Group Title: BMC Cell Biology
Title: Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin
CITATION PDF VIEWER THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00099970/00001
 Material Information
Title: Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin
Physical Description: Book
Language: English
Creator: Padua, Maria
Hansen, Peter
Publisher: BMC Cell Biology
Publication Date: 2008
 Notes
Abstract: BACKGROUND:Uterine serpins are members of the serine proteinase inhibitor superfamily. Like some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most studied member of the group, ovine uterine serpin (OvUS), inhibits proliferation of several cell types including activated lymphocytes, bovine preimplantation embryos, and cell lines for lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3) cells. The goal for the present study was to evaluate the mechanism by which OvUS inhibits cell proliferation. In particular, it was tested whether inhibition of DNA synthesis in PC-3 cells involves cytotoxic actions of OvUS or the induction of apoptosis. The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL)-8 by PC-3 cells was also determined. Finally, it was tested whether OvUS blocks specific steps in the cell cycle using both PC-3 cells and lymphocytes.RESULTS:Recombinant OvUS blocked proliferation of PC-3 cells at concentrations as low as 8 µg/ml as determined by measurements of 3Hthymidine incorporation or ATP content per well. Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8 secretion into medium. Results from flow cytometry experiments showed that OvUS blocked the entry of PC-3 cells into S phase and the exit from G2/M phase. In addition, OvUS blocked entry of lymphocytes into S phase following activation of proliferation with phytohemagglutinin.CONCLUSION:Results indicate that OvUS acts to block cell proliferation through disruption of the cell cycle dynamics rather than induction of cytotoxicity or apoptosis. The finding that OvUS can regulate cell proliferation makes this one of only a few serpins that function to inhibit cell growth.
General Note: Start page 5
General Note: M3: 10.1186/1471-2121-9-5
 Record Information
Bibliographic ID: UF00099970
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: Open Access: http://www.biomedcentral.com/info/about/openaccess/
Resource Identifier: issn - 1471-2121
http://www.biomedcentral.com/1471-2121/9/5

Downloads

This item has the following downloads:

PDF ( PDF )


Full Text



BMC Cell Biology


Research article


Regulation of DNA synthesis and the cell cycle in human prostate
cancer cells and lymphocytes by ovine uterine serpin
Maria B Padua and Peter J Hansen*


Address: Department of Animal Sciences, University of Florida, Gainesville, FL 32611-0910, USA
Email: Maria B Padua mpadua@ufl.edu; Peter J Hansen* hansen@animal.ufl.edu
* Corresponding author


Published: 24 January 2008
BMC Cell Biology 2008, 9:5 doi:10. 1186/1471-2121-9-5


Received: 17 August 2007
Accepted: 24 January 2008


This article is available from: http://www.biomedcentral.com/1471-2121/9/5
2008 Padua and Hansen; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Uterine serpins are members of the serine proteinase inhibitor superfamily. Like
some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most
studied member of the group, ovine uterine serpin (OvUS), inhibits proliferation of several cell
types including activated lymphocytes, bovine preimplantation embryos, and cell lines for
lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3) cells. The goal for the
present study was to evaluate the mechanism by which OvUS inhibits cell proliferation. In
particular, it was tested whether inhibition of DNA synthesis in PC-3 cells involves cytotoxic
actions of OvUS or the induction of apoptosis. The effect of OvUS in the production of the
autocrine and angiogenic cytokine interleukin (IL)-8 by PC-3 cells was also determined. Finally, it
was tested whether OvUS blocks specific steps in the cell cycle using both PC-3 cells and
lymphocytes.
Results: Recombinant OvUS blocked proliferation of PC-3 cells at concentrations as low as 8 lIg/
ml as determined by measurements of [3H]thymidine incorporation or ATP content per well.
Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8
secretion into medium. Results from flow cytometry experiments showed that OvUS blocked the
entry of PC-3 cells into S phase and the exit from G2/M phase. In addition, OvUS blocked entry of
lymphocytes into S phase following activation of proliferation with phytohemagglutinin.
Conclusion: Results indicate that OvUS acts to block cell proliferation through disruption of the
cell cycle dynamics rather than induction of cytotoxicity or apoptosis. The finding that OvUS can
regulate cell proliferation makes this one of only a few serpins that function to inhibit cell growth.


Background
Serine proteinase inhibitors (serpins) inactivate their tar-
get proteinases through a suicide substrate-like inhibitory
mechanism. The proteinase binds covalently to the reac-
tive center loop (RCL) of the serpin and cleaves the scissile
bond at the P1-PI' site. The RCL then moves to the oppo-
site side to form the 3-sheet A and a distortion in the struc-
ture of the proteinase that results in its inactivation [1-3].


Not all serpins, however, exert proteinase inhibitory activ-
ity. Some examples are corticosteroid and thyroxine bind-
ing globulins, which function as hormone transport
proteins [4], the chaperone heat shock protein 47 [5],
mammary serine protease inhibitor (Maspin), which
increases the sensitivity of cancer cells to undergo apopto-
sis [6], and pigment epithelium derived factor (PEDF),


Page 1 of 10
(page number not for citation purposes)


0
B.olled Central








http://www.biomedcentral.com/1471-2121/9/5


which has neurotrophic, neuroprotective, antiangiogenic,
and proapoptotic actions [7].

