Transcript CADMIUM_STRESS

CADMIUM STRESS AND MT
GENE EXPRESSION IN THE
PLHC-1 FISH CELL LINE
By: James Plymale
Introduction
Cheung et al. [1] conducted studies on metal concentrations
and metallothionein (MT) gene expression in the Tilapia fish
cell line liver tissues. MT has been used as a biomarker of
metal contaminations in polluted waters. MT is a small metalbinding protein that regulates cellular response to essential
metals (Zn2+, Cu2+) and non-essential metal ions (Cd2+,
Hg2+) [1]. However, only a few studies reported the study of
other fish cell lines for this particular gene and its regulation by
heavy metal ions in fish cell lines such as Zebrafish and
Goldfish. The fish hepatoma cell line (PLHC-1) is a tool used
to study cytotoxicity, but MT expression in this cell like has not
been characterized. Cheung et al. [1] has reported that the
metallothionein gene becomes activated once exposed to
metals and induces mRNA expression levels.
Previous Research Objectives
1) To evaluate the role of the MT gene in
the PLHC-1 cell line through differential
computational methods
 2) To evaluate RNA expression levels in
the MT gene in PLHC-1 cell line by RTPCR
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Methods
Gene Identification
Selected MT sequences were researched
from NCBI GenBank
 Those selected sequences were grouped by
similar mRNA reference sites and aligned in
ClustalW to find a conserved sequence
 An aligned sequence of the conserved region
was selected and primers and a biotinylated
probe was designed for the detection of the
conserved region
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MT RNA Expression Studies
RNA Purification
Step 1: Cells were grown for 3 days and
treated with and without 10 uM
Cadmium for 24 hours
Step 2: Cells were then washed with 5
mL PBS and then collected by scrapping
the petri dish
Step 3: Purified RNA using Promega RNA
Purification protocol
RNA Wet Lab Analysis
Figure 1. RNA
Concentration Diagram
RNA concentration was
measured using the
NanoDrop in Dr. Norton’s
lab. The concentrations
were as followed:
Control (not shown) was
given by Zachary
Tackett, RNA 1-black
(29.1 ng/ml; OD 260/280
1.90), RNA 2-red ( 51.6
ng/ml; OD 260/280
2.02), RNA 3-green (14.4
ng/ml; OD 260/280
1.70).
(Reverse Transcriptase) RT-PCR
Figure 2. RT-PCR Method
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RNA samples followed the Promega
protocol (Figure 2) and ran under
these thermocycler conditions:
 1 cycle 45 degrees C, 45 min.
 1cycle 94 degrees C, 2 min.
 45 cycles
 94 degrees C, 30 sec
 60 degrees C, 1 min
 68 degrees C, 2 min
 1 cycle 68 degrees C, 7 min
 1 cycle 4 degrees, hold
Samples were then loaded into a 2.5%
Agarose gel in order to determine
molecular weight
Samples are also prepared to
complete a quantitative analysis
(qPCR)
(Designed by: Mia Brown)
Previous Research Results
RT-PCR Analysis
A
B
C
D
E
Figure 3. RT-PCR Gel Electrophoresis
A 157 bp RT-PCR product complementary
to the MT gene in Zebrafish was amplified
using designated primers and the indicated
amounts of RNA. RT-PCR was performed
according to the Promega System protocol.
RT-PCR profile: 48°C for 45 minutes, 94°C
for 2 minutes, 45 cycles (94°C for 30
seconds, 60°C for 1 minute, 68°C for 2
minutes), 68°C for 7 minutes. To analyze
the reactions, 10μl of each reaction were
subjected to electrophoresis on a 2.5%
agarose gel and DNA was detected by
ethidium bromide staining. Lane A, DNA
Ladder, Lane B, Control, Lanes C-E,
RNA1-3 respectively.
Major Conclusions from
Previous Research
The MT gene in the PLHC-1 cell line has
been identified computationally at around
~150 bp.
 RNA Expression levels of the MT gene in
the PLHC-1 cell line has not been
determined specifically but has shown
great potential in being the desired MT
gene sequence through RT-PCR
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Future Research and
Questions
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Located gene computationally
Tested gene through RT-PCR and results
suggested that this could be a MT gene of
interest
With the PLHC-1 cell line being treated with
10µM of Cadmium at 30ºC at 24 hours which
indicated that our potential gene to be over 150
base pairs
At what point in time would we see the earliest
expression once exposed to 10µM of Cadmium
(determined over graduation of time)
Results indicated that we had a maximum of
51.6 ng per ml
Future Research and
Questions (cont’d)
With this data, we would like to determine
the earliest form of expression (by
repeating the experiment through different
time points)
 What is the earliest form of MT gene
expression and the PLHC-1 cell line
 What is the earliest form of RNA
expression in the PLHC-1 cell line at 10µM
of Cadmium
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Objectives

