Nrf1b-MafG complex binds the ARE.

<p>(<b>A</b>) EMSA studies were performed with Nrf1b and MafG proteins generated by in vitro transcription and translation, and biotinylated DNA probe containing a consensus antioxidant response element described in Materials and Methods. Rabbit anti-Nrf1, anti-MafG, and IgG were used for super-shifts. Chevrons and arrows indicate shifted and super-shifted bands, respectively. (<b>B</b>) HEK293 cells transfected with Nrf1b-V5 were harvested 48 h after, and lysates were immunoprecipitated with anti-V5 antibody. Immunoprecipitates were then subjected to immunoblotting with anti-V5 or anti-MafG antibodies.</p

Nrf1b expression is widely distributed.

<p>Nrf1a and Nrf1b mRNA expression patterns were analyzed by RT-PCR in various cell lines (<b>A</b>) and mouse tissues (<b>B</b>)<b>.</b> Nrf1a and Nrf1b cDNA was amplified by PCR for 30 cycles and 18S was amplified for 20 cycles. Histograms show relative Nrf1a and Nrf1b expression normalized against 18S. (<b>C</b>)<b>.</b> Western blot of different mouse tissues probed with Nrf1 antibody. HEK293 cells transfected with pEF1-Nrf1a (lane 1), and pEF1-Nrf1b (lane 2) were used as controls for detection of the Nrf1a and Nrf1b isoforms by the Nrf1 antibody. Beta-actin was used as a loading control.</p

Nrf1b protein is localized in the nucleus.

<p>(<b>A</b>)<b>.</b> Epifluorescent micrographs showing COS7 cells transfected with Nrf1b-EGFP. (<b>B</b>) Quantitative analysis of the results in (B). The subcellular localization of EGFP and Nrf1b-EGFP was scored as follows: N>C, predominantly nuclear; N  =  C, evenly distributed between the nucleus and cytoplasm; NC)<b>.</b> Distribution of Nrf1b-V5 in cells. V5-tagged Nrf1b was harvested from HEK293 cells 48hr after transfection as described in the methods section, and analyzed by Western blot. The antibodies used for Western blotting are indicated on the right. Pyruvate kinase was used as a cytoplasmic marker, calnexin as an ER membrane marker, and lamina-associated polypeptide 2α (LAP2α) as a nuclear marker.</p

Nrf1b is derived by alternative promoter usage.

<p>(<b>A</b>)<b>.</b> Nucleotide sequence and identification of putative cis-acting elements in the 5′-flanking region of Nrf1b exon 1. Numbering is relative to the first nucleotide of the initiation codon (ATG) designated as +1. Vertical arrow represents the transcription start site identified by primer extension analysis, and coding region is underlined. The cis-acting elements containing consensus sequences are boxed. (<b>B</b>)<b>.</b> Primer extension result with NIH3T3 mRNA. The peak corresponding to a 108-bp elongation product (FAM-labeled cDNA) is indicated by an arrow. Labeled cDNA was aligned with the sequence electropherogram to identify the base at which transcription starts for Nrf1b. Nucleotides are indicated on the x-axis. (<b>C</b>)<b>.</b> Relative luciferase activities of the mouse Nrf1b promoter construct in HEK293 and Hepa1c1c7 cells. Negative control consisted of the pGL3-Basic control. Luciferase activities were normalized to <i>Renilla</i> luciferase from pRL-TK. Histograms show mean ± SD of three independent experiments with triplicate samples per experiment. *P<0.05.</p

Nrf1b encodes a novel Nrf1 protein.

<p>Schematic diagram of human and mouse Nrf1 genomic sequences, and depiction of Nrf1a and Nrf1b transcripts. Solid and open boxes represent coding regions and untranslated regions, respectively. Solid lines represent introns and 5′-flanking regions.</p

Nrf1b activates ARE-driven genes.

<p>Luciferase reporter bearing 3 copies of ARE (<b>A</b>)<b>,</b> or GCLM-luciferase reporter (<b>B</b>) was co-transfected with either Nrf1a or Nrf1b expression plasmid. Luciferase activities were normalized to <i>Renilla</i> luciferase from pRL-TK. Results are expressed relative to luciferase activities observed with vector alone. Histograms show the means of three separate experiments ± SD carried out in triplicates. *P<0.05 (<b>C</b>) Induction of endogenous ARE target genes. NIH3T3 cells were transfected with the indicated plasmids, and mRNA was harvested and analyzed 2 days after transfection by qRT-PCR. Histograms show mean ± SD (n = 4).</p

Copy Number Gains at 8q24 and 20q11-q13 in Gastric Cancer Are More Common in Intestinal-Type than Diffuse-Type

