Carboxyfullerenes Protect Human Keratinocytes from Ultraviolet-B-Induced Apoptosis  Cristiana Fumelli, Alessandra Marconi, Stefano Salvioli, Elisabetta Straface, Walter Malorni, Anna Maria Offidani, Roberto Pellicciari, Gennaro Schettini, Alberto Giannetti, Daniela Monti, Claudio Franceschi, Carlo Pincelli  Journal of Investigative Dermatology  Volume 115, Issue 5, Pages 835-841 (November 2000) DOI: 10.1046/j.1523-1747.2000.00140.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Structure of CF (water-soluble hexacarboxylic acid derivatives of C60, C60[C(COOH)2]3) showing the paired carboxyl groups on the C60 sphere. The two regioisomers shown are in C3 and D3 symmetry. Journal of Investigative Dermatology 2000 115, 835-841DOI: (10.1046/j.1523-1747.2000.00140.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 CF prevent UVB-induced inhibition of keratinocyte proliferation. Cells were seeded at a density of 9 × 103 per well and cultured in KGM. At subconfluency, 10 μM CF (striped bars) or diluent (closed bars) were added to the medium before sham or UVB irradiation. 3[H]-thymidine incorporation was determined 24 h after irradiation. Each point represents the mean cpm ± SD of separate determinations in six different wells from six different experiments. Student's t test was used for comparison of the means. Journal of Investigative Dermatology 2000 115, 835-841DOI: (10.1046/j.1523-1747.2000.00140.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 CF protect human keratinocytes from UVB-induced apoptosis. Dose–response curve. (A) Subconfluent keratinocytes were given different doses of CF (1, 10, 25, 50, 100 μM) before UVB (100 mJ per cm2) or sham irradiation. At 24 h cells were trypsinized and, after incubation with PI (50 mg per ml) solution, analyzed by flow cytometry. Results are expressed as the mean ± SD of three experiments. Student's t test was used for comparison of the means. 1 μM CF + UVB vs UVB, not significant; 10 μM CF + UVB vs UVB, p < 0.05; 25 μM CF + UVB vs UVB, p < 0.05; 50 μM CF + UVB vs UVB, p < 0.05; 100 μM CF + UVB vs UVB, p < 0.05. (B) Keratinocyte cultures 24 h after sham (a) or UVB (100 mJ per cm2) irradiation with diluent (b) or with the addition of different doses of CF (c, d, e, f, g). Scale bar: 0.1 μm. Journal of Investigative Dermatology 2000 115, 835-841DOI: (10.1046/j.1523-1747.2000.00140.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 CF protect human keratinocytes from UVB-induced apoptosis. (A) Subconfluent keratinocytes were given 10 μM CF or diluent before UVB (100 mJ per cm2) or sham irradiation. At 24 h cells were directly stained with TUNEL. Around 100 cells were counted in randomly selected fields for each point and percentages are expressed as the means ± SD of seven experiments. Student's t test was used for comparison of the means. (B) Cells were treated as in (A), trypsinized at 24 h, stained with PI, and analyzed by flow cytometry. Results are expressed as the mean ± SD of four experiments. Student's t test was used for comparison of the means. (C) Histograms from one representative flow cytometry experiment illustrating sham-irradiated keratinocytes, UVB (100 mJ per cm2) irradiated cells, UVB-irradiated cells after the addition of 10 μM CF. Data from the original histograms were used to generate the mean values expressed in (B). (D) Subconfluent keratinocytes were sham or UVB (50 mJ per cm2) irradiated after the addition of 10 μM CF or diluent. Cells were stained with TUNEL in situ at different time points. About 100 cells were counted in randomly selected fields for each point and percentages are expressed as the means ± SD of three experiments. Student's t test was used for comparison of the means. 10 μM CF + UVB vs UVB at 8 h, not significant; 10 μM CF + UVB vs UVB at 12 h, p < 0.05; 10 μM CF + UVB vs UVB at 24 h, p < 0.01; 10 μM CF + UVB vs UVB at 36 h, p < 0.05; 10 μM CF + UVB vs UVB at 48 h, p < 0.01; 10 μM CF + UVB vs UVB at 72 h, p < 0.05. Journal of Investigative Dermatology 2000 115, 835-841DOI: (10.1046/j.1523-1747.2000.00140.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 CF reduce UVB-induced PARP cleavage. (A) Keratinocytes were cultured in KGM and sham or UVB (50, 100 mJ per cm2) irradiated after the addition of 10 μM CF or diluent. Cells were lyzed at different time points and proteins were analyzed by Western blotting using anti-PARP monoclonal antibody. The 85 kDa fragment represents the cleaved form of PARP. A non-specific band is present immediately below the 85 kDa band. (B) Relative intensity of the band on autoradiograms was quantified by scanning laser densitometry (TN-Image program; Copyright © T. Nelson 1995–97, Version 2.27). Values of 85 kDa bands are expressed as fold variations, compared with UVB 50 irradiated cells at 12 h. Journal of Investigative Dermatology 2000 115, 835-841DOI: (10.1046/j.1523-1747.2000.00140.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Bcl-2 protein levels in CF-treated keratinocytes. (A) Keratinocytes were cultured in KGM and sham or UVB (100 mJ per cm2) irradiated after the addition of either 10 μM CF or diluent. (C) As a control, keratinocytes were UVB or sham irradiated after the addition of NAC (10 mM). Cells were lyzed at 24 h and proteins were analyzed by Western blotting using anti-Bcl-2 monoclonal antibody. (B), (D) Relative intensity of the bands on autoradiograms was quantified by scanning laser densitometry, as in Figure 5. Values are expressed as fold variations compared with sham cells. (E) Keratinocytes were UVB or sham irradiated after pretreatment with NAC (10 mM), and analyzed by flow cytometry. Results are expressed as the mean ± SD of three experiments. Student's t test was used for comparison of the means. UVB + NAC vs UVB, p < 0.05; UVB vs sham, p < 0.01; sham + NAC vs sham, not significant. Journal of Investigative Dermatology 2000 115, 835-841DOI: (10.1046/j.1523-1747.2000.00140.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Cytofluorimetric analysis of mitochondrial membrane potential. (A) Subconfluent keratinocytes were sham or UVB (50 mJ per cm2) irradiated after the addition of 10 μM CF or diluent and trypsinized at 24 h. Cells were stained with JC-1 probe as described in Materials and Methods. (a) Sham-irradiated cells; (b) UVB-irradiated cells; (c) sham-irradiated cells + CF; (d) UVB-irradiated cells + CF (depolarized mitochondria are located in the lower quadrants, and polarized mitochondria in the upper quadrants). (B) The percentage of cells with depolarized mitochondria was evaluated as the mean ± SD of three different experiments. Student's t test was used for comparison of the means. UVB + CF vs UVB, p < 0.01; UVB vs sham, p < 0.01; sham + CF vs sham, not significant. Journal of Investigative Dermatology 2000 115, 835-841DOI: (10.1046/j.1523-1747.2000.00140.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 CF protect human keratinocytes from dRib-induced apoptosis. Subconfluent keratinocytes were treated with 40 mM dRib with the addition of either 10 μM CF or diluent. At 48 h cells were trypsinized and, after incubation with PI (50 mg per ml) solution, analyzed by flow cytometry. Results are expressed as the mean ± SD of three experiments. Student's t test was used for comparison of the means. Journal of Investigative Dermatology 2000 115, 835-841DOI: (10.1046/j.1523-1747.2000.00140.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions