Cultures of yeast strains and human UROtsa cells
The wild-type BY4743 yeast strain was purchased from Invitrogen (Carlsbad, CA), and the MTQ2 deletion strain has the same background as the wild-type strain. Growth was conducted in rich media [yeast extract-peptone-dextrose (YPD)] at 30°C with shaking at 200 rpm. UROtsa cells (generously provided by P. Simeonova, National Institute for Occupational Safety and Health, Morgantown, WV) were cultured at a starting cell density of 4–5 × 104 cells/mL in RPMI 1640 (Mediatech, Inc., Manassas, VA) with l-glutamine, 10% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin (Omega Scientific, San Diego, CA), under standard culturing conditions.
We purchased sodium arsenite [NaAsO2 (iAsIII); purity > 99%] from Sigma-Aldrich (St. Louis, MO). Diiodomethylarsine [MMAIII iodide (MMAIII)] was a generous gift from J. Gandolfi (University of Arizona, Tucson, AZ). iAsIII and MMAIII solutions were freshly prepared using sterile water (Milli-Q; Millipore, Billerica, MA) and protected from light before use. Yeast cells were treated with either iAsIII or MMAIII at concentrations ranging from 0 to 300 μM.Once UROtsa cells reached 70–80% confluence in culture, they were treated with iAsIII at concentrations from 0 to 100 μM or MMAIII at 0–5 μM.
Yeast growth assay
Yeast strains were pregrown in YPD media to mid-log phase, diluted in fresh media to an optical density at 595 nm (OD595) of 0.0165, and inoculated into a 48-well microplate. Stock solutions of arsenicals were added to each culture with at least three replicate wells per dose.
Plates were incubated in a Tecan GENios spectrophotometer (Tecan Systems Inc., San Jose, CA) set to 30°C with intermittent shaking, and OD595 measurements were taken at 15-min intervals for 24 hr. Raw absorbance data were averaged for all replicates, corrected for background, and plotted as a function of time. The area under the curve (AUC) was calculated for the cultures in each well using Prism software (version 5.01; GraphPad Software, Inc., La Jolla, CA), and the treatments were averaged and expressed as a percentage of the control.
Human tissue array and real-time quantitative polymerase chain reaction (PCR) assay
We used TaqMan-based real-time quantitative polymerase chain reaction (rt-qPCR) to quantify N6AMT1 and AS3MT expression on a panel of 48 normal human tissues using the Human Rapid-Scan Plate (OriGene Technologies, Inc., Rockville, MD). The human tissues were selected from multiple individuals of different ethnicity and pooled together. We obtained the primers and probes used for amplification of N6AMT1, AS3MT, and ACTB (β-actin; control) from Applied Biosystems (Foster City, CA). Gene expression of N6AMT1 and AS3MT was calculated relative to ACTB using the ΔΔCT method.
N6AMT1 gene expression vector constructs and stable cell lines
cDNA (GenBank accession no. NM_013240; National Center for Biotechnology Information 2011
) was PCR amplified with primers 5′-AACGCAGCGAAGGACTAT-3′ and 5′-CAGTAGTTCTGGGCACAC-3′. The PCR product was gel purified (Qiagen, Valencia, CA) and cloned into pcDNA 2.1 vector (Invitrogen) according to the manufacturer’s instructions, and the sequence was confirmed. The pcDNA 2.1 vector containing the N6AMT1
gene was excised using Not
HI restriction enzymes (New England Biolabs, Ipswich, MA) and subjected to gel purification. The nucleotides of the N6AMT1
gene containing Bam
HI and Not
I overhangs were annealed and ligated to a linearized pRetro X-IRES-ZsGreen vector (Clonetech, Mountain View, CA) digested with Bam
HI and Not
I (New England Biolabs). The pRetro X-IRES-ZsGreen vector is a fluorescent retroviral expression vector that allows both a gene of interest and the ZsGreen gene to be expressed. The resultant constructs were amplified, purified, and sequenced. UROtsa cells were transfected with this constructed vector or a control vector using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. After incubation at 37°C for 8 hr, the supernatant fraction containing the retroviral vector was removed and replaced with normal growth medium. Cells grown for 48–72 hr were assessed by fluorescence microscopy. The ZsGreen fluorescent marker yields a bright green fluorescence, permitting direct monitoring of the delivery efficiency. Finally, the cell populations were sorted by the DAKO-Cytomation MoFlo High Speed Sorter (Dako North America, Carpinteria, CA), and the green fluorescent cells were purified and collected for continuing culture. The green fluorescent cells were used for additional experimentation.
