Valiability of Radioactive Thymidine Labeling in the Rat Brain Tumor Model

Valiabilities in labeling indices (LI) by 3H-thymidine (TdR) were analysed using the 9L rat brain tumor model with the following procedures. 1. Continuous labeling was carried out delivering 3H-TdR by means of a minipump implanted in the rat flank. Tumors were excised 2, 6, 12, 18, 24, 30 and 34 hours after the implantation of the pump. Differences in LI were computed in terms of a) geometry of the tumor (from the periphery to the center of each tumor), b) time interval of infusion and c) population of the tumor cells in each unit area (4.84×104 u2). 2. Other groups of rats with tumors were infused firstly with 3H-TdR and secondly with 14C-TdR 0, 30, 60 and 180 minutes later, and were killed 5 minutes after the 14C-TdR injection. Several normal brain tissues and 4-5 pieces of tumor (ca. 20 mg) were excised and lyophilized and activities of 3H and 14C were measured with a liquid scintillation counter. 3. DNA from tumor and normal brain, double labeled as above, was extracted by the Schmidt-Thannhauser method. In this group, the rats were sacrificed 30 minutes after administration of 14C-TdR. LI's remained less than 20% up to 12 hours after implant of the pump, but the LI's increased to 43% in 18-24 hours and decreased thereafter. There were no differences in LI obtained from the peripheries and central part of each tumor. Also, no correlation was observed between the LI and geometry or population of tumor cells per unit area (400-1, 000 cells/unit area). Lyophilized normal tissue and tumor pieces showed variable amounts of 3H and 14C activities when they were normalized to counts per gram. However, the ratio 3H activity to 14C activity was constant throughout the specimens obtained from each rat. DNA extracted from normal brain showed almost no 3H and 14C uptake, whereas DNA extracted from tumors showed variable uptake of each isotope when compared in terms of counts per gram. However, 3H/14C values were fairly constant in pieces obtained from each tumor. These results were interpreted to indicate that variabilities of LI in tumors were assumed to represent variabilities in the actual proliferating population in each tumor and were not due to abnormalities in availability of exogenous TdR caused by abnormal blood flow or permiability. Thus, average LI's of such a tumor should be computed by screening a considerable number of tumor cells.