Detection of heterogeneity of apoptotic fragments of poly (ADP-ribose) polymerase in MDA-MB-468 breast cancer cells: two-dimensional gel analysis.
Caspace-mediated proteolysis of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) (EC 2.4, 2.30) is a biochemical marker of cell death in response to various apoptotic stimuli. Anti-PARP antibodies identifying the 89 kDa polypeptide from the C-terminus as well as the 113 kDa native enzyme are often used to demonstrate evidence of apoptosis-associated, interleukin converting enzyme (ICE)-mediated limited cleavage. Recent evidence points to redundancy of caspases, heterogeneity of their cleavage sites, and a possibility of generating distinct context-specific, and cell-specific PARP fragments. In the present study, we employed antibodies directed to multiple sites in PARP and probed two-dimensionally resolved proteins of the estrogen receptor negative MDA-MB-468 breast tumor cells, induced to undergo apoptosis by ionizing radiation (IR). Our results revealed that the 24 kDa apoptotic fragment of PARP, from the N-terminus, consists of at least three isoforms, located at a p/more basic than the full length enzyme. We also report a hitherto unrecognized feature of an anti-PARP antiserum, VIC-5, detecting both the 89 kDa and the 24 kDa caspase-generated fragments of PARP. Thus, application of two-dimensional electrophoresis combined with antisera directed to multiple sites would be valuable in distinguishing PARP cleavage site- and inhibitor specificities of proteases during apoptosis.
Prasad
S C
SC
Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20007, USA. prasads@gunet.georgetown.edu
Soldatenkov
V
V
Notario
V
V
Smulson
M
M
Dritschilo
A
A
D017209:Apoptosis; D001943:Breast Neoplasms / Q000201:enzymology*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005260:Female; D006801:Humans; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis*; D014407:Tumor Cells, Cultured
D011065:Poly(ADP-ribose) Polymerases