Another class of serpins without apparent proteinase
activity is the uterine serpins. These proteins, which are
produced by the endometrial epithelium of the pregnant
cow, sow, sheep, and goat [8-13], have been classified as
either a separate clade of the serpin superfamily [14] or as
a highly-diverge group of the a 1-antitrypsin clade [ 1]. The
best characterized protein of this unique group of serpins
is ovine uterine serpin (OvUS). This basic glycoprotein is
a weak inhibitor of aspartic proteinases (pepsin A and C)
[12,151, but it does not inhibit a broad range of serine
proteinases [9,16]. Additionally, amino acids in the hinge
region of inhibitory serpins are not conserved in uterine
serpins and OvUS behaves different in the presence of
guanidine HC1 than for inhibitory serpins [13,15 ].

The biological function of OvUS during pregnancy may
be to inhibit immune cell proliferation during pregnancy
and provide protection for the allogeneically-distinct con-
ceptus [17]. Ovine US decreases proliferation of lym-
phocytes stimulated with concanavalin A,
phytohemagglutinin (PHA), Candida albicans, and the
mixed lymphocyte reaction [18-22]. In addition, OvUS
decreases natural killer cell cytotoxic activity, abortion
induced by poly(I)poly(C) in mice [23] and the produc-
tion of antibody in sheep immunized with ovalbumin
[21]. The antiproliferative actions of OvUS are not limited
to lymphocytes. Ovine US decreases development of the
bovine embryos and proliferation of mouse lymphoma,
canine primary osteogenic sarcoma and human prostate
cancer cell lines [24,25].

The mechanism by which OvUS inhibits proliferation of
cells is unknown. The protein could block activation of
cell proliferation, inhibit the cell cycle at other points or
induce apoptosis or other forms of cell death. For the PC-
3 prostate cancer line, inhibition of cell proliferation by
OvUS might involve reduction in interleukin-8 (IL-8)
secretion because of the importance of autosecretion of
this cytokine for cell androgen-independent proliferation
[26]. The goal of the present study was to evaluate the
mechanism by which OvUS inhibits cell proliferation.
Using PC-3 cells as a model system, it was tested whether
inhibition of DNA synthesis involves cytotoxic action of
OvUS, induction of apoptosis or disruption of the IL-8
autocrine loop. It was also tested whether OvUS blocks
specific steps in the cell cycle for PC-3 cells and lym-
phocytes.

Results and Discussion
Proliferation of PC-3 cells
The antiproliferative effects of rOvUS on proliferation of
PC-3 cells were evaluated by two different assays. In the


first experiment, it was shown that rOvUS caused a con-
centration-dependent decrease in incorporation of
pH]lthymidine into DNA (P < 0.001) with the minimum
effective concentration being 8 lig/ml (Figure 1). The anti-
proliferative actions of OvUS using [H]lthymidine uptake
as the measure of proliferation has been demonstrated
previously for PC-3 cells and other cell types [18-
22,24,25]. To confirm this effect of rOvUS reflected an
inhibition in cell proliferation and not a disruption in
[ 3H]thymidine uptake by the cells, antiproliferative effects
were also evaluated by an assay in which the relative total
number of cells per well was estimated by the ATP content
per well. Treatment with rOvUS reduced ATP content per
well at all concentrations tested (50, 100 and 200 pg/ml)
(Figure 2). In contrast, the control serpin, ovalbumin, did
not cause effect in the ATP content per well. The finding
that rOvUS reduced ATP content per well confirms that
the effects of OvUS to reduce [3H]thymidine incorpora-
tion reflect a reduction in cell proliferation rather than
interference with [pH]thymidine transport into the cell.

Lactate dehydrogenase release
Possible cytotoxic effects of rOvUS on PC-3 cells were
evaluated by measurements of lactate dehydrogenase
release into culture medium (Figure 3). None of the con-
centrations of rOvUS or OVA tested caused an increase in
the percent of lysed cells during culture. Thus, rOvUS does
not inhibit proliferation through induction of cell death.


4500-
E
Q..
c 4000-
0

0
o
3000

E 2500
I-
' 2000

1500


4000

3000
2500
2000
0 2 4 6 8 10
Protein concentration (pg/iml)


0 50 100 150 200 250
rOvUS concentration (pg/ml)


Figure I
Inhibition of [3H]thymidine incorporation of PC-3 cells by
recombinant ovine uterine serpin (rOvUS). The inset graph is
provided to clarify the effects of rOvUS at lower concentra-
tions (< 8 ig/ml). Data represent least-squares means SEM.
Values that differ from untreated cells are indicated by aster-
isks (***P < 0.001).



Page 2 of 10
(page number not for citation purposes)


BMC Cell Biology 2008, 9:5








http://www.biomedcentral.com/1471-2121/9/5


1600-

1400-

3 1200-

i 1000-

S- 800 -

600 -

400 -

200-

0
Control 50 100 200 50 100 200
rOvUS (pg/ml) OVA (pg/ml)

Figure 2
Inhibition of proliferation of PC-3 cells by recombinant ovine
uterine serpin (rOvUS) as determined by ATP content/well.
Ovalbumin (OVA) was used as a negative control. Data rep-
resent least-squares means SEM. Means that differ from
untreated cells are indicated by symbols (tP < 0.1; **P <
0.01; ***P < 0.001).



DNA fragmentation (apoptosis)
The TUNEL procedure was used to test whether rOvUS
decreased cell proliferation by induction of DNA frag-
mentation characteristic of apoptosis and other forms of
cell death. Representative images of TUNEL labeled cells
are shown in Figure 4 and the average percent of cells that
were TUNEL positive is shown in Figure 5. Treatment of
PC-3 with either rOvUS or the control protein OVA did

30-

25

20

15-

o 10
0.
5-


Control 50 100 200 50 100 200
rOvUS (pg/ml) OVA (pg/ml)

Figure 3
Lack of cytotoxic effect of recombinant ovine uterine serpin
(rOvUS) on PC-3 cells was measured by the release of lac-
tate dehydrogenase. Ovalbumin (OVA) was used as a control
protein. Data represent least-squares means SEM.


