at 4 C. This working solution is stable for few hours
10. For a quantitative analysis, it is important to always use a nega- tive control as a baseline to compare to, as close as possible to
the cell type of the sample to be studied. Growing or quiescent cells should be negative for all the senescence-specifi c markers.
It is also recommended to use as a positive control a sample in which most of the cells would be senescent (classic examples are fi broblasts subjected to oxidative stress or Ras expression, or inducible cell lines [ 19 – 23 ]). The fl uorescence values of these positive controls would determine the top MFI that can be achieved in any experiment.
Acknowledgments
This work was supported by an MRC New Blood Fellowship and an Innovation Fellowship from the University of Leicester, as well as a Saudi Government Doctoral Scholarship (to MA).
References
1. Perez-Mancera PA, Young AR, Narita M (2014) Inside and out: the activities of senes- cence in cancer. Nat Rev Cancer 14:547–558 2. Campisi J (2005) Senescent cells, tumor sup-
pression, and organismal aging: good citizens, bad neighbors. Cell 153:1194–1217
3. Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G (2013) The hallmarks of aging. Cell 153:1194–1217
4. Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B, Kirkland JL, van Deursen JM (2011) Clearance of p16Ink4a- positive senescent cells delays ageing- associated disorders. Nature 479:232–236
5. Zhu Y, Armstrong JL, Tchkonia T, Kirkland JL (2014) Cellular senescence and the senescent secretory phenotype in age-related chronic dis- eases. Curr Opin Clin Nutr Metab Care 17:324–328
6. Munoz-Espin D, Serrano M (2014) Cellular senescence: from physiology to pathology. Nat Rev Mol Cell Biol 15:482–496
7. Chinta SJ, Lieu CA, Demaria M, Laberge RM, Campisi J, Andersen JK (2013) Environmental stress, ageing and glial cell senescence: a novel mechanistic link to Parkinson's disease?
J Intern Med 273:429–436
8. Yoshimoto S, Loo TM, Atarashi K, Kanda H, Sato S, Oyadomari S, Iwakura Y, Oshima K, Morita H, Hattori M, Honda K, Ishikawa Y, Hara E, Ohtani N (2013) Obesity-induced gut micro- bial metabolite promotes liver cancer through senescence secretome. Nature 499:97–101 9. Krizhanovsky V, Yon M, Dickins RA, Hearn S,
Simon J, Miething C, Yee H, Zender L, Lowe SW (2008) Senescence of activated stellate cells limits liver fi brosis. Cell 134:657–667
10. Kang TW, Yevsa T, Waller N, Hoenicke L, Wuesterfeld T, Dauch D, Hohmeyer A, Gereke M, Rudalska R, Potapova A, Iken M, Vucur M, Weiss S, Heikenwalder M, Khan S, Gil J, Bruder D, Manns M, Schirmacher P, Tacke F, Ott M, Luedde T, Longerich T, Kubicka S, Zender L (2011) Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature 479:547–551
11. Collado M, Serrano M (2006) The power and the promise of oncogene-induced senescence markers. Nat Rev Cancer 6:472–476
12. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O et al (1995) A biomarker that identifi es senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92:9363–9367
13. Yang NC, Hu ML (2005) The limitations and validities of senescence associated-beta- galactosidase activity as an aging marker for human foreskin fi broblast Hs68 cells. Exp Gerontol 40:813–819
14. Yegorov YE, Akimov SS, Hass R, Zelenin AV, Prudovsky IA (1998) Endogenous beta- galactosidase activity in continuously nonpro- liferating cells. Exp Cell Res 243:207–211 15. Collado M, Gil J, Efeyan A, Guerra C,
Schuhmacher AJ, Barradas M, Benguria A, Zaballos A, Flores JM, Barbacid M, Beach D, Serrano M (2005) Tumour biology: senescence in premalignant tumours. Nature 436:642 16. Althubiti M, Lezina L, Carrera S, Jukes-Jones
R, Giblett SM, Antonov A, Barlev N, Saldanha GS, Pritchard CA, Cain K, Macip S (2014) Characterization of novel markers of senes- cence and their prognostic potential in cancer.
