PLoS Med. 2006 October; 3(10): e420.

Real-Time RT-PCRTotal RNA was extracted from cells using RNeasy kit (Qiagen) and was quantified by UV absorbance spectrophotometry. The reverse transcription reaction was performed by using the Superscript First-Strand Synthesis System (Invitrogen) in a final volume of 20 μl containing 2 μg of total RNA, 100 ng of random hexamers, 1× reverse transcription buffer, 2.5 mM MgCl2, 1 mM dNTP, 10 U of RNaseOUT, 20 U of Superscript reverse transcriptase, and DEPC-treated water. Quantitative real-time RT-PCR analyses of human KEAP1, NRF2, GCLc, GCLm, GSR, PRDX1, GSTA3, GSTA2, NQO1, MRP1, and MRP2 were performed by using Assay-on-Demand primers and probe sets from Applied Biosystems. Assays were performed by using the ABI 7000 Taqman system (Applied Biosystems). β-actin was used for normalization.

PLoS Med. 2006 October; 3(10): e420.

Plasmid ConstructionPlasmid encoding human KEAP1 cDNA in pCMV6-XL5 was purchased from Origene Technologies (Rockville, Maryland, United States). Glycine-to-serine mutations, leucine-to-arginine mutations, and a single nucleotide deletion were introduced into the KEAP1 expression vector by using a site-directed mutagenesis kit (Stratagene, La Jolla, United States). Details of the site-directed mutagenesis primers used in this study are listed in Table S2.

PLoS Med. 2006 October; 3(10): e420.

Cell Culture and ReagentsHBE4, NL20, A549, H460, H1435, H292, H23, H358, H1299, H1993, H1395, and H838 cells were purchased from American Type Culture Collection (Manassas, Virginia, United States) and cultured under recommended conditions. BEAS2B cells were provided by S. Reddy (Johns Hopkins University, Baltimore, Maryland, United States). All transfections were carried out using Lipofectamine 2000 (Invitrogen).

PLoS Med. 2006 October; 3(10): e420.

Generation of Stable TransfectantsH838 cells overexpressing ARE luciferase reporter plasmid were obtained by transfecting H838 cells with 3 μg of NQO1-ARE reporter plasmid and 0.3 μg of pUB6 empty vector (Invitrogen). Stable transfectants were selected using Blasticidin at a concentration of 6 μg/ml. Stable clones were expanded and screened for the expression of ARE luciferase.

PLoS Med. 2006 October; 3(10): e420.

Luciferase AssayH838 cells stably expressing NQO1-ARE luciferase were seeded onto a 24-well dish at a density of 0.2 × 106 cells/ml for 12 h before transfection. WT-KEAP1 cDNA constructs along with the mutant cDNA constructs (G333C and L413R) were transfected into the cells along with pRL-TK plasmid expressing Renilla luciferase as a transfection control. Twenty-four hours after transfection, cells were lysed and both firefly and Renilla luciferase activities were measured with a Dual-Luciferase Reporter Assay System (Promega).

PLoS Med. 2006 October; 3(10): e420.

siRNA Duplex Screening and TransfectionThe siRNA sequence targeting NRF2 corresponds to the coding region nucleotides 1903–1921 (5′-GTAAGAAGCCAGATGTTAA-3′) in the NRF2 cDNA. The NRF2 siRNA duplex with the following sense and antisense sequences was used: 5′-GUAAGAAGCCAGAUGUUAAdUdU-3′ (sense) and 3′-dUdUCAUUCUUCGGUCUACAATT-5′ (antisense). KEAP1 siRNA corresponds to the coding region nucleotides 1545–1563 (5′-GGGCGTGGCTGTCCTCAAT-3′) in KEAP1 transcript variant 2. The KEAP1 siRNA duplex with the following sense and antisense sequences was used: 5′-GGGCGUGGCUGUCCUCAAUdUdU-3′ (sense) and 3′-dUdUCCCGCACCGACAGGAGUUA-5′ (antisense). To confirm the specificity of the inhibition, the siCONTROL non-targeting siRNA 1 (NS siRNA; 5′-UAGCGACUAAACACAUCAAUU-3′) with microarray-defined signature was used as a negative control. All of the siRNA duplexes were synthesized by Dharmacon Research (Lafayette, Colorado, United States). Cells in the exponential growth phase were plated at a density of 0.2 × 106 cells/ml, grown for 12 h, and transfected twice at an interval of 48 h with 50 nM siRNA duplexes using Lipofectamine 2000 and OPTI-MEM reduced serum medium (Invitrogen) according to the manufacturer's recommendations. Concentrations of siRNAs were chosen on the basis of dose–response studies (data not shown).

PLoS Med. 2006 October; 3(10): e420.