Figure 4
Representative photomicrographs of PC-3 cells labeled using
the TUNEL procedure after 48 h of culture with either 100
(A) or 200 pig/ml (B) of rOvUS or 200 pig/ml of the control
protein ovalbumin (C). Cells in panel D were treated with
DNAse as a positive control.



not increase the percent of cells that were TUNEL positive
at either 24 or 48 h after treatment; the percentage of cells
that were TUNEL positive was low for all groups (< 5.7%).
The fact that rOvUS did not induce apoptosis makes the
action of this serpin distinct from that of two other serpins
that inhibit cell proliferation. Both maspin [61 and PEDF
[71 are proapoptotic serpins.

Interleukin-8 secretion
Interleukin-8 accumulation in the medium was measured
because of the autocrine effect of this chemokine on pros-
tate cell proliferation [26]. In addition, at least one class
of molecule that inhibits PC-3 cell proliferation, soy iso-
flavones, also reduces IL-8 secretion [27]. As shown in Fig-
ure 6, there was, however, no effect of rOvUS on
accumulation of IL-8 into conditioned cultured medium.
Thus, rOvUS does not block PC-3 cell proliferation
through inhibition of IL-8 secretion.

Cell cycle dynamics
Dynamics through the different phases of the cell cycle
were affected by the treatment of PC-3 cells with rOvUS.
Representative DNA histograms after treatment with vehi-
cle or 200 ig/ml rOvUS are shown in Figures 7A and 7B
while least-squares means + SEM for for results at 12 and
24 h after treatment are shown in Figures 7C and 7D,
respectively. At 12 h after addition of treatments, rOvUS
decreased (P < 0.1 and P < 0.05 for 100 and 200 lig/ml of
rOvUS respectively) the percent of cells in S phase and



Page 3 of 10
(page number not for citation purposes)


BMC Cell Biology 2008, 9:5







http://www.biomedcentral.com/1471-2121/9/5


15-


10-


5-
G)
0
. 0-
LU
z
I-
S15-

10
10-


B


Control 50


100 200 OVA


rOvUS (pg/ml)

Figure 5
Effect of recombinant ovine uterine serpin (rOvUS) on DNA
fragmentation (apoptosis) of PC-3 cells. Results show the
percent of TUNEL positive cells at 24 (A) and 48 (B) h after
addition of the treatments. Data represent least-squares
means SEM. Ovalbumin (OVA, 200 pig/ml) was used as
control protein.



increased (P < 0.01) the percent of cells in the G2/M phase
(Figure 6C). There was no effect of rOvUS on the percent
of cells in Go/G1. At 24 h after addition of treatment, 200
1ig/ml rOvUS increased the percent of cells in Go/G1 (P <
0.001), decreased the percent of cells in S phase (P <
0.01), and did not affect the percent of cells in G2/M phase
(Figure 6D).

Control of the cell cycle dynamics by rOvUS was also eval-
uated in a second cell type the peripheral blood lym-
phocyte. Representative DNA histograms for PHA-treated
lymphocytes are shown for control cells and cells treated
with 200 ig/ml rOvUS in Figures 8A and 8B, respectively
while least-squares means + SEM are shown in Figures 8C
and 8D. At both 72 (Figure 8C) and 96 h (Figure 8D) after
addition of PHA, rOvUS increased (P < 0.001) the propor-
tion of lymphocytes in the Go/G1 phase and decreased (P
< 0.05) the proportion of cells in the S phase. In contrast,
there was no effect of the control protein (OVA) on the
distribution of cells in the cell cycle.


25000


E
20000

o


C
o 10000


OVA
rOvUS


5000


0 50 100 150 200
Protein concentration (pg/ml)


Figure 6
Effect of recombinant ovine uterine serpin (rOvUS) on inter-
leukin (IL) 8 concentration in cell culture supernatants of
PC-3 cells. Ovalbumin (OVA) was used as control serpin.
Data represent least-squares means SEM.



These results indicate that OvUS block cell proliferation
through cell cycle arrest in both PC-3 cells and lym-
phocytes. The differences in specific stages at which the
cell cycle was blocked between PC-3 cells and lym-
phocytes is likely to be caused by differences in activation
and regulation pathways for these two cell types. Unlike
PC-3 cells, lymphocytes are arrested at Go until prolifera-
tion is induced by addition of PHA. Inhibition at points
in the cell cycle other than Go/G, are less likely to be seen
since few cells progress to later stages of the cell cycle. In
addition, it is possible that genetic mutations in PC-3 cells
compromise some potential regulatory mechanisms. In
particular, unlike lymphocytes, PC-3 cells lack functional
p53 [28] which causes cell cycle arrest at G1/S by inducing
p21cip1 that inhibits cyclin dependent kinases [29,30].

