Arsenic and Apoptosis in PHLC-1 cells
Yeong-Nam Jeong and Dr. Elizabeth Murray Marshall University, Integrated Science and Technology Department
Abstract
Methods
Background:
Arsenic has been well characterized as a carcinogen in eukaryotic cells. Many researches propose that arsenic
caused tumor or various cancer types using mammalian cells. We are studying using the PHLC-1 (Poeciliopsis
lucida hepatocellular carcinoma) cell lines which are derived from a liver tumor from Poeciliopsis lucida because
fish can be affected very directly by environmental contamination in water. The previous study, we detected DNA
damage from comet assay. The cells were exposed in low levels of arsenic (5uM and 10uM of As2O3 and 25uM
and 50uM of As2O5) for one and two hours and compared with 1X PBS controls. Lau et al. reported that arsenic
trioxide caused apoptosis in rat lung epithelial cells. Their results suggest low arsenic levels may cause cancer
and high arsenic may cure cancer, because of induction of apoptosis.
Methods:
Confocal Assay: PHLC-1 cells were grown in WillCo glass bottom dishes. Cells were exposed to 5uM As2O3 for
varying lengths of time (1 to 6 hr). Cells were treated with the Calbiochem Fluorescein-FragELTM DNA
Fragmentation Detection Kit. It is a fluorescence system for labeling of DNA breaks in apoptotic nuclei in cell
preparations directly fixed in WillCo glass bottom dishes. We detected and analyzed the apoptotic cells using
the Bio-Rad Confocal Microscope.
Agarose gel DNA fragmentation assay: PHLC-1 cells exposed to 5uM As2O3 for varying lengths of time (1 to 6
hr) were lysed, and the DNA was purified by phenol–chloroform extraction followed by ethanol precipitation. In
1.0% TBE–agarose gel, the DNA was electrophoresed and the gel was stained with ethidium bromide and
imaged using the Bio-Rad GelDoc System.
Cell culture
P. lucida cell lines were obtained from ATCC and cultured in humidified CO2 incubator at 30°
C. Cells were grown in Minimal Essential Medium Eagle with 1mM Sodium pyruvate, 2 mM
L-glutamine and 1500 mg sodium bicarbonate/L supplemented with 5% Fetal Calf serum and
1% Pen Strep. Cells were split every 2-3 days. Experiments were performed on confluent
cells.
Cell Growth Assay
Cells were seeded at 12 well. 1x105 cells/ml and 3x104 cells/ml were seeded in each of 6
wells. This assay was duplicated. For counting the cells, these cells were trypsinized. A
standard Hemocytometer Cell Count Calculator was used to count cells at intervals after
initial plating. These cells were stained with trypan blue to determine cell viability. Initial
concentrations of cells plated were varied to determine optimal growth rate.
A.
B.
Apoptosis Kit
For apoptosis assay we used the FragELTM DNA fragmentation Detection Kit, Fluorescent
TdT Enzyme (QIA 39) from CalBiochem. There are four steps and they are:
1. Fixation 2. Permeabilization 3. Enzymatic labeling reaction 4. Termination
Results:
Apoptosis is a highly regulated pathway that maintains cell proliferation in balance with cell death. If apoptosis is
stimulated or suppressed inappropriately, cancer or abnormal development could result. Arsenic induces
apoptosis in mammals. This metal is a commonly found in waters in West Virginia, Ohio and Kentucky that are
inhabited by fishes. Although apoptosis occurs in fishes exposed to metals, the specific effects of this element
on the apoptotic cascade is unknown. PHLC-1 cells are fish cells used to study toxicology. This research will
report the use of these cells to monitor apoptosis induced by As2O3.
Significance:
Our laboratory will use these research results on the effects of arsenic on the fish apoptosis, to develop speedy,
cost-effective cytotoxicity tests to examine apoptosis in fishes inhabiting polluted streams in Appalachia.
First, cells were fixed with 4% formaldehyde in PBS. To allow the enzyme and substrates to
enter the cell, we permeabilized them by Proteinase K treatment 20ug/ml in 10mM pH 8.0
Tris buffer for 5 minutes. Enzymatic addition of labeled nucleotides was carried out by using
Terminal Deoxynucleotidyl Transferase (TdT) and fluorescein labeled deoxynucleotides.
