PROBING CHROMATIN STRUCTURE IN THE EARLY PHASES OF APOPTOSIS

A typical flow cytometric marker of apoptosis is the appearance of a h ypodiploid peak. This phenomenon is related to the chromatin fragmenta tion and loss that occurs during the late stages of the process. We de scribe herein the changes occurring at the chromatin level in purified nuclei preparations obtained from human peripheral blood mononuclear cells in a time-course study, including the simultaneous evaluation of nuclear proteins and DNA stainability, light-scattering properties, a nd spectrophotometric determination of the protein content. An augment ation of fluoroscein isothiocyanate (FITC) stainability was noticed as early as Ih after irradiation. As this phenomenon is not correlated t o changes in actual protein content, one can conclude that modificatio ns of bas-c protein accessibility occur from the early phases of the a poptotic process. Also DNA stainability augmented with time, generatin g the transient appearance of a hyperdiploid peak that preceded the ap pearance of the hypodiploid peak typical of the late stages of the pro cess, and that shared with the latter the same light-scattering proper ties. Chromatin status was further explored by staining apoptotic nucl ei using DNA probes with peculiar molecular weight. Propidium iodide ( PI) and ethidium bromide (EB), but not the much bulkier 7-aminoactinom ycin D (7-AAD), identified the nuclei with a transient increase in DNA stainability confirming that an increased dye accessibility to bindin g sites was responsible for the phenomenon. Remarkably, all dyes ident ified the same proportion of hypodiploid nuclei when an apoptotic nucl eus shed its fragmented chromatin. Control experiments included differ ential interference contrast and fluorescence microscopy that showed t he purity of nuclei preparations and the typical morphological apoptot ic features. Finally, the simultaneous evaluation of DNA by PI and nuc lear proteins by FITC in a time course study allowed a thorough assess ment of changes occurring at the chromatin level in the diverse stages of apoptosis. It is suggested that proteolysis precedes endonucleolys is and probably renders it easier the final endonucleolytic step leadi ng to DNA fragmentation and loss.