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.