Abstract
One way to study the mechanisms of cytotoxicity of toxin conjugates1 (TC) is to analyze cell mutants that are resistant to the conjugates and to compare the properties of the mutant cells with those of the parental cells. This approach has an important advantage over comparative analysis of randomly chosen sensitive and resistant cell lines. Because of their different origin, nonrelated cell lines may have a number of phenotypic differences, and it may be difficult to determine which is linked to the mechanism of resistance. Therefore, cells of different origin are not useful for comparative studies. Because of low frequencies of mutations in eukaryotic cells, in the range of 10-4 to 10-7 or less [1, 2], most of the mutant cell lines selected from a wild type cell line will be the result of a single mutation in the parental DNA. This genetic change would be translated into a single phenotypic change, although pleiotropic mutations are also possible. In other words, mutants may be similar to the parental cell line except for a difference in a single gene product that is involved in the interaction of the toxin conjugate with cells. Thus, comparative analysis of TC-resistant mutants with the parental cells may identify single steps in the internalization and toxic action of the conjugates. Similar approaches have been widely used for analysis of the interaction of various drugs with cells and for studies of the phenomena of drug resistance [1, 3]. This chapter describes methodology for isolation of TC-resistant mutants and results obtained from studies of these mutants.
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References
Lewin, B. (1080) Gene Expression, Vol. 2. John Wiley and Sons, New York, Chapter 6.
Morrow, J. (1983) Eukaryotic Cell Genetics. Academic Press, New York, Chapter 1.
Wright, J.A., Lewis, W.H., and Parfett, C.L. (1980) Somatic cell genetics. A review of drug resistance, lectin resistance and gene transfer in mammalian cells in culture. Can. J. Gen. Cytol., 22, 443–496.
Siminovitch, L. (1976) On the nature of hereditable variation in cultured somatic cells. Cell, 7, 1–11.
Chu, E.H., Brimer, P., Jacobson, K.B., and Merriam, E.V. (1969) Mammalian cell genetics. I. Selection and characterization of mutations auxotrophic for L-glutamine or resistant to 8-azaguanine in Chinese hamster cells in vitro. Genetics, 62, 359–377.
Caboche, M. (1974) Comparision of the frequencies of spontaneous and chemically induced 5-bromodeoxyuridine-resistance mutations in wild type and revertant BHK 21.13 cells. Genetics, 77, 309–322.
Baker, R.M., Brunette, D.M., Mankovitz, R., Thompson, L.H., Whitmore, G.F., Siminovitch, L., and Till, J.E. (1974) Ouabain-resistant mutants of mouse and hamster cells in culture. Cell, 1, 9–21.
DeLuca, J.G., Kaden, D.A., Krolewski, J.J., Skopek, T.R., and Thilly, W.G. (1977) Comparative mutagenicity of ICR-191 to S. Typhimurium and diploid human lymphoblasts. Mutat. Res., 46, 11–17.
Slapikoff, S.A., Andon, B.M., and Thilly, W.G. (1980) Comparison of toxicity and mutagenicity of methylnitrosourea, methylnitrosoguanidine and ICR-191 among human lymphoblast lines. Mutat. Res., 70, 365–371.
Calos, M.P., and Miller, J.H. (1981) Genetic and sequence analysis of frameshift mutations induced by ICR-191. J. Mol. Biol., 153, 39–66.
Gupta, R.S., and Siminovitch, L. (1980) Genetic markers for quantitative mutagenesis studies in Chinese hamster ovary cells: Characteristics of some recently developed selective systems. Mutat. Res., 69, 113–126.
Goldmacher, V.S., Anderson, J., Schulz, M.-L., Blättler, W.A., and Lambert, J.M. (1987) Somatic cell mutants resistant to ricin, diphtheria toxin, and to immunotoxins. J. Biol. Chem., 262, 3205–3209.
