Bystander effects of PC12 cells treated with Pb²⁺ depend on ROS-mitochondria-dependent apoptotic signaling via gap-junctional intercellular communication.

The demonstration of bystander effect, which means injured cells propagate damage to neighboring cells, in whole organisms has clear implication of the potential relevance of the non-targeted response to human health. Here we show that 10 μM lead acetate, the optimum concentration for inducing apoptosis confirmed by the expression levels of Bax and Bcl-2, can also induce rat pheochromocytoma (PC12) cells to exert bystander effects to neighboring cells. In a novel co-culture system, GFP-PC12 (Pb(2+)) cells, which were stable transfected with EF1A-eGFP and pre-exposed with lead acetate, were co-cultured with unexposed PC12 cells at a 1:5 ratio. Parachute assays demonstrated the functional gap-junctional intercellular communication (GJIC) formed between Pb(2+)-exposed and unexposed cells. The Pb(2+)-exposed cells induced very similar effects on neighboring unexposed cells to apoptosis coincide with intracellular ROS generation and the collapse of mitochondrial membrane potential (Δψm). Furthermore, carbenoxolone (CBX), a blocker of GJIC, inhibited the bystander effects. The results indicate that the Pb(2+)-induced insults propagate through GJIC between PC12 cells, while inducing the bystander cells to apoptosis via ROS-mitochondria-dependent apoptotic signaling.