Summary
A series of experiments have been carried out to identify those factors that affect the number of altered populations detected in control, nonexposed, and radiation-exposed primary cultures of rat tracheal epithelial cells. The number of colony forming cells per milliliter of culture medium and the frequency with which the culture medium is changed seemed to be the most critical factors regulating the emergence of induced and spontaneous variants. Increasing the number of cells plated so that of colony forming cells increase from 25 to 200 per ml, regardless of the dish size used, was associated with a 200-fold decline in the frequency of spontaneous variants and a 40-fold decline in X-ray-induced variants. Increasing the interval between medium changes from 3 to 7 days after the first week of culture was associated with a 10-fold decrease in the frequency of spontaneous variants. The frequency of spontaneous and induced variants is markedly less dependent on culture density at densities between 150 and 600 colony forming cells per ml. The type of medium used to establish primary cultures had little effect on the frequency of variants detected. Similarly, when assays were performed at densities in excess of 150 colony forming cells per ml the frequency of spontaneous and x-ray-induced variants was not affected by the absence of epidermal growth factor, increased levels of calcium (final concentration, 0.8 mM), or by removal of pyruvate from the selection medium.
Similar content being viewed by others
References
Bertolero, F.; Kaighn, M. E.; Gonda, M. A., et al. Mouse epidermal keratinocytes. Exp. Cell Res. 155:64–80; 1984.
Brenner, D. J. Radon: current challenges in cellular radiobiology. Int. J. Radiat. Biol. 61(1):3–13; 1992.
Elkind, M. M.; Hill, C. K.; Han, A. Repair and misrepair in radiation-induced neoplastic transformation. In: Huberman, E.; Barr, S. H., eds. Carcinogenesis, vol. 10. New York: Raven Press; 1985:317–361.
Ferriola, P. C.; Steigerwalt, R.; Robertson, A. T., et al. Abnormalities in growth regulation of transformed rat tracheal epithelial cells. Pathobiology 58:28–36; 1990.
Ford, J. R.; Terzaghi-Howe, M. Basal cells are the progenitors of primary tracheal epithelial cell cultures. Exp. Cell. Res. 198:69–77; 1992.
Gray, T.; Rundhaug, J.; Nettesheim, P. Critical variables controlling cell proliferation in primary cultures of rat tracheal epithelial cells. In Vitro Cell. Dev. Biol. 27A:805–814; 1991.
Gray, T. E.; Thomassen, D. G.; Mass, M. J., et al. Quantitation of cell proliferation, colony formation, and carcinogen induced cytotoxicity of rat tracheal epithelial cells grown in culture on 3T3 feeder layers. In Vitro 19:559–569; 1983.
Harris, C. C.; Brash, D. E.; Lechner, J. F., et al. Aberrations of growth and differentiation pathways during neoplastic transformation of human epithelial cells. In: Kakunaga, T.; Sugimura, T.; Tomatis, L., et al., eds. Cell differentiation, genes and cancer. Geneva, Switzerland: International Agency for Research on Cancer; 1988:139–147.
Hei, T. K.; Komatsu, K.; Hall, E. J., et al. Oncogenic transformation by charged particles of defined LET. Carcinogenesis 9:747–750; 1988.
Johnson, N. F.; Margiotta, E. A.; Wilson, J. S., et al. Preparation of viable single cell suspensions of tracheal epithelial cells. Br. J. Exp. Pathol. 68:157–165; 1987.
Kitamura, H.; Fitzgerald, D. J.; Li, L-A., et al. Morphological characterization of transformed colonies in rat tracheal epithelial cell cultures exposed to carcinogen. Can. Res. 46:4631–4641; 1986.
Marchok, A. C.; Rhoton, J. C.; Griesemer, R. A., et al. Increased in vitro growth capacity of tracheal epithelium exposed in vivo to 7, 12-dimethylbenz(a)anthracene. Can. Res. 37:1811–1821; 1977.
Marchok, A. C.; Huang, S. F.; Martin, D. H. Selection of carcinogen-altered rat tracheal epithelial cells preexposed to 7, 12-dimethylbenz[a]anthracene by their loss of a need for pyruvate to survive in culture. Carcinogenesis 5:789–796; 1984.
Pai, S. B.; Steele, V. E.; Nettesheim, P. Neoplastic transformation of primary tracheal epithelial cell cultures. Carcinogenesis 4:369–374; 1983.
Terzaghi, M.; Nettesheim, P. Dynamics of neoplastic development in carcinogen-exposed tracheal mucosa. Can. Res. 39:4003–4010; 1979.
Terzaghi-Howe, M. Inhibitor production by normal rat tracheal epithelial cells influences the frequency of spontaneous and X-ray-induced enhanced growth variants. Carcinogenesis 10:967–971; 1989.
Terzaghi-Howe, M. Changes in response to, and production of, transforming growth factory typeβ during neoplastic progression in cultured rat tracheal epithelial cells. Carcinogenesis 10:973–980; 1989.
Terzaghi-Howe, M. Interactions between cell populations influence expression of the transformed phenotype in irradiated rat tracheal epithelial cells. Radiat. Res. 121:242–247; 1990.
Terzaghi-Howe, M. Induction of preneoplastic alterations by X rays and neutrons in exposed rat tracheas and isolated tracheal epithelial cells. Radiat. Res. 120:352–363; 1989.
Thomassen, D. C. Role of spontaneous transformation in carcinogenesis: development of preneoplastic rat tracheal epithelial cells at a constant rate. Can. Res. 46:2344–2348; 1986.
Thomassen, D. G.; Seiler, F. A.; Shyr, L.-J., et al. Alpha-particles induce preneoplastic transformation of rat tracheal epithelial cells in culture. Int. J. Radiat. Biol. 57:395–405; 1990.
Thomassen, D. G.; Saffiotti, U.; Kaighn, M. E. Clonal proliferation of rat tracheal epithelial cells in serum-free medium and their responses to hormones, growth factors and carcinogens. Carcinogenesis 7:2033–2039; 1986.
Thomassen, D. G.; Harkema, J. R.; Stephens, N. D., et al. Preneoplastic transformation of rat tracheal epithelial cells by ozone. Toxicol. Appl. Pharmacol. 109:137–148; 1991.
Thomassen, D. G.; Hubbs, A. F.; Kelly, G. Changes in cellular responses to inducers of differentiation, growth factors, and oncogenes during neoplastic progression of respiratory epithelial cells. In: Thomassen, D. G.; Nettesheim, P., eds. Biology, toxicology, and carcinogenesis of respiratory epithelium. New York, New York: Hemisphere Publishing Co.; 1990:205–216.
Steele, V. E.; Kelloff, G. J.; Wilkinson, B. P., et al. Inhibition of transformation in cultured rat tracheal epithelial cells by potential chemopreventive agents. Can. Res. 50:2068–2974; 1990.
Wasilenko, W. J.; Marchok, A. C. Pyruvate regulation of growth and differentiation in primary cultures of rat tracheal epithelial cells. Exp. Cell Res. 155:507–517; 1984.
Zar, J. H. Biostatistical analysis. Englewood Cliffs, NJ: Prentice-Hall, Inc.; 1984:408–409.
Zhu, S.; Cunningham, M. L.; Gray, T. E., et al. Cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat tracheal epithelial cells. Mut. Res. 261:249–259; 1991.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Terzaghi-Howe, M. Factors regulating the emergence of spontaneous and X-ray-induced variants in primary rat tracheal epithelial cell cultures. In Vitro Cell Dev Biol - Animal 29, 120–126 (1993). https://doi.org/10.1007/BF02630942
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02630942