Detection of apoptosis‐associated DNA strand breaks in fine‐needle aspiration biopsies by in situ end labeling of fragmented DNA

Open Access
Publisher Website
Google Scholar
Abstract
The predominant mode of either spontaneous or drug‐induced death of cells in tumors is apoptosis. A flow cytometric method was developed in our laboratory to identify apoptotic cells, based on labeling DNA strand breaks, which appear as a result of extensive DNA cleavage by the apoptosis‐associated endonuclease, with biotinylated dUTP in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The aim of this study was to reveal whether this methodology can be applied to human solid tumors sampled by fine‐needle biopsy. Twentytwo tumors, consisting of 11 breast carcinomas; three metastatic anaplastic carcinomas; three adenocarcinomas of colon, endometrium, and lung; two metastatic lymphnode squamous cell carcinomas of the larynx; and three malignant lymphomas were examined. It was possible to identify cells with DNA strand breaks in all these tumors Extremely high variability in the proportion of cells with DNA strand breaks Was observed between the individual tumors. In diploid tumors (n=12) the percentage of cells with DNA strand breaks varied from 1% to 43%, and the mean value, was 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 with DNA strand breaks was limited to the DNA aneuploid cell population; very few diploid, presumably tumor infiltrating or stromal cells, showed the presence of DNA strand breaks. No correlation was observed between the percent of cells in S phase and those with DNA strand breaks. The data indicate that apoptosis is more frequent in populations of tumor cells than among normal cells of the same organs. The higher percent of cells with DNA strand breaks compared to the percent of cells with morphology typical of apoptosis suggests that the early phase of apoptosis, characterized by the appearance of DNA strand breaks, which precedes morphological changes (chromatin condensation, nuclear fragmentation), 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 early during treatment.