Dual role of SIRT1 in UVB-induced skin tumorigenesis

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1 Dual role of SIRT1 in UVB-induced skin tumorigenesis Mei Ming 1, Keyoumars Soltani 1, Christopher R. Shea 1, Xiaoling Li 2, and Yu-Ying He 1 * 1 Section of Dermatology, Department of Medicine, University of Chicago, Chicago, IL, USA 2 Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, NC, USA Supplemental Information: Methods Supplemental Figure s1: Related to Figure 1 Supplemental Figure s2: Related to Figure 3 Supplemental Figure s3: Related to Figure 4 sirna/plasmids transfectioin p53, p53 K382R mutant, and control plasmids were kindly provided by Dr. Xiaobing Shi (1). Cells were transfected with negative control (sinc) or sirna (ON-TARGETplus SMARTpool, Dharmacon) targeting SIRT1 (sisirt1) and/or p53 (sip53), p53, p53 mutant, or their combination with sinc or sisirt1, using Amaxa Nucleofector according to the manufacturer's instructions as described previously (2). Western blotting Protein concentrations were determined using the BCA assay (Pierce, Rockford, IL, USA) and equal amounts of protein were subjected to electrophoresis. Western blotting was performed as described previously (3). Antibodies used included XPC, SIRT1, p53, GAPDH (Santa Cruz), active Caspase 3 (Cell Signaling Technology), XPA (Kamiya Biomedical Company), and acetylated-p53 k382 (Ac-p53, Abcam). Real-time PCR Quantitative real time PCR assays were performed using ABI7300 (Applied Biosystems, Foster City, CA) in 96-well plates with the SYBR Green PCR Master Mix (Applied Biosystems) as described previously (2, 4). Amplification primers were 5'- TGGCTGGGAAGGCCTCCTCTC-3'(forward), 5'-AGATGGTGAGCGAGGCGGTGA- 3'(reverse) for mouse Bax; 5'-GCGGTGAACGATGCCTGCCT-3' (forward) and 5'- ACCTATGCAATGAGATGGATGGGGA-3' (reverse) for mouse Puma; 5'- ACTCAGGAAGATCGGAGACAAAGTG-3'(forward) and 5'-

2 ACACTCGTCCTTCAAGTCTGCTGG-3' (reverse) for mouse Noxa (5); and 5'- AGGTCGGTGTGAACGGATTTG-3 (forward) and 5 - TGTAGACCATGTAGTTGAGGTCA-3 (reverse) for mouse GAPDH. The threshold cycle number (Ct) for each sample was determined in triplicate. The Ct values for Bax, Puma and Noxa were normalized against GAPDH, and the XPC primers were used as described previously (6). Determination of two major forms of UVB-induced DNA damage in genomic DNA by slot blot assay Slot blot assays of CPD were performed as described previously (7, 8). Briefly, mouse skin or cells were collected at different time points post-uvb, and DNA was isolated using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA). The DNA concentration was calculated from the absorbance at 260 nm using NanoDrop 1000 (NanoDrop products, Wilmington, DE, USA). The CPD in DNA were quantified by slot blot (Bio-Rad, Hercules, CA, USA) with monoantibodies (TDM-2 for CPD, COSMO BIO Co., Tokyo, Japan) as describled previously (7). The chemiluminescence was detected with a Carestream Imaging Station (Carestream, Rochester, NY, USA). For examining repair kinetics, the percentage of repair was calculated by comparing the optical density at the indicated time with that of the corresponding absorbance at time zero when there was no opportunity for repair and 100% of CPDs were present post-uvb. TUNEL assay DNA fragmentation characteristic of apoptotic cells was evaluated by Tdt-mediated dutp nick end labeling (TUNEL) with an in situ cell detection kit (Roche) according to the manufacturer's instructions. To calculate the percentage of TUNEL-positive cells, ten random microscopic fields at 400x magnifications were examined, and apoptosis expressed as percentage of TUNEL-positive cells in the epidermis. sub-g1 flow cytometric analysis Sub-G1 flow cytometric analysis was performed to determine apoptosis as described previously (8). Briefly, cells were fixed and stained with propidium iodide. The percentage of cells at the sub-g1 phase was quantified by flow cytometry. SIRT1 activity assay in mouse skin SIRT1 activity was determined with a SIRT1 Fluorometric Kit (Biomol International) as described previously (9). This assay uses a small lysine-acetylated peptide, corresponding to K382 of human p53, as a substrate. The lysine residue is deacetylated by SIRT1, and this process is dependent on the addition of exogenous NAD +. Basically, samples were homogenized in IP buffer. The homogenates were incubated in SIRT1 assay buffer in the presence of 100uM Fluor de Lys-SIRT1 substrate and 200uM NAD + to determine the SIRT1-dependent activity. After 60 minutes of incubation at 37C, the reaction was terminated by adding a solution containing Fluor de Lys Developer and

