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Pc 4, a photosensitizer first synthesized at Case Western Reserve University and now in clinical trial at University
Hospitals of Cleveland, has been shown to bind preferentially and with high affinity to mitochondrial and endoplasmic
reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components are photodamaged. In most
cancer cells, apoptosis is triggered by the initial photodamage; however, in cells deficient in one of the critical
intermediates of apoptosis, this process does not occur, although the cells remain as sensitive to the lethal effects of Pc 4-PDT as the apoptosis-competent cells, when cell death is determined by colony formation. Here we report that an
alternative death process, autophagy, is induced in all cells tested and becomes the dominant pathway for elimination of
lethally damaged cells when apoptosis is compromised. The anti-apoptotic protein Bcl-2, when overexpressed, protects
only apoptosis-competent cells against loss of clonogenicity, while the autophagy inhibitor 3-methyladenine provides a
markedly greater protection to apoptosis-deficient cells. The results suggest that the primary determinant of cell death is
not the final pathway for elimination of the cells but the initial photodamage to critical membrane targets. In attempts to
identify those targets, we have studied the role of different membrane phospholipids in the localization of Pc 4.
Cardiolipin (CL) is a phospholipid found exclusively in the mitochondrial inner membrane and at the contact sites
between the inner and outer membranes. Previous fluorescence resonance energy transfer studies revealed
colocalization of Pc 4 and CL, which points to CL as a possible binding site and target for Pc 4. Unilamellar liposomes
with different lipid compositions were used as membrane models to test the affinity of Pc 4. As revealed by the binding
constants, Pc 4 does not display preferential binding to CL in these systems. Moreover, binding affinities appear to be
independent of lipid composition. Localization of Pc 4 in mitochondrial membranes is likely determined by proteins or
other factors not replicated in the liposomes. Studies in cells with modified CL content could report modified binding
affinities.
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