Olsnes, S., and Pihl, A. (1982) Toxic lectins and related proteins. In: Molecular Action of Toxins and Viruses. P. Cohen and S.V. Heyningen, eds. Elsevier, Amsterdam, pp 51–105.
Goldmacher, V.S., Lambert, J.M., Young, A.Y., Anderson, J., Tinnel, N.L., Kornacki, M., Ritz, J., and Blättler, W.A. (1986) Expression of the common acute lymphoblastic leukemic antigen (CALLA) on the surface of individual cells of human lymphoblastoid lines. J. lmmunol., 136, 320–325.
Robbins, A.R., Myerowitch, R., Youle, R.J., Murray, G.J., and Neville, D.M. (1981) The mannose 6-phosphate receptor of Chinese hamster ovary cells. Isolation of mutants with altered receptors. J. Biol. Chem., 256, 10618–10622.
Goldmacher, V.S., Cuzick, R.A., and Thilly, W.G. (1986) Isolation and partial characterization of human cell mutants differing in sensitivity to killing and mutation by methylnitrosourea and N-methyl-N’-nitro-N-nitrosoguanidine. J. Biol. Chem., 261, 12462–12471.
Basilico, C. (1977) Temperature-sensitive mutations in animal cells. Adv. Cancer Res., 24, 223–266.
Thilly, W.G., DeLuca, J.G., Furth, E.E., Hoppe, H., Kaden, D.A., Krolewski, J.J., Liber, H.L., Skopek, T.R., Slapikoff, S.A., Tizard, R.J., and Penman, B.W. (1980) Gene-locus mutation assays in diploid human lymphoblast lines. In: Chemical Mutagens. A.E. Hollaender and F.J. DeSerres, eds. Plenum, New York, pp 331–364.
Thilly, W.G., DeLuca, J.G., Hoppe, H., and Penman, B.W., (1978) Phenotypic lag and mutation to 6-thioguanine resistance in diploid human lymphoblasts. Mutat. Res., 50, 137–144.
O’Neil, J.P., and Hsie, A.W. (1979) Phenotypic expression time of mutagen-induced 6-thioguanine resistance in Chinese hamster ovary cells (CHO/HGPRT system). Mutat. Res., 59, 109–118.
Sato, K., and Hieda, N. (1980) Mutation induction in a mouse lymphoma cell mutant sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation. Mutat. Res., 71, 233–241.
Rath, H., Tlsty, T., and Schimke, R.T. (1984) Rapid emergence of methotrexate resistance in cultured mouse cells. Cancer Res., 44, 3303–3306.
Goldmacher, V.S., Anderson, J., Blättler, W.A., Lambert, J.M., and Senter, P.D. (1985) Antibody-complement-mediated cytotoxicity is enhanced by ribosome-inactivating proteins. J. Immunol., 135, 3648–3651.
Lambert, J.M., Yau-Young, A., Senter, P.D., Blättler, W.A., and Goldmacher, V.S. (1985) Purified immunotoxins that are reactive with human lymphoid cells. Monoclonal antibodies conjugated to the ribosome-inactivating proteins gelonin and the pokeweed antiviral proteins. J. Biol. Chem., 260, 12035–12041.
Goldmacher, V.S., Tinnel, N.L., and Nelson, B.C. (1986) Evidence that pinocytosis in lymphoid cells has a low capacity. J. Cell Biol., 102, 1311–1319.
Ono, M., Kuwano, M., Watanabe, K.-I., and Funatsu, G. (1982) Chinese hamster cell variants resistant to the A-chain of ricin carry altered ribosome function. Mol. Cell. Biol., 2, 599–606.
Leppla, S.H., Dorland, R.B., and Middlebrook, L. (1980) Inhibition of diphtheria toxin degradation and cytotoxic action by chloroquine. J. Biol. Chem., 255, 2247–2250.