The mechanism by which OvUS inhibits cell cycle dynam-
ics is not understood. One serpin has been identified
which can affect cell cycle regulatory proteins. This serpin,
myeloid and erythroid nuclear termination stage-specific
protein (MENT), is a nuclear protein that inhibits cell pro-
liferation through interactions with a nuclear protein with
papain-like cysteine proteinase activity [31]. Inhibition of
the proteinase prevents degradation of the cell cycle pro-
tein Rb although antiproliferative effects may depend
more on other actions of MENT to mediate euchromatin
condensation in an Rb-independent manner [31]. In any
case, OvUS, is apparently without proteinase inhibitory
activity and is an extracellular protein that is unlikely to
achieve a nuclear localization. The antipepsin activity of


Page 4 of 10
(page number not for citation purposes)


11 06-


BMC Cell Biology 2008, 9:5







http://www.biomedcentral.com/1471-2121/9/5


A G /M 20%



Channels (FL2-A)

rOvUS-12 h O

G o/G 61% ..


S14%
A G /M 25%


Channels (FL2-A)


G01G1 G2IM S


Figure 7
Cell cycle dynamics of PC-3 cells as affected by recombinant ovine uterine serpin (rOvUS). Controls included vehicle (control)
and ovalbumin (OVA). Representative DNA histograms for analysis at 12 h after treatment with vehicle or 200 pig/ml rOvUS
are shown in panels A and B, respectively. The least-squares means for results of three separate assays are shown in panels C
and D for analysis at 12 h (C) and 24 h (D) after treatment. Bars with different superscripts differ (t P < 0.10, others at P <
0.05 or less).


uterine serpin is probably not biologically significant.
OvUS is a very weak inhibitor of pepsin C (a 35 and 8 fold
molar excess of OvUS was required to inhibit pepsin A
and C [12]) C and the binding of OvUS to pepsin is elec-
trostatic [15]. Moreover, pepsin shows an acidic pH opti-
mum and is unlikely to be involved in cell proliferation
under the conditions utilized.

A major point in the cell cycle regulated by OvUS is tran-
sition from Go/GI to S phase: rOvUS decreased the pro-
portion of cells in S phase in all experiments and
increased the proportion of cells in Go/GI at 24 h after
treatment for PC-3 cells and at both times examined for
lymphocytes. Ovine uterine serpin can bind to cell mem-
branes [32] and, perhaps, OvUS inhibits proliferation by
activation of signal transduction systems that inhibit tran-


sition from Go/GI to S phase or prevents pro-proliferative
molecules in culture medium from binding their recep-
tors. Experiments with Rp-8-Cl-cAMPS, a selective inhibi-
tor of cAMP-dependent type-I protein kinase A, indicated
that effects of OvUS on proliferation of PHA-stimulated
lymphocytes are not dependent on this kinase [24]. Stud-
ies to determine activation of other anti-proliferative sig-
nal transduction systems by OvUS are warranted.

Conclusion
The present study indicates that the mechanism by which
OvUS inhibits proliferation of PC-3 cells and lym-
phocytes involves cell cycle arrest and not, at least for PC-
3 cells, apoptosis, cytotoxicity or inhibition of IL-8 secre-
tion.



Page 5 of 10
(page number not for citation purposes)


BMC Cell Biology 2008, 9:5







http://www.biomedcentral.com/1471-2121/9/5


A PHA-Control-72 h '
100. a
Go/Gi 75% 80 b

E 60

.~ 40
S 25% b b
SG2/M 0% 0 20 -

Channels (FL2-A) a


B PHA-rOvUS-72 h 80b b
I-
60
S G/G1 91%
40 b b

Z.- 202 a






Go/G1 G2/M S
0 0 100 150 00
Channels (FL2-A) O S1O
GolG, G2/M S

Figure 8
Cell cycle dynamics of lymphocytes as affected by recombinant ovine uterine serpin (rOvUS). Controls included vehicle (con-
trol), phytohemagglutinin (PHA) and ovalbumin (OVA). Representative DNA histograms for analysis at 72 h after treatment
with PHA or 200 pig/ml rOvUS are shown in panels A and B, respectively. The least-squares means for results of four separate
assays are shown in panels C and D for analysis at 72 h (C) and 96 h (D) after treatment. Bars with different superscripts differ
(P < 0.05 or less).


Methods
Materials
The human prostate cancer (PC-3) cell line was purchased
from ATCC (Rockville, MD), the FreeStyleTM 293 expres-
sion medium, Dulbecco's Modified Eagle Medium Nutri-
ent Mixture F-12 (DMEM-F12) and 0.25% Trypsin-EDTA
were obtained from Gibco-Invitrogen (Carlsbad, CA), the
RQ1 RNase-free DNase and the CellTiter-Glo' Lumines-
cent Cell Viability Assay kit were obtained from Promega,
(Madison, WI), the DHLU Cell Cytotoxicity Assay kit was
from Anaspec (San Jose, CA) and the ELISA MAXTM Set
Deluxe kit for human IL-8 was obtained from BioLegend
(San Diego, CA). The in situ cell death detection kit [termi-
nal deoxynucleotidyl transferase-mediated dUTP nick end
labeling (TUNEL)] was purchased from Roche (Indianap-
olis, IN), the DNase-free RNase A was obtained from Qia-


gen (Valencia, CA), Precast Tris-HCl gradient Ready gels'
were from BioRad (Richmond, CA) and [H]lthymidine
(6.7 Ci/mmol) was from ICN (Irvine, CA). The Prolong
Antifade' kit was purchased from Molecular Probes
(Eugene, OR), Geneticin was from Research products
international (Mount Prospect, IL), Centricon filter
devices were from Millipore Corporation (Bedford, TX),
niquel Sepharose chromatography medium (high per-
formance) was from Amersham Biosciences (Piscataway,
NJ), fetal bovine and horse serum from Atlanta Biologi-
cals (Norcross, GA). Other reagents were obtained from
either Fisher (Pittsburg, PA) or Sigma-Aldrich (St. Louis,
MO).


