Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells.
During apoptosis, the activation of a family of cysteine proteases, or caspases, results in proteolytic cleavage of numerous substrates. Antibody probes specific for neoepitopes on protein fragments generated by caspase cleavage provide a means to monitor caspase activity at the level of the individual cell. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis. Here, we demonstrate that an affinity-purified polyclonal antibody to the p85 fragment of PARP is specific for apoptotic cells. Western blots show that the antibody recognizes the 85-kDa (p85) fragment of PARP but not full-length PARP. We demonstrate a time course of PARP cleavage and DNA fragmentation in situ using the PARP p85 fragment antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in Jurkat cells treated with anti-Fas. Furthermore, our results indicate that the p85 fragment of PARP resulting from caspase cleavage during apoptosis is rapidly localized outside the condensed chromatin but not in the cytoplasm.
O'Brien
M A
MA
Promega Corporation, Madison, WI, USA. mobrien@promega.com
Moravec
R A
RA
Riss
T L
TL
D000906:Antibodies; D000918:Antibody Specificity; D017209:Apoptosis; D015153:Blotting, Western; D003593:Cytoplasm / Q000737:chemistry; D053938:DNA Fragmentation; D005456:Fluorescent Dyes; D006801:Humans; D020287:In Situ Nick-End Labeling; D007211:Indoles; D019169:Jurkat Cells; D011065:Poly(ADP-ribose) Polymerases / Q000276:immunology / Q000378:metabolism*; D019014:fas Receptor / Q000032:analysis / Q000276:immunology
D000906:Antibodies; D005456:Fluorescent Dyes; D007211:Indoles; D019014:fas Receptor; C007293:DAPI; D011065:Poly(ADP-ribose) Polymerases