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Manipulation of Expression of Arsenic (+3 Oxidation State) Methyltransferase in Cultured Cells

Zuzana Drobna¹, Miroslav Styblo^2,1, David J. Thomas³

¹University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
²Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
³Pharmacokinetics Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina

Publication Name:

Current Protocols in Toxicology

Unit Number:

Unit 4.35

DOI:

10.1002/0471140856.tx0435s43

Online Posting Date:

February, 2010

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Abstract

Methylation of inorganic arsenic to produce mono-, di-, or trimethylated products is the central process in the cellular metabolism of arsenic. Identification of arsenic (+3 oxidation state) methyltransferase (As3mt) as the enzyme that could catalyze all the steps in the pathway for arsenic methylation suggests that expression of this enzyme could be a useful target for manipulation. Here, methods are described for heterologous expression of the rat As3mt gene in a human urothelial cell line that normally does not express this enzyme and for silencing of the AS3MT gene by RNA interference in a human hepatoma cell line. These tools can be applied to elucidating the role of methylation in the toxic and carcinogenic effects of arsenicals. Curr. Protoc. Toxicol. 43:4.35.1-4.35.24. © 2010 by John Wiley & Sons, Inc.

Keywords: arsenic; arsenic (+3 oxidation state) methyltransferase; UROtsa cells (human urothelial cells); heterologous expression; human hepatoma cells (HepG2 cells); RNA interference; shRNA

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Introduction
Basic Protocol 1: Heterologous Expression of Rat As3mt in a Cultured Human Cell Line
Basic Protocol 2: Silencing of Human AS3MT Gene Expression by RNA Interference
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Heterologous Expression of Rat As3mt in a Cultured Human Cell Line

Materials

Rat liver cDNA
rAs3mt cDNA: full-length rAs3mt cDNA is amplified from rat liver cDNA using PCR Primers (5¢-GATCGACTCGAGTTCCTGAGACCCTCTGCCAAC-3¢ and 5¢-GATCGAGAATTCCTAGCAGCTTTTCCTTTTGCC-3¢) (Walton et al., 2003)
Expression vector: retroviral vector, e.g., pLEGFP-N1, pRetroQ-AcGFP1-N1/C1 (Clontech)
XhoI and EcoRI
pRSET vector (Invitrogen)
Buffer 2 (New England Biolabs)
Molecular biology–grade water
T4 DNA ligase
QIAprep Spin Miniprep kit
Ultra-competent DH5 E. coli strain (Stratagene) for transformation with a rAs3mt expressing vector
SOC medium (Invitrogen) for E. coli transformation
LB-agar plates containing 100 µg/ml ampicillin (see recipe)
10 mM dNTP mix (Invitrogen, cat. no. 18427-013)
Taq polymerase kit (Invitrogen, cat. no. 10342-020) containing:
- 10× PCR buffer
- MgCl₂
Primers for sequencing of the rAs3mt insert in the expression vector of choice—the primers specific for pLEGFP-N1/rAS3MT vector are as follows:
- CMV/forward 5¢-GGATAGCGGTTTGACTCACGC-3¢
- EGFP-N/reverse 5¢-CGTCGCCGTCCAGCTCGACCAG-3¢
Luria-Bertani (LB) broth (Invitrogen) containing 100 µg/ml ampicillin
6-well collagen I–coated culture plates (BD Biosciences, cat. no. 356400)
AmphoPack-293 retrovirus packaging cell line (Clontech) for production of recombinant retroviral particles
AmphoPack-293 medium (see recipe)
OPTI-MEM medium (Invitrogen)
Fugene6 (Roche)
100 µg/ml ampicillin (Sigma)
250 µg/ml geneticin (G418, Invitrogen)
UROtsa cell line (Rossi et al., 2001)
UROtsa medium (see recipe)
Polybrene (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide, hexadimethrine bromide, Sigma-Aldrich)
Primers for analysis of rAs3mt mRNA expression in transduced UROtsa/F35 cells:
- Forward 5¢-ATTTTGGATCTGGGCAGTGGGAGT-3¢
- Reverse 5¢-AGTGACCAAACGTGGAGGGCAGA-3¢
Primers for analysis of -actin mRNA expression in cells:
- Forward 5¢-TCATGAAGTGTGACGTTGACATCCGT-3¢
- Reverse 5¢-CCTAGAAGCATTTGCGGTGCACGATG-3¢
Primary anti-As3mt antibody/antiserum

RIPA lysis buffer (see recipe)
Corresponding secondary antibody conjugated with horseradish peroxidase [HRP; e.g., anti-rabbit HRP-conjugated antibody (Santa Cruz Biotechnology, cat. no. sc-2004)]
Purified or recombinant rAs3mt to be used as a positive control (unit 4.34)
Enhanced chemiluminescence (ECL) reagent for detection of the antibody-antigen complexes (Super Signal West Pico ECL reagent; Pierce, cat. no. 34077)

Laminar-flow level-2 biological safety cabinet for sterile cell manipulations and safe handling of viral particles
37°C shaking incubator
37°C humidified cell culture incubator with 95% air, 5% CO₂ atmosphere
Conventional or real-time PCR cycler
200-µl PCR tubes
60-mm culture plates
0.45-µm low-protein-binding cellulose acetate or polysulfonic filter
Electroblot module for transfer of proteins separated by PAGE to PVDF membranes (e.g., Mini Trans-Blot Cell; Bio-Rad, cat. no. 170-3930)
X-ray or autoradiography films and cassettes
Imaging system for detection and quantification of the HRP signal (e.g., GeneGnome imaging system; Syngene)

