Characterization of antibodies specific for the caspase cleavage site on poly(ADP-ribose) polymerase: specific detection of apoptotic fragments and mapping of the necrotic fragments of poly(ADP-ribose) polymerase.
Intracellular cysteine proteases are important mediators of apoptosis. Indeed, some nuclear proteins and enzymes are cleaved during apoptosis, in particular poly(ADP-ribose) polymerase (PARP), which is activated by DNA strand interruptions and is involved in DNA repair. PARP is cleaved into two fragments of 29 and 85 kDa (apparent molecular mass) in human promyelomonocytic leukemia cells, HL-60, treated with etoposide to induce apoptosis. These cells possess protease activities, caspases, that share many features with the ICE/CED-3 family. The cleavage occurs between Asp-214 and Gly-215, a site that is conserved in human, bovine, and chicken PARP. This cleavage has been shown to be an early marker of apoptosis. To monitor apoptosis, to understand the role of PARP cleavage by caspases, and to study the role of the two fragments in DNA repair, members of our laboratory have developed two polyclonal antipeptide antibodies directed against the two human PARP sequences: [196-214] for LP96-22 and [215-228] for LP96-24. Moreover, these antibodies will be useful to map the necrotic cleavage of PARP, which generates fragments different from those obtained during apoptosis, and thus to discriminate between apoptotic and necrotic cell death.
Sallmann
F R
FR
Health and Environment Unit, CHUL Research Center, CHUQ, Ste-Foy, QC, Canada.
Bourassa
S
S
Saint-Cyr
J
J
Poirier
G G
GG
D000595:Amino Acid Sequence; D000906:Antibodies / Q000097:blood; D000918:Antibody Specificity; D017209:Apoptosis / Q000276:immunology; D016923:Cell Death / Q000276:immunology; D003546:Cysteine Endopeptidases / Q000276:immunology* / Q000378:metabolism; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D015473:Leukemia, Promyelocytic, Acute / Q000201:enzymology / Q000473:pathology*; D008969:Molecular Sequence Data; D009336:Necrosis; D010446:Peptide Fragments / Q000097:blood / Q000138:chemical synthesis / Q000276:immunology*; D010449:Peptide Mapping; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D010446:Peptide Fragments; D011065:Poly(ADP-ribose) Polymerases; D003546:Cysteine Endopeptidases