An Interaction between Arsenic-Induced Epigenetic Modification and Inflammatory Promotion in a Skin Equivalent during Arsenic Carcinogenesis  Wei-Ting Liao, Jian-He Lu, Chih-Hung Lee, Cheng-Che E. Lan, Jan-Gowth Chang, Chee-Yin Chai, Hsin-Su Yu  Journal of Investigative Dermatology  Volume 137, Issue 1, Pages 187-196 (January 2017) DOI: 10.1016/j.jid.2016.08.017 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Morphological abnormalities in arsenic-treated SEs. (a) Histopathological features of normal and As-BD skin specimens (upper), PBMC-free SEs (middle; 10-day, with or without 1-μmol/L arsenic treatment), and PBMC-treated SEs (lower; 10-day, with or without 1-μmol/L arsenic treatment) were observed using hematoxylin and eosin staining (original magnification ×200). Bar = acanthosis, dotted circle = dysplasia, arrows = dyskeratosis. Scale bar (yellow) = 100 μm. (b) Epidermal thickness of SEs was measured using DP-Controller software (OLYMPUS, Tokyo, Japan) with a microscope (n = 55). (c) PCNA, CK14, and CK10 immunofluorescence staining (red) were performed in As-BD lesions and SEs. Scale bar (yellow) = 100 μm. (d) DNA histogram of As-BD specimens (n = 15) was analyzed by flow cytometry. Arrow = aneuploidy. (e) Cell cycle and sub-G1 apoptotic histogram of SEs was analyzed by flow cytometry. ∗P < 0.05, arsenic treated vs. untreated; #P < 0.05, PBMC-free vs. PBMC-treated. (f) DNA histogram of epidermal cells from SEs. Triangle = sub-G1 peak, arrow = aneuploidy. As, arsenic; As-BD, arsenic-induced Bowen’s disease; CK, cytokeratin; M, mol/L; NS, not significant; PBMC, peripheral blood mononuclear cells; PCNA, proliferating cell nuclear antigen; SE, skin equivalent. Journal of Investigative Dermatology 2017 137, 187-196DOI: (10.1016/j.jid.2016.08.017) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Epigenetic modification of decreased trimethyl H3K9 in the E2F1 promoter induces centrosome amplification in arsenic-treated KCs. (a) Immunofluorescence staining for centrosomes (α-tubulin, green) and Aurora-A (red) with DNA counterstaining (DAPI, blue) was observed using a fluorescence microscope (original magnification ×400). Scale bar (yellow) = 10 μm. (b) Binding of trimethyl H3K9 to the E2F1 promoter in 48-hour cultured KCs was analyzed by chromatin immune precipitation assay. ∗P < 0.05, n = 3. (c) The expression of E2F1 in 48-hour arsenic-treated KCs was tested by Western blotting. The activity of E2F1 was detected by pE2F1-TA-Luc luciferase activity assay. ∗P < 0.05, n = 6. (d) Binding of E2F1 on the Aurora-A promoter was analyzed by chromatin immune precipitation assay. ∗P < 0.05, n = 3. (e) The percentage of centrosome amplification (more than two centrosomes) was analyzed by counting 100 cells in arsenic-treated KCs (48 hours). ∗P < 0.05, n = 6. VX680 is an Aurora-A inhibitor. As, arsenic; As-BD, arsenic-induced Bowen’s disease; KC, keratinocyte; M, mol/L; SE, skin equivalent. Journal of Investigative Dermatology 2017 137, 187-196DOI: (10.1016/j.jid.2016.08.017) Copyright © 2016 The Authors Terms and Conditions

Figure 3 TNF-α shows epidermal growth-promoting effects in arsenic-PBMC co-treated SEs. (a) IL-6, TGF-β, and TNF-α in supernatants from 10-day PBMC-treated SEs (with or without 1 μmol/L arsenic treatment) were measured by ELISA. ∗P < 0.05, n = 12. (b) Effects of recombinant IL-6, TGF-β, or TNF-α on epidermal thickness in 10-day PBMC-free SEs were measured using DP-Controller software (OLYMPUS, Tokyo, Japan). ∗P < 0.05, cytokine-treated vs. untreated in arsenic-free groups (hollow bars); #P < 0.05, cytokine-treated vs. untreated in arsenic-treated groups (solid bars); n = 6. (c) The effects of anti–IL-6, anti–TGF-β, and TNF-Ab on epidermal thickness in 10-day PBMC-treated SEs were measured using DP-Controller software (OLYMPUS). ∗P < 0.05, neutralizing antibody treated vs. control antibody treated; n = 6. (d) TNF-Ab treatment reduced epidermal thickness in 10-day arsenic-PBMC co-treated SEs (by hematoxylin and eosin staining, original magnification ×200), n = 3. Scale bar (yellow) = 100 μm. As, arsenic; conc., concentration; M, mol/L; PBMC, peripheral blood mononuclear cell; SE, skin equivalent; TGF, transforming growth factor; TNF, tumor necrosis factor; TNF-Ab, anti-tumor necrosis factor neutralizing antibody. Journal of Investigative Dermatology 2017 137, 187-196DOI: (10.1016/j.jid.2016.08.017) Copyright © 2016 The Authors Terms and Conditions

