Bystander effects in cell death induced by photodynamic treatment, UVA radiation and inhibitors of ATP synthesis

Confluent layers of MDCK LI cells were treated with four different photosen sitizers (a purified version of hematoporphyrin derivative [Photofrin], tet ra(3-hydroxyphenyl)porphine [3-THPP], meso-tetra(4-sulphonatophenyl)porphin e [TPPS4] and ALA-induced Protoporphyrin M) and irradiated with blue light, with UVA without exogenous photosensitizers, or incubated with the metabol ic inhibitors carbonyl cyanide m-chlorophenylhydrazone and a-deoxy-D-glucos e, Necrotic and apoptotic cells were detected about 4 h later by fluorescen ce microscopy. Dead cells appeared in distinct clusters in the confluent la yers. The number of dead cells in these clusters was determined by manual c ounting and image analysis, Forty-one of the 43 experimental distributions of dead cells in clusters were found to be significantly different from a M onte Carlo simulation of the distribution of independently inactivated cell s. However, a Monte Carlo simulation model, assuming that each dead cell in creased the probability of inactivation of adjacent cells, fitted 34 of the 43 observed distributions of dead cells in clusters, indicating a signific ant bystander effect for all the investigated treatments. The bystander-eff ect model parameter, defined as a cell's increase in probability of dying w hen it has dead neighbors, was significantly lower for 3-THPP-PDT and TPPS4 -PDT than for Photofrin-PDT, ALA-PDT and treatment with metabolic inhibitor s.