3 2mM nicotinamide. Plates were incubated at 37 C for 1 hour. Values were determined by reading fluorescence on a plate reader with an excitation wavelength of 360nm and an emission wavelength of 460nm. SIRT1-dependent activity was calculated after subtracting of a blank consisting of buffer containing no NAD+ and expressed as a percentage of control. In all cases, we confirmed the linearity of the reaction over time. References 1. Shi X, Kachirskaia I, Yamaguchi H, West LE, Wen H, Wang EW, et al. Modulation of p53 function by SET8-mediated methylation at lysine 382. Molecular cell Aug 17;27(4): PubMed PMID: Pubmed Central PMCID: Ming M, Feng L, Shea CR, Soltani K, Zhao B, Han W, et al. PTEN positively regulates UVB-induced DNA damage repair. Cancer Res. Aug 1;71(15): PubMed PMID: Epub 2011/07/21. eng. 3. Ming M, Han W, Maddox J, Soltani K, Shea CR, Freeman DM, et al. UVBinduced ERK/AKT-dependent PTEN suppression promotes survival of epidermal keratinocytes. Oncogene. Jan 28;29(4): PubMed PMID: Epub 2009/11/03. eng. 4. Ming M, Shea CR, Guo X, Li X, Soltani K, Han W, et al. Regulation of global genome nucleotide excision repair by SIRT1 through xeroderma pigmentosum C. Proc Natl Acad Sci U S A. Dec 28;107(52): PubMed PMID: Epub 2010/12/15. eng. 5. Matsumoto A, Susaki E, Onoyama I, Nakayama K, Hoshino M, Nakayama KI. Deregulation of the p57-e2f1-p53 axis results in nonobstructive hydrocephalus and cerebellar malformation in mice. Mol Cell Biol. Oct;31(20): PubMed PMID: Epub 2011/08/17. eng. 6. Ming M, Shea CR, Guo X, Li X, Soltani K, Han W, et al. Regulation of global genome nucleotide excision repair by SIRT1 through xeroderma pigmentosum C. Proceedings of the National Academy of Sciences of the United States of America Dec 28;107(52): PubMed PMID: Pubmed Central PMCID: Maeda T, Chua PP, Chong MT, Sim AB, Nikaido O, Tron VA. Nucleotide excision repair genes are upregulated by low-dose artificial ultraviolet B: evidence of a photoprotective SOS response? J Invest Dermatol Dec;117(6): PubMed PMID: Epub 2002/03/12. eng. 8. Han W, Ming M, Zhao R, Pi J, Wu C, He YY. Nrf1 CNC-bZIP protein promotes cell survival and nucleotide excision repair through maintaining glutathione homeostasis. The Journal of biological chemistry May 25;287(22): PubMed PMID: Pubmed Central PMCID: Escande C, Chini CC, Nin V, Dykhouse KM, Novak CM, Levine J, et al. Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice. The Journal of clinical investigation Feb;120(2): PubMed PMID: Pubmed Central PMCID:

4 Supplemental Figure s1 A B Fig. s1. Skin tumors developed in UVB-irradiated and mice. A, representative skin tumors from and mice. B, histological analysis of tumors from SIRT1 and mice. Scale bar, 100 µm. Supplemental Figure s2 cko Fig. s2. Representative pictures of SKH1 hairless mice irradiated with UVB for 25 weeks.

5 Supplemental Figure s3 cko Fig. s3. Immunohistochemical analysis of the XPC protein levels in,, and cko mice. Scale bar, 25 µm.