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Protease Activation and Cleavage of Poly(ADP-ribose) Polymerase: An Integral Part of Apoptosis in Response to Photodynamic Treatment¹

Departments of Radiology [J. H., L-Y. X., N. L. O.] and Medicine [C. M. W., N. A. B.] and the CWRU/Ireland Cancer Center [N. A. B., N. L. O.], Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4942

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 90% cell death, as judged by a clonogenic assay. The rate of entry of cells into apoptosis was dose dependent. For 0.5 µM 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 M_r 116,000 PARP into fragments of M_r 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 M_r 90,000 PARP cleavage products, in contrast to the parent enzyme. This analysis also revealed that levels of a poly(ADP-ribosylated) M_r 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.

¹ This work was supported by USPHS Grants P01 CA48735, P01 CA51183, P30 CA63200, and P30 CA43703 from the National Cancer Institute.

² To whom requests for reprints should be addressed, at Division of Radiation Biology, School of Medicine (BRB-324), Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4942. Phone: (216) 368-1117; Fax: (216) 368-1142; E-mail: nlo@po.cwru.edu.

Received 11/27/97. Accepted 1/ 6/98.

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