SPONTANEOUS APOPTOSIS OF THYMOCYTES IS UNCOUPLED WITH PROGRESSION THROUGH THE CELL-CYCLE

Most developing lymphocytes spontaneously die in the thymus during pos itive and negative selection of the T cell repertoire, By evaluating t he expression of the proliferation antigens Ki-67 and PCNA, we demonst rated here that more than 95% of thymocytes are potentially proliferat ing. The coincidence within the same cell population of death and prol iferation is thus apparent in developing thymocytes, Using dual-parame ter cytometric techniques to evaluate in single cells the amount of DN A versus Light-scattering values, we found that spontaneous thymocyte apoptosis occurs with similar frequency in all the cycle phases, where as apoptosis induced by the anti-topoisomerase-II, etoposide (which is the consequence of irreversible DNA damage), takes place with higher frequency in S and G(2) phases (i.e., in those cycle phases in which D NA is subjected to torsional constraints). The capability of thymocyte s to enter apoptosis was also monitored by digesting DNA in situ with DNase I (a nuclease that cleaves DNA mimicking the nuclear damage comm on to most apoptotic suicides), We also show that endonuclease-mediate d DNA digestion occurs to a similar extent in cells with different DNA contents, i.e., in cycle phases in which the superstructural organiza tion of chromatin is markedly different. (C) 1996 Academic Press, Inc.