Unique effects of zinc protoporphyrin on HO-1 induction and apoptosis by Guang Yang, Xuandai Nguyen, Judy Ou, Prasad Rekulapelli, David K. Stevenson, and Phyllis A. Dennery Blood Volume 97(5):1306-1313 March 1, 2001 ©2001 by American Society of Hematology

HO-1 expression in HA-1 cells incubated with 10 μM MPs for 24 hours HO-1 expression in HA-1 cells incubated with 10 μM MPs for 24 hours.Upper panel shows a representative example of 3 Northern blots of HO-1 mRNA. HO-1 expression in HA-1 cells incubated with 10 μM MPs for 24 hours.Upper panel shows a representative example of 3 Northern blots of HO-1 mRNA. Lower panel shows a representative example of 3 Western blots of HO-1 immunoreactive protein. Lane HO-1 is HO-1 standard obtained from rat livers injected with COCl2; lane C, untreated control; lane ZP, cells incubated with ZnPP; lane SP, cells incubated with SnPP; lane CP, cells incubated with CrPP. Guang Yang et al. Blood 2001;97:1306-1313 ©2001 by American Society of Hematology

Densitometric evaluation of HO-1 mRNA in HA1 cells incubated with ZnPP or ZnCl2.HO-1 mRNA was measured by Northern analysis and normalized to the β-actin mRNA obtained from the same blot. Densitometric evaluation of HO-1 mRNA in HA1 cells incubated with ZnPP or ZnCl2.HO-1 mRNA was measured by Northern analysis and normalized to the β-actin mRNA obtained from the same blot. Lane C is untreated control; lane ZP, cells incubated with 10 μM ZnPP for 24 hours; lane ZC, cells incubated with 100 μM ZnCl2 for 24 hours; lane ZP/ZC, cells incubated with 10 μM ZnPP for 6 hours then with 100 μM ZnCl2 for an additional 18 hours; lane ZC/ZP, cells incubated with 100 μM ZnCl2 for 6 hours then with 10 μM ZnPP for 18 hours. Each bar represents the mean ± SE of 3 experiments. *P < .05 versus controls; †P < .05 versus ZnPP- or ZnCl2-treated cells. Insert: Determining the maximum HO-1 mRNA induction with ZnPP incubation. Densitometric evaluation of HO-1 mRNA normalized to the β-actin mRNA. Lane C is untreated control; lane 1, cells incubated with 1 μM ZnPP for 24 hours; lane 10, cells incubated with 10 μM ZnPP for 24 hours; lane 50, cells incubated with 50 μM ZnPP for 24 hours. Maximal induction was achieved with 10 μM ZnPP. Guang Yang et al. Blood 2001;97:1306-1313 ©2001 by American Society of Hematology

Effect of cycloheximide on HO-1 mRNA induction by ZnPP Effect of cycloheximide on HO-1 mRNA induction by ZnPP.HA-1 cells were preincubated with various doses of cycloheximide for 15 minutes prior to incubation with 10 μM ZnPP. Effect of cycloheximide on HO-1 mRNA induction by ZnPP.HA-1 cells were preincubated with various doses of cycloheximide for 15 minutes prior to incubation with 10 μM ZnPP. HO-1 mRNA was evaluated by Northern analysis and normalized to the β-actin mRNA obtained from the same blot. Lane Cyc, cells incubated with 2 mM cycloheximide alone for 15 minutes as control; lane ZP, cells incubated with 10 μM ZnPP for 24 hours. Lanes 0.05, 0.1, 0.3, 1, and 2 show cells preincubated with 0.05, 0.1, 0.3, 1, or 2 mM cycloheximide and then incubated with ZnPP for 24 hours. Guang Yang et al. Blood 2001;97:1306-1313 ©2001 by American Society of Hematology

AP-1, SP-1, and Egr-1 binding in nuclear extracts from HA-1 cells incubated with 100 μM ZnCl2 or 10 μM MPs for 24 hours.Each panel represents an electrophoretic mobility shift gel from 3 separate experiments. AP-1, SP-1, and Egr-1 binding in nuclear extracts from HA-1 cells incubated with 100 μM ZnCl2 or 10 μM MPs for 24 hours.Each panel represents an electrophoretic mobility shift gel from 3 separate experiments. (A) AP-1 binding. Lane F, free probe; lane C; untreated control; lane ZC, cells incubated with ZnCl2; lane ZP, cells incubated with ZnPP; lane SP, cells incubated with SnPP; lane CP, cells incubated with CrPP; lane ZC+AP-1, ZnCl2treated cells incubated with a 100-fold excess of cold AP-1 probe to demonstrate specificity of binding. (B) SP-1 binding. Lane F, free probe; lane C, untreated control; lane ZC, cells incubated with ZnCl2; lane ZP, cells incubated with ZnPP; lane CP, cells incubated with CrPP; lane SP, cells incubated with SnPP; lane ZC+SP-1, ZnCl2 treated cells incubated with a 100-fold excess of cold SP-1 probe to demonstrate specificity of binding. (C) Egr-1 binding. Lane F, free probe; lane C, untreated control; lane ZP, cells incubated with ZnPP; lane ZC, cells incubated with ZnCl2; lane CP, cells incubated with CrPP; lane SP, cells incubated with SnPP; lane ZP+Egr-1, ZnPP-treated cells incubated with a 100-fold excess of cold Egr-1 probe to demonstrate specificity of binding. In each gel, cold competition was performed with the compound leading to the highest nuclear protein binding. Guang Yang et al. Blood 2001;97:1306-1313 ©2001 by American Society of Hematology

