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Characterization of Apoptosis Pathways and Cytoskeleton Structures in HTR-8 Immortalized Human Placental Cells Exposed to Porphyromonas gingivalis and Benzo[a]pyrene.

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Characterization of Apoptosis Pathways and Cytoskeleton Structures in HTR-8 Immortalized Human Placental Cells Exposed to Porphyromonas gingivalis and Benzo[a]pyrene.
Creator:
Torres, Diogo
Shiverick, Kathleen ( Mentor )
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Gainesville, Fla.
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University of Florida
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English

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Characterization of Apoptosis Pathways and Cytoskeleton Structures in HTR-
8 Immortalized Human Placental Cells Exposed to Porphyromonas
gingivalis and Benzo[a]pyrene.

Diogo Torres


ABSTRACT


Proper trophoblast differentiation is critical in normal placental development. Physical or chemical agents

that interfere with the key trophoblast cell signaling pathways may induce miscarriages21, preeclampsia4, pre-

term birth, and fetal growth restrictions8,9,12,25. Porphyromonas gingivalis, an anaerobic periodontal pathogen,
may have an underlying role in these key pathways. Recent human studies have detected the presence of

P. gingivalis sensitized T-cells in the peripheral blood of patients with oral infections15,20In addition, animal
studies provide evidence that P. gingivalis impairs placental functioning by inducing local immune responses

and altering placental anti-inflammatory pathways18. My study used fluorescent microscopy to demonstrate
P. gingivalis infection in HTR-8 immortalized human placenta extravillous trophoblast cells. Cells exposed to

P. gingivalis for 24 hours showed a decrease in phospho-p53 expression, while Bcl-2 and total p53 levels

remained unchanged relative to control.



In a second protocol, HTR-8 cells were also treated with benzo[a]pyrene, a carcinogen found in cigarette

smoke. Placental cells exposed to benzo[a]pyrene are prone to irregularities that can cause spontaneous

abortions and fetal growth retardation. RL95-2 human uterine endometrial cells exposed to benzo[a]pyrene

showed actin disaggregation, impaired cellular attachment and invasion5. In my study,
immunocytochemistry analysis was used to examine the effects of the carcinogen on HTR-8 cell attachment.

Control cells had continuous membrane actin filaments, whereas 10pM and 20pM benzo[a]pyrene treated cells

had actin aggregates.



INTRODUCTION


Porphyromonas gingivalis


Periodontal disease is a chronic oral infection caused by oral pathogens. Maternal periodontal disease occurs in

5-40% of pregnant women and is associated with second-trimester miscarriage21, preeclampsia8, pre-term
birth, and fetal growth restriction, possibly triggered by placental inflammatory responses following utero-

placental exposure to oral pathogens8,9,12,18'25. Among oral pathogens, Porphyromonas gingivalis is a





gram-negative, anaerobic bacterium prominent in subgingival plaque31. P. gingivalis is a major contributor to

chronic inflammation of the gingivia26 and may be an underlying factor in pre-term births25. Recent human

studies have detected the presence of P. gingivalis sensitized T-cells in the peripheral blood of patients with

oral infections15,20.



Recent animal studies provide evidence that P. gingivalis impairs placental functioning by inducing local

immune responses and altering placental anti-inflammatory pathways. Successful pregnancies consist of an

important Thl/Th2 cytokine placental balance. P. gingivalis infection in pregnant mice has been associated

with placental Thl/Th2 imbalances18.



Benzo[a]pyrene


P. gingivalis is not the only mutagen altering placental protein expression. Benzo[a]pyrene, a

carcinogenic polyaromatic hydrocarbon found in cigarette smoke, also alters placental protein levels. Exposure

to benzo[a]pyrene is associated with a reduction in epidermal growth factor receptors (EGF-R) in human

placental choriocarcinoma cells, resulting in uncoordinated expression of the genes responsible for normal

trophoblast proliferation, differentiation, and invasiveness. Thus, benzo[a]pyrene is a cigarette smoke toxicant

that likely interferes with normal placental development, resulting in spontaneous abortions and fetal

growth retardation5.



