Skip to main content
SpringerLink
Log in

Staurosporine-induced differentiation of the RGC-5 cell line leads to apoptosis and cell death at the lowest differentiating concentration

  • Glaucoma
  • Published:
Graefe's Archive for Clinical and Experimental Ophthalmology Aims and scope Submit manuscript

Abstract

Background

Supplementation of staurosporine is the method of choice for differentiating the solely existing retinal ganglion cell (RGC)-5 cell line. This differentiation was initially claimed to be in the absence of apoptosis, but some publications supposed the induction of apoptosis during staurosporine induced RGC-5 differentiation. In respect to these inconsistencies in the literature, we investigated in detail whether RGC-5 cell differentiation by staurosporine induces apoptosis or not.

Methods

Amounts of 50 nM, 200 nM, 300 nM, and 600 nM of staurosporine were supplemented on RGC-5 cells for 24 h. Cell morphology and cell death, via propidium iodide staining, were evaluated with phase contrast and fluorescence microscopy, respectively. Cell amount, cell proliferation, and cell viability were analyzed by crystal violet staining, CFSE flow cytometry, and MTS assay, respectively. Apoptosis was determined by analyzing caspase 3/7 activity, Annexin-V+/ 7AAD- cells and the quotient of Bax to Bcl-2 mRNA expression via caspase 3/7 activity assay, flow cytometry, and real-time PCR, respectively.

Results

RGC-5 cells started to change their morphology and their expression of neuronal markers at 50 nM of staurosporine. This was associated with apoptosis and cell death, as indicated by a 2.1-fold (p < 0.0005) increase in caspase 3/7 activity, a 1.2–fold (p < 0.05) induction of Annexin-V+/ 7AAD- cells, and a 12–fold (p < 0.0005) increase in propidium iodide positive cells, respectively. Furthermore, staurosporine led to a dose-dependent increase in apoptosis and reduction in cell viability, cell density, and cell proliferation.

Conclusions

The lowest staurosporine concentration inducing RGC-5 cell differentiation is accompanied by apoptosis and cell death.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

CFSE:

Succinimidyl ester of carboxyfluorescein diacetate

PI:

Propidium iodide

RGC:

Retinal ganglion cell

7-AAD:

7-Amino-actinomycin

NF:

Neurofilament

References

  1. Krishnamoorthy RR, Agarwal P, Prasanna G, Vopat K, Lambert W, Sheedlo HJ, Pang IH, Shade D, Wordinger RJ, Yorio T, Clark AF, Agarwal N (2001) Characterization of a transformed rat retinal ganglion cell line. Brain Res Mol Brain Res 86:1–12

    Article  PubMed  CAS  Google Scholar 

  2. Moorhouse AJ, Li S, Vickery RM, Hill MA, Morley JW (2004) A patch-clamp investigation of membrane currents in a novel mammalian retinal ganglion cell line. Brain Res 1003:205–208

    Article  PubMed  CAS  Google Scholar 

  3. Van Bergen NJ, Wood JP, Chidlow G, Trounce IA, Casson RJ, Ju WK, Weinreb RN, Crowston JG (2009) Recharacterization of the RGC-5 retinal ganglion cell line. Invest Ophthalmol Vis Sci 50:4267–4272

    Article  PubMed  Google Scholar 

  4. Wood JP, Chidlow G, Tran T, Crowston J, Casson RJ (2010) A comparison of differentiation protocols for Rgc-5 cells. Invest Ophthalmol Vis Sci 51(7):3774–3783

    Article  PubMed  Google Scholar 

  5. Frassetto LJ, Schlieve CR, Lieven CJ, Utter AA, Jones MV, Agarwal N, Levin LA (2006) Kinase-dependent differentiation of a retinal ganglion cell precursor. Invest Ophthalmol Vis Sci 47:427–438

    Article  PubMed  Google Scholar 

  6. Schallenberg M, Charalambous P, Thanos S (2009) GM-CSF regulates the ERK1/2 pathways and protects injured retinal ganglion cells from induced death. Exp Eye Res 89:665–677

    Article  PubMed  CAS  Google Scholar 

  7. Schwechter BR, Millet LE, Levin LA (2007) Histone deacetylase inhibition-mediated differentiation of RGC-5 cells and interaction with survival. Invest Ophthalmol Vis Sci 48:2845–2857

    Article  PubMed  Google Scholar 

  8. Ganapathy PS, Dun Y, Ha Y, Duplantier J, Allen JB, Farooq A, Bozard BR, Smith SB (2010) Sensitivity of staurosporine-induced differentiated RGC-5 cells to homocysteine. Curr Eye Res 35:80–90

    Article  PubMed  CAS  Google Scholar 

  9. Lieven CJ, Millet LE, Hoegger MJ, Levin LA (2007) Induction of axon and dendrite formation during early RGC-5 cell differentiation. Exp Eye Res 85:678–683

    Article  PubMed  CAS  Google Scholar 

  10. Nieto PS, Acosta-Rodriguez VA, Valdez DJ, Guido ME (2010) Differential responses of the mammalian retinal ganglion cell line RGC-5 to physiological stimuli and trophic factors. Neurochem Int 57:216–226