Cell Death Dis 5:e1528
17. Cui H, Kong Y, Xu M, Zhang H (2013) Notch3 functions as a tumor suppressor by controlling cellular senescence. Cancer Res 73:3451–3459
18. Gorgoulis VG, Pratsinis H, Zacharatos P, Demoliou C, Sigala F, Asimacopoulos PJ, Papavassiliou AG, Kletsas D (2005) p53- dependent ICAM-1 overexpression in senes- cent human cells identifi ed in atherosclerotic lesions. Lab Invest 85:502–511
19. Chen Q, Ames BN (1994) Senescence-like growth arrest induced by hydrogen peroxide in human diploid fi broblast F65 cells. Proc Natl Acad Sci U S A 91:4130–4134
20. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW (1997) Oncogenic ras provokes pre- mature cell senescence associated with
accumulation of p53 and p16INK4a. Cell 88:593–602
21. Sugrue MM, Shin DY, Lee SW, Aaronson SA (1997) Wild-type p53 triggers a rapid senes- cence program in human tumor cells lacking functional p53. Proc Natl Acad Sci U S A 94:9648–9653
22. Chang BD, Xuan Y, Broude EV, Zhu H, Schott B, Fang J, Roninson IB (1999) Role of p53 and p21waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs. Oncogene 18:4808–4818
23. Macip S, Igarashi M, Fang L, Chen A, Pan ZQ, Lee SW, Aaronson SA (2002) Inhibition of p21-mediated ROS accumulation can rescue p21- induced senescence. EMBO J 21:2180–2188
155
Mikhail A. Nikiforov (ed.), Oncogene-Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 1534, DOI 10.1007/978-1-4939-6670-7_15, © Springer Science+Business Media New York 2017
Chapter 15
Metabolic Changes Investigated by Proton NMR
Spectroscopy in Cells Undergoing Oncogene-Induced Senescence
Claudia Gey and Karsten Seeger
Abstract
Investigating metabolic changes during different organismal or cellular states is of increasing interest. The combination of a data-rich analytical method like mass spectrometry or NMR spectroscopy with a statisti- cal analysis identifi es metabolites that are affected by a certain stimulus. Thus, important information on the underlying molecular pathways can be obtained. Here, we describe how to investigate metabolic changes in a model of oncogene-induced senescence. The water-soluble metabolites are isolated by a chloroform-methanol treatment and subsequently analyzed by NMR spectroscopy.
Key words Metabolomics , NMR spectroscopy , Cellular senescence , Metabolite extraction , Choline metabolism
1 Introduction
Investigating changes in metabolism in an omics-setting has gained much attention during the last years. Metabolic profi les of cells, tis- sues, or bodily fl uids are determined by combining modern analyti- cal techniques like mass spectrometry or NMR spectroscopy with an advanced statistical analysis. Much data on metabolic changes during malignant transformation is available and the lipid and cho- line metabolism have been identifi ed as key players during malig- nant transformation [ 1 , 2 ]. Recently, Quijano et al. and our group investigated cell models of oncogene-induced senescence (OIS) [ 3 , 4 ]. These cell models are based on human lung fi broblasts, the most extensively investigated cell type for cellular senescence .
Investigation of intracellular metabolites requires an initial extraction step that was done in earlier times with perchloric acid.
Comparison of different extraction protocols identifi ed the usage of chloroform, methanol, and water for metabolite extraction from wet tissues to be superior over other solvent systems like
acetonitrile/water when using NMR spectroscopy as an analytical technique [ 5 ]. Hence, the combination of chloroform, methanol, water is recommended for isolation of metabolites from tissues [ 6 ].
Extraction with methanol, chloroform, and water has additional advantages as, for example, the concomitant isolation of the lipid fraction [ 7 ]. For investigating changes in metabolism during senes- cence we adapted a protocol published by Lee and colleagues [ 8 ].
After metabolite extraction the methanol-water phase is trans- ferred to a new test tube and the solvent is removed by using a cen- trifugal vacuum concentrator. The dried metabolites are fi nally dissolved in a buffer that should have a suffi cient high buffer capacity.
We have used 0.1 M sodium phosphate buffer in deuterated water for NMR measurements. To ensure a high signal-to-noise ratio and nar- row line widths of the signals 3 mm Shigemi tubes were used. This allowed us to record spectra of very good quality within 1 h. After processing of the data, equidistant regions of the spectra were inte- grated and are referred to as buckets. Statistical analysis of the data with a principal component analysis showed a clear discrimination of control cells and senescent cells, with the most signifi cant differences in the levels of α- L -glycerophosphocholine (GPC). However, for anal- ysis of the data also other statistical methods should be considered [ 9 ].
2 Materials
All chemicals used should be of analytical grade. Safety and waste disposal regulations must be obeyed. Also follow the safety regulations when using an ultrasonic homogenizer or an NMR spectrometer .
In order to induce senescence we used fi broblasts (WI-38) stably transfected with a plasmid containing hyperactive human Raf-1, the hormone binding domain of the human estrogen receptor and GFP. Expression of Raf-1 is induced by addition of 4-hydroxy- tamoxifen (4-HT) and within 72 h standard markers for senes- cence can be monitored [ 10 , 11 ].