Abstract
1. We hypothesized that non-neuronal cells could be eliminated from primary dorsal root ganglion (DRG) cultures by including a DNA topoisomerase inhibitor (camptothecin) during culture.
2. Exposure to 20 μM camptothecin for 48 h, beginning at 3 days in vitro, reliably eliminates proliferating non-neuronal cells.
3. Following camptothecin treatment, neurons survived and continued to extend neurites for several weeks without obvious defects in morphology or viability.
4. Transient camptothecin exposure is therefore an efficient and fast-acting method to purify DRG neurons in culture.
This is a preview of subscription content, log in via an institution to check access.
References
Beidler, D. R., and Cheng, Y.-C. (1995). Camptothecin induction of a time-and concentration-dependent decrease of topoisomerase I and its implication in camptothecin activity. Mol. Pharmacol. 47:907–914.
Bunge, R. P., and Wood, P. M. (1987). Tissue culture studies of interactions between axons and myelinating cells of the central and peripheral nervous system. In Seil, F. J., Herbert, E., and Carlson, B. M. (eds.), Progress in Brain Research, Vol. 71, Elsevier, New York, pp. 143–152.
Desai, S. D., Li, T.-K., Rodriguez-Bauman, A., Rubin, E. H., and Liu, L. F. (2001). Ubiquitin/26S proteasome-mediated degradation of topoisomerase I as a resistance mechanism to camptothecin in tumor cells. Cancer Res. 61:5926–5932.
Desai, S. D., Liu, L. F., Vazquez-Abad, D., and D'Arpa, P. (1997). Ubiquitin-dependent destruction of topoisomerase I is stimulated by the antitumor drug camptothecin. J. Biochem. Chem. 272:24159–24164.
Devon, R., and Doucette, R. (1992). Olfactory ensheathing cells myelinate dorsal root ganglion neurites. Brain Res. 589:175–179.
Goldenberg, S. S., and De Boni, U. (1983). Pure population of viable neurons from rabbit dorsal root ganglia, using gradients of Percoll. J. Neurobiol. 14:195–206.
Horie, H., and Kim, S. U. (1984). Improved survival and differentiation of newborn and adult mouse neurons in F12 defined medium by fibronectin. Brain Res. 294:178–181.
Kaufmann, S. H. (1998). Cell death induced by topoisomerase-targeted drugs: More questions than answers. Biochim. Biophys. Acta 1400:195–211.
Liu, L. F., Desai, S. D., Li, T.-K., Mao, Y., Sun, M., and Sim, S.-P. (2000). Mechanism of action of camptothecin. Ann. N.Y. Acad. Sci. 922:1–10.
Masuko, S., Kuromi, H., and Shimada, Y. (1979). Isolation and culture of motor neurons from embryonic chicken spinal cords. Proc. Natl. Acad. Sci. U.S.A. 76:3537–3541.
Morris, E. J., and Geller, H. M. (1996). Induction of neuronal apoptosis by camptothecin, an inhibitor of DNA topoisomerase-1: Evidence for cell-cycle-independent toxicity. J. Cell Biol. 134:757–770.
Mosesso, P., Pichierri, P., Franchitto, A., and Palitti, F. (2000). Evidence that camptothecin-induced aberrations in the G2 phase of the cell cycle of Chinese hamster ovary (CHO) cell lines is associated with transcription. Mutat. Res. 452:189–195.
Nakajima, M., Kashiwagi, K., Ohta, J., Furukawa, S., Hayashi, K., Kawashima, T., and Hayashi, Y. (1994). Etoposide induces programmed cell death in neurons cultured from the fetal rat central nervous system. Brain Res. 641:350–352.
Ryan, A. J., Squires, S., Strutt, H. L., and Johnson, R. T. (1991). Camptothecin cytotoxicity in mammalian cells is associated with the induction of persistent double strand breaks in replicating DNA. Nucleic Acids Res. 19:3295–3300.
Scott, B. S. (1977). Adult mouse dorsal root ganglia neurons in cell culture. J. Neurobiol. 8:417–427.
Tomkins, C. E., Edwards, S. N., and Tolkovsky, A. M. (1994). Apoptosis is induced in postmitotic rat sympathetic neurons by arabinosides and topoisomerase II inhibitors in the presence of NGF. J. Cell Sci. 107:1499–1507.
Tsutsui, K., Tsutsui, K., Sano, K., Kikuchi, A., and Tokunaga, A. (2001). Involvement of DNA topoisomerase IIβ in neuronal differentiation. J. Biol. Chem. 276:5769–5778.
Wang, J. C. (1985). DNA topoisomerases. Annu. Rev. Biochem. 54:665–697.
Wood, P. M., and Bunge, R. P. (1986). Myelination of cultured dorsal root ganglion neurons by oligodendrocytes obtained from adult rats. J. Neurol. Sci. 74:153–169.
Wood, P. M., and Williams, A. K. (1984). Oligodendrocyte proliferation and CNS myelination in cultures containing dissociated embryonic neuroglia and dorsal root ganglion neurons. Brain Res. 314:225–241.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Andersen, P.L., Doucette, J.R. & Nazarali, A.J. A Novel Method of Eliminating Non-Neuronal Proliferating Cells from Cultures of Mouse Dorsal Root Ganglia. Cell Mol Neurobiol 23, 205–210 (2003). https://doi.org/10.1023/A:1022902006434
Issue Date:
DOI: https://doi.org/10.1023/A:1022902006434