    Article  PubMed  CAS  Google Scholar 

  11. Schnichels S, Heiduschka P, Julien S (2011) RGMA and neogenin protein expression are influenced by lens injury following optic nerve crush in the rat retina. Graefes Arch Clin Exp Ophthalmol; Epub ahead of print

  12. Schnichels S, Heiduschka P, Julien S (2011) Different spatial and temporal protein expressions of repulsive guidance molecule a and neogenin in the rat optic nerve after optic nerve crush with and without lens injury. J Neurosci Res 89:490–505

    Article  PubMed  CAS  Google Scholar 

  13. Heiduschka P, Schnichels S, Fuhrmann N, Hofmeister S, Schraermeyer U, Wissinger B, Alavi MV (2010) Electrophysiological and histologic assessment of retinal ganglion cell fate in a mouse model for OPA1-associated autosomal dominant optic atrophy. Invest Ophthalmol Vis Sci 51:1424–1431

    Article  PubMed  Google Scholar 

  14. Van Linthout S, Spillmann F, Riad A, Trimpert C, Lievens J, Meloni M, Escher F, Filenberg E, Demir O, Li J, Shakibaei M, Schimke I, Staudt A, Felix SB, Schultheiss HP, De Geest B, Tschope C (2008) Human apolipoprotein A-I gene transfer reduces the development of experimental diabetic cardiomyopathy. Circulation 117:1563–1573

    Article  PubMed  Google Scholar 

  15. Harper MM, Adamson L, Blits B, Bunge MB, Grozdanic SD, Sakaguchi DS (2009) Brain-derived neurotrophic factor released from engineered mesenchymal stem cells attenuates glutamate- and hydrogen peroxide-mediated death of staurosporine-differentiated RGC-5 cells. Exp Eye Res 89:538–548

    Article  PubMed  CAS  Google Scholar 

  16. Thompson AF, Levin LA (2010) Neuronal differentiation by analogs of staurosporine. Neurochem Int 56:554–560

    Article  PubMed  CAS  Google Scholar 

  17. Marzo I, Brenner C, Zamzami N, Jurgensmeier JM, Susin SA, Vieira HL, Prevost MC, Xie Z, Matsuyama S, Reed JC, Kroemer G (1998) Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281:2027–2031

    Article  PubMed  CAS  Google Scholar 

  18. Antonsson B, Montessuit S, Sanchez B, Martinou JC (2001) Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J Biol Chem 276:11615–11623

    Article  PubMed  CAS  Google Scholar 

  19. Antonsson B, Montessuit S, Lauper S, Eskes R, Martinou JC (2000) Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria. Biochem J 345(Pt 2):271–278

    Article  PubMed  CAS  Google Scholar 

  20. Broughton BR, Reutens DC, Sobey CG (2009) Apoptotic mechanisms after cerebral ischemia. Stroke 40:e331–e339

    Article  PubMed  Google Scholar 

  21. Love S (2003) Apoptosis and brain ischaemia. Prog Neuropsychopharmacol Biol Psychiatry 27:267–282

    Article  PubMed  CAS  Google Scholar 

  22. Aoun P, Simpkins JW, Agarwal N (2003) Role of PPAR-gamma ligands in neuroprotection against glutamate-induced cytotoxicity in retinal ganglion cells. Invest Ophthalmol Vis Sci 44:2999–3004

    Article  PubMed  Google Scholar 

  23. Harvey R, Chintala SK (2007) Inhibition of plasminogen activators attenuates the death of differentiated retinal ganglion cells and stabilizes their neurite network in vitro. Invest Ophthalmol Vis Sci 48:1884–1891

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Annika Koschel and Johanna Hofmann for excellent technical assistance. This work was partly funded by the Deutsche Ophthalmologische Gesellschaft.

Disclosure

There are no conflicts of interest.

Author information

Authors and Affiliations

  1. Centre of Ophthalmology, University Eye Hospital Tübingen, Tübingen, Germany

    Maximillian Schultheiss, Sven Schnichels, Peter Szurman & Martin S. Spitzer

  2. Berlin-Brandenburg Center for Regenerative Therapies, Platform Cardiovascular System, Charité, University-Medicine Berlin, Campus Virchow, Südstrasse 2, 13353, Berlin, Germany

    Kapka Miteva, Katrin Warstat & Sophie Van Linthout

  3. Department of Cardiology & Pneumology, Charité, University-Medicine Berlin, Campus Benjamin Franklin, Berlin, Germany

    Sophie Van Linthout

Authors
  1. Maximillian Schultheiss
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sven Schnichels
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kapka Miteva
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Katrin Warstat
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter Szurman
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Martin S. Spitzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sophie Van Linthout
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sophie Van Linthout.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schultheiss, M., Schnichels, S., Miteva, K. et al. Staurosporine-induced differentiation of the RGC-5 cell line leads to apoptosis and cell death at the lowest differentiating concentration. Graefes Arch Clin Exp Ophthalmol 250, 1221–1229 (2012). https://doi.org/10.1007/s00417-011-1906-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00417-011-1906-3

Keywords

Search

Navigation