Benzo[a]pyrene also has adverse effects on some of the genes directly responsible for a normal pregnancy.

Human uterine endometrial cells exposed to BaP exhibited significant decreases in plasma membrane EGF-

Rs, decreasing cell attachment. Fluorescent experiments also showed actin degradation in human uterine

endometrial cells. Since actin filaments form the cellular cytoskeleton, they are crucial for cellular morphology

and cell-cell interactions5.



Trophoblast Differentiation


Following fertilization, human placental precursor cells appear on the outer layer of the blastocyst. These

trophoblast cells are critical for proper placental development. Regulated secretion of the hormone human

chorionic gonadotropin (hCG) assures proper implantation conditions. Once endometrium conditions are suitable,

the trophoblast will attach to the uterine lining13. Subsequently, placental differentiation commences along either

the villous or the extravillous trophoblast pathway. Extravillous trophoblasts are responsible for securing

attachment of the chronic villi in the uterus3. On the other hand, villous trophoblast coat these villi and assists

in nutriment transport. Once differentiated, the placenta serves as a highly vascular, cellular barrier between

the mother and fetus. The placenta mediates pregnancy hormone production, immune protection of the fetus,

and filtration of harmful substances. It is also directly involved in the transfer of nutrients and gases to the fetus

from the mother. Due to its importance, proper placental development is critical. Altering trophoblast

differentiation can be detrimental to the fetus, neglecting it of proper nourishment13.

Preliminary experiments in Dr Shiverick's laboratory were conducted using an in vitro HTR-8 immortalized






human placenta extravillous trophoblast cells. My experiments show P. gingivalis invasion in this established

human placental cell line, which is a model for early placental development. In addition, my experiments have

shown HTR-8 cell susceptibility to benzo[a]pyrene.



Cell Cycle Regulatory Pathways


Normal placental development is balanced by cell proliferation and cell death. p53, the "ultimate suppressor

gene," codes for key regulatory proteins involved in cellular growth27. Failure to induce p53 proteins after

DNA damage may lead to cancerous growth. Once induced, p53 proteins control cell arrest and

apoptosis10,28. Suppressed growth permits DNA repair; if the cell is unable to correct the damage it enters

apoptosis. p53 expression is also regulated through phosphorylation. Once phosphorylated, the p53 pathway

is activated28.



p53 also mediates cell growth through other regulatory proteins. Apoptosis triggers release of cyctochrome C

from the inner mitochondrion membrane. Induction or inhibition of cyctochrome C is regulated by Bcl-2.

p53 activation has been linked to the transcriptional repression of the antipoptic gene Bcl-2; thus, high levels of Bcl-

2 protein suppresses apoptosis24.



p53 also regulates expression of p21CIP1, also known as cyclin-dependent kinase inhibitor 1A or CDKN1A,

which codes for a cyclin-dependent kinase inhibitor linked to gap 1 (Gl) of the cell cycle. When expressed,

p21CIP1 inhibits Cdk production28.



METHODS AND OBSERVATIONS


Cell cultures


HTR-8/SVneo cells were derived from cultures of human first-trimester placental trophoblast cells with SV40 large

T antigen. This cell line was provided by Dr C. Graham in Canada. Cells were plated and grown to confluence for

48 hours in a 5% CO2 atmosphere at 370C. Culture medium (RPMI-1640 [Invitrogen, Carlsbad, CA] contained

5% heat-inactivated fetal bovine serum [Hyclone, Logan, UT]. 100 mm cell plates were used for preparation

cell lysates and membranes for Western blot analysis. For microscopic analysis, cultured cells were grown on

either plastic or glass microscope chamber slides. It was assumed that cell count doubled daily.



Bacterial strains and P. gingivalis treatment


P. gingivalis strain ATCC 33277 was cultured anaerobically for 24 h at 370C in trypticase soy. Bacteria were

harvested after centrifugation at 6000 g and 40C for 10 min. They were prepared in HTR-8 cell medium at

random OD600 values.





