Protease activation and cleavage of poly(ADP-ribose) polymerase: an integral part of apoptosis in response to photodynamic treatment.
Apoptosis induced by numerous cancer chemotherapeutic and other toxic agents has been shown to proceed through a cascade of proteases, now termed caspases, culminating in cleavage of a set of proteins. The ability of photodynamic treatment (PDT) with the phthalocyanine Pc 4 to activate cellular caspases has been assessed during the rapid apoptosis in murine lymphoma L5178Y-R cells. Cells were exposed to combinations of Pc 4 and activating red light that result in > or =90% cell death, as judged by a clonogenic assay. The rate of entry of cells into apoptosis was dose dependent. For 0.5 microM Pc 4 and either 2.1 or 3 kJ/m2, which kill 90 or 99.9% of the cells, oligonucleosomal fragmentation was visible on agarose gels as early as 60 or 30 min after PDT, respectively. To assess caspase activation, cells were harvested at various times after PDT, and cell proteins were subjected to electrophoresis and Western blot analysis, using an antibody to poly(ADP-ribose) polymerase (PARP). The cleavage of the normally Mr 116,000 PARP into fragments of Mr approximately 90,000 was observed at approximately the same time as the earliest DNA fragmentation. An antibody to the polymer, poly(ADP-ribose), did not recognize the Mr approximately 90,000 PARP cleavage products, in contrast to the parent enzyme. This analysis also revealed that levels of a poly(ADP-ribosylated) Mr 100,000 protein, tentatively identified as topoisomerase I, were maintained in cells after PARP was fully cleaved. Caspase-3 (and/or caspase-7) activity, as measured in cell lysates with the fluorogenic substrate DEVD-AMC, was elevated almost immediately after PDT. The cell-permeable, irreversible caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoro-methylketone, inhibited PDT-induced apoptosis and PARP cleavage, whereas the inactive peptide analogue, benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone, was without effect. The results indicate that PDT-induced apoptosis is mediated by activation of caspase-3 and/or other similar caspases.
He
J
J
Department of Radiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4942, USA.
Whitacre
C M
CM
Xue
L Y
LY
Berger
N A
NA
Oleinick
N L
NL
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007211:Indoles / Q000494:pharmacology* / Q000627:therapeutic use; D054833:Isoindoles; D008223:Lymphoma / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology*; D051379:Mice; D009374:Neoplasms, Experimental / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology; D010778:Photochemotherapy; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology* / Q000627:therapeutic use; D014407:Tumor Cells, Cultured
D007211:Indoles; D054833:Isoindoles; D011838:Radiation-Sensitizing Agents; D011065:Poly(ADP-ribose) Polymerases; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; C013647:phthalocyanine