Control and target gene selection for studies on UV-induced genotoxicity in whales

BMC Research Notes

Karina Acevedo-Whitehouse¹  Robert J Knell²  Diane Gendron⁴  Laura M Martinez-Levasseur³ 

[1] Unit for Basic and Applied Microbiology. School of Natural Sciences, Autonomous University of Queretaro, Queretaro 76230, México;School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK;Current Address: Department of Biology, Trent University, 1600 West Bank Dr, Peterborough K9J 7B8, Canada;Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, Av. IPN s/n, Playa Palo de Sta Rita, La Paz, BCS 23000, México

关键词: Skin biopsy;    Whales;    Solar ultraviolet radiation;    KIN;    P53;    HSP70;    Internal control genes;    qPCR normalization;    Gene expression;   

Others  :  1142247
DOI  :  10.1186/1756-0500-6-264

 received in 2013-02-07, accepted in 2013-04-11,  发布年份 2013

【 摘 要 】

Background

Despite international success in reducing ozone-depleting emissions, ultraviolet radiation (UV) is not expected to decrease for several decades. Thus, it is pressing to implement tools that allow investigating the capacity of wildlife to respond to excessive UV, particularly species like cetaceans that lack anatomical or physiological protection. One approach is to examine epidermal expression of key genes involved in genotoxic stress response pathways. However, quantitation of mRNA transcripts requires previous standardization, with accurate selection of control and target genes. The latter is particularly important when working with environmental stressors such as UV that can activate numerous genes.

Results

Using 20 epidermal biopsies from blue, fin and sperm whale, we found that the genes encoding the ribosomal proteins L4 and S18 (RPL4 and RPS18) were the most suitable to use as controls, followed by the genes encoding phosphoglycerate kinase 1 (PGK1) and succinate dehydrogenase complex subunit A (SDHA). A careful analysis of the transcription pathways known to be activated by UV-exposure in humans and mice led us to select as target genes those encoding for i) heat shock protein 70 (HSP70) an indicator of general cell stress, ii) tumour suppressor protein P53 (P53), a transcription factor activated by UV and other cell stressors, and iii) KIN17 (KIN), a cell cycle protein known to be up-regulated following UV exposure. These genes were successfully amplified in the three species and quantitation of their mRNA transcripts was standardised using RPL4 and RPS18. Using a larger sample set of 60 whale skin biopsies, we found that the target gene with highest expression was HSP70 and that its levels of transcription were correlated with those of KIN and P53. Expression of HSP70 and P53 were both related to microscopic sunburn lesions recorded in the whales’ skin.

Conclusion

This article presents groundwork data essential for future qPCR-based studies on the capacity of wildlife to resolve or limit UV-induced damage. The proposed target genes are HSP70, P53 and KIN, known to be involved in genotoxic stress pathways, and whose expression patterns can be accurately assessed by using two stable control genes, RPL4 and RPS18.

