Summary
DNA staining methods based on aspecific interactions with dye molecules have been replaced by an immunofluorescent approach to measure DNA replication. Biotin-11-dUTP was incorporated into permeable thymocytes isolated after emetine or cyclosporin A treatment of mice. Active sites of DNA replication were amplified based on biotin-avidin interaction and verified under fluorescent microscope. Cytometry of fluorescent images allow the direct measurement of replicating DNA without aspecific detection of total cellular DNA. Cytometric analysis of replication revealed that emetine acts at the early S phase, while cyclosporin A blocks in vivo DNA synthesis at mid S phase.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Antoni F, Luat NN, Csuka I, Olah I, Sooki-Toth A, Banfalvi G (1987) The immunosuppressive effect of acute dose of emetine on murine thymic cells. Int J Immunopharmacol 9:333–340
Banfalvi G, Wiegant J, Sarkar N, Duijn P van (1989) Immunofluorescent visualization of DNA replication sites within nuclei of Chinese hamster ovary cells. Histochemistry 93:81–86
Banfalvi G, Tanke H, Raap AK, Slats J, Ploeg M van der (1990) Early replication signals in nuclei of Chinese hamster ovary cells. Histochemistry 94:435–440
Basnakyan A, Banfalvi G, Sarkar N (1989) Contribution of DNA polymerase δ to DNA replication in permeable CHO cells synchronized in S phase. Nucleic Acids Res 17:4757–4767
Beschorner WE, Hess AD, Shinn CA, Santos GW (1988) Transfer of cyclosporine associated syngeneic graft-versus-host disease by thymocytes. Resemblance to chronic graft-versus-host disease. Transplantation 45:209–215
Blow JJ, Watson JV (1987) Nuclei act as independent and integrated units of replication in Xenopus cell-free DNA replication system. EMBO J 6:1997–2002
Fakan S, Hancock R (1974) Localization of newly-synthesized DNA in mammalian cell as visualized by high resolution autoradiography. Exp Cell Res 83:95–102
Furue M, Katz SI, Kawakami Y, Kawakami T (1990) Coordinate expression of src family protooncogenes in T cell activation and its modulation by cyclosporine. J Immunol 144:736–739
Gao E-K, Lo D, Cheney R, Kanagawa O, Sprent J (1988) Abnormal differentiation of thymocytes in mice treated with cyclosporin A. Nature 336:176–179
Glazier A, Tutschka PJ, Farmer ER, Santos GW (1983) Graftversus-host disease in cyclosporin A-treated rats after syngeneic and autologous bone marrow reconstitution. J Exp Med 158:1–8
Grollman AP (1966) Structural basis for inhibition of protein synthesis by emetine and cycloheximide based on an analogy between ipecac alkaloids and glutarimide antibiotics. Proc Natl Acad Sci USA 56:1867–1874
Grollman AP (1968) Inhibitors of protein biosynthesis. V. Effects of emetine on protein and nucleic acid biosynthesis in HeLa cells. J Biol Chem 243:4089–4094
Handeli S, Klar A, Menth M, Cedar H (1989) Mapping replication units in animal cells. Cell 57:909–920
Hay ED, Revel JP (1966) The fine structure of the DNP component of the nucleus. An electron microscopic study utilizing an autoradiography to localize DNA synthesis. J Cell Biol 16:29–51
Hiriyanna KT, Varkey J, Beer M, Benbow RM (1988) Electron microscopic visualization of sites of nascent DNA synthesis by streptavidin-gold binding to biotinylated nucleotides incorporated in vivo. J Cell Biol 107:33–34
Huberman JA, Tsai A, Reich RA (1973) DNA replication sites within nuclei of mammalian cells. Nature 241:32–36
Hunting DJ, Dresler SL, Murcia G (1985) Incorporation of biotinlabeled deoxyuridine triphosphate into DNA during excision repair and electron microscopic visualization of repair patches. Biochemistry 24:5729–5734
Ladanyi A, Timar J, Paku S, Molnar G, Lapis K (1990) Selection and characterization of human melanoma lines with different liver colonizing capacity. Int J Cancer 46:456–461
Langer PR, Waldrop AA, Ward DC (1981) Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci USA 78:6633–6637
Miller MR, Castellot JJ Jr, Pardee AB (1978) A permeable animal cell preparation for studying macromolecular synthesis. DNA synthesis and the role of deoxyribonucleotides in S phase initiation. Biochemistry 17:1073–1080
Nakamura H, Morita T, Sato C (1986) Structural organizations of replicon domains during DNA synthetic phase in mammalian nucleus. Exp Cell Res 165:291–297
Nakayasu H, Bereznay RJ (1989) Mapping replicational sites in the eukaryotic nucleus. J Cell Biol 108:1–11
Olah I, Antoni F, Banfalvi G, Csuka I, Luat NN, Toro I (1988) Structural changes in the mouse thymus and lymph node after emetine treatment. J Dev Comp Immunol 12:385–395
Sakaguchi S, Sakaguchi N (1988) Thymus and autoimmunity. Transplantation of the thymus from cyclosporin A treated mice causes specific autoimmune disease in athymic nude mice. J Exp Med 167:1479–1485
Smith HC, Puvion E, Buckholtz LA, Bereznay R (1984) Spatial distribution of DNA loop attachment and replicational sites in the nuclear matrix. J Cell Biol 99:1794–1802
Sooki-Toth A, Banfalvi G, Szollosi J, Kirsten E, Staub M, Antoni F, Kun E (1989) Cellular regulation of ADP-ribosylation of proteins. III. Selective augmentation of in vitro ADP-ribosylation in histon H3 in murine thymic cells after in vivo emetine treatment. Exp Cell Res 184:44–52
Sorokin R, Kimura H, Schroder K, Wilson DH, Wilson DB (1986) Cyclosporine-induced autoimmunity. Conditions for expressing disease, requirement for intact thynus and potency estimates of autoimmune lymphocytes in drug-treated rats. J Exp Med 164:1615–1625
Sprent J, Webb SR (1987) Function and specificity of T cell subsets in the mouse. Adv Immunol 41:39–133
Wagenaar EB, Mazia D (1978) In: Dirksen ER, Prescott DM, Fox CF (eds) ICN-UCLA Symposium on Molecular and Cell Biology, vol XII. Cell reproduction. Academic Press, New York, pp 539–545
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schweighoffer, T., Schweighoffer, E., Apati, A. et al. Cytometric analysis of DNA replication inhibited by emetime and cyclosporin A. Histochemistry 96, 93–97 (1991). https://doi.org/10.1007/BF00266767
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00266767