Equilibration of cells was done in the 1X TdT (Terminal Deoxynucleotidyl Transferase) buffer
for 10-30 minutes at room temperature. After equilibration, enzyme and nucleotides were
added (60ul TdT Labeling Reaction); the mixture was incubated at 37oC for 1-1.5 hours.
The fixed and labeled cells were washed in 1x TBS 3 times for 1 minute and mounted with a
glass cover slip using Mounting Media.
Introduction
Confocal Microscopy
For this assay, the Bio-Rad MRC1024 Confocal Scanning Microscope was used. Since we
used fluorescein as our fluorescent label (ex. max 490nm, em. Max 525nm), we set up our
microscope with the 488nm excitation line and the 522/329nm band pass emission line. The
image was gotten on 40X with oil. We use Carl Zeiss AIM software and Image J for making
images better.
Arsenic is a well-characterized as a carcinogen in mammalian cells. Arsenic is soluble in
water and is a colorless, flavorless, and unscented solution. The U.S. EPA had established
the maximum contaminant level (MCL) for arsenic in drinking water of 50 ppb (50 ug/L) which
changed to 10 ppb (10 ug/L) in January 2006 [8]. Arsenic has been linked to several forms of
cancer (bladder, lungs, skin, kidney, nasal passages, liver, and prostate). Arsenic exposure
is associated with cardiovascular, pulmonary, immunological, and neurological, and
endocrine problems.
DNA Fragmentation
Cells were transferred 5X105 cells/ ml into the Eppendorf tubes and centrifuge at 2000 rpm,
4oC for 5 minutes. After remove supernatant, 20ul of lysis buffer was added and then 10ul of
RNase was added at 37oC for 30-120 minutes. 10ul of proteinase K and cells were added
and incubated at 50oC for overnight. Cells were electrophoresis in 1% agarose gel at 35V,
over 4 hours. Gel was stained in ethidium bromide and taken picture by Bio-Rad GelDoc
System.
Inorganic arsenic has both acute (short-term) and chronic (long-term) toxicity [8]. How
arsenic causes cancer is not well understood. We chose the PLHC-1 cell lines because fish
are informative for environmental carcinogenesis research, since fish populations live in
polluted habitats [7]. Liver cells are also useful for toxicity assays. Jia et al. reports that the
two most common forms of inorganic arsenic forms found in drinking water, As2O3 and As2O5,
cause DNA damage to rat astroglia cells (primary nerve cells) as detected by the Comet
Assay [9]. We have already detected DNA damage from Comet Assay using PLHC-1 cells,
but we could not determine if this was from necrosis or apoptosis. So, we decided to do this
experiment using the Bio-Rad MRC 1024 Confocal Scanning Microscope to figure out if
Arsenic causes apoptosis in fish.
Apoptosis is a highly regulated pathway that maintains cell proliferation in balance with cell
death. If apoptosis is stimulated or suppressed inappropriately, cancer or abnormal
development could result. The arsenic induced apoptosis in mammals are well studied.
Apoptosis occurs in living fishes exposed to metals, but the specific effects of this element on
the apoptotic cascade is not understood well. Cell culture cells are a useful model for
apoptosis since they are easier to work with than whole fish. This research will report the use
of PLHC-1 cells to monitor apoptosis induced by As2O3.
We detected apoptosis for treat terminal deoxynucleotidyl-mediated transferase (TdT)
fluorescent Enzyme labeling using FragEL DNA fragmentation Detection Kit from
CalBiochem. To obtain apoptosis images, we used the Bio-Rad MRC1024 Confocal
Scanning Microscope.
C.
Fig 8. A. No arsenic treatment control cells. B. 5um As2O3 1hr. C. 5um As2O3 3hr
Occasional apoptotic cells are seen in the untreated cells. On the 1 hour treatment
cells, note blobbing and shrinking apoptotic cells. Many apoptotic cells are visible. On
the 3 hours treatment cells, these cells look like they are almost dead from apoptosis.
DNase Fragmentation Assay did
not show significant DNA
degradation in apoptotic ladders.
Liu et al. suggested that DNA
fragmentation assays were for 48
or 72 hours, so we plan to repeat
these experiments [6].
Results
Fig 9. DNA Fragmentation Assay.
Discussion
Fig 5. Normal appearance of cultured PHLC-1 cells from liver tumor of the topminnow. Note, the
morphology seen here is somewhat different than that seen in our experimental groups that were
grown to confluence.
