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The dark side of BrdU in neural stem cell biology: detrimental effects on cell cycle, differentiation and survival

Abstract

5-Bromo-2′-deoxyuridin (BrdU) is frequently used in anaylsis of neural stem cell biology, in particular to label and to fate-map dividing cells. However, up to now, only a few studies have addressed the question as to whether BrdU labeling per se affects the cells to be investigated. Here, we focused on the potential impact of BrdU on neurosphere cultures derived from the adult rat brain and on proliferation of progenitors in vivo. In vitro, neurospheres were pulsed for 48 h with BrdU, and cell proliferation, cell cycle, differentiation, survival and adhesion properties were subsequently analyzed. BrdU inhibited the expansion of neural progenitors as assessed by MTS assay and increased the fraction of cells in the G0/G1-phase of the cell cycle. Moreover, BrdU increased cell death and dose-dependently induced adherence of NPCs. Cell adherence was accompanied by a reduced amount of active matrix-metalloproteinase-2 (MMP-2). Furthermore, BrdU repressed neuronal and oligodendroglial differentiation, whereas astroglial fate was not affected. In contrast to the in vitro situation, BrdU apparently did not influence endogenous proliferation of NPCs or neurogenesis in concentrations that are typically used for labeling of neural progenitors in vivo. Our results reveal so far uncharacterized effects of BrdU on adult NPCs. We conclude that, because of its ubiquitous use in stem cell biology, any potential effect of BrdU of NPCs has to be scrutinized prior to interpretation of data.

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References

  1. Agnish ND, Kochhar DM (1976) Direct exposure of postimplantation mouse embryos to 5-bromodeoxyuridine in vitro and its effect on subsequent chondrogenesis in the limbs. J Embryol Exp Morphol 36:623–638

    PubMed  CAS  Google Scholar 

  2. Bannigan JG (1985) The effects of 5-bromodeoxyuridine on fusion of the cranial neural folds in the mouse embryo. Teratology 32:229–239

    PubMed  Article  CAS  Google Scholar 

  3. Barasch JM, Bressler RS (1977) The effect of 5-bromodeoxyuridine on the postnatal development of the rat testis. J Exp Zool 200:1–8

    PubMed  Article  CAS  Google Scholar 

  4. Berger S (2001) Untersuchung der Wirkung von Schwerionenstrahlen auf menschliche Hautfibroblasten unter besonderer Berücksichtigung chromosomaler Veränderungen. Dissertation, Universität Mainz

  5. Burns TC, Ortiz-Gonzalez XR, Gutierrez-Perez M, Keene CD, Sharda R, Demorest ZL, Jiang Y, Nelson-Holte M, Soriano M, Nakagawa Y, Luquin MR, Garcia-Verdugo JM, Prosper F, Low WC, Verfaillie CM (2006) Thymidine analogs are transferred from prelabeled donor to host cells in the central nervous system after transplantation: a word of caution. Stem Cells 24:1121–1127

    PubMed  Article  CAS  Google Scholar 

  6. Caldwell MA, He X, Svendsen CN (2005) 5-Bromo-2′-deoxyuridine is selectively toxic to neuronal precursors in vitro. Eur J Neurosci 22:2965–2970

    PubMed  Article  Google Scholar 

  7. Cameron HA, McKay RD (2001) Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol 435:406–417

    PubMed  Article  CAS  Google Scholar 

  8. Cooper-Kuhn CM, Kuhn HG (2002) Is it all DNA repair? Methodological considerations for detecting neurogenesis in the adult brain. Brain Res Dev Brain Res 134:13–21

    PubMed  Article  CAS  Google Scholar 

  9. Davidson RL, Kaufman ER (1979) Resistance to bromodeoxyuridine mutagenesis and toxicity in mammalian cells selected for resistance to hydroxyurea. Somatic Cell Genet 5:873–885

    PubMed  Article  CAS  Google Scholar 

  10. Diermeier S, Schmidt-Bruecken E, Kubbies M, Kunz-Schughart LA, Brockhoff G (2004) Exposure to continuous bromodeoxyuridine (BrdU) differentially affects cell cycle progression of human breast and bladder cancer cell lines. Cell Prolif 37:195–206

    PubMed  Article  CAS  Google Scholar 

  11. Eisch AJ, Mandyam CD (2007) Adult neurogenesis: can analysis of cell cycle proteins move us "Beyond BrdU"? Curr Pharm Biotechnol 8:147–165

    PubMed  Article  CAS  Google Scholar 

  12. Elkington PT, O'Kane CM, Friedland JS (2005) The paradox of matrix metalloproteinases in infectious disease. Clin Exp Immunol 142:12–20

    PubMed  Article  CAS  Google Scholar 

  13. Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH (1998) Neurogenesis in the adult human hippocampus. Nat Med 4:1313–1317

