DETECTION OF APOPTOSIS-ASSOCIATED DNA STRAND BREAKS IN FINE-NEEDLE ASPIRATION BIOPSIES BY IN-SITU END LABELING OF FRAGMENTED DNA

The predominant mode of either spontaneous or drug-induced death of ce lls in tumors is apoptosis. A Bow cytometric method was developed in o ur laboratory to identify apoptotic cells, based on labeling DNA stran d breaks, which appear as a result of extensive DNA cleavage by the ap optosis-associated endonuclease, with biotinylated dUTP in the reactio n catalyzed by exogenous terminal deoxynucleotidyl transferase. The ai m of this study was to reveal whether this methodology can be applied to human solid tumors sampled by fine-needle biopsy. Twenty-two tumors , consisting of 11 breast carcinomas; three metastatic anaplastic carc inomas; three adenocarcinomas of colon, endometrium, and lung; two met astatic lymph node squamous cell carcinomas of the larynx; and three m alignant lymphomas were examined. It was possible to identify cells wi th DNA strand breaks in all these tumors. Extremely high variability i n the proportion of cells with DNA strand breaks was observed between the individual tumors. In diploid tumors (n = 12) the percentage of ce lls with DNA strand breaks varied from 1% to 43%, and the mean value w as 19%. In aneuploid tumors this percentage varied from 15% to 51% and the mean value was 37%. In the latter tumors the presence of cells wi th DNA strand breaks was limited to the DNA aneuploid cell population; very few diploid, presumably tumor infiltrating or stromal cells, sho wed the presence of DNA strand breaks. No correlation was observed bet ween the percent of cells in S phase and those with DNA strand breaks. The data indicate that apoptosis is more frequent in populations of t umor cells than among normal cells of the same organs. The higher perc ent of cells with DNA strand breaks compared to the percent of cells w ith morphology typical of apoptosis suggests that the early phase of a poptosis, characterized by the appearance of DNA strand breaks, which precedes morphological changes (chromatin condensation, nuclear fragme ntation), may be very long in individual cells of the tumors studied. The present method may by applicable to estimate DNA strand breaks in solid tumors, either during spontaneous apoptosis or during apoptosis induced by chemo or radiotherapy, to evaluate tumor cell response earl y during treatment. (C) 1994 Wiley-Liss, Inc.