INVESTIGATION OF THE ROLE OF LIGHT-INDEPENDENT DYE UPTAKE IN MC540-MEDIATED PHOTOSENSITIZATION OF L1210 LEUKEMIA CELLS.
Linda R. Jones (Presently at the Department of Physics and Astronomy, College of Charleston, Charleston, SC 29424) and Fritz Sieber (Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226).
It has been shown that in the absence of light, leukemia cells take up an amount of MC540 that is insufficient to initiate photosensitized cell killing. However, the amount of light-independent dye uptake in MC540-sensitive cells is on the same order of magnitude as light-enhanced uptake. This study examines the role of light-independent cellular dye uptake in MC540-mediated photosensitization of leukemia cells. Confocal imaging and spectroscopic methods confirm the existence of temperature-dependent light-indpendent MC540 uptake in L1210 leukemia cells. Light-independent MC540 uptake appears as punctate perinuclear fluorescence that colocalizes with simultaneous lysosome and golgi probes (acridine orange and NBD-ceramide). Confocal illumination of pre-incubated, washed cells results in bleaching of the punctate fluorescence with no subsequent reappearance of subcellular fluorescence. Illumination of pre-incubated, unwashed cells results in blurring and lightening of the punctate fluorescence followed by a dramatic time-delayed influx of MC540 and staining of the nuclear envolope. Lysosomal involvement is confirmed with biochemical assays. Delivery of a broad-band light dose sufficient to kill 50% of the cells results in disruption of the lysosomal membrane, as shown by increased UMBG fluorescence. The same light dose causes a significant decrease in punctate perinuclear fluorescence, an increase in diffuse perinuclear fluorescence and nuclear envelope staining. While not lethal in itself, lysosomal damage may initiate further dye uptake in MC540-sensitive cells.
email Linda Jones
Back to the homepage