PROTOPORPHYRIN BIOSYNTHESIS IN MELANOMA B16 CELLS STIMULATED BY 5-AMINOLEVULINIC ACID AND CHEMICAL INDUCERS - CHARACTERIZATION OF PHOTODYNAMIC INACTIVATION

The stimulation of protoporphyrin (PP) biosynthesis in B16 melanoma ce lls in order to facilitate photodynamic cell killing was studied. Bios ynthesis and accumulation of PP in the melanoma cells was increased fr om 8 to 15 pmol/mg protein by the use of dimethyl-sulfoxide (DMSO), a differentiation-inducer. Treatment of the cells with the porphyrogenic agent allyl-isopropyl-acetamide (AIA) stimulated an additional PP inc rease. The most remarkable enhancement of intracellular PP was achieve d by the supplementation of 5-aminolevulinic acid (5-ALA) to the growt h medium following the addition of DMSO and AIA during the induction p hase. The intracellular concentration of PP exceeded 21 950 pmol/mg pr otein following combined stimulation by DMSO/AIA and 5-ALA. The porphy rins produced in the incubated cells, in serum-depleted medium, consis ted of 95% PP; 88% of it was recovered from the cells and only 7% was excreted into the medium. Photosensitization of the B16 melanoma cells containing high PP concentrations was effective even at low light dos es. Potassium (K) efflux was the first measurable sign of cell damage determined by X-ray microanalysis (XRMA) following fast liquid-nitroge n fixation. During a 1 min interval, 70% of cellular K was lost. After 5 min illumination, complete cell destruction was detected by scannin g electron microscopy (SEM) and XRMA. The photodamaged cells showed in flux of Na, Cl and Ca ions accompanying the immediate K losses. Ultras tructural cell damage was manifested by disintegration of the outer me mbrane. Total cell death of B16 melanoma cells was achieved by chemica l induction of endogenous PP and photosensitization. (C) 1994 Wiley-Li ss, Inc.