Page 6 of 10
(page number not for citation purposes)


BMC Cell Biology 2008, 9:5







http://www.biomedcentral.com/1471-2121/9/5


Purification of rOvUS
The His-tagged rOvUS was purified from conditioned
medium of FreeStyleTM human embryonic kidney (HEK)-
293F cells (Gibco-Invitrogen, Carlsbad, CA) transfected
with a plasmid construct containing the gene for OvUS.
Details of the cell line are provided elsewhere [25]. Cells
were cultured continuously in selective medium [Free-
StyleTM 293 expression medium containing 700 lig/ml of
Geneticin] at 37 C in a humidified 8% (v/v) CO2 incuba-
tor according to the manufacturer's recommendations.
Conditioned medium containing rOvUS was diluted 1:1
(v/v) in binding buffer [20 mM sodium phosphate buffer,
35 mM imidazole, 0.3 M NaC1, pH 8.0] and loaded into a
nickel Sepharose column that was pre-equilibrated with
binding buffer. The His-tagged rOvUS was eluted with 20
mM phosphate buffer, 500 mM imidazole, 0.3 M NaC1,
pH 8.0, concentrated and buffer-exchanged into Dul-
becco's phosphate buffered saline (DPBS) using Centri-
con plus-20 concentration devices. Purity of the rOvUS
was assessed by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis using precast 4-15% polyacrylamide
Tris-HCl gradient gels. The protein concentration was
determined by Bradford assay [33] using bovine serum
albumin as standard.

For each experiment, rOvUS and the control protein,
OVA, were added to culture wells of PC-3 cells or lym-
phocytes dissolved in DPBS. The vehicle control included
addition of DPBS at the same volume as for rOvUS and
OVA. The actual volume of protein or vehicle added var-
ied between experiments but was generally 14-25 il and
never more than 50 il. Cultures were set up so that the
volume of DPBS was the same in all wells.

PC-3 cell culture
The PC-3 cell line was cultured continuously in Dul-
becco's Modified Eagle Medium Nutrient Mixture F-12
(DMEM-F12) supplemented with 10% (v/v) heat-inacti-
vated fetal bovine serum, 200 U/ml penicillin and 2 mg/
ml streptomycin at 37C in a humidified 50% (v/v) CO2
incubator. For the IL-8 experiment only, the medium was
modified to reduce the fetal bovine serum concentration
to 4% (v/v). For all the experiments, cells were cultured in
75 cm2 flasks until they reached 50-70% of confluence.
Cells were then trypsinized, centrifuged at 110 x g for 5
min and resuspended in fresh complete medium. Cell via-
bility was assessed by trypan blue exclusion and cell con-
centration was adjusted according to the requirements of
each experiment.

PHJThymidine incorporation by PC-3 cells
PC-3 cells (100 il) were plated overnight at a final con-
centration of 1 x 105 cell/ml in a 96-well plate. Afterwards,
various concentrations of rOvUS (0, 0.5, 1, 2, 4, 8, 16, 32,
64, 125 and 250 lig/ml) or vehicle were added to each


well in a total volume (including additional culture
medium) of 200 il. After 48 h of culture, 0.1 |iCi [3Hlthy-
midine in 10 il of culture medium were added. Cells were
harvested 24 h after [3H]thymidine addition onto fiber-
glass filters using a cell harvester (Brandel, Gaithersburg,
MD). Filters were counted for radioactivity using scintilla-
tion spectrometry (Beckman Coulter Inc., Fullerton, CA).
Each concentration of protein was tested in triplicate and
the experiment was performed in six different replicates
using a different batch of rOvUS for each replicate.

Cell proliferation based on ATP content
Aliquots of 50 tl of PC-3 cells (1 x 105 cells/ml) were cul-
tured for 24 h in a dark wall-clear bottom 96 well plate.
Then, treatments consisting of vehicle (DPBS) or three dif-
ferent concentrations (50, 100 and 200 lig/ml) of rOvUS
or a control protein (OVA) and culture medium added to
bring the final volume to 100 Al. Additional control wells
without cells were prepared to determine background. At
48 h after addition of treatments, ATP content per well
was determined using the CellTiter-Glo Luminescent Cell
Viability Assay kit according to the manufacturer's instruc-
tions. Briefly, 100 il of the CellTiter-Glo reagent were
added to each well, contents of the plate were mixed on a
shaker for 2 min and then incubated at room temperature
for 10 min. Chemiluminescence was quantified using a
multi-detection microplate reader (FLX-800, BioTek,
Winooski, VT). All treatments were performed in tripli-
cates and the assay was performed on three different occa-
sions using a different batch of rOvUS for each replicate.

Cytotoxicity assay
The assay was based on the release of lactate dehydroge-
nase into culture medium following loss of cell mem-
brane integrity accompanying cell death [34]. Procedures
for cell culture and treatments were similar to those
described for the ATP assay. At 48 h after addition of the
treatments, release of lactate dehydrogenase into the
medium was determined using the DHL' Cell Cytotoxic-
ity Assay kit following the vendor's instructions. Briefly,
the plate was equilibrated at room temperature for 20 min
before adding 10 Al of lysis solution or DPBS. To facilitate
cell lysing, the plate was placed on a shaker for 2 min. A
total of 50 |il lactate dehydrogenase assay solution was
then added to each well. After 10 min at room tempera-
ture, the reaction was stopped using 20 |il of the stop solu-
tion and the fluorescence intensity was measured using a
multi-detection microplate reader (FLX-800) with excita-
tion and emission wavelengths of 530-560 nm and 590
nm, respectively. Percent cytotoxicity was calculated by
dividing 100 x fluorescence from the unlysed cells by flu-
orescence of the lysed cells. For each assay, each treatment
was performed in six wells. The assay was replicated five
different times using a different batch of rOvUS for each
replicate.


