Double Labeling Autoradiography..

Examples of the multiple applicability of the double labeling method with 3H- and 14C-TdR are demonstrated. Double labeling with 3H- and 14C-TdR makes it possible to determine the cycle and its phases with high precision by modifying the usual percent labeled mitoses method with a single injection of 3H-TdR. This results in a considerable improvement of the resolution of the percent labeled mitoses curve. In addition, data is provided on the variances of the transit times of the cells through the cycle phases. For example, in the case of the jejunal crypt cells of the mouse, the transit times through successive cycle phases are uncorrelated. In the case of glial cells the double labeling method provides cell kinetic parameters despite the paucity of proliferating glial cells. In the adult untreated animal, glial cell mitoses are so rare that the percent labeled mitoses method can not be utilized. However, the S-phase duration can be measured by double labeling and the cycle time can be determined by the so-called method of labeled S phases. With the latter method the passage through the next S phase of the 3H-TdR-labeled S phase cells can be registered by injecting 14C-TdR at different time intervals following 3H-TdR application. In this way an S-phase duration of about 10 hr and a cycle time of about 20 hr was found for glial cells in the adult untreated mouse. An exchange of glial cells between the growth fraction and the nongrowth fraction has also been shown by double labeling. A quite different application of the double labeling method with 3H- and 14C-TdR is the in vivo study of the cell cycle phase-specific effect of drugs used in chemotherapy of tumors. Up to now studies of the effect of cytotoxic drugs on cells in different cycle phases were confined to in vitro experiments, since such studies need synchronized cells. However, the double labeling method, which leads to well-defined subpopulations of differently labeled cells in different phases of the cycle, allows such studies to be carried out under in vivo conditions. The effect of vincristine on these cells has been studied. Vincristine affects cells in S and G2 in such a manner that they are arrested during the next metaphase and subsequently become necrotic. It has no effect on G1 cells.