Altered hypoxia- and redox-related transcriptional signatures in mitochondrial-DNA-depleted PC-3 cells

Abstract

Rho 0 (ρ0) cells are widely used as a tool to investigate how the absence of respiring mitochondria affects a variety of physiological and pathological processes. Prominently, ρ0 cells have been used to study the role of mitochondrial reactive oxygen species (ROS) production and/or mitochondrial respiration in the stabilization of the hypoxia-inducible factor (HIF) in hypoxia. In this study, we cultured ρ0 and WT PC-3 cells in 5% O2 (physioxia) and Plasmax medium for 2 weeks prior to transcriptomic and functional analyses. RNA-seq showed that ρ0 PC-3 cells exhibit impaired induction of HIF-regulated genes when exposed to hypoxia, compared to wild-type (WT) cells. Surprisingly, when comparing the transcriptomes of ρ0 and WT cells in physioxia (5% O2), we found a strong presence of HIF-related gene signatures in ρ0 cells compared to WT. Among the HIF targets found to be upregulated in ρ0 cells are CA9, EGLN3, EPAS1, HK2, ENO2, and SLC2A1. Moreover, several Nrf2 targets were upregulated in ρ0 cells, including NQO1, HMOX1, GPX2, and SLC7A11, which is in line with ρ0 cells showing a significantly higher H2O2 efflux rate than WT cells. Given the alterations in HIF-dependent and Nrf2-dependent gene expression and basal ROS production observed in ρ0 PC-3 cells, we conclude that caution should be taken when interpreting the results from experiments that focus on ROS production and HIF signaling using ρ0 cells as a model.