In Vitro Incorporation of Tritiated Thymidine in Normal and Neoplastic Tissues

Abstract

The present work is part of a broad study on quantitative aspects of cell proliferation in normal and neoplastic human tissues. Normal and tumor growth in vivo has been estimated quantitatively in experimental animals by a variety of methods; the most valuable for analyzing cell population kinetics has been the incorporation of tritiated thymidine (TdR-3H) (1, 4). Thymidine is a specific DNA precursor, and is incorporated into cells synthesizing DNA and preparing for division. The resulting nuclear labeling permits identification of sites of cell proliferation and provides information on the proliferative capacity of the cell population. A method has now been developed whereby surgical and biopsy specimens may be labeled with TdR-3H in vitro but under conditions in which the incorporation of the label occurs only in those cells which were synthesizing DNA in the patient, thereby providing a pattern of labeling similar to that obtained by in vivo methods. Materials and Methods Small tissue samples (1 × 3 mm) obtained in the operating room are immediately placed in ice-cold M-199 (Earle base) medium (20.0 ml) with 20 per cent fetal calf serum (4.0 ml) and transported to the laboratory, and TdR-3H (0.24 ml, 1.0 µCi/ml, specific activity 14.5 Ci/mmole) is added. The container is placed in a specially designed hyperbaric oxygen chamber, and the tissue is then incubated in the agitated medium at 37.5°C, pH ∼7.5, and at 2,280 mm Hg pO2 for sixty minutes. Normally, anoxic cells are present within the specimen, and the increased oxygen pressure permits utilization of the available thymidine (7). Since TdR-3H is available to the proliferating cell population for a short interval relative to the duration of synthesis, labeling occurs only in those cells in DNA synthesis in vivo. The tissue is then fixed in ethanol-formalin-acetic acid mixture for twenty-four hours, histological sections (4 µ thick) from wax-embedded tissue are prepared for high resolution autoradiography (liquid-emulsion-dipping technic using Ilford K-5 nuclear emulsion). Autoradiographs are exposed at 4°C for three weeks, developed (Kodak D-19 developer), fixed (Kodak acid fixer), and stained with hematoxylin and eosin. Figures 1 and 2 illustrate the typical autoradiographic appearance of these tissues. Labeled cells are readily identified, and in slides with background counts of less than 1 grain per nucleus, cell nuclei with more than 5 grains are scored as labeled. Percentage-labeling indexes are obtained in counts of 1,000 cells or more in a proliferating population of the same type. Results and Discussion In most mammalian tissues, cells proceed through the generative cycle within a few hours of completing DNA synthesis (5).