Page 7 of 10
(page number not for citation purposes)


BMC Cell Biology 2008, 9:5







http://www.biomedcentral.com/1471-2121/9/5


TUNEL labeling
An aliquot of 100 il of PC-3 cells were cultured overnight
in chamber slides at a final concentration of 1 x 104 cells/
ml. Then, treatments consisting of vehicle (DPBS), 50,
100 or 200 ig/ml rOvUS, or 200 |tg/ml OVA were added
and additional culture medium added to produce a final
volume of 300 Al. After 24 and 48 h in culture with treat-
ments, cells were washed with PBS/PVP [100 mM sodium
phosphate pH 7.4, 0.9% (w/v) NaCl, 1 mg/ml polyvinyl
pyrrolidone] and fixed with 4% (w/v) paraformaldehyde
for 1 h at room temperature. Cells then were washed in
PBS/PVP and stored at 4 o C for the TUNEL (terminal deox-
ynucleotidyl transferase and fluorescein isothiocyanate-
conjugated dUTP nick end labeling) procedure.

For TUNEL labeling, fixed cells were incubated for 1 h at
room temperature with permeabilization solution [PBS,
pH 7.4, 0.1 (v/v) Triton X-100, 0.1% (w/v) sodium cit-
rate). After washing with PBS/PVP, slides were incubated
with 50 Al of TUNEL reaction mixture containing terminal
deoxynucleotidyl transferase and fluorescein isothiocy-
anate-conjugated dUTP, for 1 hour at 37 o C. Positive con-
trols were preincubated with RQ1 RNase-free DNase (50
U/ml) and negative controls were incubated without
transferase. Slides were washed with PBS/PVP, incubated
for 1 h with 50 pg/ml of RNase A and then for 30 min with
propidium iodide (2.5 pig/ml) at room temperature.
Slides were washed with PBS/PVP and Prolong Antifade
was used to mount coverslips. Samples were observed
using a Zeiss Axioplan 2 fluorescence microscope with
dual filter (Carl Zeiss, Inc., Gottingen, Germany). Percent
of cells with DNA fragmentation was determined by
counting the total number of nuclei and total number of
TUNEL-labeled nuclei at 10 different sites on the slide.
The experiment was performed using three different
batches of rOvUS.

Secretion of IL-8
PC-3 cells (100 il) were cultured in wells of a 96-well
plate overnight at a final concentration of 1 x 105 cells/ml.
Treatments were then added including vehicle (DPBS,
similar volume as for rOvUS and OVA treatments), and
three different concentrations of rOvUS and OVA (50,
100 and 200 ig/ml). The volume of each well was
brought to 200 il with culture medium. At 48 h after addi-
tion of treatments, cell culture supematants were col-
lected, centrifuged and stored at -20 C until ELISA for IL-
8. Treatments were performed in triplicate for each assay;
the experiment was repeated on three different occasions
using three different batches of the recombinant protein.
For the measurement of IL-8, the ELISA MAXTM Set Deluxe
kit for human IL-8 was used according to the manufac-
turer's instructions using 100 Al of conditioned medium.


Cell cycle analysis
PC-3 cells (100 il) were cultured in 4 well plates at a final
concentration of 4 x 105 cells/ml. After 24 h, treatments
consisting of vehicle, 100 and 200 lig/ml rOvUS, and 200
lig/ml OVA were added with additional culture medium
for a total volume of 400 |il. At 12 and 24 h after addition
of treatments, cells were collected by trypsinization and
washed with DPBS. Cells were fixed overnight in 70% (v/
v) ethanol at 4C, washed with DPBS and resuspended
with 500 il of staining solution [DPBS pH 7.4, 0.1% (v/
v) Triton X-100, 0.05 mg/ml DNase-ftee RNase A, 50 |ig/
ml propidium iodide]. Cells were then analyzed by flow
cytometry using a FACSort flow cytometer (Becton Dick-
inson, Franklin Lakes, NJ) and the red fluorescence of sin-
gle events was recorded at wavelengths of 488 nm
(excitation) and 600 nm (emission). Data were gated
using pulse width and pulse area to exclude doublets, and
the percent of cells present in each phase of the cell cycle
was calculated using ModFITLT V3.1 software (Verity Soft-
ware, Topsham, ME). The experiment was performed on
three occasions with five different batches of rOvUS.

For the sheep lymphocyte experiment, mononuclear cells
were purified by density gradient centrifugation from the
buffy coat of heparinized peripheral blood collected by
jugular venipuncture from non pregnant Rambouillet
ewes [35]. After removing red blood cells by incubation
with red cell lysis buffer (0.01 M Tris-HCl pH 7.5 contain-
ing 8.3 g/L of ammonium chloride), cell viability was
assessed by trypan blue exclusion, and concentration
adjusted to 4 x 106 cells/ml. Cells were then suspended in
a culture medium consisting of Tissue Culture Medium-
199 containing 5% (v/v) horse serum, 200 U/ml penicil-
lin, 0.2 mg/ml streptomycin, 2 mM glutamine and 10-5 M
3-mercaptoethanol and aliquots of 100 |il cells cultured in
4 well plates in the presence or absence of 4 lig/ml PHA
and with treatments of DPBS vehicle, 200 lig/ml rOvUS,
and 200 ig/ml OVA. Total culture volume was 400 pl.
After 72 and 96 h in culture at 37C in a humidified 5%
(v/v) CO2 incubator, lymphocytes were collected and
washed with DPBS. Thereafter, lymphocytes were fixed
and treated as described above. The experiment was per-
formed separately for lymphocytes from four different
sheep. Three different batches of rOvUS were tested for
each sheep.