Cell plates were cultured to confluence (80-90%) for 48 hours and then infected with P. gingivalis. Fresh media

was added to the plates and every HTR-8 cell was infected with 100 P. gingivalis bacteria. The cells were

then returned to a 5% CO2 incubator at 370C. Treatment duration varied for each sample. Cells were cultured for

2 hours in the absence or presence of P. gingivalis, then collected immediately for lysates and membranes; or

were washed with PBS and cultured an additional 22 hours in medium without P. gingivalis and then collected.

All samples were collected using Brandel cell harvester.



Plastic and glass chamber slides were first treated with 0.5ml/chamber of Poly L-Lysine for 5 minutes to enhance

cell attachment. Poly L-Lysine was then removed, and the slides were air-dried under the hood. Cells were

then cultured to confluence (80-90%) for 48 hours. New media was added and cells were exposed to P. gingivalis

for 30 minutes.



Benzo[a]pyrene treatment


60-80% confluent HTR-8 cells were treated with varying stock solutions of benzo[a]pyrene prepared in DMSO

(100 mm cell plates or chamber slides). Negative controls were treated with DMSO vehicle only.



Cell lysate preparation


HTR-8 media was discarded from each cell plate. PBS washes minimized media proteins which may interfere in

the examination of the protein of interest. A solution consisting of lysis buffer and protease stocks was prepared

and vortexed. Proteases are essential in protein protection during cellular explosion; lysis buffer contains

protein denaturing detergents. Thus, imL of the solution was applied to each cell sample prior to collection

using sterile Brandel cell harvesters. Microfuge tubes (1.5 mL) were prepared and labeled according to

treatments. Once scrapped thoroughly, cell samples were pipetted into appropriate microfuge tube (since

absorption readings were usually on the low side, cell samples were combined; ex. Three 24-hour cell plates

were scraped and pipetted into the same microfuge tube). A 3 ml syringe was used to disrupt any

inappropriate lysate chunks. Once in solution, the cell lysate samples were placed on ice for 30 minutes. The

samples were then removed from ice, vortexed, and centrifuged at 4?C. Supernatants were then transferred to

new microfuge tubes and stored at -80?C.



Cell membrane preparation


Membrane samples were prepared similarly to cell lysates with the exception of protease stocks. One milliliter of

PBS was pipetted onto each cell plate prior to collection. Cell samples were inserted in 1.5 ml microfuge tubes

and placed on ice. A freezing solution of ethyl alcohol and dry ice was prepared under the hood. After the

vapors cleared, samples were frozen for 1 minute. Sequentially, the samples were thawed in room

temperature water. The samples were then centrifuged in a 4?C room for 5 minutes. The supernatants were

poured out, leaving the membranes as pellets. Five-hundred microliters of PBS was added to each tube. The

samples were then vortexed and centrifuged for 5 minutes at 4?C. PBS (300 pl) was added to each tube, and






the samples were stored at -80?C.


BCA protein assay for lysate preparation


Total lysate protein concentration was measuring using a Bovine Serum Albumen (BSA) standard curve. Six

different glass tubes were prepared with different BSA concentrations: 10 pl, 20 pl, 30 pl, 40 pl, and 50 pl. Cell

lysate samples were removed from the -80?C freezer, and three test tubes were prepared for each sample (10 pl,

20 pl, 30 pl of cell lysate). Lysis buffer was added to each tube to bring all volumes to 50 pl. Once each tube

had equal volumes, 1 ml of a 1:50 diluted solution of protein assay reagent A and protein reagent B was added

to each tube. Samples were then covered with saran wrap and incubated in a 37?C water bath for 30

minutes. Samples were then transferred to plastic cuvettes and inserted into a spectrophotometer. Absorbance

of each sample was measured at 562A. Results were then entered into Microsoft Excel and a regression line

was computed. A regression formula provided by Excel was used to find protein concentrations of each sample.