There have not been many studies of apoptosis in tissue cultured fish cells. Recently
there have been two papers published on this topic. Liu et al. discusses using grass
carp cell lines in environmental toxicology using atrazine, an herbicide which pollutes
water [6]. They found that there was a dose response to increased amounts of atrazine.
Embry et al. shows that apoptosis mechanism in fish cells may be very different that in
mammalian cell lines. P53 expression in apoptosis is not induced by chemotherapy
agents in PLHC-1 cell and in primary liver cells from rainbow trout. Arsenic induced
apoptosis is reported to be p53 independent in mammalian cell lines [5]. Arsenic induces
AP-I activations through activation of MAP (Mitogen Activated Protein) kinases and PKC
(protein kinase C) [7].
This previous research shows that arsenic is a carcinogen, but also induces apoptosis in
tumor cells. We are interested in continuing this research. We plan a cytotoxicity assay,
arsenic induced-apoptosis assay using As2O3 and As2O5, repeat our DNA fragmentation
assay for longer exposure, an arsenic exposure cell growth assay, and a Western blot for
detecting p53 in PLHC-1 cell lines exposed to arsenic.
References
1. www.nativefish.org/Gallery
Fig 1. Arsenic-induced signal transduction pathways and their role
in cell transformation and apoptosis [5].
Fig 2. High magnification image of PHLC-1 cell
nucleas showing typical apoptotic bodies
composed of nuclear chromatin. Here, as in Fig 7
& 8, the green fluorescence indicates enzymatic
labeling of fragmented DNA (scale bar is related
to grey image, fluorescent inset is slightly
enlarged).
Fig 6. These are representative cell growth assay plots for PHLC-1 cells. The different plot
lines are a measure of growth rate resulting from variation in the initial concentration of cells
plated in medium. We plan to determine the difference in Cell Growth using arsenic exposed
cells with standard 96 well cytotoxicity assay.
2. X. Chris Le, Xiufen Lu, Xing-Fang Li. Arsenic Spectation. Americal Chemical Society. 2004; 26A-33A
3. Andy T.Y. Lau, Muyai Li, Ronglin Xie, Quing-Yu He, Jen-Fu Chiu. Opposed arsenic-induced signaling
pathways promote cell proliferation or apoptosis in cultured lung cells. Carcinogenesis. 2004; 25(1):21-28
4. Susan M. Dibartolomeis, James P. Mone. Apoptosis: A Four-Week Laboratory Investigation for
Advanced Molecular and Cellular Biology Students. Cell Biology Education. 2003; 2: 275-295
5. Zigang D. The Molecular Mechanisms of Arsenic-Induced Cell Transformation and Apoptosis.
Environmental Health Perspectives. 2002; 110(5): 757-759
6. Xin-Mei Liu, Jian-Zhong Shao, Li-Xin Xian, Xian-Yong Chen. Cytotoxic Effect and Apoptosis Induction of
Atrazine in a Grass Carp (Ctenopharyngodon idellus) Cell Line. WILEY InterScience. 2006. 80-89
7. M Rau Embry, SM Billiard, RT Di Giulio. Lack of p53 induction in fish cells by model chemotherapeutics.
Oncogene. 2006; 25: 2004-2010
8. http://www.epa.gov/watersecurity/guide/chemicalsensorforarsenic.html
9. Jin Y, Sun G, Li X, Li G, Lu C, Qu L. Study on the toxic effects induced by different arsenicals in primary
cultured rat astroglia. Toxicol Appl Pharmacol. 2004;196:396-403.
A.
Fig 3. Poeciliopsis lucida, Desert topminnow [1].
Fig 4. Comet Assay. This assay represents
the DNA damages.
B.
Fig 7. Apoptotic endonucleases have a fragmenting effect on the cellular DNA by producing the
typical DNA ladder and also generate free 3’-OH groups at the ends of DNA fragments. These
3’-OH ends are the target for our labeling reaction. A. These HL-60 cells were prepared as a
positive control for apoptosis as indicated by the TdT assay (cells were provided in assay kit).
B. These PHLC-1 cells are untreated but show a low rate of apoptosis that normally occurs cell
cultures. Notice the clearly defined ‘blebs’ of nuclear material.
Acknowledgements
Thanks to David Neff and Dr. Norton for assisting with use of the Bio-Rad MRC 1024 Confocal
Scanning Microscope to figure out the apoptosis assay.
This is a project for CHM 583 class.