CADMIUM STRESS AND MT CADMIUM STRESS AND MT GENE EXPRESSION IN THE GENE EXPRESSION IN THE

PLHC-1 FISH CELL LINEPLHC-1 FISH CELL LINE

By: James PlymaleBy: James Plymale

IntroductionIntroduction

Cheung et al. [1] conducted studies on metal concentrations Cheung et al. [1] conducted studies on metal concentrations and metallothionein (MT) gene expression in the Tilapia fish and metallothionein (MT) gene expression in the Tilapia fish cell line liver tissues. MT has been used as a biomarker of cell line liver tissues. MT has been used as a biomarker of

metal contaminations in polluted waters. MT is a small metal-metal contaminations in polluted waters. MT is a small metal-binding protein that regulates cellular response to essential binding protein that regulates cellular response to essential metals (Zn2+, Cu2+) and non-essential metal ions (Cd2+, metals (Zn2+, Cu2+) and non-essential metal ions (Cd2+,

Hg2+) [1]. However, only a few studies reported the study of Hg2+) [1]. However, only a few studies reported the study of other fish cell lines for this particular gene and its regulation by other fish cell lines for this particular gene and its regulation by

heavy metal ions in fish cell lines such as Zebrafish and heavy metal ions in fish cell lines such as Zebrafish and Goldfish. The fish hepatoma cell line (PLHC-1) is a tool used Goldfish. The fish hepatoma cell line (PLHC-1) is a tool used to study cytotoxicity, but MT expression in this cell like has not to study cytotoxicity, but MT expression in this cell like has not

been characterized. Cheung et al. [1] has reported that the been characterized. Cheung et al. [1] has reported that the metallothionein gene becomes activated once exposed to metallothionein gene becomes activated once exposed to

metals and induces mRNA expression levels. metals and induces mRNA expression levels.

1) To evaluate the role of the MT gene in 1) To evaluate the role of the MT gene in the PLHC-1 cell line through differential the PLHC-1 cell line through differential computational methodscomputational methods

2) To evaluate RNA expression levels in 2) To evaluate RNA expression levels in the MT gene in PLHC-1 cell line by RT-the MT gene in PLHC-1 cell line by RT-PCRPCR

Previous Research ObjectivesPrevious Research Objectives

MethodsMethods

Gene IdentificationGene IdentificationSelected MT sequences were researched Selected MT sequences were researched

from NCBI GenBankfrom NCBI GenBankThose selected sequences were grouped by Those selected sequences were grouped by

similar mRNA reference sites and aligned in similar mRNA reference sites and aligned in ClustalW to find a conserved sequenceClustalW to find a conserved sequence

An aligned sequence of the conserved region An aligned sequence of the conserved region was selected and primers and a biotinylated was selected and primers and a biotinylated probe was designed for the detection of the probe was designed for the detection of the conserved regionconserved region

MT RNA Expression Studies

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 Purification

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).

RNA Wet Lab Analysis

(Reverse Transcriptase) (Reverse Transcriptase) RT-PCRRT-PCR

RNA samples followed the Promega RNA samples followed the Promega protocol (Figure 2) and ran under protocol (Figure 2) and ran under these thermocycler conditions:these thermocycler conditions: 1 cycle 45 degrees C, 45 min.1 cycle 45 degrees C, 45 min. 1cycle 94 degrees C, 2 min.1cycle 94 degrees C, 2 min. 45 cycles45 cycles

94 degrees C, 30 sec94 degrees C, 30 sec 60 degrees C, 1 min60 degrees C, 1 min 68 degrees C, 2 min68 degrees C, 2 min

1 cycle 68 degrees C, 7 min1 cycle 68 degrees C, 7 min 1 cycle 4 degrees, hold1 cycle 4 degrees, hold

Samples were then loaded into a 2.5% Samples were then loaded into a 2.5% Agarose gel in order to determine Agarose gel in order to determine molecular weightmolecular weight

Samples are also prepared to Samples are also prepared to complete a quantitative analysis complete a quantitative analysis (qPCR)(qPCR)

(Designed by: Mia Brown)(Designed by: Mia Brown)

Figure 2. RT-PCR Method

Previous Research ResultsPrevious Research Results

RT-PCR AnalysisFigure 3. RT-PCR Gel ElectrophoresisA 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.

A EC DB

Major Conclusions from Major Conclusions from Previous ResearchPrevious Research

The MT gene in the PLHC-1 cell line has The MT gene in the PLHC-1 cell line has been identified computationally at around been identified computationally at around ~150 bp.~150 bp.

RNA Expression levels of the MT gene in RNA Expression levels of the MT gene in the PLHC-1 cell line has not been the PLHC-1 cell line has not been determined specifically but has shown determined specifically but has shown great potential in being the desired MT great potential in being the desired MT gene sequence through RT-PCR gene sequence through RT-PCR

Future Research and Future Research and QuestionsQuestions

Located gene computationallyLocated gene computationally Tested gene through RT-PCR and results Tested gene through RT-PCR and results

suggested that this could be a MT gene of suggested that this could be a MT gene of interestinterest

With the PLHC-1 cell line being treated with With the PLHC-1 cell line being treated with 1010µM of Cadmium at 30ºC at 24 hours which µM of Cadmium at 30ºC at 24 hours which indicated that our potential gene to be over 150 indicated that our potential gene to be over 150 base pairsbase pairs

At what point in time would we see the earliest At what point in time would we see the earliest expression once exposed to 10µM of Cadmium expression once exposed to 10µM of Cadmium (determined over graduation of time)(determined over graduation of time)

Results indicated that we had a maximum of Results indicated that we had a maximum of 51.6 ng per ml 51.6 ng per ml

Future Research and Future Research and Questions (cont’d)Questions (cont’d)