【 授权许可】

   
2013 Martinez-Levasseur et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Hader DP, Helbling EW, Williamson CE, Worrest RC: Effects of UV radiation on aquatic ecosystems and interactions with climate change. Photoch Photobio Sci 2011, 10:242-260.
• [2]Manney GL, Santee ML, Rex M, Livesey NJ, Pitts MC, Veefkind P, Nash ER, Wohltmann I, Lehmann R, Froidevaux L, Poole LR, Schoeberl MR, Haffner DP, Davies J, Dorokhov V, Gernandt H, Johnson B, Kivi R, Kyro E, Larsen N, Levelt PF, Makshtas A, McElroy CT, Nakajima H, Parrondo MC, Tarasick DW, von der Gathen P, Walker KA, Zinoviev NS: Unprecedented Arctic ozone loss in 2011. Nature 2011, 478:469-475.
• [3]McKenzie RL, Aucamp PJ, Bais AF, Bjorn LO, Ilyas M, Madronich S: Ozone depletion and climate change: impacts on UV radiation. Photoch Photobio Sci 2011, 10:182-198.
• [4]Martinez-Levasseur LM, Gendron D, Knell RJ, O’Toole EA, Singh M, Acevedo-Whitehouse K: Acute sun damage and photoprotective responses in whales. Proc R Soc B 2011, 278:1581-1586.
• [5]Dahms HU, Lee JS: UV radiation in marine ectotherms: molecular effects and responses. Aquat Toxicol 2010, 97:3-14.
• [6]Evans TG, Hofmann GE: Defining the limits of physiological plasticity: how gene expression can assess and predict the consequences of ocean change. Trans R Soc Lond B Biol Sci 2012, 367(1596):1733-45.
• [7]Zhou BB, Elledge SJ: The DNA damage response: putting checkpoints in perspective. Nature 2000, 408:433-439.
• [8]Week DW: Tumour suppression by p53: a role for the DNA damage response? Nat Rev Cancer 2009, 9:714-723.
• [9]Menendez D, Inga A, Resnick MA: The expanding universe of p53 targets. Nat Rev Cancer 2009, 9:724-737.
• [10]Derveaux S, Vandesompele J, Hellemans J: How to do successful gene expression analysis using real-time PCR. Methods 2010, 50:227-230.
• [11]Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M: A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines. Methods 2010, 50:S1-5.
• [12]Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002., 3(7) research0034.1–0034.11. http://genomebiology.com/content/3/7/RESEARCH0034 webcite
• [13]Spinsanti G, Panti C, Lazzeri E, Marsili L, Casini S, Frati F, Maria Fossi CM: Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies. BMC Mol Biol 2006, 7:32. BioMed Central Full Text
• [14]Spinsanti G, Panti P, Bucalossi D, Marsili L, Casini S, Frati F, Fossi MC: Selection of reliable reference genes for qRT-PCR studies on cetacean fibroblast cultures exposed to OCs, PBDEs, and 17[beta]-estradiol. Aquat Toxicol 2008, 87:178-186.
• [15]Noutsias M, Rohde M, Block A, Klippert K, Lettau O, Blunert K, Hummel M, Kühl U, Lehmkuhl H, Hetzer R, Rauch U, Poller W, Pauschinger M, Schultheiss HP, Volk HD, Kotsch K: Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies. BMC Mol Biol 2008, 9:3. BioMed Central Full Text
• [16]Fossi MC, Urban J, Casini S, Maltese S, Spinsanti G, Panti C, Porcelloni S, Panigada S, Lauriano G, Niño-Torres C, Rojas-Bracho L, Jimenez B, Muñoz-Arnanz J, Marsili L: A multi-trial diagnostic tool in fin whale (Balaenoptera physalus) skin biopsies of the Pelagos Sanctuary (Mediterranean Sea) and the Gulf of California (Mexico). Mar Environ Res 2010, 69:S17-S20.
• [17]Lin M, Morrison CD, Jones S, Mohamed N, Bacher J, Plass C: Copy number gain and oncogenic activity of YWHAZ/14-3-3ζ in head and neck squamous cell carcinoma. Int J Cancer 2009, 125(3):603-611.
• [18]Sirover MA: On the functional diversity of glyceraldehyde-3-phosphate dehydrogenase: Biochemical mechanisms and regulatory control. Biochim Biophys Acta 2011, 1810:741-751.
• [19]Wu YY, Rees JL: Variation in epidermal housekeeping gene expression in different. Acta Derm Venereol 2000, 80:2-3.
• [20]Garmyn M, Yaar M, Holbrook N, Gilchrest BA: Immediate and delayed molecular response of human keratinocytes to solar-simulated irradiation. Lab Invest 1991, 65(4):471-478.
• [21]Balogh A, Paragh G, Juhász A, Köbling T, Törocsik D, Mikó E, Varga V, Emri G, Horkay I, Scholtz B, Remenyik E: Reference genes for quantitative real time PCR in UVB irradiated keratinocytes. J Photochem Photobiol B 2008, 93:133-139.
• [22]Mayer MP, Bukau B: Hsp70 chaperones: Cellular functions and molecular mechanism. CMLS Cell Mol Life Sci 2005, 62:670-684.
• [23]Guzhova IV, Lazarev VF, Kaznacheeva AV, Ippolitova MV, Muronetz VI, Kinev AV, Margulis BA: Novel mechanism of Hsp70 chaperone-mediated prevention of polyglutamine aggregates in a cellular model of huntington disease. Hum Mol Genet 2011, 20:3953-3963.
