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 t ermed caspases, culminating in cleavage of a set of proteins, The abil ity of photodynamic treatment (PDT) with the phthalocyanine Pc 4 to ac tivate 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 greater than or equal to 90% cell death, as judged by a clonogenic asset;, The rate of entry of cells into apoptosis was dose dependent, For 0.5 mu M Pc 4 and eit her 2.1 or 3 kJ/m(2), which kill 90 or 99.9% of the cells, oligonucleo somal fragmentation was risible on agarose gels as early as 60 or 30 m in after PDT, respectively. To assess caspase activation, cells were h arvested at various times after PDT, and cell proteins were subjected to electrophoresis and Western blot analysis, using an antibody to pol y(ADP-ribose) polymerase (PARP). The cleavage of the normally M-r 116, 000 PARP into fragments of M-r similar to 90,000 was observed at appro ximately the same time as the earliest DNA fragmentation. An antibody to the polymer, poly(ADP-ribose), did not recognize the M-r similar to 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 II were maintain ed in cells after PARP was fully cleared, 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-permeabl e, irreversible caspase inhibit-or, arbonyl-Val-Ala-Asp(O-methyl)-fluo ro-methylketone, inhibited PDT-induced apoptosis and PARP cleavage, wh ereas the inactive peptide analogue, benzyloxycarbonyl-Phe-Ala-fluorom ethyl ketone, was without effect, The results indicate that PDT-induce d apoptosis is mediated by activation of caspase-3 and/or other simila r caspases.