Leucine Incorporation and Thymidine Incorporation

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 (³ H), and leucine, which is incorporated into most eukaryotic protein molecules. Leucine can be labeled with either tritium or carbon-14 (¹⁴ C) and, although in this discussion [³ H]-labeled leucine will be considered, the same principles apply to [¹⁴ C]-leucine, apart from requiring different programming of the scintillation counter.