Downregulation of BRCA1 in A375 Melanoma Cell Line Increases Radio-Sensitivity and Modifies Metastatic and Angiogenic Gene Expression Cédric Hesling

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Downregulation of BRCA1 in A375 Melanoma Cell Line Increases Radio-Sensitivity and Modifies Metastatic and Angiogenic Gene Expression Cédric Hesling Journal of Investigative Dermatology Volume 122, Issue 2, Pages 369-380 (February 2004) DOI: 10.1046/j.0022-202X.2004.22212.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Rbz3′-RNA expression.Rbz3′-RNA expression in parental A375 cells (A375), a clone transfected with LXSN (A375LXSN) as negative control and A375 clones transfected with LXSN-rbz3′ was checked by nonquantitative reverse transcription–PCR. Amplification generates a 350 bp fragment and was performed using VLXF and RBVXR primers. Amplification without cDNA was performed as a negative control (T–). Journal of Investigative Dermatology 2004 122, 369-380DOI: (10.1046/j.0022-202X.2004.22212.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 BRCA1 expression in A375, A375LXSN, c8.1, and c9.1. (a) BRCA1 mRNA expression in A375LXSN, c9.1, and c8.8 were determined by real-time quantitative PCR. At least, Three RNA extractions and three reverse transcriptions were performed for each clone. Data are expressed as percentages of the level in A375. Values represent mean±SD. Significant differences between A375 and c9.1 or c8.8 were identified by *p<0.05. (b) BRCA1 protein expression: 35 μg of total proteins were loaded and separated on 5% SDS–PAGE and transferred to ImmobilonP membrane (Millipore). Immunostaining was performed using 1/200 diluted anti-BRCA1 K18 (Santa-Cruz Biotechnology) or 1/5000 diluted anti-actin A4700 (Sigma). Σ: c8.8 and c9.1 clones cultured 4 mo after transfection. Journal of Investigative Dermatology 2004 122, 369-380DOI: (10.1046/j.0022-202X.2004.22212.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Clonogenic survival assay. A375LXSN, c8.8, and c9.1 cells were irradiated with 1, 3, or 5 Gy. Results are mean±SD of three experiments. Significant differences were observed between A375LXSN and c8.8 or c9.1 (c8.8 or c9.1 vs A375LXSN: *p<0.02; **p<0.046).Σ: c8.8 and c9.1 clones cultured 4 mo after transfection. Journal of Investigative Dermatology 2004 122, 369-380DOI: (10.1046/j.0022-202X.2004.22212.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Proliferation assay. Quantification of proliferation was performed using the SRB proliferation test. Three experiments using A375 parental cells, A375LXSN, c8.8, and c9.1 were carried out to 264 h (c8.8 vs A375: *p=0.046). Journal of Investigative Dermatology 2004 122, 369-380DOI: (10.1046/j.0022-202X.2004.22212.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 SYBRGreen specificity. Amplification specificity was checked, loading PCR products on a 2% agarose gel. Only one product with the excepted size was obtained. Journal of Investigative Dermatology 2004 122, 369-380DOI: (10.1046/j.0022-202X.2004.22212.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of angiogenic gene.THBS1, THBS2, VEGF, and FGF-2 expression in A375LXSN, c9.1, and c8.8 were determined by real-time quantitative PCR using SYBRGreen and 18S as parallel control. Three RNA extractions and three reverse transcriptions were performed for each clone and for each time (T0: 2 h before 3 Gy irradiation, T2 and T4: 5 h and 29 h after irradiation). Data are expressed as percentages of the level in A375LXSN at T0. Values represent mean±SD. Significant differences between A375LXSN and c9.1 or c8.8 were identified by *p<0.05. Journal of Investigative Dermatology 2004 122, 369-380DOI: (10.1046/j.0022-202X.2004.22212.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 THBS1, THBS2, VEGF, and FGF-2 protein expression. Forty-five micrograms of total proteins were loaded and separated on 5% (THBS1) and 15% (VEGF and FGF-2) SDS–PAGE and transferred to ImmobilonP membrane (Millipore). Immunostaining were performed using 1/100 diluted anti-THBS H300, anti-VEGF C1, and anti-FGF-2 147 (Santa Cruz Biotechnology) or 1/5000 diluted anti-actin A4700 (Sigma). Journal of Investigative Dermatology 2004 122, 369-380DOI: (10.1046/j.0022-202X.2004.22212.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Expression of metastasis gene.MMP1, MMP2, MMP9, TIMP1, and KiSS1 expression in A375LXSN, c9.1, and c8.8 were determined by real-time quantitative PCR using SYBRGreen and 18S as parallel control. Three RNA extractions and three reverse transcriptions were performed for each clone and for each time (T0: 2 h before 3 Gy irradiation, T2 and T4: 5 h and 29 h after irradiation). Data are expressed as percentages of the level in A375LXSN at T0. Values represent mean±SD. Significant differences between A375LXSN and c9.1 or c8.8 were identified by *p<0.05. Journal of Investigative Dermatology 2004 122, 369-380DOI: (10.1046/j.0022-202X.2004.22212.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 MMP1 protein expression Forty-five micrograms of total proteins were loaded and separated on 9% SDS–PAGE and transferred to ImmobilonP membrane (Millipore). Immunostaining were performed using 1/100 diluted anti-MMP1 3B6 (Santa Cruz Biotechnology) or 1/5000 diluted anti-actin A4700 (Sigma). Journal of Investigative Dermatology 2004 122, 369-380DOI: (10.1046/j.0022-202X.2004.22212.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 10 Activity of MMP2, MMP9, and TIMP1. Forty-eight hours before T0, cells were plated in T175 cm2 flask at a density of 2.106 cells per flask. Medium was replace by serum-free medium 24 h before T0 (2 h before irradiation), T2 (5 h after irradiation), or T4 (29 h after irradiation). Proteins of the conditioned medium (CM) were quantified using Bradford assays. (a) Gelatin zymography: equal protein mass was loaded on to SDS–9% polyacrylamide gel electrophoresis. Pro-MMP2 0.1 ng and Pro-MMP9 0.1 ng were loaded on each gel as positives controls. Pro-form and mature form of MMP2 are indicated on the left. (b) Zymography reverse: equal protein mass was loaded on to SDS–15% polyacrylamide gel electrophoresis. Two hundred nanograms of pro-MMP2 was added to the gel. TIMP1 was loaded as the positive control. Quantification of clear (MMP1 and 2) or dark (TIMP1) bands volumes was obtained with Bio-Capt and Bio-1D software (Vilbert Lourmat, Marne La Vallée) and values are indicated in graphs. Journal of Investigative Dermatology 2004 122, 369-380DOI: (10.1046/j.0022-202X.2004.22212.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 11 In vitro invasion assays. Invasion was assayed in modified Boyden chambers (tissue culture treated, 6.5 mm diameter, 8 μm pore, Transwell, Dutscher, Brumath, France). Data are expressed as percentages of the level in A375LXSN. Values represent mean±SD. Significant differences between A375LXSN and c9.1 was identified by *p< 0.05. Journal of Investigative Dermatology 2004 122, 369-380DOI: (10.1046/j.0022-202X.2004.22212.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions