Molecular Responses to Photogenotoxic Stress Induced by the Antibiotic Lomefloxacin in Human Skin Cells: From DNA Damage to Apoptosis Laurent Marrot,

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Molecular Responses to Photogenotoxic Stress Induced by the Antibiotic Lomefloxacin in Human Skin Cells: From DNA Damage to Apoptosis Laurent Marrot, Jean Phillipe Belaïdi, Christophe Jones, Phillipe Perez, Jean Roch Meunier Journal of Investigative Dermatology Volume 121, Issue 3, Pages 596-606 (September 2003) DOI: 10.1046/j.1523-1747.2003.12422.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Photocytotoxic impact of lomefloxacin on fibroblasts, keratinocytes, and melanocytes assessed by the decrease of cell viability 24 h post treatment (MTT colorimetric assay). Journal of Investigative Dermatology 2003 121, 596-606DOI: (10.1046/j.1523-1747.2003.12422.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 DNA breakage induced by lomefloxacin in nuclei of cells exposed to UVA. (a) Typical comets observed immediately after exposure to UVA (15 min) of cells treated by lomefloxacin (20 μM). The figure shows aspects of nuclei in unexposed control (identical for all cell types and for UVA alone), fibroblasts, keratinocytes, and melanocytes. (b) Quantification by image analysis (mean tail moments of 50 nuclei, bars represent standard deviation) of comets observed after exposure to UVA (15 min) of skin cells treated with increasing lomefloxacin concentrations. Journal of Investigative Dermatology 2003 121, 596-606DOI: (10.1046/j.1523-1747.2003.12422.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Detection of thymine dimers by dot-blot analysis in genomic DNA isolated from fibroblasts (upper part) or from keratinocytes (lower part) immediately after treatment by lomefloxacin (50 and 100 μM) and exposure to UVA (30 min). The amounts of DNA spotted on the membrane were 1.4, 1.6, 1.8, and 2 μg. Journal of Investigative Dermatology 2003 121, 596-606DOI: (10.1046/j.1523-1747.2003.12422.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Expression of HO-1 gene in fibroblasts treated by lomefloxacin and exposed to UVA (30 min) assessed at 4 h and 24 h post treatment. Quantification (mean value from two independent experiments, bars represent standard deviation) was standardized with GAPDH expression measured simultaneously. Journal of Investigative Dermatology 2003 121, 596-606DOI: (10.1046/j.1523-1747.2003.12422.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 P53 status in cells treated by lomeflaxacin and exposed to UVA. (a) Western blot detection of p53 and p21 proteins in fibroblasts, keratinocytes, and melanocytes treated with lomefloxacin and exposed to UVA (30 min). Proteins were extracted from nuclei 4 h, 8 h (except for melanocytes), and 24 h post exposure. (b) Densitometric analysis of p53 and p21 bands scanned from the western blot shown in (a). Values represent the ratio between assays and untreated control for each cell type. Journal of Investigative Dermatology 2003 121, 596-606DOI: (10.1046/j.1523-1747.2003.12422.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of the MDM2 gene in fibroblasts and keratinocytes 24 h after treatment with lomefloxacin and UVA (30 min). Quantification (mean value from two independent experiments, bars represent standard deviation) was standardized with GAPDH expression measured simultaneously. Journal of Investigative Dermatology 2003 121, 596-606DOI: (10.1046/j.1523-1747.2003.12422.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Expression of GADD45 gene in fibroblasts and keratinocytes 4 h and 24 h after treatment with lomefloxacin and UVA (30 min). Quantification (mean value from two independent experiments, bars represent standard deviation) was standardized with GAPDH expression measured simultaneously. Journal of Investigative Dermatology 2003 121, 596-606DOI: (10.1046/j.1523-1747.2003.12422.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Activation of caspase-3 in keratinocytes treated with lomefloxacin and exposed to UVA (30 min). Proteins were extracted from cells 8 h and 24 h post exposure and assessed for caspase-3 activity using a colorimetric assay as described in Materials and Methods. Journal of Investigative Dermatology 2003 121, 596-606DOI: (10.1046/j.1523-1747.2003.12422.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Expression of the FAS-L gene in keratinocytes treated with lomefloxacin and exposed to UVA (30 min) assessed 8 h and 24 h post exposure. Quantification (mean value from two independent experiments, bars represent standard deviation) was standardized with GAPDH expression measured simultaneously. Journal of Investigative Dermatology 2003 121, 596-606DOI: (10.1046/j.1523-1747.2003.12422.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 10 Impact of lomefloxacin and UVA (30 min) on cell growth and dopa-oxidase activity of endogenous tyrosinase in melanocytes from two Caucasian donors 5 d post exposure. Journal of Investigative Dermatology 2003 121, 596-606DOI: (10.1046/j.1523-1747.2003.12422.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions