Arsenic suppresses gene expression in promyelocytic leukemia cells partly through Sp1 oxidation by Wen-Chien Chou, Hsuan-Yu Chen, Sung-Liang Yu, Linzhao Cheng, Pan-Chyr Yang, and Chi V. Dang Blood Volume 106(1):304-310 July 1, 2005 ©2005 by American Society of Hematology

Expression profile of arsenic-responsive genes. Expression profile of arsenic-responsive genes. (Left) Gene symbols, UniGene numbers, and change ratios of expression as revealed by microarray analysis of the 26 highly responsive genes for RT-PCR after hydrogen peroxide treatment or NAC rescue experiment. Three genes, denoted with asterisks, were not changed more than 6-fold; hence, they are omitted from the heat map. u indicates up-regulated; d, down-regulated. (Right) Heat map demonstrating the arsenicinduced changes in expression (at least 6-fold) of 106 down-regulated and 177 up-regulated genes. Wen-Chien Chou et al. Blood 2005;106:304-310 ©2005 by American Society of Hematology

Arsenic induces ROS production. Arsenic induces ROS production. (A) ROS detection by flow cytometry using dihydrorhodamine 123 as a probe. Pretreatment with NAC significantly blunts ROS production after arsenic treatment. (B) ROS detection by luminol chemiluminescence. Cotreatment with NAC significantly blunts arsenic-induced ROS. Experiments were repeated twice with similar results. The y-axis represents arbitrary units of signal intensity, and the x-axis represents duration of measurement in minutes. Wen-Chien Chou et al. Blood 2005;106:304-310 ©2005 by American Society of Hematology

Genes responsive to hydrogen peroxide and arsenic. Genes responsive to hydrogen peroxide and arsenic. Nineteen of 26 genes are represented in this figure. Among 9 arsenic up-regulated genes, 2 (CDw52 and ALOX5AP) are also up-regulated by hydrogen peroxide. Among 17 arsenic down-regulated genes, 7 (C17, TERT, MYC, GJA1, SOX18, CCNA1, and STAB1) are also down-regulated by hydrogen peroxide (only 5 were presented in this figure); see Figure 4. Values are averages of triplicates. Independent experiments were performed at least twice with similar results. Wen-Chien Chou et al. Blood 2005;106:304-310 ©2005 by American Society of Hematology

Effect of NAC on arsenic-induced gene expression. Effect of NAC on arsenic-induced gene expression. (A-B) Certain arsenic-induced gene expression changes can be blunted by at least 50% with NAC cotreatment (reversed). There are also genes whose expression is altered by arsenic but that remain unaffected by NAC (not reversed). Some genes were responsive (more than 2-fold) to NAC alone without arsenic and hence were categorized as being “not informative.” Values are averages of triplicates. Independent experiments were performed at least twice with similar results. (C) Schematic diagram showing that 6 of 26 genes tested are directly related to ROS production (common area of the 3 circles). Blue highlights down-regulated genes, and red highlights up-regulated genes by arsenic treatment. The 9 genes in the area common to the red and blue circles denote those with consensus change of expression after treatment with arsenic or hydrogen peroxide. The 12 genes in the area common to the red and black circles are those whose expression change after arsenic treatment could be reversed by NAC. Wen-Chien Chou et al. Blood 2005;106:304-310 ©2005 by American Society of Hematology

In vivo Sp1 binding to the promoters of hTERT, c-Myc, and C17, as determined by ChIP assay. In vivo Sp1 binding to the promoters of hTERT, c-Myc, and C17, as determined by ChIP assay. (A, left) Nuclear extracts from the same samples used in ChIP were immunoblotted with c-Myc and Sp1 antibodies. Coomassie blue staining served as loading control. (Right) SYBR green real-time PCR shows diminished Sp1 and c-Myc binding on the hTERT promoter after arsenic (0.75 μM for 12 days) exposure. Mock indicates no nuclear extract in the reaction; No Ab, omission of antibody in the immunoprecipitation procedures. HGF antibody was used as a control IgG. PCR products obtained at 35 cycles and resolved on agarose gel are shown below the graph. (B-C) SYBR green real-time PCR quantification of ChIP using anti-Sp1 antibody shows a significant decrease in Sp1 binding in the promoters of MYC (B) and C17 (C) after arsenic treatment. Error bars represent standard deviations from triplicate experiments. Wen-Chien Chou et al. Blood 2005;106:304-310 ©2005 by American Society of Hematology

Sp1 deglycosylation after arsenic treatment. Sp1 deglycosylation after arsenic treatment. (A) Sp1 glycosylation was decreased after arsenic treatment, as detected by immunoblotting (IB) with O-linked GlcNAc-specific antibody (110.6 Ab) after immunoprecipitation (IP) with an anti-Sp1 antibody. The Sp1 antibody-detected signals serve as loading controls. (B) Sp1 deglycosylation was induced by glucose starvation (24 hours) or treatment with DON (40 μM for 24 hours). (C) Decrease in Sp1 glycosylation did not diminish in vivo Sp1 binding on the hTERT promoter, as determined by chromatin immunoprecipitation. (D) Deglycosylation of Sp1 did not result in decreased hTERT expression, as determined by quantitative RT-PCR. Error bars represent standard deviations from triplicate experiments. Wen-Chien Chou et al. Blood 2005;106:304-310 ©2005 by American Society of Hematology

Hydrogen peroxide treatment decreases in vivo Sp1 binding to MYC and C17 promoters. Hydrogen peroxide treatment decreases in vivo Sp1 binding to MYC and C17 promoters. (A) ChIP assay showed decrease of Sp1 binding on the promoters of c-Myc and C17 after oxidation by hydrogen peroxide treatment. (B) Cell fractionation after hydrogen peroxide treatment showed no decrease in the level of Sp1, which was exclusively nuclear. Cytosolic heat shock protein 90 (Hsp90) was used as a control for fractionation. γ-Tubulin was used as a loading control. Wen-Chien Chou et al. Blood 2005;106:304-310 ©2005 by American Society of Hematology

Arsenic and hydrogen peroxide treatments result in oxidation of Sp1 protein. Arsenic and hydrogen peroxide treatments result in oxidation of Sp1 protein. Immunoprecipitated (IP) Sp1 from control, arsenic, or hydrogen peroxide-treated samples was incubated with BIAM, and this was followed by detection by immunoblotting (IB) with streptavidin-conjugated HRP. The same immunoprecipitate was subjected to detection by Sp1 antibody as a loading control. BIAM/Sp1 signal ratios represent relative BIAM incorporation. Wen-Chien Chou et al. Blood 2005;106:304-310 ©2005 by American Society of Hematology