With this data, we would like to determine With this data, we would like to determine the earliest form of expression (by the earliest form of expression (by repeating the experiment through different repeating the experiment through different time points)time points)

What is the earliest form of MT gene What is the earliest form of MT gene expression and the PLHC-1 cell lineexpression and the PLHC-1 cell line

What is the earliest form of RNA What is the earliest form of RNA expression in the PLHC-1 cell line at 10expression in the PLHC-1 cell line at 10µM µM of Cadmiumof Cadmium

ObjectivesObjectives

To evaluate RNA expression levels in the To evaluate RNA expression levels in the MT gene in PLHC-1 cell line over a MT gene in PLHC-1 cell line over a supplemented time period by RT-PCRsupplemented time period by RT-PCR

MethodsMethodsStep 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 DiagramRNA Concentration Diagram

Figure 4. RNA Concentration DiagramRNA 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 ResultsGel Results

LANESLANESA – LadderA – LadderB – ControlB – ControlC – 6 hoursC – 6 hoursD – 12 hoursD – 12 hoursE – 18 hoursE – 18 hoursF – 24 hoursF – 24 hoursG - LadderG - Ladder

A B C D E F G

Major ConclusionsMajor Conclusions Previous research indicated that the expression Previous research indicated that the expression

is as originally thought (150 base pairs/size of is as originally thought (150 base pairs/size of insert)insert)

B) Control – ok B) Control – ok

C) 6 hours – ok C) 6 hours – ok

D) 12 hours – okD) 12 hours – ok

E) 18 hours –E) 18 hours – gel electrophoresis gel electrophoresis suggested suggested that there was a large that there was a large amount of amount of expression expression between between hours 18-24hours 18-24

Major Conclusions (cont’d)Major Conclusions (cont’d)

Gel electrophoresis does not suggest that Gel electrophoresis does not suggest that we indicated the MT gene specifically. But we indicated the MT gene specifically. But it does indicate a 150 base pair sequencer it does indicate a 150 base pair sequencer that can be sequenced for further analysis that can be sequenced for further analysis to determine whether or not we have MT to determine whether or not we have MT gene expressiongene expression

Questions/Future ResearchQuestions/Future Research

Future analysis for taking fish exposed for Future analysis for taking fish exposed for a specific time and amount and then a specific time and amount and then looking at different pathways to the looking at different pathways to the exposal of metal internal affects.exposal of metal internal affects.

Does cadmium have a certain affect on Does cadmium have a certain affect on humans such as mercury in tuna?humans such as mercury in tuna?

ReferencesReferences Cheung, Andrews Pok Lap, Vincent Kwok Lim Lam, King Ming Chan, Cheung, Andrews Pok Lap, Vincent Kwok Lim Lam, King Ming Chan,

"Tilapia metallothionein genes: PCR-cloning and gene expression studies." "Tilapia metallothionein genes: PCR-cloning and gene expression studies." Biochimica et Biophisica ActaBiochimica et Biophisica Acta 1731(2005): 191-201. 1731(2005): 191-201.

Scudiero, Carginale, Capasso, Riggio, Filosa, Parisi, Rosaria, Vincenzo, Scudiero, Carginale, Capasso, Riggio, Filosa, Parisi, Rosaria, Vincenzo, Clement, Marilisa, Stefania, Elio. "Structural and functional analysis of metal Clement, Marilisa, Stefania, Elio. "Structural and functional analysis of metal regulatory elements in the promoter region of genes encoding regulatory elements in the promoter region of genes encoding metallothionein isoforms in the Antarctic fish Chionodraco hamatus metallothionein isoforms in the Antarctic fish Chionodraco hamatus (icefish)." (icefish)." GeneGene 274(2001): 199-208. 274(2001): 199-208.

Li-Hua Shen, Kwok Lim Lam, Po Wai Ko and King Ming Chan, "Metal Li-Hua Shen, Kwok Lim Lam, Po Wai Ko and King Ming Chan, "Metal Concentrations and Analysis of Metal Binding Protein Fractions from the Concentrations and Analysis of Metal Binding Protein Fractions from the Liver of Tilapia Collected from Shing Mun River." Liver of Tilapia Collected from Shing Mun River." Marine Enviornmental Marine Enviornmental ResearchResearch 46(1998): 597-600. 46(1998): 597-600.

Carol Hiu Mei Yan, King Ming Chan, "Cloning of zebrafish metallothionein Carol Hiu Mei Yan, King Ming Chan, "Cloning of zebrafish metallothionein gene and characterization of its gene promoter region in HepG2 cell line." gene and characterization of its gene promoter region in HepG2 cell line." Biochimica et Biophisica ActaBiochimica et Biophisica Acta 1679(2004): 47-58. 1679(2004): 47-58.

King Ming Chan, "Metallothionein: Potenial Biomarker for Monitoring Heavy King Ming Chan, "Metallothionein: Potenial Biomarker for Monitoring Heavy Metal Pollution in Fish Around Hong Kong." Metal Pollution in Fish Around Hong Kong." Marine Pollution BulletinMarine Pollution Bulletin 31(1995): 411-415. 31(1995): 411-415.

AcknowledgementsAcknowledgementsDr. Elizabeth Murray for all her guidanceDr. Elizabeth Murray for all her guidanceMia Brown for taking her summer out to Mia Brown for taking her summer out to

advise me through this experiment advise me through this experiment Dr. Norton’s lab, Ms. Valerie, (NanoDrop Dr. Norton’s lab, Ms. Valerie, (NanoDrop

and qPCR machine)and qPCR machine)Yung Nahm for her PLCH-1 cell line from Yung Nahm for her PLCH-1 cell line from

the previous research conductedthe previous research conducted