    PubMed  Article  CAS  Google Scholar 

  14. Garner W (1974) The effect of 5-bromodeoxyuridine on early mouse embryos in vitro. J Embryol Exp Morphol 32:849–855

    PubMed  CAS  Google Scholar 

  15. Gomez DE, Alonso DF, Yoshiji H, Thorgeirsson UP (1997) Tissue inhibitors of metalloproteinases: structure, regulation and biological functions. Eur J Cell Biol 74:111–122

    PubMed  CAS  Google Scholar 

  16. Gould E, Gross CG (2002) Neurogenesis in adult mammals: some progress and problems. J Neurosci 22:619–623

    PubMed  CAS  Google Scholar 

  17. Goz B (1977) The effects of incorporation of 5-halogenated deoxyuridines into the DNA of eukaryotic cells. Pharmacol Rev 29:249–272

    PubMed  CAS  Google Scholar 

  18. Hancock A, Priester C, Kidder E, Keith JR (2009) Does 5-bromo-2′-deoxyuridine (BrdU) disrupt cell proliferation and neuronal maturation in the adult rat hippocampus in vivo? Behav Brain Res 199:218–221

    PubMed  Article  CAS  Google Scholar 

  19. Hayes NL, Nowakowski RS (2000) Exploiting the dynamics of S-phase tracers in developing brain: interkinetic nuclear migration for cells entering versus leaving the S-phase. Dev Neurosci 22:44–55

    PubMed  Article  CAS  Google Scholar 

  20. Herrup K, Arendt T (2002) Re-expression of cell cycle proteins induces neuronal cell death during Alzheimer's disease. J Alzheimers Dis 4:243–247

    PubMed  CAS  Google Scholar 

  21. Hume WJ, Saffhill R (1986) Iodo- and bromodeoxyuridine are excised at different rates from DNA of mouse tongue keratinocytes in vitro. Chem Biol Interact 60:227–232

    PubMed  Article  CAS  Google Scholar 

  22. Katchanov J, Harms C, Gertz K, Hauck L, Waeber C, Hirt L, Priller J, von Harsdorf R, Bruck W, Hortnagl H, Dirnagl U, Bhide PG, Endres M (2001) Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death. J Neurosci 21:5045–5053

    PubMed  CAS  Google Scholar 

  23. Kaufman ER, Davidson RL (1978) Biological and biochemical effects of bromodeoxyuridine and deoxycytidine on Syrian hamster melanoma cells. Somatic Cell Genet 4:587–601

    PubMed  Article  CAS  Google Scholar 

  24. Kuan CY, Schloemer AJ, Lu A, Burns KA, Weng WL, Williams MT, Strauss KI, Vorhees CV, Flavell RA, Davis RJ, Sharp FR, Rakic P (2004) Hypoxia-ischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain. J Neurosci 24:10763–10772

    PubMed  Article  CAS  Google Scholar 

  25. Kuhn HG, Cooper-Kuhn CM (2007) Bromodeoxyuridine and the detection of neurogenesis. Curr Pharm Biotechnol 8:127–131

    PubMed  Article  CAS  Google Scholar 

  26. Levkoff LH, Marshall GP 2nd, Ross HH, Caldeira M, Reynolds BA, Cakiroglu M, Mariani CL, Streit WJ, Laywell ED (2008) Bromodeoxyuridine inhibits cancer cell proliferation in vitro and in vivo. Neoplasia 10:804–816

    PubMed  CAS  Google Scholar 

  27. Michishita E, Nakabayashi K, Suzuki T, Kaul SC, Ogino H, Fujii M, Mitsui Y, Ayusawa D (1999) 5-Bromodeoxyuridine induces senescence-like phenomena in mammalian cells regardless of cell type or species. J Biochem 126:1052–1059

    PubMed  CAS  Google Scholar 

  28. Miller MW, Nowakowski RS (1988) Use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system. Brain Res 457:44–52

    PubMed  Article  CAS  Google Scholar 

  29. Nagao T, Kuwagata M, Saito Y (1998) Effects of prenatal exposure to 5-fluoro-2′-deoxyuridine on developing central nervous system and reproductive function in male offspring of mice. Teratog Carcinog Mutagen 18:73–92

    PubMed  Article  CAS  Google Scholar 

  30. Nowakowski RS, Hayes NL (2001) Stem cells: the promises and pitfalls. Neuropsychopharmacology 25:799–804

    PubMed  Article  CAS  Google Scholar 

  31. Nowakowski RS, Lewin SB, Miller MW (1989) Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and the DNA-synthetic phase for an anatomically defined population. J Neurocytol 18:311–318

    PubMed  Article  CAS  Google Scholar 

  32. Packard DS Jr, Menzies RA, Skalko RG (1973) Incorporation of thymidine and its analogue, bromodeoxyuridine, into embryos and maternal tissues of the mouse. Differentiation 1:397–404