Statistical analysis
Data were analyzed by least-squares means analysis of var-
iance using the General Linear Models Procedures of SAS
(SAS System for Windows, Version 9.0; SAS Institute,
Cary, NC, USA). Error terms were determined based on
calculation of expected mean squares with replicate con-
sidered random and other main effects considered fixed.
For the cytotoxicity and IL-8 data, orthogonal polynomial
contrasts were used to determine the linear and quadratic


Page 8 of 10
(page number not for citation purposes)


BMC Cell Biology 2008, 9:5








http://www.biomedcentral.com/1471-2121/9/5


effects of rOvUS and OVA. In other analysis, the pdiff
mean separation test of SAS was used to distinguish the
difference of various levels of a treatment.


Abbreviations
IL-8: Interleukin-8; Maspin: Mammary serine proteinase
inhibitor; MENT: Myeloid and erythroid nuclear termina-
tion stage-specific protein; OVA: Ovalbumin; PC-3: Pros-
tate cancer-3; PEDF: Pigment epithelium derived factor;
PHA: Phytohemagglutinin; TUNEL: Terminal deoxynucle-
otidyl transferase (TdT) and fluorescein isothiocyanate-
conjugated dUTP nick end labeling; US: Uterine serpin.


Authors' contributions
MBP carried out all of the studies described, participated
in experimental design and drafted the manuscript. PJH
conceived of the study, participated in its design and coor-
dination, and helped to draft the manuscript. Both
authors read and approved the final manuscript.


Acknowledgements
The authors want to thank Neal Bensen and Steve McClellan from the Flow
Cytometry Core Laboratory, Facility of the Interdisciplinary Center of Bio-
technology Research, University of Florida and to Luciano Bonilla for help
with the sheep. Thanks are also extended to the following University of
Florida people for providing access to laboratory equipment: Dr. Owen
Rae and Shelley Lanhart from the Department of Large Animal Clinical Sci-
ences, Dr. Lori Warren and Jan Kivilpeto from the Department of Animal
Sciences, and Dr. Pushpa Kalra from the Dept. of Physiology and Functional
Genomics.

References
I. Irving JA, Pike RN, Lesk AM, Whisstock JC: Phylogeny of the ser-
pin superfamily: Implications of patterns of amino acid con-
servation for structure and function. Genome Res 2000,
10:1845-1864.
2. Van Gent D, Sharp P, Morgan K, Kalsheker N: Serpins: structure,
function and molecular evolution. Int J Biochem Cell Biol 2003,
35:1536-1547.
3. Law RHP, Zhang Q, McGowan S, Buckle AM, Silverman GA, Wong
W, Rosado CJ, Langendorf CG, Pike RN, Bird PI, Whisstock JC: An
overview of the serpin superfamily. Genome Biol 2006, 7:216.
4. Pemberton PA, Stein PE, Pepys MB, Potter JM, Carrell RW: Hor-
mone binding globulins undergo serpin conformational
change in inflammation. Nature 1988, 336:257-258.
5. Sauk JJ, Nikitakis N, Siavash H: Hsp47 a novel collagen binding
serpin chaperone, autoantigen and therapeutic target. Front
Biosci 2005, 10:107-118.
6. Sheng S: A role of novel serpin maspin in tumor progression:
the divergence revealed through efforts to converge. j Cell
Physiol 2006, 209:631-635.
7. Fernandez-Garcia NI, Volpert OV,Jimenez B: Pigment epithelium-
derived factor as a multifunctional antitumor factor. j Mol
Med 2007, 85:15-22.
8. Moffatt J, Bazer FW, Hansen PJ, Chun PW, Roberts RM: Purifica-
tion, secretion and immunocytochemical localization of the
uterine milk proteins, major progesterone-induced proteins
in uterine secretions of the sheep. Biol Reprod 1987, 36:419-430.
9. Ing NH, Roberts RM: The major progesterone-induced pro-
teins secreted into the sheep uterus are members of the ser-
pin superfamily of protease inhibitors. J Biol Chem 1989,
264:3372-3379.
10. Malathy PV, Imakawa K, Simmen RC, Roberts RM: Molecular clon-
ing of the uteroferrin-associated protein, a major progester-
one-induced serpin secreted by the porcine uterus, and