Effect of Egr-1 on HO-1 mRNA induction by ZnPP Effect of Egr-1 on HO-1 mRNA induction by ZnPP.(A) Relative HO-1 mRNA induction by ZnPP in Egr-1−/−mouse fibroblast cells. Effect of Egr-1 on HO-1 mRNA induction by ZnPP.(A) Relative HO-1 mRNA induction by ZnPP in Egr-1−/−mouse fibroblast cells. The HO-1 mRNA value for nontreated cell controls was assigned as 1. 3T3 indicates wild-type mouse fibroblast cells (NIH 3T3) incubated with 10 μM ZnPP for 24 hours. Egr+/− indicates Egr-1 heterozygote mouse fibroblasts incubated with 10 μM ZnPP for 24 hours. Egr−/−indicates Egr-1 homozygote null mutant mouse fibroblasts incubated with 10 μM ZnPP for 24 hours. (B) Effect of Egr-1 antisense oligonucleotides on ZnPP-induced HO-1 mRNA in HA-1 cells. Upper panel shows Egr-1 immunoreactive protein levels after incubation with Egr-1 antisense oligomer. Lower panel is the densitometric evaluation of HO-1 mRNA normalized to β-actin mRNA. Lane S, cells treated with 40 μM Egr-1 sense oligomers prior to incubation with ZnPP; lane A, cells treated with 40 μM Egr-1 antisense oligomers prior to incubation with ZnPP. Values represent the mean ± SE of 2 experiments; *P < .05 versus sense. Guang Yang et al. Blood 2001;97:1306-1313 ©2001 by American Society of Hematology

S/E binding in nuclear extracts from HA-1 cells incubated with 100 μM ZnCl2 or 10 μM MPs for 24 hours.The 12-bp oligomer (S/E) from the mouse HO-1 gene DE2 enhancer region has 85% similarity to the consensus sequence for an SP-1 and Egr-1 overlapping bindin... S/E binding in nuclear extracts from HA-1 cells incubated with 100 μM ZnCl2 or 10 μM MPs for 24 hours.The 12-bp oligomer (S/E) from the mouse HO-1 gene DE2 enhancer region has 85% similarity to the consensus sequence for an SP-1 and Egr-1 overlapping binding site. Lane F, free probe; lane ZC, cells incubated with ZnCl2; lane ZP, cells incubated with ZnPP; lane SP, cells incubated with SnPP; lane CP, cells incubated with CrPP. Guang Yang et al. Blood 2001;97:1306-1313 ©2001 by American Society of Hematology

ZnPP and ZnCl2 responsive elements in the mouseHO-1 gene transfected to HA-1 cells.A partial restriction map and structural organization of the mouseHO-1 gene and the 5′-flanking region are shown. ZnPP and ZnCl2 responsive elements in the mouseHO-1 gene transfected to HA-1 cells.A partial restriction map and structural organization of the mouseHO-1 gene and the 5′-flanking region are shown. Position +1 represents the transcription initiation site. The location of the EH and SX2 enhancer elements are indicated by solid bars. B indicates BamHI; E, EcoRI; H, HindIII; X, XhoI. HA-1 cells were transiently transfected with the indicated CAT constructs and incubated with 10 μM ZnPP or 100 μM ZnCl2 for 24 hours. The CAT assay was carried out as described under “Materials and methods.” The data represent the average value from 3 independent experiments and the mean fold induction of CAT activity in extracts of cells incubated with ZnPP or ZnCl2 compared to that in extracts of cells incubated with the constructs alone. Guang Yang et al. Blood 2001;97:1306-1313 ©2001 by American Society of Hematology

Localization of transcription enhancer activity of the EH subfragment in HA-1 cells.The indicated fragments of EH were subcloned into the vector pMHO1CATΔ-33, which contains a minimal HO-1 promoter (−33 to +73). Localization of transcription enhancer activity of the EH subfragment in HA-1 cells.The indicated fragments of EH were subcloned into the vector pMHO1CATΔ-33, which contains a minimal HO-1 promoter (−33 to +73). E indicates EcoRI; A, AflII; B, BsrBI; T, TaqI; R, RsaI; H,HindIII. HA-1 cells were transiently transfected with the indicated CAT constructs and incubated with 10 μM ZnPP or 100 μM ZnCl2 for 24 hours. The CAT assay was carried out as described under “Materials and methods.” The data represent the average value from 3 independent experiments and the mean fold induction of CAT activity in extracts of cells incubated with ZnPP or ZnCl2 compared to that in extracts of cells incubated with the constructs alone. Guang Yang et al. Blood 2001;97:1306-1313 ©2001 by American Society of Hematology

