We are investigating the molecular mechanisms of apoptosis (programmed cell death), a critically important process of cell suicide. In a cell-free system, the addition of cytosol from condemned cells (e.g. Jurkat human T-leukemia cells treated with anti-Fas to initiate apoptosis), to nuclei from healthy cells results in the classical nuclear changes of apoptosis: internucleosomal DNA fragmentation, chromatin condensation, and nuclear fragmentation. This proapoptotic cytosolic activity, provisionally termed """"""""apoptase,"""""""" is a protein with an apparent MW of 100 kDa; a larger protein complex is also detected. Apoptase activity is chromatographically distinguishable from endodeoxyribonuclease activity, which is often elevated in apoptotic cells. Apoptase activities (nuclear DNA fragmentation and chromatin condensation) are sensitive to thiol and chloromethylketone serine-protease inhibitors, but these inhibitors may react with a cysteine in the final endonuclease. Apoptase is insensitive to inhibitors of the caspase family of proteases which, when activated, irreversibly commit the cell to apoptosis. Thus during apoptosis apoptase may be proteolytically activated by the active caspases and may elicit DNA fragmentation and other changes in nuclei via endogenous endonucleases. We have also discovered in the cytosol of healthy cells a novel activity that inhibits the apoptase activity of condemned-cell cytosol in the cell-free system. This activity, provisionally termed """"""""antiapoptase,"""""""" is a protein which chromatographs as two peaks having apparent MW values of 50 kDa and over 200 kDa. Antiapoptase levels are particularly high in tumor lines such as Jurkat and HeLa cells but are generally lower or absent in normal mouse tissues. Both apoptase and antiapoptase proteins are being purified further and characterized with respect to function. We hypothesize that they play important roles in apoptosis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL000155-05
Application #
6162629
Study Section
Special Emphasis Panel (LBG)
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
1997
Total Cost
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
United States
Zip Code