SUBCELLULAR-LOCALIZATION OF PHOTOFRIN(R) AND AMINOLEVULINIC ACID AND PHOTODYNAMIC CROSS-RESISTANCE IN-VITRO IN RADIATION-INDUCED FIBROSARCOMA CELLS SENSITIVE OR RESISTANT TO PHOTOFRIN-MEDIATED PHOTODYNAMIC THERAPY

The subcellular and, specifically, mitochondrial localization of the p hotodynamic sensitizers Photofrin and aminolevulinic acid (ALA)-induce d protoporphyrin-IX (PpIX) has been investigated in vitro in radiation -induced fibrosarcoma (RIF) tumor cells. Comparisons were made of pare ntal RIF-1 cells and cells (RIF-8A) in which resistance to Photofrin-m ediated photodynamic therapy (PDT) had been induced. The effect on the uptake kinetics of Photofrin of coincubation with one of the mitochon dria-specific probes 10N-Nonyl acridine orange (NAG) or rhodamine-123 (Rh-123) and vice versa was examined. The subcellular colocalization o f Photofrin and PpIX with Rh-123 was determined by double-label confoc al fluorescence microscopy, Clonogenic cell survival after ALA-mediate d PDT was determined in RIF-I and RIF-8A cells to investigate cross-re sistance with Photofrin-mediated PDT, At long (18 h) Photofrin incubat ion times, stronger colocalization of Photofrin and Rh-123 was seen in RIF-I than in RIF-8A cells, Differences between RIF-1 and RIF-8A. in the competitive mitochondrial binding of NAO or Rh-123 with Photofrin suggest that the inner mitochondrial membrane is a significant Photofr in binding site. The differences in this binding may account for the P DT resistance in RIF-8A cells. With ALA, the peak accumulations of PpI X occurred at 5 h for both cells, and followed a diffuse cytoplasmic d istribution compared to mitochondrial localization at 1 h ALA incubati on. There was rapid efflux of PpIX from both RIF-1 and RIF-8A. As with Photofrin, ALA-induced PpIX exhibited weaker mitochondrial localizati on in RIF-8A than in RIF-1 cells. Clonogenic survival demonstrated cro ss-resistance to incubation in PpIX but not to ALA-induced PpIX, imply ing differences in mitochondrial localization and/or binding, dependin g on the source of the PQIX within the cells.