By multiplying these concentrations by dilution factors, concentrations in pg/ml of the original samples were derived.



BCA protein assay for membrane preparation


Total membrane protein concentration was also measured using a BCA protein assay. PBS was used instead of

lysis buffer.



Loading lysate and membrane samples


In order to provide an optimum environment for the proteins, a buffer solution was prepared. Solution buffer (900

pl) was added to 100 pl of 2-Mecaptoethanol. The solution buffer was used to trace protein progress

during electrophoresis. It also contained glycerol, responsible for weighing down light proteins while in solution.

2-Mercaptoethanol, characterized by its strong sulfur smell, reduced sulfide bridges allowing the proteins to uncoil.



Microfuge tubes were then prepared and labeled according to sample type. Equal amounts of cell lysate and

loading buffer were pipetted into each tube (70 pl and 70 pl loading buffer). Membrane samples were dispersed

using a 3 ml syringe. All samples were vortexed and placed in boiling water for three minutes. Calculations were

then conducted to determine how much of each sample should be used to yield equal protein concentrations.



Using a thin pipette, 10 ml of Sigma molecular-weight marker was inserted into the first well. Samples were

loaded individually into their respective wells.



Gel Preparation


Dividers and glass sheets were disinfected with ethanol. Sandwich-like devices were made by placing a small

sheet on top of a big sheet, using a divider on each side for separation. Using a regular ml pipette, each

sandwich was filled with running gel (acrylamide, 20% SDS, DH20, running buffer, 0.28% APS) and solidified for






20 minutes. Once the running gel polymerized, stacking gel (acrylamide, 20% SDS, DH20, stacking buffer,

0.28% APS) was pipetted until 1 cm from the top of the sandwich. White combs were inserted for well formation

and the stacking gel was given 20 minutes to polymerized. Gel thickness depended on the molecular weight of

the proteins to be analyzed.



Electrophoresis


Once the stacking gel polymerized, the white combs were removed, and chamber buffer was used to prevent the

gels from drying out. Samples were loaded into the wells using a pipette and electrophoresed at 50 V for 1 hour.

The 50 V electric current successfully forced the proteins across the gel and separated the molecules based

on molecular size.



Transferring proteins from gels to membranes


A membrane for each gel was prepared using membrane paper (4.5 cm x 7 cm). Six filters were also prepared

from filter paper for each gel (6 cm x 9 cm). Each gel was inserted into a sandwich holder, supported by a

sponge and three filters on each side. The sandwiches were then placed into electrode holders with the black side

of the sandwich facing the black side of the electrode holder. The samples were electrophoresed at 4?C overnight

at 30 V.



Membranes were removed from electrode holders and rinsed with distilled water. They were then briefly stained

with Ponceau Red to assure uniform protein loading and transfer. Once confirmed, Ponceau Red was returned to

its original container and the membranes were rinsed with distilled water. Membranes were then washed with

tris buffered saline (TBS).



Imm unostaining


A 5% nonfat milk solution was prepared by mixing 50 ml of TBS and Tween (TTBS) with 2.5 g of nonfat milk.

In order to block protein colonies which might attack the primary antibody, each membrane was incubated in 10

mL of 5% nonfat milk solution for one hour on a rocker. The milk was then washed off and the membranes

were rinsed with TBS. The membranes were then incubated in their respective diluted antibody for one

hour (1:1000). Control membranes were incubated in their respective monoclonal IgG serum. Membranes were

then rinsed with TBS+Tween (contains detergents) and incubated in secondary antibodies (1:2000) for one hour.

The nonfat milk was then discarded, and each membrane was thoroughly rinsed (2x) with TTBS on the

shaker. Membranes were then rocked in TBS overnight at 4?C.