Semiquantitative reverse-transcription (RT)-PCR
UROtsa cells with either N6AMT1 or plasmid vectors were collected, and total RNA was isolated from these cells using the Qiagen RNAEasy Mini kit. We performed a reverse transcription reaction using SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. The PCR conditions for DNA amplification in the linear range were established on the GeneAmp PCR System 7600 (PerkinElmer, Inc., Wellesley, MA). The primers for DNA amplification were as follows: N6AMT1, 5′-AACGCAGCGAAGGACTAT-3′ and 5′-CAGTAGTTCTGGGCACAC-3′; AS3MT, 5′-GTGTCTGGGTGGTGCTTTATACTG-3′ and 5′-TGGAGGGCAGAACCCAATT-3′; and the housekeeping gene ACTB, 5-TCACCCACACTGTGCCCATCTACGA-3 and 5-CAGCGGAACCGCTCATTGCCAATGG-3. RT-PCR products were analyzed on 1% agarose gels.
We performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay to assess cell viability after arsenic treatment. Cells were cultured in 96-well plates in a volume of 100 μL medium/well at a density of 5 × 104 cells/mL. Twenty-four hours after incubation with iAsIII or MMAIII (six replicates/arsenical concentration), 10 μL sterile MTT dye (Sigma-Aldrich; 5 mg/mL) was added to each well and plates were incubated at 37°C for 4 hr. The culture medium was then removed, and 200 μL dimethyl sulfoxide was added and thoroughly mixed for 10 min. Spectrophotometric absorbance at 570 nm was measured in a microplate reader.
Arsenic species profile analysis by high-performance liquid chromatography/inductively coupled plasma–mass spectrometry (HPLC-ICP-MS) methods
UROtsa cells with N6AMT1
and UROtsa cells with vector were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics. Culture medium was collected after exposure to iAsIII
for 24 hr or 3 days and stored at −80°C until analysis. Cells treated with iAsIII
for 3 days were collected, lysed in RIPA buffer, and extracted in methanol by incubating overnight at 4°C in a rotational shaker. After centrifugation at maximum speed for 5 min at 4°C, supernatants were transferred to microcentrifuge tubes and stored at −80°C until analysis. Before analysis, samples were diluted 1:5 with water–methanol to bring the methanol concentration to 2.5%, incubated at 5°C to precipitate poorly soluble material, and filtered (0.45 μm). Analysis was performed by HPLC-ICP-MS (Agilent 1090 HPLC and Agilent 7500CE ICP-MS run in normal mode; both from Agilent Technologies, Santa Clara, CA) under conditions that resolved neutral, trivalent, and pentavalent iAs species. The ion-pairing method (Le et al. 2000
) was used with major modifications to improve the resolution of the species. Briefly, calibrants were prepared from neat materials [As2
(Aldrich) and As2
(Acros), Sigma-Aldrich; DMAV
O, Chem Service, West Chester, PA] in deionized water (≥ 18 MΩ). For speciation, we used a Phenomenex Gemini-NX column (3 μm, C18, 110Å, 150 × 4.6 mm; Torrance, CA) with a corresponding guard column at 40°C. Concentrations of arsenic species in stock solutions were standardized against NIST traceable commercial ICP-MS standards (VWR BDH Aristar Plus; Ultra Scientific, Kingstown, RI). Serial dilutions were made into deionized water. iAsIII
species were quantified by separate calibrant series, and iAsIII
concentration in the calibrants was corrected for any conversion to iAsV
. The HPLC conditions were isocratic (5 mM tetrabutylammonium hydroxide, 10 mM ammonium carbonate, 2.5% methanol, pH 9.2, 1 mL/min) for 5 min; then a step gradient (5 mM tetrabutylammonium hydroxide, 30 mM ammonium carbonate, 2.5% methanol, pH 8.75, 1.2 mL/min) for 5 min to elute iAsV
was followed by step gradients (5 mM tetrabutylammonium hydroxide, 30 mM ammonium carbonate, 2.5% methanol, pH 9.2, 1.2 mL/min) for a 5-min equilibration to the initial pH and finally to the initial mobile phase for 5 min (1.2 mL/min). The data were analyzed by LC ChemStation A.09.03 and ICP-MS ChemStation B.03.03 software (Agilent Technologies).
Statistical analyses were performed using one-way analysis of variance. Data represent mean ± SE of at least three independent experiments.