    PubMed  Article  CAS  Google Scholar 

  33. Phuphanich S, Levin VA (1985) Bioavailability of bromodeoxyuridine in dogs and toxicity in rats. Cancer Res 45:2387–2389

    PubMed  CAS  Google Scholar 

  34. Pollard DR, Baran MM, Bachvarova R (1976) The effect of 5-bromodeoxyuridine on cell division and differentiation of preimplantation mouse embryos. J Embryol Exp Morphol 35:169–178

    PubMed  CAS  Google Scholar 

  35. Poot M, Hoehn H, Kubbies M, Grossmann A, Chen Y, Rabinovitch PS (1994) Cell-cycle analysis using continuous bromodeoxyuridine labeling and Hoechst 33358-ethidium bromide bivariate flow cytometry. Methods Cell Biol 41:327–340

    PubMed  Article  CAS  Google Scholar 

  36. Rakic P (2002) Adult neurogenesis in mammals: an identity crisis. J Neurosci 22:614–618

    PubMed  Google Scholar 

  37. Rivera FJ, Couillard-Despres S, Pedre X, Ploetz S, Caioni M, Lois C, Bogdahn U, Aigner L (2006) Mesenchymal stem cells instruct oligodendrogenic fate decision on adult neural stem cells. Stem Cells 24:2209–2219

    PubMed  Article  CAS  Google Scholar 

  38. Ross HH, Levkoff LH, Marshall GP 2nd, Caldeira M, Steindler DA, Reynolds BA, Laywell ED (2008) Bromodeoxyuridine induces senescence in neural stem and progenitor cells. Stem Cells 26:3218–3227

    PubMed  Article  CAS  Google Scholar 

  39. Shinin V, Gayraud-Morel B, Gomes D, Tajbakhsh S (2006) Asymmetric division and cosegregation of template DNA strands in adult muscle satellite cells. Nat Cell Biol 8:677–687

    PubMed  Article  CAS  Google Scholar 

  40. Steffenhagen C, Kraus S, Dechant FX, Kandasamy M, Lehner B, Poehler AM, Furtner T, Siebzehnrubl FA, Couillard-Despres S, Strauss O, Aigner L, Rivera FJ (2011) Identity, Fate and Potential of Cells Grown as Neurospheres: Species Matters. Stem Cell Rev: Mar 24. [Epub ahead of print]

  41. Sternlicht MD, Lochter A, Sympson CJ, Huey B, Rougier JP, Gray JW, Pinkel D, Bissell MJ, Werb Z (1999) The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis. Cell 98:137–146

    PubMed  Article  CAS  Google Scholar 

  42. Suzuki T, Minagawa S, Michishita E, Ogino H, Fujii M, Mitsui Y, Ayusawa D (2001) Induction of senescence-associated genes by 5-bromodeoxyuridine in HeLa cells. Exp Gerontol 36:465–474

    PubMed  Article  CAS  Google Scholar 

  43. Taupin P (2007) BrdU immunohistochemistry for studying adult neurogenesis: paradigms, pitfalls, limitations, and validation. Brain Res Rev 53:198–214

    PubMed  Article  CAS  Google Scholar 

  44. Tempfer H, Gehwolf R, Lehner C, Wagner A, Mtsariashvili M, Bauer HC, Resch H, Tauber M (2009) Effects of crystalline glucocorticoid triamcinolone acetonide on cultered human supraspinatus tendon cells. Acta Orthop 80:357–362

    PubMed  Article  Google Scholar 

  45. Wachs FP, Winner B, Couillard-Despres S, Schiller T, Aigner R, Winkler J, Bogdahn U, Aigner L (2006) Transforming growth factor-beta1 is a negative modulator of adult neurogenesis. J Neuropathol Exp Neurol 65:358–370

    PubMed  Article  CAS  Google Scholar 

  46. Zhu Y, Yang GY, Ahlemeyer B, Pang L, Che XM, Culmsee C, Klumpp S, Krieglstein J (2002) Transforming growth factor-beta 1 increases bad phosphorylation and protects neurons against damage. J Neurosci 22:3898–3909

    PubMed  CAS  Google Scholar 

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Correspondence to Ludwig Aigner.

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The authors would like to thank the following funding agencies for their support: the Bavarian State Ministry of Sciences, Research and the Arts (ForNeuroCell grant), the Germany Federal Ministry of Education and Research (BMBF grants #0312134, #01GG0706 and #01GN0505), the EU-FP6-Programm "DiMI", LSHB-CT-2005-512146, and the state government of Salzburg.

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Lehner, B., Sandner, B., Marschallinger, J. et al. The dark side of BrdU in neural stem cell biology: detrimental effects on cell cycle, differentiation and survival. Cell Tissue Res 345, 313 (2011). https://doi.org/10.1007/s00441-011-1213-7

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Keywords

  • Neural progenitors
  • Neurogenesis
  • Cell culture
  • Cell labeling
  • Rat (Fischer 344)