To evaluate RNA expression levels in the
MT gene in PLHC-1 cell line over a
supplemented time period by RT-PCR
Methods
Step 1: Cells were grown for 3 days and
treated with and without 10 uM Cadmium
for 6,12,18, and 24 hours
Step 2: Cells were then washed with 5 mL
PBS and then collected by scrapping the
petri dish
Step 3: Purified RNA using Promega RNA
Purification protocol
RNA Concentration Diagram
Figure 4. RNA
Concentration Diagram
RNA concentration was
measured using the
NanoDrop in Dr. Norton’s
lab. The concentrations
were as followed:
0 hours -11.5 ng/ml (OD
260/280: 2.29)
6 hours - 12.5 ng/ml (OD
260/280: 1.90)
12 hours - 16.0 ng/ml (OD
260/280: 1.97)
18 hours - 25.5 ng/ml (OD
260/280 1.94)
24 hours - 10.2 ng/ml (OD
260/280: 1.25)
Gel Results
LANES
A – Ladder
B – Control
C – 6 hours
D – 12 hours
E – 18 hours
F – 24 hours
G - Ladder
A
B C D
E
F
G
Major Conclusions
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Previous research indicated that the expression
is as originally thought (150 base pairs/size of
insert)
B) Control – ok
C) 6 hours – ok
D) 12 hours – ok
E) 18 hours – gel electrophoresis suggested
that there was a large amount of
expression between hours 18-24
Major Conclusions (cont’d)

Gel electrophoresis does not suggest that
we indicated the MT gene specifically. But
it does indicate a 150 base pair sequencer
that can be sequenced for further analysis
to determine whether or not we have MT
gene expression
Questions/Future Research
Future analysis for taking fish exposed for
a specific time and amount and then
looking at different pathways to the
exposal of metal internal affects.
 Does cadmium have a certain affect on
humans such as mercury in tuna?
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References
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Cheung, Andrews Pok Lap, Vincent Kwok Lim Lam, King Ming Chan,
"Tilapia metallothionein genes: PCR-cloning and gene expression studies."
Biochimica et Biophisica Acta 1731(2005): 191-201.
Scudiero, Carginale, Capasso, Riggio, Filosa, Parisi, Rosaria, Vincenzo,
Clement, Marilisa, Stefania, Elio. "Structural and functional analysis of metal
regulatory elements in the promoter region of genes encoding
metallothionein isoforms in the Antarctic fish Chionodraco hamatus
(icefish)." Gene 274(2001): 199-208.
Li-Hua Shen, Kwok Lim Lam, Po Wai Ko and King Ming Chan, "Metal
Concentrations and Analysis of Metal Binding Protein Fractions from the
Liver of Tilapia Collected from Shing Mun River." Marine Enviornmental
Research 46(1998): 597-600.
Carol Hiu Mei Yan, King Ming Chan, "Cloning of zebrafish metallothionein
gene and characterization of its gene promoter region in HepG2 cell line."
Biochimica et Biophisica Acta 1679(2004): 47-58.
King Ming Chan, "Metallothionein: Potenial Biomarker for Monitoring Heavy
Metal Pollution in Fish Around Hong Kong." Marine Pollution Bulletin
31(1995): 411-415.
Acknowledgements
Dr. Elizabeth Murray for all her guidance
 Mia Brown for taking her summer out to
advise me through this experiment
 Dr. Norton’s lab, Ms. Valerie, (NanoDrop
and qPCR machine)
 Yung Nahm for her PLCH-1 cell line from
the previous research conducted
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