expression of its mRNA during pregnancy. Mol Endocrinol 1990,
4:428-440.
I I. Leslie MV, Hansen PJ, Newton GR: Uterine secretions of the cow
contain proteins that are immunochemically related to the
major progesterone-induced proteins of the sheep uterus.
Domest Anim Endocrinol 1990, 7:517-526.
12. Mathialagan N, Hansen TR: Pepsin-inhibitory activity of uterine
serpins. Proc Natt Acad Sci 1996, 93:13653-13658.
13. Tekin S, Padua MB, Newton GR, Hansen PJ: Identification and
cloning of caprine uterine serpin. Mol Reprod Dev 2005,
70:262-270.
14. Peltier MR, Raley LC, Liberles DA, Benner SA, Hansen PJ: Evolution-
ary history of the uterine serpins. j Exp Zool 2000, 288:165-174.
15. Peltier MR, Grant TR, Hansen PJ: Distinct physical and structural
properties of the ovine uterine serpin. Biochim Biophys Acta
2000, 1479:37-51.
16. Liu WJ, Hansen PJ: Progesterone-induced secretion of dipepti-
dyl peptidase-IV (cell differentiation antigen 26) by the uter-
ine endometrium of the ewe and cow that costimulates
lymphocyte proliferation. Endocrinology 1995, 136:779-787.
17. Hansen PJ: Regulation of uterine immune function by proges-
terone-lessons from the sheep. J Reprod Immunol 1998, 40:63-79.
18. Segerson EC, Moffatt RJ, Bazer FW, Roberts MR: Suppression of
phytohemagglutinin-stimulated lymphocyte blastogenesis
by ovine uterine milk protein. Biol Reprod 1984, 30:1175-1186.
19. Hansen PJ, Segerson EC, Bazer FW: Characterization of immu-
nosuppressive substances in the basic protein fraction of
uterine secretions of pregnant ewes. Biol Reprod 1987,
36:393-403.
20. Skopets B, Hansen PJ: Identification of the predominant pro-
teins in uterine fluids of unilaterally pregnant ewes that
inhibit lymphocyte proliferation. Biol Reprod 1993, 49:997-1007.
21. Skopets B, Liu WJ, Hansen PJ: Effects ofendometrial serpin-like
proteins on immune responses in sheep. Am J Reprod Immunol
1995, 33:86-93.
22. Peltier MR, Liu WJ, Hansen PJ: Regulation of lymphocyte prolif-
eration by uterine serpin: interleukin-2 mRNA production,
CD25 expression and responsiveness to interleukin-2. Exp
Biol Med 1999, 223:75-8 1.
23. Liu WJ, Hansen PJ: Effect of the progesterone-induced serpin-
like proteins of the sheep endometrium on natural-killer cell
activity in sheep and mice. Biol Reprod 1993, 49:1008-1014.
24. Tekin S, Padua MB, Brad AM, Hansen PJ: Antiproliferative actions
of ovine uterine serpin. Am J Reprod Immunol 2005, 53:136-143.
25. Tekin S, Padua MB, Brad AM, Rhodes ML, Hansen PJ: Expression
and properties of recombinant ovine uterine serpin. Exp Biol
Med 2006, 231:1313-1322.
26. Araki S, Omori Y, Lyn D, Singh RK, Meinbach DM, Sandman Y,
Lokeshwar VB, Lokeshwar BL: Interleukin-8 is a molecular
determinant on androgen independence and progression in
prostate cancer cells. Cancer Res 2007, 67:6854-6862.
27. Handayani R, Rice L, Cui Y, Medrano TA, Samedi VG, Baker HV,
Szabo NJ, Shiverick KT: Soy isoflavones alter expression of
genes associated with cancer progression, including inter-
leukin-8, in androgen-independent PC-3 human prostate
cancer cells. j Nutr 2006, 136:75-82.
28. Isaacs WB, Carter BS, Erwing CM: Wild-type p53 supresses
growth of human prostate cancer cells containing mutant
p53 alleles. Cancer Res 1991, 51:4716-4720.
29. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ: The p21
Cdk-interacting protein Cip I is a potent inhibitor of G I cyc-
lin dependent kinases. Cell 1993, 75:805-816.
30. Dulix V, Kaufmann WK, Wilson SJ, TIsty TD, Lees E, Harper JW,
Elledge SJ, Reed SI: p53-dependent inhibition of cyclin-depend-
ent kinase activities in human fibroblasts during radiation-
induced GI arrest. Cell 1994, 76:1013-1023.
31. Irving JA, Shushanov SS, Pike RN, Popova EY, Bromme D, Coetzer
THT, Bottomley SP, Boulynko IA, Grigoryev SA, Whisstock JC:
Inhibitory activity of a heterochromatin-associated serpin
(MENT) against papain-like cysteine proteinases affects
chromatin structure and can regulate cell proliferation. j Biol
Chem 2002, 277:13192-13201.
32. Liu WJ, Peltier MR, Hansen PJ: Binding of ovine uterine serpin to
lymphocytes. Am J Reprod Immunol 1999, 41:428-432.





Page 9 of 10
(page number not for citation purposes)


BMC Cell Biology 2008, 9:5








http://www.biomedcentral.com/1471-2121/9/5


33. Bradford MM: A rapid and sensitive method for the quantita-
tion of microgram quantities of protein utilizing the princi-
ple of protein-dye binding. Anal Biochem 1976, 78:248-254.
34. Decker T, Lohmann-Matthes ML: A quick and simple method for
the quantitation of lactate dehydrogenase release in meas-
urements of cellular cytotoxicity and tumor necrosis factor
(TNF) activity. j Immunol Methods 1988, I 15:61-69.
35. Tekin S, Hansen PJ: Natural-killer like cells in the sheep: Func-
tional characterization and regulation by pregnancy-associ-
ated proteins. Exp Biol Med (Maywood) 2002, 227:803-811 I.


Page 10 of 10
(page number not for citation purposes)


Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours you keep the copyright
Submit your manuscript here: BioMedcentral
http://www.biomedcentral.com/info/publishing adv.asp


BMC Cell Biology 2008, 9:5




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - - mvs