MTT Cell Viability AssayThe in vitro drug sensitivity to etoposide and carboplatin was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Cells were plated at a density of 10,000 cells for BEAS2B, 5,000 cells for A549, H460, and H838, and 20,000 cells for H1435 in 96-well plates. They were allowed to recover for 12 h and then exposed to various concentrations of etoposide and carboplatin for 72 h. After 72 h, drug cytotoxicity was evaluated by using a MTT reduction conversion assay (Sigma, Saint Louis, Missouri, United States). Forty microliters of MTT at 5 mg/ml concentration was added to each well, and incubation was continued for 4 h. The formazan crystals resulting from mitochondrial enzymatic activity on MTT substrate were solubilized with 200 μl of dimethyl sulfoxide, and absorbance was measured at 570 nm by using a SpectraMAX microplate reader (Molecular Devices, Sunnyvale, California, United States). Each combination of cell line and drug concentration was set up in eight replicate wells, and the experiment was repeated three times. Cell survival was expressed as absorbance relative to that of untreated controls. Results are presented as means ± standard deviation (SD).

PLoS Med. 2006 October; 3(10): e420.

Figure 3 Dysfunctional KEAP1朜RF2 Interaction in NSCLC Tumors (A) Immunohistochemical analysis of NRF2 in NSCLC tissues. Part a shows a patient (PT-18) with mutation in KEAP1 showing strong nuclear and cytoplasmic staining. Part b shows a patient negative for mutation (PT-28) showing weak cytoplasmic staining. Part c shows a patient negative for mutation (PT-20) showing increased nuclear and cytoplasmic staining in tumor tissue. Part d shows weakly staining normal bronchus from the same patient (PT-20). (B) Total GSH and enzyme activities of NQO1 and total GST in NSCLC and matched normal tissues. Raw data for the heat maps are presented in Table S4. *, samples harboring KEAP1 mutation; ? nmol/mg protein; ? nmol DCPIP reduced/min/mg protein; ? nmol of product formed/min/mg protein.

PLoS Med. 2006 October; 3(10): e420.

Figure 4 Status of KEAP1 and NRF2 Is Altered in Cancer Cells (A) Immunoblot showing increased nuclear localization of NRF2 in nuclear extracts (NE) from cancer cells. Cancer cells showed lower levels of KEAP1 (~69 kDa) and higher levels of NRF2 (~110 kDa) in total protein lysates (TP). NIVT and KIVT indicate NRF2 and KEAP1 in vitro transcribed/translated product, respectively. (B and C) Quantification of NRF2 and KEAP1 protein in immunoblots. For band densitometry, bands in nuclear extract blot (B) were normalized to Lamin B1, and those in total protein (C) were normalized to GAPDH. (D) Heat map showing relative expression of KEAP1, NRF2, and NRF2-dependent genes by real-time RT-PCR. Raw data for the heat maps are presented in Table S5.

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Figure 7 Increased NRF2 Activity Confers Chemoresistance BEAS2B cells and cancer cells were exposed to etoposide (A) or carboplatin (B) for 72 h, and viable cells were determined by MTT assay. BEAS2B cells displayed enhanced sensitivity whereas cancer cells with dysfunctional KEAP1 activity demonstrated reduced chemosensitivity to etoposide and carboplatin treatment. Data are presented as percentage of viable cells relative to the vehicle-treated control. Data are the mean of eight independent replicates, combined to generate the mean 卤 SD for each concentration.

PLoS Med. 2006 October; 3(10): e420.

We thank Laura Kasch at the DNA fragment analysis facility at Johns Hopkins University for microsatellite-based genotyping services. We also thank the Statistics Core, Bloomberg School of Public Health, Johns Hopkins University, for assistance with statistical analyses.

PLoS Med. 2006 December; 3(12): e486.

A Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies

PLoS Med. 2006 December; 3(12): e486.

Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The “rules” governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets.

PLoS Med. 2006 December; 3(12): e486.

Methods and FindingsIn an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5′ CpG islands, are induced from undetectable levels by 5-aza-2′-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors.

PLoS Med. 2006 December; 3(12): e486.

Quantitative RT-PCRExpression of LOX, NRCAM, BNC1, CCNA1, MAF, ALDH1A3, CTSZ, IRX4, MSX1, KLF11, SERPINB5, TKTL1, GAPDH, r18s, and CDKN2A was analyzed by quantitative real-time RT-PCR. Primers and probes were purchased from Applied Biosystems assay-on-demand, with the exception of p16, which was an assay-by-design (Hs00923893_m1) (http://www.appliedbiosystems.com). All samples were run on the Chromo 4 Real Time Detector (MJ Research [http://www.bio-rad.com]) twice, each time in duplicate. We averaged expression of GAPDH and r18s as internal reference genes to normalize input cDNA. Quantitative real-time reverse-transcriptase-PCR (QPCR) was performed in a reaction volume of 20 μl including 1 μl of cDNA. We used the comparative Ct method to compute relative expression values.