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Effect of protease inhibitors on early events of apoptosis
1Cancer Research Institute, New York Medical College, Valhalla, New York 10595, USA.
Abstract:
Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization of in situ DNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence of in situ DNA strand breaks. DNA stability was estimated by the measure of its sensitivity in situ to denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4',6-diamidino-2- phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of >=50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNA in situ, and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to >=50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.
Insights
Protease activity is crucial for internucleosomal DNA cleavage and nuclear envelope breakdown during apoptosis. However, it is not required for initial DNA fragmentation or chromatin condensation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Proteolysis is an early event in apoptosis, linked to endonuclease activation and DNA cleavage.
- Early apoptotic events include chromatin condensation, nuclear breakdown, and DNA destabilization.
Purpose of the Study:
- To investigate the role of proteolysis in early apoptotic events beyond DNA cleavage.
- To determine if proteases are involved in chromatin condensation, nuclear breakdown, and DNA structural destabilization.
Main Methods:
- Apoptosis was induced in HL-60 cells and rat thymocytes using various agents.
- DNA degradation assessed via gel electrophoresis and in situ strand breaks.
- DNA stability evaluated by denaturation sensitivity; morphology by microscopy.
Main Results:
- Serine protease inhibitors blocked internucleosomal DNA degradation, nuclear breakdown, and DNA destabilization.
- Inhibitors did not prevent initial chromatin condensation or DNA cleavage into large fragments (>=50 kb).
- Protease activity is necessary for internucleosomal DNA cleavage and potentially nuclear envelope dissolution.
Conclusions:
- Serine proteases are essential for internucleosomal DNA cleavage and nuclear envelope breakdown during apoptosis.
- These proteases likely degrade DNA-stabilizing proteins like histones and nuclear matrix proteins.
- Protease activity is not required for early chromatin condensation or initial large DNA fragment generation.