• [24]Li Y, Zou L, Li Q, Haibe-Kains B, Tian R, Li Y, Desmedt C, Sotiriou C, Szallasi Z, Iglehart JD, Richardson AL, Wang ZC: Amplification of LAPTM4B and YWHAZ contributes to chemotherapy resistance and recurrence of breast cancer. Nat Med 2010, 16:214-218.
• [25]Hightower LE: Heat shock, stress proteins, chaperones, and proteotoxicity. Cell 1991, 66:191-197.
• [26]Tsang MF, Gao B: Heat shock proteins and immune system. J Leukocyte Biol 2009, 85:905-910.
• [27]Matsuda M, Hoshino T, Yamashita Y, Tanaka K, Maji D, Sato K, Adachi H, Sobue G, Ihn H, Funasaka Y, Mizushima T: Prevention of UVB radiation-induced epidermal damage by expression of heat shock protein 70. J Biol Chem 2010, 285:5848-5858.
• [28]Kucab JE, Phillips DH, Arlt VM: Linking environmental carcinogen exposure to TP53 mutations in human tumours using the human TP53 knock-in (Hupki) mouse model. FEBS J 2010, 277:2567-2583.
• [29]Latonen L, Laiho M: Cellular UV damage responses–functions of tumor suppressor p53. Biochim Biophys Acta 2005, 1755:71-89.
• [30]Oren M, Bartek J: The sunny side of p53. Cell 2007, 128:826-828.
• [31]Biard DS, Saintigny Y, Maratrat M, Paris F, Martin M, Angulo JF: Enhanced expression of the Kin17 protein immediately after low doses of ionizing radiation. Radiat Res 1997, 147:442-450.
• [32]Kannouche P, Mauffrey P, Pinon-Lataillade G, Mattei MG, Sarasin A, Daya-Grosjean L, Angulo JF: Molecular cloning and characterization of the human KIN17 cDNA encoding a component of the UVC response that is conserved among metazoans. Carcinogenesis 2000, 21:1701-1710.
• [33]Biard DS, Miccoli L, Despras E, Frobert Y, Creminon C, Angulo JF: Ionizing radiation triggers chromatin-bound kin17 complex formation in human cells. J Biol Chem 2002, 277:19156-19165.
• [34]Masson C, Menaa F, Pinon-Lataillade G, Frobert Y, Chevillard S, Radicella JP, Sarasin A, Angulo JF: Global genome repair is required to activate KIN17, a UVC-responsive gene involved in DNA replication. PNAS 2003, 100:616-621.
• [35]Ierardi JL, Mancia A, McMillan J, Lundqvist ML, Romano TA, Wise JP Sr, Warr GW, Chapman RW: Sampling the skin transcriptome of the North Atlantic right whale. Comp Biochem Phy D 2009, 4:154-158.
• [36]Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative CT method. Nat Protoc 2008, 3:1101-1108.
• [37]Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP: Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations. Biotechnol Lett 2004, 26:509-515.
• [38]Andersen CL, Jensen JL, Orntoft TF: Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004, 64:5245-5250.
• [39]R Development Core Team: R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2008. URL http://www.R-project.org webcite
• [40]Zuur A, Ieno E, Walker N, Saveliev A, Smith G: Mixed effects models and extensions in ecology with R. New York: Springer; 2009.
• [41]Pinheiro J, Bates D, DebRoy S, Sarkar D, Coreteam R: nlme: Linear and Nonlinear Mixed Effects Models. 2008. R package version 3.1-89
• [42]Jean S, Bideau C, Bellon L, Halimi G, De Meo M, Orsiere T, Dumenil G, Berge-Lefranc JL, Botta A: The expression of genes induced in melanocytes by exposure to 365-nm UVA: study by cDNA arrays and real-time quantitative RT-PCR. Biochim Biophys Acta 2001, 1522:89-96.
• [43]Helmbrecht K, Zeise E, Rensing L: Chaperones in cell cycle regulation and mitogenic signal transduction: a review. Cell Prolif 2000, 33:341-365.
• [44]Zylicz M, King FW, Wawrzynow A: Hsp70 interactions with the p53 tumour suppressor protein. EMBO J 2001, 20:4634-463.
• [45]Fourie AM, Hupp TR, Lane DP, Sang BC, Barbosa MS, Sambrook JF, Gething MJH: HSP70 Binding sites in the tumor suppressor protein p53. J Biol Chem 1997, 272:19471-19479.
• [46]Gudkov AV, Gurova KV, Komarova EA: Inflammation and p53: A Tale of Two Stresses. Genes Cancer 2011, 2(4):503-16.
• [47]De Jong PR, Schadenberg AW, Jansen NJ, Prakken BJ: Hsp70 and cardiac surgery: molecular chaperone and inflammatory regulator with compartmentalized effects. Cell Stress Chaperon 2009, 14(2):117-31.
• [48]Ziegler A, Jonason AS, Leffell DJ, Simon JA, Sharma HW, Kimmelman J, Remington L, Jacks T, Brash DE: Sunburn and p53 in the onset of skin cancer. Nature 1994, 372(6508):773-6.
• [49]Sperandio S, De Belle I, Bredesen DE: An alternative, nonapoptotic form of programmed cell death. P Natl Acad Sci USA 2000, 97(26):14376-14381.
• [50]Garrido C, Schmitt E, Candé C, Vahsen N, Parcellier A, Kroemer G: HSP27 and HSP70: potentially oncogenic apoptosis inhibitors. Cell Cycle 2003, 2(6):579-84.