Wilhelm bernhard workshop on the cell nucleus

MODELING APOPTOTIC CHROMATIN CONDENSATION IN NUCLEI ISOLATED FROM NORMAL CELLS

Widlak P.

Department of Experimental and Clinical Radiobiology, Center of Oncology, Gliwice, Poland
Hallmarks of the terminal stages of apoptosis are genomic DNA fragmentation and chromatin condensation. Nuclease primarily responsible for apoptotic oligonucleosomal DNA fragmentation is DFF40 (DNA Fragmentation Factor 40 kD), also termed CAD (Caspase-activated DNase). DFF40/CAD triggers one of the pathways of apoptotic chromatin condensation. Another pathway of apoptotic chromatin condensation is triggered by mitochondrial AIF (Apoptosis-inducing Factor), which induce large-scale DNA fragmentation possibly activating other nucleases. Proteolysis of nuclear proteins, like lamin A cleavage by caspase-6, is required for complete breakdown of nuclei in cells undergoing apoptosis. Here, the mechanism of chromatin condensation triggered by DFF40/CAD has been studied. We have found that DNA fragmentation per se of isolated nuclei from non-apoptotic cells induces chromatin condensation under physiological ionic strengths that closely resembles the morphology seen in apoptotic cells. This phenomenon is not specific for an apoptotic nuclease DFF40/CAD. Although there is no energy requirement for this process, such condensation can be inhibited without affecting DNA fragmentation by two different pretreatment of nuclei: (i) exposing to reagents that bind to the DNA minor groove, which disrupt native nucleosomal DNA wrapping, and (ii) stabilizing internal nuclear components. Interestingly, inhibition of nuclear F-actin depolymerization or promoting its formation also reduces chromatin condensation. Therefore, the ability of chromatin fragments with native nucleosomal DNA wrapping to form large clumps of condensed chromatin during apoptosis requires the apparent disassembly of internal nuclear structures that may normally constrain chromosome subdomains in non-apoptotic cells. Consistently, chromatin condensation triggered by DNA fragmentation was greatly enhanced when isolated nuclei were first incubated with caspase-3 and caspase-6. In conclusion, morphological changes that closely resembles apoptotic chromatin condensation in vivo can be induced by well defined factors in purified normal cell nuclei. Such factors include nucleases that fragmentize chromatin and proteases that cleave nuclear proteins and enhance intranuclear mobility of chromatin fragments.