Immunocytochemistry: fluorescent staining


Fluorescent staining washes followed the same restrictions as above: two brief washes followed by 1 thru 5

minute wash on the shaker. Culture medium was removed from each chamber slide. Cells were washed 2x with





HTR-8 cell medium and then 3x with PBS. They were then fixed with fresh 4% Paraformaldehyde + PBS solution

for 6 minutes under the hood with their slide-covers off (Note: a stock solution of 8% PFA was diluted using 2X

PBS). The cells were washed 3x in PBS; the first 2 washes were brief and the 3rd was for 5 minutes on the

shaker. 0.1% Triton-X-100 in PBS or acetone was used to permeablize the cells. Acetone was only used on

glass slides. When used, it was prepared at -25?C; cells were incubated for 3 minutes at -20?C.

Following permeablization, the cells were washed 3x in PBS; the first 2 washes were brief and the 3rd was for

5 minutes on the shaker. Nonspecific binding sites were blocked by incubating with PBS + 1% Bovine

Serum Albumen (BSA) for 1 hour at 24?C. Respective primary antibody incubation lasted 1 hour at 24?C.

The antibodies were diluted in PBS + 1% BSA. Negative controls were incubated in PBS + 1% BSA alone. Fresh

PBS + 0.1% Tween-20 was prepared for cell washes (3x). Respective secondary antibody incubation lasted 1 hour

at 24?C. Tubulin and P. gingivalis antibodies were prepared in solution with PBS + 1% BSA. Phalloidin (5 pL/

slide) was heated in a disposable culture tube for 5 minutes at 75?C (evaporates the methanol). A solution was

then prepared using PBS + 1% BSA. Chamber slides were treated with phalloidin 30 minutes after

secondary antibody incubation began (Note: due to secondary antibody photosensitivity, slides were covered

in aluminum foil during the entire incubation). The cells were then washed 3x in PBS + 0.1% Tween-20; the first

2 washes were brief and the 3rd was for 5 minutes on the shaker. Chambers were removed, and cover-slips

were mounted using ProLong gold anti-fade reagent (100pl/chamber). Slides were left in the incubator (5% C02,

370C) overnight.Cover-slip edges were covered with nail polish and stored in aluminum foil at -20?C.



RESULTS


Cell cycle regulatory proteins: Western immunoblot blot



Western blot results of HTR-8 cells exposed to P. gingivalis bacteria


Lysate/Membrane samples 1: collected 2/9/06 and 2/10/06

Lysate/Membrane samples 2: collected 4/13/06 and 4/14/06

Lysate/Membrane samples 3: collected 6/5/06 and 6/6/06



P. gingivalis treatment had no effects on p21 protein expression. As shown in Figure 1, there was no change

in protein expression for the 2-hour samples. Samples cultured for an extra 24 hours showed an increase in

p21 expression (Fig. 1A). However,protein expression decreased in the 24 hour samples (Fig. 1B); thus,

no conclusions were drawn.





A Cell Lvsates 1
C Pg C Pg
2h 2h 24h 24h


moo


B Cell Lysates 2
C Pg C Pg
2h 2h 24h 2h


Figure 1. Western immunoblots of HTR-8/SVneo whole cell lysates probed with p21 antibody
(Santa Cruz, mouse anti-human monoclonal). Cells in 100mm dishes were cultures for 2 hours in
the absence (C) or presence (Pg) of P. gingivalis (100 MOI), then collected immediately for lysates (2
h); or washed with PBS and cultured an additional 22 hours in medium without P. gingivalis and
collected (24 h). p21 was detected at the expected weight of approximate 20 kDA.


Western immunoblot analysis (Fig. 2A-C) of the p53 pathway showed a decrease in phospo-p53 expression
following P. gingivalis treatment. Protein expression was up-regulated in samples cultured for an extra 24
hours. Total p53 concentration remained the same (Fig.3A,B). Bcl-2 protein (Fig. 4A,B) concentration showed
no signs of change.


