Abstract
The photosensitivity of bromodeoxyuridine (BrdU)-substituted cells is known to be markedly enhanced by the fluorochrome Hoechst 33258. Since the incorporation of BrdU into nucleic acids depends upon its prior phosphorylation via thymidine kinase (TK; EC 2.7.1.21), cells deficient in TK activity are refractory to photoinduced killing. These observations suggested that combined treatment with BrdU, Hoechst 33258, and visible light would constitute an efficient selective strategy for the recovery of TK− mutant cells. In this report we describe a single-step selection protocol which reduced the survival of TK+ cells by a factor of 105 without affecting the viability of TK− mutants. This procedure was used to isolate H4IIEC3-derived rat hepatoma cells deficient in TK activity. The properties of several TK− hepatoma clones are discussed.
Similar content being viewed by others
References
Barile, M. F., Bodey, G. P., Snyder, J., Riggs, D. B., and Grabowski, M. W. (1966). Isolation of Mycoplasma orale from leukemic bone marrow and blood by direct culture. J. Natl. Cancer Inst. 36155.
Brown, J. E., and Weiss, M. C. (1975). Activation of production of mouse liver enzymes in rat hepatoma-mouse lymphoid cell hybrids. Cell 6481.
Chen, T. R. (1977). In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res. 104255.
Deitrich, R. A. (1971). Genetic aspects of increase in rat liver aldehyde dehydrogenase induced by phenobarbital. Science 173334.
Deschatrette, J., and Weiss, M. C. (1974). Characterization of differentiated and dedifferentiated clones from a rat hepatoma. Biochimie 561603.
Diamondstone, T. I. (1966). Assay of tyrosine transaminase activity by conversion of p-hydroxyphenylpyruvate to p-hydroxybenzaldehyde. Anal. Biochem. 16395.
Fournier, R. E. K., and Frelinger, J. A. (1982). Construction of microcell hybrid clones containing specific mouse chromosomes: Application to autosomes 8 and 17. Mol. Cell. Biol. 2526.
Fujiwara, Y., Oki, T., and Heidelberger, C. (1970). Fluorinated pyrimidines. XXVI. Effects of 5-fluoromethyl-2′-deoxyuridine on the synthesis of deoxyribonucleic acid of mammalian cells in culture. Mol. Pharmacol. 6273.
Furlong, N. B. (1963). A rapid assay for nucleotide kinases using C14- or H3-labeled nucleotides. Anal. Biochem. 5515.
Grosveld, F. G., Lund, T., Murray, E. J., Mellor, A. L., Dahl, H. H. M., and Flavell, R. A. (1982). The construction of cosmid libraries which can be used to transform eukaryotic cells. Nucl. Acids Res. 106715.
Holmes, R. S. (1978). Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, sorbitol dehydrogenase, and xanthine oxidase from mouse tissues. Comp. Biochem. Physiol. 61B339.
Kit, S., Dubbs, D. R., Piekarski, L. J., and Hsu, T. C. (1963). Deletion of thymidine kinase activity from L-cells resistant to bromodeoxyuridine. Exp. Cell. Res. 31297.
Norgard, M. V. (1981). Rapid and simple removal of contaminating RNA from plasmid DNA without the use of RNase. Anal. Biochem. 11334.
Pellicer, A., Robins, D., Wold, B., Sweet, R., Jackson, J., Lowy, I., Roberts, J. M., Sim, G. K., Silverstein, S., and Axel, R. (1980). Altering genotype and phenotype by DNA-mediated gene transfer. Science 2091414.
Pitot, H. C., Peraino, C., Morse, P. A., and Potter, V. A. (1964). Hepatoma in tissue culture compared with adapting liver in vivo. Natl. Cancer Inst. Monogr. 13229.
Segal, H. S., and Matsuzawa, T. (1970). L-Alanine aminotransferase (rat liver). Meth. Enzymol. XVII A153.
Skopek, T. R., Liber, H. L., Penman, B. W., and Thilly, W. G. (1978). Isolation of a human lymphoblastoid line heterozygous at the thymidine kinase locus: Possibility for a rapid human cell mutation assay. Biochem. Biophys. Res. Comm. 84411.
Stetten, G., Davidson, R. L., and Latt, S. A. (1977). 33258 Hoechst enhances the selectivity of the bromodeoxyuridine-light method of isolating conditional lethal mutants. Exp. Cell Res. 108447.
Stetten, G., Latt, S. A., and Davidson, R. L. (1976). 33258 Hoechst enhancement of the photosensitivity of bromodeoxyuridine-substituted cells. Somat. Cell Genet. 2285.
Szybalski, W., Szybalska, E. H., and Ragni, G. (1962). Genetic studies with human cell lines. Natl. Cancer Inst. Monogr. 775.
Vallee, B. L., and Hoch, F. L. (1955). Zinc, a component of yeast alcohol dehydrogenase. Proc. Natl. Acad. Sci. USA 41327.
Weiss, M. C., and Chaplain, M. (1971). Expression of differentiated functions in hepatoma cell hybrids: Reappearance of tyrosine aminotransferase inducibility after the loss of chromosomes. Proc. Natl. Acad. Sci. USA 683026.
Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S., and Axel, R. (1979a). Tranformation of mammalian cells with genes from procaryotes and eucaryotes. Cell 16777.
Wigler, M., Pellicer, A., Silverstein, S., Axel, R., Urlaub, G., and Chasin, L. (1979b). DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc. Natl. Acad. Sci. USA 761373.
Author information
Authors and Affiliations
Additional information
The valuable technical assistance of J. M. Courvall, F. R. Parker, and M. M. Smith is acknowledged. These studies were supported by grant GM26449 from the National Institutes of Health. A.M.K. was supported by a postdoctoral fellowship from the American Cancer Society, California Division. T.G.L. is a Leukemia Society of America postdoctoral fellow. R.E.K.F. is the recipient of an American Cancer Society Faculty Research Award.
Rights and permissions
About this article
Cite this article
Killary, A.M., Lugo, T.G. & Fournier, R.E.K. Isolation of thymidine kinase-deficient rat hepatoma cells by selection with bromodeoxyuridine, Hoechst 33258, and visible light. Biochem Genet 22, 201–213 (1984). https://doi.org/10.1007/BF00484224
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00484224