Photodynamic therapy and cell death pathways

Abstract

Photodynamic therapy (PDT) is the term used to describe the irradiation of photosensitized cells or tissue with phototoxic consequences. This process can result in the rapid initiation of not only apoptosis, an irreversible death pathway, but also autophagy. The procedures described here are designed to characterize the correlation between the PDT dose vs. survival of cells in vitro, the apoptotic effects of photodamage, and the extent of an autophagic response. These are assessed by clonogenic assays, observation of condensed chromatin characteristic of apoptosis, activation of "executioner" caspases, and the autophagic flux as indicated by comparing accumulation of the LC3-II protein under conditions where processing of autophagosomes is retarded vs. is not retarded.

Fig. 3.1

Fluorogenic effects of PDT in the caspase 3/7 assay system. Numbers in each cell reflect the position of the sample in the 96-well plate. A₁, A₂, and C₁ are controls; other samples received different PDT doses.

Fig. 3.2

Typical patterns of chromatin labeled with HO342. Left, control L1210 cells; right, 60 min after an LD₉₀ PDT dose using BPD.

Fig. 3.3

Conversion of LC3-I to LC3-II during PDT. Cells were treated with BPD and given a 25 mJ/cm² light dose. Lysates were prepared for Western blots 15 min later. Where shown, 15 mM ammonium chloride was present during all incubations. A probe for actin was used to confirm that equal levels of protein were applied to each lane.

Fig. 3.4

Dose–response curve for L1210 cells using BPD and 690 ± 10 nm light. Cells were incubated with 2 μM BPD for 30 min, then irradiated as described in the text. Data indicate numbers of colonies/plate after 10 days. Approximately 100 cells were plated using light doses of 0–50 mJ/cm² and 1,000 cells for greater light doses.