ASSOCIATION OF CERAMIDE ACCUMULATION WITH PHOTODYNAMIC TREATMENT-INDUCED CELL-DEATH

Ceramide, a stress-induced second messenger, has been associated with apoptosis in several malignant and nonmalignant cell lines. We have sh own that photodynamic treatment (PDT), using the phthalocyanine photos ensitizer Pc 4 (HOSiPcOSi[CH3](2)[CH2](3)N[CH3](2)), causes increased ceramide generation and subsequent induction of apoptosis in L5178Y-R (LY-R) mouse lymphoma cells. To test further if ceramide generation ac companies photocytotoxicity, we treated various cell lines with a PDT dose producing a 99-99.9% loss of clonogenicity, Like LY-R cells, huma n leukemia (U937) cells underwent rapid DNA fragmentation initiating w ithin 1 h after PDT, Similarly, Chinese hamster ovary (CHO) cells show ed rapid DNA laddering, beginning 1 h following the treatment. In cont rast, mouse radiation-induced fibrosarcoma (RIF-1) cells showed no apo ptosis within 24 h post-PDT, as judged by the absence of 50 kbp and ol igonucleosome-size DNA fragments, as well as no annexin V binding to c ells with preserved membrane integrity. Using the same doses of PDT, w e observed a time-dependent ceramide accumulation in all three cell li nes. While a significant increase in ceramide levels was reached withi n 1 and 10 min in U937 and CHO cells, respectively, elevated ceramide production was measured only after 30 min in RIF-1 cells. In addition, exogenous N-acetyl-sphingosine was able to mimic PDT-induced apoptosi s in U937 and CHO cells. We suggest that ceramide accumulation is asso ciated with PDT-induced apoptosis and photocytotoxicity.