Figure 4 TNF-α exhibits anti-apoptotic effects in both arsenic-PBMC co-treated KCs and in arsenic-PBMC co-treated SEs. (a) A significant sub-G1 apoptotic peak (arrow) was noticed in 10-day arsenic-treated (1 μmol/L) KCs by flow cytometry (n = 6). (b) Arsenic induced apoptosis in 8–14 day arsenic-treated (1 μmol/L) KCs by TUNEL staining. ∗P < 0.05 (n = 6). (c) An anti-TNF neutralizing antibody (TNF-Ab) inhibition test confirmed the anti-apoptotic effect of TNF-α in 10-day 1 μmol/L arsenic-PBMC co-treated KCs (n = 6). Arrow = sub-G1 peak, triangle = G2/M arrest. (d) Western blotting of phospho-NF-κB p65, FLIP, and caspase-8 in 10-day arsenic-treated (1 μmol/L) KCs. Right panel shows the quantified results of Western blotting using Bio-Profil-Bio-1D software (Bio-Rad, Hercules, CA). ∗P < 0.05, n = 4. (e) By immunofluorescence staining, TNF-α showed anti-apoptotic effects on SEs (10 days), and TNF-Ab reversed this effect (n = 6). Arrows = apoptotic cells (TUNEL positive). Scale bar = 100 μm. (f) NF-κB p65 knockdown (using small interfering RNA) reduced the anti-apoptotic effects by PBMC or TNF-α (n = 5) in SEs (10 days). Vehicle was nontargeting small interfering RNA. Arrows = apoptotic cells. Scale bar = 100 μm. As, arsenic; Bcl-xl, B-cell lymphoma-extra large; FLIP, FLICE-inhibitory protein; h, hour; KC, keratinocyte; M, mol/L; PBMC, peripheral blood mononuclear cell; pNF-κB, phospho-NF-κB; TNF, tumor necrosis factor; TNF-Ab, anti-tumor necrosis factor neutralizing antibody. Journal of Investigative Dermatology 2017 137, 187-196DOI: (10.1016/j.jid.2016.08.017) Copyright © 2016 The Authors Terms and Conditions

Figure 5 TNF-α enhances both centrosome amplification and chromosomal abnormalities in 10-day arsenic-treated KCs. (a) TNF-α enhances centrosome amplification in 10-day arsenic-treated (1 μmol/L) KCs. The percentage of KCs with more than two centrosomes was counted using α-tubulin staining under a fluorescence microscope. ∗P < 0.05 by the Mann-Whitney test, n = 8. (b) TNF-α enhances chromosomal abnormalities in 10-day arsenic-treated (1 μmol/L) KCs. The percentage of cells with abnormal chromosome numbers (more than 46 chromosomes) in 10-day cultured KCs was counted using DAPI staining under a fluorescence microscope. ∗P < 0.05 by the Mann-Whitney test, n = 3. ND indicates not detectable (i.e., 0%). (c) Scheme of arsenic carcinogenesis in SEs. In arsenic-PBMC co-treated SEs, arsenic induced centrosome amplification in 2 days via SUV39H2-mediated epigenetic modification in E2F1 and the Aurora-A pathway. However, arsenic also induced caspase-8–mediated apoptosis in 10 days in KCs. In parallel, arsenic stimulated TNF-α release mainly from PBMCs. TNF-α was able to induce NF-κB activation and FLIP-associated anti-apoptotic signals in KCs, which contributed to cell survival, which, in turn, promoted centrosome amplification and aneuploidy. The interaction between the arsenic-induced epigenetic tumor-initiating effect and the tumor-promoting effect by TNF-α in epidermal KCs resulted in the generation of the As-BD–like SE. As, arsenic; As-BD, arsenic-induced Bowen’s disease; FLIP, FLICE-inhibitory protein; KC, keratinocyte; M, mol/L; ND, not detectable; PBMC, peripheral blood mononuclear cell; SE, skin equivalent; TNF, tumor necrosis factor; TNF-Ab, anti-tumor necrosis factor neutralizing antibody. Journal of Investigative Dermatology 2017 137, 187-196DOI: (10.1016/j.jid.2016.08.017) Copyright © 2016 The Authors Terms and Conditions