Quantitative Analysis of Pc 4 Localization in Mouse Lymphoma (LY-R) Cells via Double-label Confocal Fluorescence Microscopy

Abstract

Photodynamic therapy (PDT) is a novel cancer therapy that uses light-activated drugs (photosensitizers) to destroy tumor tissue. Reactive oxygen species produced during PDT are thought to cause the destruction of tumor tissue. However, the precise mechanism of PDT is not completely understood. To provide insight into the in vitro mechanisms of PDT, we studied the subcellular localization of the photosensitizer HOSiPcOSi(CH3)2-(CH2)3N(CH3)2 (Pc 4) in mouse lymphoma (LY-R) cells using double-label confocal fluorescence microscopy. This technique allowed us to observe the relative distributions of Pc 4 and an organelle-specific dye within the same cell via two, spectrally distinct, fluorescence images. To quantify the localization of Pc 4 within different organelles, linear correlation coefficients from the fluorescence data of Pc 4 and the organelle-specific dyes were calculated. Using this measurement, the subcellular spatial distributions of Pc 4 could be successfully monitored over an 18 h period. At early times (0-1 h) after introduction of Pc 4 to LY-R cells, the dye was found in the mitochondria, lysosomes and Golgi apparatus, as well as other cytoplasmic membranes, but not in the plasma membrane or the nucleus. Over the next 2 h, there was some loss of Pc 4 from the lysosomes as shown by the correlation coefficients. After an additional incubation period of 2 h Pc 4 slowly increased its accumulation in the lysosomes. The highest correlation coefficient (0.65) was for Pc 4 and BODIPY-FL C5 ceramide, which targets the Golgi apparatus, and also binds to other cytoplasmic membranes. The correlation coefficient was also high (0.60) for Pc 4 and a mitochondria-targeting dye (Mitotracker Green FM). Both of these correlation coefficients were higher than that for Pc 4 with the lysosome-targeting dye (Lysotracker Green DND-26). The results suggest that Pc 4 binds preferentially and strongly to mitochondria and Golgi complexes.

Enhanced Lysosomal Activity Is Involved in Bax Inhibitor-1-induced Regulation of the Endoplasmic Reticulum (ER) Stress Response and Cell Death against ER Stress

“…These probes have high selectivity for acidic organelles and are effective for the labeling of live cells (21). Cells were grown in a cell culture dish, rinsed with PBS, and stained with 100 nM LysoTracker Green DND-26 (Molecular Probes, Eugene, OR) in serum-free medium for 30 min at room temperature.…”

Photodynamic Therapy-induced Apoptosis in Epidermoid Carcinoma Cells

“…Fluorescence data were analyzed using Adobe Photoshop (Adobe Systems, San Jose, CA). Clusters of several mitochondria (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) in each experiment were identified as regions of interest, and fields not containing cells were taken as the background. Statistical analyses were performed with Student's t test, using p Ͻ 0.01 as a criterion of significance.…”

“…The phthalocyanine photosensitizer Pc 4 preferentially binds to the endoplasmic reticulum, Golgi complex, and mitochondria in L5178Y-R (LY-R) mouse lymphoma cells (28) and induces rapid apoptotic cell death after exposure to light (29). The critical early events and specific subcellular targets triggering PDT-induced apoptotic death remain to be determined.…”

Differential responses of Mcl-1 in photosensitized epithelial vs lymphoid-derived human cancer cells

“…This difference is unlikely to be due to different Pc 4 localization or uptake, because we have found similar Pc 4 uptake in A431 and Jurkat cells and similar distribution of the photosensitizer in mitochondria and ER/Golgi in both lymphoma cells (L5178Y-R) and carcinoid cells (A431, MCF-7, DU-145) (Trivedi et al, 2000;Lam et al, 2001;Usuda et al, 2003).…”

“…Pc 4 is found in intracellular membranes, especially mitochondria, endoplasmic reticulum (ER), and the nuclear periphery (Trivedi et al, 2000;Lam et al, 2001;Usuda et al, 2003). Bcl-2 has been demonstrated to localize to the nuclear envelope, ER, and mitochondrial membranes (Hockenbery et al, 1990;Krajewski et al, 1993), whereas Bcl-xL is found in mitochondria as well as cytosol (Wang et al, 2001).…”