A Cell Lysats 1
C Pg C Pg
2h 2h 24h 24h

v- - -


B Cell Lyates 2
C Pg C
2h 2h 24h


C
C
2h


Cell Lysates 3
Pg C
2h 24h


m -// -


Figure 2. Western immunoblots of HTR-8/SVneo whole cell lysates probed with phosphorylated-
p53 (ser15, PhosphoDetect rabbit anti-human polyclonal). Cells in 100 mm dishes were cultures for
2 hours in the absence (C) or presence (Pg) of P. gingivalis (100 MOI), then collected immediately
for lysates (2 h); or washed with PBS and cultured an additional 22 hours in medium without P.
gingivalis and collected (24 h). phosp-p53 was detected at the expected weight of approximate 37 kDA.


Cell Lykates 1 B
C Pg C Pg
2h 2h 24h 24h


Cell Lysates 2
C Pg C Pg
2h 2h 24h 2h
VvW


Pg
24h






Figure 3. Western immunoblots of HTR-8/SVneo whole cell lysates probed with p53 antibody (Ab-
6, mouse anti-human monoclonal). Cells in 100 mm dishes were cultures for 2 hours in the absence
(C) or presence (Pg) of P. gingivalis (100 MOI), then collected immediately for lysates (2 h); or
washed with PBS and cultured an additional 22 hours in medium without P. gingivalis and collected
(24 h). p53 was detected at the expected weight of approximate 52 kDA.


Cell Lysates 1
C Pg C Pg
h 2h 24h 24h

*a


Cell Lysates 2
Pg C Pg
2h 24h 2h


Figure 4. Western immunoblots of HTR-8/SVneo whole cell lysates probed with Bcl-2 antibody
(Santa Cruz mouse anti-human monoclonal). Cells in 100mm dishes were cultures for 2 hours in
the absence (C) or presence (Pg) of P. gingivalis (100 MOI), then collected immediately for lysates (2
h); or washed with PBS and cultured an additional 22 hours in medium without P. gingivalis and
collected (24 h). Bcl-2 was detected at the expected weight of approximate 27 kDA.



P. gingivalis cell attachment


Figure 5 shows no change in Toll Like Receptor 4 expression in cell lysates. Although TLR-4 showed a
clear immunoreactive band at its expected weight of 95 kDA, there was also non-specific cross-reactivity with
the antibody (not shown). There was no detection of TLR-4 in membrane preparations (Fig. 5).


Cell Lysates 2
C Pg C Pg
h 2h 24h 24h


Membranes 2
Pg C Pg
2h 24h 24h


Figure 5. Western immunoblots of HTR-8/SVneo whole cell lysate and cell membrane
preparations probed with TLR-4 (Santa Cruz, rabbit polyclonal) antibody. Cells in 100 mm dishes
were cultured for 2 hours in the absence (C) or presence (Pg) of P. gingivalis (100 MOI), then
collected immediately for lysates (2 h); or washed with PBS and cultured an additional 22 hours


� -"NNW MM*MMW --- '101





in medium without P. gingivalis and collected (24 h). TLR-4 was detected at the expected weight

of approximate 95 kDA. Note: there was plenty of P. gingivalis cross-reactivity with anti-TLR-4 at

65kDA (not shown).



P. gingivalis invasion


Fluorescent microscopy confirmed P. gingivalis uptake in HTR-8 cells. Bacteria were found inside the cell and

in extracellular spaces (Fig. 6).


Figure 6. Fluorescent immunocytochemical analysis of P.gingivalis invading HTR-8/SVneo cells.

Cells were plated on poly-L lysine coated glass chamber slides with four-wells and treated for 30

minutes with P.gingivalis. Cells were fixed in freshly prepared 4% Paraformaldehyde and incubated

in primary and secondary antibodies: 33277 rabbit polyclonal anti-P. gingi (1:500), mouse

monoclonal anti-tubulin (1:400), anti-mouse rhodamine TRITC secondary anti-mouse (1:300),

and fluorescein FITC secondary anti-rabbit (1:500). Green fluorescein specs represent clustered

P. gingivalis incapable of cellular invasion. Rhodamine tubulin spread the entire cell.



Cell cycle regulatory proteins and actin & tubulin: fluorescent immunocytochemistry


Using fluorescein labeled phalloidin and rhodamine labeled tubulin, cytoskeletal structures of HTR-8 cells

were examined. Control cells showed abundant continuous subcortical actin filaments. Conversely, 10 pM benzo

[a]pyrene treated cells, showed clear signs of actin degradation along the cellular membrane. Subcortical

actin aggregates were also present in the cells treated with 20 pM benzo[a]pyrene.


