting purified hormone, the tumor environment was
exposed continuously to the antiangiogenic hormone and presumably to a
reasonably constant concentration of hormone relative to tumor size.
Exogenous administration of the hormone, or of an agonist ligand, to
block tumor growth may pose greater challenges in terms of efficiently
targeting the protein to the tumor site and preventing rapid clearance
of the hormone from the circulation. Nevertheless, the inhibition of
growth of the SVT2-PRP1 line as a tumor, a line that secretes only
modest amounts of PRP compared with the other three SVT2-PRP cell lines
that were tested, gives some reason to expect that it would be possible
to deliver sufficient doses of this hormone or a hormone agonist to
limit tumor growth.
In contrast to the intermediate effect seen with the SVT2-PRP1 cell
line, only C6 cell lines expressing high levels of PRP were able to
inhibit tumor growth. We were unable to extend the tumor growth studies
to a point at which the effects of a lower dose of PRP might become
evident, because the tumors had already reached the maximum size
allowed by the animal care guidelines. The ability of a high dose, but
not a lower dose, of PRP to inhibit C6 tumor growth is consistent with
the idea that the net angiogenic activity, rather than a dominant
effect of any single factor, drives tumor vascularization and growth.
In the C6 model, very high levels of PRP are likely to be required to
overcome the amount of angiogenic factor [vascular endothelial growth
factor (VEGF)] produced by these tumor cells.
PRP can act directly on human microvascular endothelial cells to
inhibit their response to bFGF, suggesting that the effects of PRP on
tumor growth in mice may translate into similar effects of binding to
the PRP receptor in humans. Although no evidence for a human PRP
homolog has been obtained, the ability of PRP to act on human (and rat
and bovine) endothelial cells indicates that the receptor and cell
response are conserved among mammals. These results further argue that
identifying placental hormones in model systems such as mice and rats
can represent a useful approach to defining activities relevant to
human physiology. Because the physiological changes during pregnancy
are so dramatic, the characterization of these placental hormones is
likely to reveal new mechanisms of cell regulation that are not evident
in the nonpregnant animal. Furthermore, if PRP and other antiangiogenic
factors are found to act on endothelial cells through distinct
signaling pathways or to affect distinct stages of angiogenesis that
contribute to tumor growth (27), then it is tempting to speculate that
combinations of factors may provide an even more effective blockade of
tumor angiogenesis than would be achieved by a single factor alone.
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MATERIALS AND METHODS
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Cell Culture and Migration Assay
SV40 transformed BALB/c 3T3 cells (SVT2) (17), provided by
Arnold Levine, and rat glioma C6 cells (18) were cultured in DMEM
(Life Technologies, Inc., Gaithersburg, MD) supplemented
with 10% calf serum and 2 mM L-glutamine in a
humidified atmosphere of 5% CO2. SVT2 and C6
cells were transfected using calcium phosphate precipitation of either
pMSXND (vector) or the pMSXND-PRP expression construct (5). Individual
colonies were selected based on resistance to treatment with G418
(Sigma, St. Louis, MO) and then screened for PRP
expression by immunoblotting. Clones expressing PRP were recloned by
growth in soft agar, after which individual colonies were selected,
passaged, and analyzed again for protein expression. For cell growth
assays, SVT2 or C6 cells transfected with vector alone (SVT2-V and
C6-V) or vector containing the PRP coding region (SVT2-PRP or C6-PRP)
were plated at 7,000 cells per well in 12-well plates and grown under
standard culture conditions; cell counts were performed with a
hemocytometer on triplicate samples on a daily basis for 7 days.
Primary human dermal microvascular endothelial cells were obtained from
Clonetics/BioWhittaker, Inc. (Walkersville, MD), and grown
in EGM-MV medium (Clonetics/BioWhittaker, Inc.). To
monitor migration, 50,000 endothelial cells were transferred per well
of a Boyden chamber onto Nucleopore membranes with a pore size of 8
µm (Fisher Scientific, Pittsburgh, PA) and that had been
coated with collagen type IV (Sigma). Medium lacking
growth factors was added to the upper wells, and the identical medium
with 10 ng/ml bFGF (R&D Systems, Minneapolis, MN), 1 µg/ml PRP, 1 or
10 µM arachidonic acid (Sigma), 20 µg/ml
anti-PRP monoclonal antibody (22), or control mouse IgG
(PharMingen, San Diego, CA), or various combinations of
these factors was added to the lower wells. PRP was purified from the
conditioned medium of Chinese hamster ovary cells stably transfected
with a PRP expression construct, as described previously (5);
monoclonal antibody was purified from hybridoma conditioned medium by
Protein G affinity chromatography (Pharmacia Biotech,
Piscataway, NJ). Six hours after treatment, filters were removed and
stained with Dade Diff-Quick (Fisher Scientific) and then
rinsed in PBS, and the number of cells that had migrated to the lower
side of the filter were counted. Only cells in which the nucleus had
moved through the membrane were counted.