Nuclear protein binding analysis of the RH enhancer fragment by DNase I protection assays.The noncoding strand of the RH fragment was labeled with32P at the 3′ end by an end-fill reaction. Nuclear protein binding analysis of the RH enhancer fragment by DNase I protection assays.The noncoding strand of the RH fragment was labeled with32P at the 3′ end by an end-fill reaction. DNase I footprint analysis was carried out using 15 μg nuclear extract from HA-1 cells incubated with MPs. The DNase I digestion products were coelectrophoresed with the G+A chemical sequencing ladder of the RH probe on a denaturing 6% polyacrylamide gel and autoradiographed for 48 hours. Lane G+A is G+A chemical sequencing ladder; lane RH, RH probe alone digested with DNase I; lane BSA, RH probe incubated with BSA prior to DNase I digestion; lane ZC, nuclear extract from ZnCl2-treated cells incubated with RH probe prior to DNase I digestion; lane SP, nuclear extract from SnPP-treated cells incubated with RH probe prior to DNase I digestion; lane ZP, nuclear extract from ZnPP-treated cells incubated with RH probe prior to DNase I digestion. The region protected from DNase I digestion is indicated by the arrows and the 9-bp sequence, which has 78% homology with Egr-1 is indicated with the parentheses. Guang Yang et al. Blood 2001;97:1306-1313 ©2001 by American Society of Hematology

Markers of apoptosis in MP-incubated cells Markers of apoptosis in MP-incubated cells.(A) A representative slide of immunoreactive annexin V detection in cultured HA-1 cells incubated with 10 μM MPs for 24 hours. Markers of apoptosis in MP-incubated cells.(A) A representative slide of immunoreactive annexin V detection in cultured HA-1 cells incubated with 10 μM MPs for 24 hours. Four slides in each group were incubated with annexin V antibody and FITC as described in “Materials and methods.” All images were obtained at the same intensity to allow for comparison. Lane C, untreated controls; lane CP, cells incubated with CrPP; lane SP, cells incubated with SnPP; lane ZP, cells incubated with ZnPP. (B) Upper panel shows representative example of 2 Western analyses for immunoreactive p53 protein content in HA-1 cells after 10 μM MP incubation. Lane p53, p53 protein standard obtained from human 293 cells; lane C, untreated controls; lane ZC, cells incubated with 100 μM ZnCl2 ; lane ZP, cells incubated with ZnPP;lane SP, cells incubated with SnPP; lane CP, cells incubated with CrPP. Lower panel indicates that p53 protein induction was dose dependent on ZnPP concentration. Lane p53, p53 protein standard obtained from human 293 cells; lane C, untreated controls; lane 1, cells incubated with 1 μM ZnPP; lane 10, cells incubated with 10 μM ZnPP; lane 50, cells incubated with 50 μM ZnPP. Guang Yang et al. Blood 2001;97:1306-1313 ©2001 by American Society of Hematology

Nuclear localization of ZnPP and SnPP in HA-1 cells Nuclear localization of ZnPP and SnPP in HA-1 cells.Representative pseudoimages of fluorescent signal for MPs and immunoreactive p53. Nuclear localization of ZnPP and SnPP in HA-1 cells.Representative pseudoimages of fluorescent signal for MPs and immunoreactive p53. Three slides in each group were incubated with ZnPP or SnPP then incubated with p53 antibody and FITC-labeled secondary antibody as described in “Materials and methods” and analyzed at various time points. The MPs were visualized using their endogenous fluorescence by setting excitation at 568 nm and emission at more than 590 nm, and FITC was detected by setting the excitation at 488 nm and the emission at 515 to 545 nm. Upper panel is ZnPP-incubated HA-1 cells. Lower panel shows SnPP-incubated HA-1 cells. Lane 1h, 1-hour incubation with MP; lane 4h, 4-hour incubation with MP; lane 12h, 12-hour incubation with MP; lane 24h, 24-hour incubation with MP. The yellow arrows represent colocalization of MPs and p53; the orange arrow represents the endogenous fluorescence of the MPs; the green arrow represents the p53 signal. In the SnPP-treated cells, the FITC signal was further enhanced to allow for visualization of p53 within the nucleus. Note the nuclear colocalization of ZnPP and p53 and the lack of nuclear colocalization of SnPP and p53. CrPP could not be visualized due to a lack of endogenous fluorescence. Guang Yang et al. Blood 2001;97:1306-1313 ©2001 by American Society of Hematology