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

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.