Apoptosis and necrosis induced with light and 5-aminolaevulinic acid-derived protoporphyrin IX

Abstract

The mode of cell death induced by photodynamic treatment (PDT) was studied in two cell lines cultured in monolayer, V79 Chinese hamster fibroblasts and WiDr human colon adenocarcinoma cells. The cells were incubated with 5-aminolaevulinic acid (5-ALA) as a precursor for the endogenously synthesised protoporphyrin IX, which was activated by light. Free DNA ends, owing to internucleosomal DNA cleavage in apoptotic cells, were stained specifically with a fluorescent dye in the terminal deoxynucleotidyl transferase (TdT) assay. The free DNA ends were measured by flow cytometry and the fractions of apoptotic cells determined. Total cell death was measured in a cell survival assay to determine the necrotic fraction after subtraction of the apoptotic fraction. V79 cells did undergo apoptosis while WiDr cells were killed only through necrosis. With time, the apoptotic fraction of V79 cells increased until a maximum was reached about 3-4 h after ALA-PDT treatment. For increasing ALA-PDT doses, a maximal apoptotic fraction 75-85% of the cells was measured at about 85% of total cell death. The flow cytometric assay of apoptosis was confirmed by the typical ladder of oligonucleosomal DNA fragments obtained from agarose gel electrophoresis, by fluorescence micrographs visualising the induced free DNA ends and by electron micrographs showing the typical morphology of apoptotic cells.