Immunoblot Analysis
Conditioned media from stably transfected cell lines or from
dispersed tumor cells that were placed into culture were collected and
concentrated by Centricon-30 filtration (Millipore Corp.,
Bedford, MA). Fifteen micrograms of total protein were loaded per lane
onto SDS-polyacrylamide gels. After electrophoresis, proteins were
transferred to nitrocellulose membranes, which were then washed in 20
mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05% Triton
X-100 (buffer A) containing 5% dried milk fat powder and then
incubated for 2 h at room temperature with a 1:1,000 dilution of
anti-PRP antiserum (5). After a 30-min wash in buffer A, membranes were
exposed for 2 h at room temperature to a 1:1,000 dilution of
horseradish peroxidase-conjugated, donkey-antirabbit immunoglobulin
(Amersham Pharmacia Biotech, Arlington Heights, IL). After
washing, bound antibody complexes were detected using the Super Signal
chemiluminescence reagent (Pierce Chemical Co., Rockford,
IL) and exposure to XAR film (Kodak, Rochester, NY).
In Vivo Tumor Analysis
Five-week-old female BALB/c mice were injected subcutaneously
with 105 SVT2, SVT2-V, or SVT2-PRP cells
resuspended in a volume of 100 µl of serum-free cell culture medium
containing 0.5% methylcellulose (Fisher Scientific).
Five-week-old nude mice were similarly injected with C6, C6-V, or
C6-PRP cells. Three weeks after injection of SVT2 cells, tumors were
excised and tumor weights and volumes were determined. For C6 cells,
tumor growth was monitored throughout the 3-week period, with tumor
volume calculated as length x width2 x
0.5. All procedures were approved by the Northwestern University Animal
Care and Use Committee.
RNA Filter Hybridization
Ten micrograms of tumor RNA, isolated using Tri Reagent
(Sigma), were loaded per lane of 2% agarose gels
containing 6% formaldehyde. After electrophoresis, the RNA was
visualized by ethidium bromide staining under UV light and then
transferred to a nylon membrane. Membranes were hybridized overnight at
42 C with a 32P-labeled PRP cDNA probe and then
washed and exposed to XAR film.
Immunohistochemistry
Excised tumors were fixed in 10% formalin and embedded in
paraffin. Sections (15 µm) were permeabilized with 0.01% trypsin for
30 min at 37 C and washed in PBS. Endogenous peroxidase activity was
quenched by incubation with 0.3%
H2O2 in PBS for 15 min at 4
C followed by three PBS washes. Sections were then incubated with
blocking buffer (2% rabbit serum and 5% BSA in PBS) for 30 min at
room temperature, followed by 1 h at room temperature with
rat-antimouse PECAM-1 monoclonal antibody (PharMingen)
diluted 1:6 in blocking buffer. After washing in PBS, sections were
exposed to biotinylated rabbit-antirat immunoglobulin secondary
antibody (Vector Laboratories, Inc. Burlingame, CA)
diluted 1:1,000 in blocking buffer for 1 h at room temperature,
washed again, and treated for 1 h at room temperature with the ABC
kit (Vector Laboratories, Inc.) followed by three PBS
washes. Substrate solution of 0.06% (wt/vol) diaminobenzidine-0.03%
(vol/vol) H2O2 in PBS was
then added to the sections for 15 min at room temperature, and the
sections were counterstained with Harriss hematoxylin.
To count vessels, PECAM-1-positive cells and structures were totaled
from complete cross-sections of several independent tumors for
different tumor lines. Vessels were divided into either larger
structures with obvious lumenal spaces, or a range of smaller
PECAM-1-positive multicellular clusters, not all of which had evident
lumenal spaces. With the inclusion of all PECAM-1-positive structures,
numbers for "small vessels" may therefore underestimate the effects
of PRP on vascular density.
 |
ACKNOWLEDGMENTS
|
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We thank Diane Mayer and Janelle Roby for expert technical
assistance and Noël Bouck for helpful comments.
 |
FOOTNOTES
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Address requests for reprints to: Daniel I. H. Linzer, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208. E-mail:
dlinzer{at}northwestern.edu
This work was supported by NIH Grant R01 HD-24518, by the Robert H.
Lurie Comprehensive Cancer Center (P30 CA-60553), and by the NIH
Research Center on Fertility and Infertility at Northwestern University
(P30 HD-28048).
Received for publication July 18, 2000.
Revision received August 24, 2000.
Accepted for publication September 7, 2000.
 |
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