Additional reagents and equipment for digesting DNA with restriction enzymes (Bloch and Grossman, 1995), PCR (Kramer and Coen, 2001), real-time PCR (Drobna et al., 2004), immunoblot analysis (Drobna et al., 2005), SDS-PAGE (Gallagher, 2006), and quantitation of iAs (unit 4.32)

Basic Protocol 2: Silencing of Human AS3MT Gene Expression by RNA Interference

Materials

Anti-hAS3MT shRNA construct (the construct shown in Fig. 4.35.5 was custom synthesized by Operon Biotechnologies and purified by PAGE)
Expression vector: pSIREN-RetroQ retroviral vector encoding for ampicillin and puromycin resistance genes (Clontech)
TE buffer
10× T4 DNA ligase buffer
Bovine serum albumin (BSA)
Nuclease-free water
T4 DNA ligase
XL10-Gold Ultracompetent E. coli cells (Stratagene)
SOC medium (Invitrogen)
10-cm LB-agar plates containing 100 µg/ml ampicillin (see recipe)
Luria-Bertani (LB) broth (Invitrogen) containing 100 µg/ml ampicillin
QIAprep Spin Miniprep Kit (Qiagen)
MluI and XhoI (New England Biolabs)
NEB buffer #3
Molecular biology–grade water
U6 promoter–specific primer (5¢-GGGCAGGAAGAGGGCCTAT-3¢)
AmphoPack-293 cells for production of recombinant retroviral particles encoding for shRNA (Clontech)
6-well collagen I–coated plates (BD Biosciences, cat. no. 356400)
AmphoPack-293 medium (see recipe)
OPTI-MEM medium (Invitrogen)
GeneJuice (Novagen, Darmstad, Germany)
Puromycin (10 mg/ml; Sigma) for selection of transfected AmphoPack-293 and transduced HepG2 cells
HepG2 cells (ATCC HB8065)
HepG2 medium (see recipe)
Polybrene
Primers for analysis of hAS3MT expression:
- Forward: 5¢-CGTCTATACGAGCCTTGAA-3¢
- Reverse: 5¢-TTAGCAGCTTTTCTTTGTGC-3¢ that amplify a 616-bp cDNA sequence

37°C shaking incubator
37°C, 5% CO₂ incubator

Additional reagents and equipment for restriction enzyme digestion (Bloch and Grossman, 1995), conventional (Drobna et al., 2006) or real-time PCR (Drobna et al., 2004), and immunoblot analysis (Drobna et al., 2006)

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Figures

Figure 4.35.1

Flowchart for the development of a UROtsa cell line stably expressing rat arsenic (+3 oxidation state) methyltransferase (As3mt).

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Figure 4.35.2

Mechanism of rAs3mt expression in UROtsa cells using retroviral particles containing the cDNA encoding rAs3mt. After incorporation of rAs3mt into the genome of UROtsa cells, high levels of rAs3mt mRNA are transcribed under control of the CMV promoter and translated to the protein, which is enzymatically active.

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Figure 4.35.3

Expression and activity of rAs3mt in UROtsa/F35 cells. (A) RT-PCR analysis: (1) positive control (pLEGFP-N1/rAs3mt plasmid); (2) UROtsa cells; (3) UROtsa/F35 cells; (4) negative control. (B) Immunoblot analysis: (1) recombinant His-tagged rAs3mt (Mr ~45 kDa); (2) UROtsa cells; (3) UROtsa/F35 cells expressing native rAs3mt (Mr ~41 kDa). (C) TLC analysis of ⁷³As-metabolites in cell cultures exposed to carrier-free [⁷³As]iAs^III for 63 hr: (1) UROtsa cells (2) UROtsa/F35 cells.

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Figure 4.35.4

Mechanism of hAS3MT silencing in HepG2 cells using retroviral particles containing the shRNA construct designed to target and cleave hAS3MT mRNA. After incorporation of target shRNA sequence into the genome of HepG2 cells, a high level of shRNA is transcribed under control of the U6 promoter. D-stranded RNA molecules are cleaved by Dicer and siRNA molecules are released. The hAS3MT specific (antisense) siRNA enters into a nuclease complex to form an RNA-induced silencing complex (RISC). Active RISC complex associates with target hAS3MT transcript and causes its cleavage.

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Figure 4.35.5

Sequences and predicted folding for an shRNA oligonucleotide designed to silence human arsenic (+3 oxidation state) methyltransferase expression in HepG2 cells. Red, target sense sequence; blue, target antisense sequence (siRNA); black, loop sequence.

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Figure 4.35.6

Flowchart for silencing of arsenic (+3 oxidation state) methyltransferase gene expression in HepG2 cells by shRNA.

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Figure 4.35.7

Analyses of hAS3MT expression and the arsenic methylation activity in parental HepG2 cells (1), HepG2 cells transduced with an empty expression vector (2), or with a vector containing the shRNA oligonucleotide (3). (A) The touch-down PCR analysis of hAS3MT and -actin mRNA; for negative control (NC) cDNA was replaced with water; pRSET/hAS3MT expression vector was used in positive control (PC). (B) hAS3MT mRNA levels quantified by a real-time PCR. (C) Immunoblot images of hAS3MT and -actin in cell lysates. (D) hAS3MT protein levels normalized for -actin contents. (E) Radiolabeled metabolites in cell lysates after a 72-hr exposure to carrier-free [⁷³As] labeled arsenite. (F) Radiolabeled metabolites in cell medium after a 72-hr exposure to carrier-free [⁷³As] labeled arsenite. Bar charts show mean values for 3 independent measurements; SD values are also shown in panels B and D; *Mean value significantly different (p < 0.05) from parental HepG2 cells.

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Literature Cited

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