Abstract
Although their precise role is poorly understood, polyamines appear to be important in cell growth. This is probably through their ready interaction with cell membrane components, nucleic acids, and proteins. One consequence of these interactions is that polyamines are able to modulate cell proliferation (1). Moreover, regulation of polyamine levels within cells appears to be closely related to cell proliferation (2), so assessment of polyamine levels and of the activity of ornithine decarboxylase activity, a key enzyme involved in polyamine synthesis, has been used in tissue extracts as an index of cell growth (3). A corollary of this association between proliferation and polyamine metabolism is that basic techniques for assessing cell growth and proliferation of cells are valuable tools in the field of polyamine research (4). In this chapter two methods of growth assessment that measure incorporation of radiolabeled precursors into cellular macromolecules will be described. These techniques employ the principle that cells will incorporate radiolabeled precursors of DNA and protein into newly synthesized macromolecules and the amount of incorporation will reflect both the synthetic activity of each cell in culture and also the number of cells. In many situations the amount of radionuclide incorporated can be used as an index of cell proliferation, although this is not always the case, as discussed in Subheading 3.6. Two of the most frequently used radiolabeled precursors are thymidine, which becomes incorporated into DNA and is generally labeled with tritium (3H), and leucine, which is incorporated into most eukaryotic protein molecules. Leucine can be labeled with either tritium or carbon-14 (14C) and, although in this discussion [3H]-labeled leucine will be considered, the same principles apply to [14C]-leucine, apart from requiring different programming of the scintillation counter.
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References
Heby, O. (1981) Roles of polyamines in the control of cell proliferation. Differentiation 19, 1–20.
Morrison, R. F. and Seidel, E. R. (1995) Vascular endothelial cell proliferation: regulation of cellular polyamines. Cardiovasc. Res. 29, 841–847.
Abrahamson, M. S. and Morris, D. R. (1990) Cell type specific mechanisms of regulating expression of the ornithine decarboxylase gene after growth stimulation. Mol. Cell. Biol. 10, 5525–5528.
Pegg, A. E. and McCann, P. P. (1982) Polyamine metabolism and function. Am. J. Physiol. 243 C212–C221.
Everhart, L. P., Hausohka, P. V., and Prescott, D. M. (1973) Measurement of growth rates and incorporation of radioactive precursors into macromolecules of cultured cells. Methods Cell Biol. 7, 329–347.
Ball, C. R., Van den Berg, H. W., and Poynter, R. W. (1973) The measurement of radioactive precursor incorporation into small monolayer cultures. Methods Cell Biol. 7, 349–360.
Uitto, J., Murray, L. W., Blumberg, B., and Shamban, A. (1986) Biochemistry of collagen in diseases (review). Ann. Intern. Med. 105, 740–756.
Dover, R. (1991) Basic methods for assessing cellular proliferation, in Assessment of Cell Proliferation in Clinical Practice (Hall, P. A., Levrson, D. A., and Wright, N. A., eds.), Springer-Verlag, London, pp. 63–81.
Beck, H. P. (1981) Radiotoxicity of incorporated [3H]thymidine as studied by autoradiography and flow cytometry. Cell Tissue Kinet. 14, 163–177.
Trosko, J. E. and Yager, J. D. (1974) A sensitive method to measure physical and chemical carcinogen induced unscheduled DNA synthesis in rapidly dividing eukaryotic cells. Exp. Cell Res. 88, 47–55.
Rew, D. A. and Wilson, G. D. (1991) Advances in cell kinetics. Br. Med. J. 303, 532–537.
Gratzner, H. G. (1982) Monoclonal antibody to 5-bromo and 5-iodo-deoxy-uridine, a new reagent for the detection of DNA replication. Science (Wash.) 218, 474, 475.
Gerecke, D. and Gross, R. (1975) Reutilisation of DNA catabolites in granulocytopoests. Blut. 31, 41–48.
Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olsen, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85.
Mossman, T. (1983) Rapid colorimetric assay for cellular growth and survival. Application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 55–63.
Maurer, H. R. (1981) Potential pitfalls of 3H-thymidine techniques to measure cell proliferation. Cell Tissue Kinetics 14, 111–120.
Schneider, W. C. and Greco, A. E. (1971) Incorporation of pyrimidine deoxyribo-nucleotides into liver lipids and other components. Biochim. Biophys. Acta 228, 610–626.
Ooi, S. O., Sim, K. Y., Chung, M. C., and Kon, O. L. (1993) Selective antiproliferative effects of thymidine. Experientia 49, 576–581.
Boulton, R. A. and Hodgson, H. F. (1995) Assessing cell proliferation a methodological review. Clin. Sci. 88, 119–130.
Simon, J. S., Baum, J. S., Moore, S. A., and Kasson, B. G. (1995) Arginine vaso-pressin stimulates protein synthesis but not proliferation of cultured vascular endothelial cells. J. Cardiovasc. Pharmacol. 25, 368–375.
Gospodarowicz, D. and Moran, J. S. (1975) Determination of 3H incorporation into DNA in vitro. Methods Cell Biol. 3, 320–329.
Stubblefield, E. (1968) Synchronisation methods for mammalian cell cultures. Methods Cell Physiol. 3, 25–44.
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Denton, C.P. (1998). Leucine Incorporation and Thymidine Incorporation. In: Morgan, D.M.L. (eds) Polyamine Protocols. Methods in Molecular Biology™, vol 79. Humana Press. https://doi.org/10.1385/0-89603-448-8:169
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DOI: https://doi.org/10.1385/0-89603-448-8:169
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