<div><p>The present study was aimed at discovering DNA copy number alterations (CNAs) involved in the carcinogenesis of stomach and at understanding their clinicopathological significances in the Korean population. DNA copy numbers were analyzed using Agilent 244K or 400K array comparative genomic hybridization (aCGH) in fresh-frozen tumor and matched normal tissues from 40 gastric cancer patients. Some of the detected CNA regions were validated using multiplex ligation-dependent probe amplification (MLPA) in six of the 40 patients and customized Agilent 60K aCGH in an independent set of 48 gastric cancers. The mRNA levels of genes at common CNA regions were analyzed using quantitative real-time PCR. Copy number gains were more common than losses across the entire genome in tumor tissues compared to matched normal tissues. The mean number of alterations per case was 64 for gains and 40 for losses, and the median aberration length was 44016 bp for gains and 4732 bp for losses. Copy number gains were frequently detected at 7p22.1 (20%), 8q24.21 (27%–30%), 8q24.3 (22%–48%), 13q34 (20%–31%), and 20q11-q13 (25%–30%), and losses at 3p14.2 (43%), 4q35.2 (27%), 6q26 (23%), and 17p13.3 (20%–23%). CNAs at 7p22.1, 13q34, and 17p13.3 have not been reported in other populations. Most of the copy number losses were associated with down-regulation of mRNA levels, but the correlation between copy number gains and mRNA expression levels varied in a gene-dependent manner. In addition, copy number gains tended to occur more commonly in intestinal-type cancers than in diffuse-type cancers. In conclusion, the present study suggests that copy number gains at 8q24 and 20q11-q13 and losses at 3p14.2 may be common events in gastric cancer but CNAs at 7p22.1, 13q34, and 17p13.3 may be Korean-specific.</p></div

CNAs according to Lauren’s classification.

<p>(A-B) Correlations of log2 ratio of DNA copy number and mRNA fold change (FC) of <i>FHIT</i> (A) and <i>BOP1</i> (B) in tumor tissues compared to matched normal tissues were analyzed using Pearson’s correlation coefficients. Different color circles indicate different samples. (C-E) The differences in prevalence of copy number alterations of multiple genes were compared between diffuse-type cancers and intestinal-type cancers using Pearson’s chi-square test. Copy number gains of <i>MYC</i> (<i>P</i> = 0.03), <i>BOP1</i> (<i>P</i> = 0.03), and <i>CD40</i> (<i>P</i> = 0.01) occurred at a high prevalence in intestinal-type cancers compared to diffuse-type cancers. In general, copy number gains tend to occur more frequently in intestinal-type cancers than in diffuse-type cancers.</p

Correlations of CNAs levels with patient’s age at diagnosis and other CNAs in different chromosomal regions.

<p>(A) Correlations of CNAs levels with patient’s age were analyzed using Pearson’s correlation coefficient. Multiple tests were corrected using the Bonferroni correction. The Bonferroni-corrected <i>P</i>-values were calculated by multiplying the observed (uncorrected) <i>P</i>-values by the number of tested genes. Violet colors indicate Bonferroni-adjusted <i>P-</i>value < 0.05. (B) Unsupervised hierarchical clustering analysis of 15 genes in regions with recurrent (>20%) CNAs was performed to investigate correlations among CNAs levels of 11 genes showing copy number gains on 8q24.21, 8q24.3, 20q11.21, and 20q13.12, and 4 genes showing copy number losses. The numbers on the right side of the figure indicate patient identification number. The color scales indicate log2 intensity ratios of CNAs at individual gene. Value “zero (= log(2/2)” indicate a copy number of 2. Green and red colors represent copy number gain and loss, respectively.</p

Copy number alterations of gastric cancer using aCGH.

<p>(A) Overall aberration frequency in gastric cancers is shown. The horizontal line represents chromosomes in order from 1 to 22 and X. The vertical line represents the frequency of gains and losses in all cases. (B) The number of aberrations in each case is shown. Magenta bars indicate gains and viridian bars indicate losses. (C) Aberration length—density plot is shown. (D) Common CNAs in 40 gastric cancer samples are shown. Within each chromosome, aberrations are expressed in order from the p telomere to q telomere. Reds on the right of the chromosomes indicate gains, and greens on the left of the chromosomes indicate losses. (E) Common aberration length with p-value plot is shown. (F) Common aberrations in chromosome 8 (upper panel) and aberrations around <i>MYC</i> gene at 8q24.21 (lower panel) are shown. Vertical lines indicate log₂-based intensity ratio values, and each colored horizontal line represents a copy number alteration. Horizontal lines above the 0.25 of log₂-based intensity ratio indicate samples with copy number gains. The large vertical blue bar in the lower panel indicates the center of the currently analyzed region.</p