Merion, M., Schlesinger, P., Brooks, R.M., Moehring, J.M., Moehring, T.J., and Sly, W.S. (1983) Defective acidification of endosomes in Chinese hamster ovary cell mutants ‘cross-resistant’ to toxins and viruses. Proc. Natl. Acad. Sci. USA, 80, 5315–5319.
Sandvig, K., and Olsnes, S. (1980) Diphtheria toxin entry into cells is facilitated by low pH. J. Cell Biol., 87, 828–832.
Olsnes, S., Sandvig, K., Eiklid, K., and Pihl, A. (1978) Properties and action mechanism of the toxic lectin modeccin: Interaction with cell lines resistant to modeccin, abrin and ricin. J. Supramol. Struct., 9, 15–25.
Moehring, J.M., and Moehring, T.H. (1983) Strains of CHO-K1 cells resistant to Pseudomonas exotoxin A and cross-resistant to diphtheria toxin and viruses. Infect. Immun., 41, 998–1009.
Raso, V., and Basala, M. (1984) A highly cytotoxic human transferrin-ricin A chain conjugate used to select receptor modified cells. J. Biol. Chem., 259, 1143–1149.
Behzadian, M.A., and Shimizu, N. (1985) Variant of A431 cells isolated by ricin A-conjugated monoclonal antibody directed to EGF receptor: Phosphorylation of EGF receptor and phosphatidylinositol. Somat. Cell Mol. Genet., 11, 579–591.
Lesley, J., and Schulte, R. (1986) Selection and characterization of transferrin receptor mutants using receptor-specific antibodies. Immunogenet., 24, 163–170.
Miskimins, W.K., Ferris, W.R., and Shimizu, N. (1981) Genetics of receptors for bioactive polypeptides: A variant of Swiss/3T3 fibroblasts resistant to a cytotoxic insulin accumulates lysosome-like vesicles. J. Supramol. Struct. Cell. Biochem., 16, 105–113.
Miskimins, W.K., and Shimizu, N. (1981) Genetics of cell surface receptors for bioactive polypeptides: Variants of Swiss/3T3 fibroblasts resistant to a cytotoxic chimeric insulin. Proc. Natl. Acad. Sci. USA, 78, 445–449.
Thorpe, P.E., Blakey, D.C., Brown, A.N., Knowles, P.P., Knyba, R.E., Wallace, P.M., Watson, G.J., and Wawrzynczak, E.J. (1987) Comparison of two anti-Thy 1.1-abrin A-chain immunotoxins prepared with different crosslinking agents: Antitumor effects, in vivo fate and tumor cell mutants. J. Natl. Canc. Inst, submitted.
Lowe, J.A., Ling, N.R., Forrester, J.A., Cumber, A.J., and Ross, W.C. (1985) Variants of lymphoid lines produced with ricin A-chain monoclonal antibody conjugates. J. Immun. Meth., 76, 93–104.
Shimizu, N. (1984) Use of hormone-toxin conjugates and serum-free media for the isolation and study of cell variants in hormone responses. In: Cell Culture Methods for Molecular and Cell Biology, Vol. 3. D.W. Barnes, D.A. Sirbashu, and G.H. Sato, eds. Alan Liss, New York, pp 233–248.
Krag, S.A., and Robbins, A.R. (1982) A Chinese hamster ovary cell mutant deficient in glycosylation of lipid-linked oligosaccharide synthesizes lysosomal enzymes of altered structure and function. J. Biol. Chem., 257, 8424–8431.
Robbins, A.R., and Myerowitz, R. (1981) The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors. J. Biol. Chem., 256, 10623–10627.
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Goldmacher, V.S. (1988). Isolation and analysis of somatic cell mutants resistant to toxin conjugates. In: Frankel, A.E. (eds) Immunotoxins. Cancer Treatment and Research, vol 37. Springer, Boston, MA. https://doi.org/10.1007/978-1-4613-1083-9_23
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DOI: https://doi.org/10.1007/978-1-4613-1083-9_23
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