DISCUSSION







Studies have shown that over 23% of women aged 30 to 54 suffer from periodontitis, the direct destruction

of periodontal tissue.29 Caused by oral pathogens such as P. gingivalis, periodontitis has been positively

correlated with pregnancy complications in 6 out 13 studies involving pregnant mothers suffering from

periodontal disease6,9,11,22,25,30. Studies also indicate a link between periodontal infection and placental-

fetal exposure. Oral pathogens may trigger fetal inflammatory responses ultimately leading to preterm birth.

The bacteria gain access to the placenta via the circulatory system8,9,21.



The p53 pathway is critical for proper placental development. Although total p53 concentration was unaffected

upon P. gingivalis treatment, phosphorylated p53 levels were reduced. Once phosphorylated, the p53 pathway

is activated and cellular proliferation is suppressed. Reduced phospo-p53 expression may prevent HTR-8

cells infected with P. gingivalis from entering cellular arrest or apoptosis. As a result, unrepaired cellular DNA

damage may accumulate. In contrast, HTR-8 cells cultured for an extra 24 hours in fresh media (no P.

gingivalis) showed normal phosp-p53 levels; thus, it may be possible that placental cells recover normal

protein expression with time.



Treatment with P. gingivalis had no effect on p53 and Bcl-2 expression levels. This observation is consistent with

the association of p53 induction and the transcriptional repression of the antipoptic gene Bcl-2. Since Bcl-2

protein levels were constant, P. gingivalis may not be an apoptotic inducer.



In contrast, p21 immunoblots were inconsistent. Lack of consistency in protein expression may be attributed to

the anti-body used or varying effects of P. gingivalis treatment.



Fluorescent immunocytochemistry confirmed P. gingivalis invasion of HTR-8 cells (Fig. 6). The bacteria were

stained with a fluorescein dye and examined under a confocal microscope. Microscopy techniques assured

that invasion was intracellular. Higher cellular confluence would yield better results. HTR-8 cells showed

extreme sensitivity to PBS washes and PFA fixation. 70-80% confluence is optimal for infection and eliminates

any suspicion that a cell retains attachment based on physical differences. The slide shown in Figure 6 lost nearly

all cells. Irregular globular morphologies can be avoided by giving the cells more time to be grown on the

chamber slides. Figure 7 shows healthy cells with the expected "stretched out" morphology. It may also be

possible that a P. gingivalis MOI was too harsh for the cells.






























B




















C

























Figure 7. Fluorescent immunocytochemical analysis of P.gingivalis invading HTR-8/SVneo cells.

Cells were plated on poly-L lysine coated plastic chamber slides with four-wells and treated for 48

hours with (A) DMSO, (B) 10pM BaP, or (C) 20pM BaP. Cells were fixed in freshly prepared





4% Paraformaldehyde and incubated in primary and secondary antibodies respectively:

mouse monoclonal anti-tubulin (1:400), rhodamine TRITC secondary (1:300), and FITC

fluorescein phalloidin. Fluorescent microscopy shows fluorescein actin localization and

rhodamine microtubules.



Finally, fluorescent immunostaining of actin in HTR-8 cells treated with 10 pM benzo[a]pyrene clearly

showed changes in actin localization (Fig. 7). Cellular attachment seems abnormal and may be attributed to benzo

[a]pyrene's toxicity. Cell morphology was also irregular. 20 pM benzo[a]pyrene treated cells did not show

greater actin degradation.






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