Abstract
Tumor cells can adapt to a hostile environment with reduced oxygen supply. The present study aimed to identify mechanisms that confer hypoxia resistance. Partially hypoxia/reoxygenation (H/R)-resistant proximal tubular (PT) cells were selected by exposing PT cultures to repetitive cycles of H/R. Thereafter, H/R-induced changes in mRNA and protein expression, inner mitochondrial membrane potential (ΔΨm), formation of superoxide, and cell death were compared between H/R-adapted and control PT cultures. As a result, H/R-adapted PT cells exhibited lower H/R-induced hyperpolarization of ΔΨm and produced less superoxide than the control cultures. Consequently, H/R triggered ΔΨm break-down and DNA degradation in a lower percentage of H/R-adapted than control PT cells. Moreover, H/R induced upregulation of mitochondrial uncoupling protein-3 (UCP-3) in H/R-adapted PT but not in control cultures. In addition, ionizing radiation killed a lower percentage of H/R-adapted as compared to control cells suggestive of an H/R-radiation cross-resistance developed by the selection procedure. Knockdown of UCP-3 decreased H/R- and radioresitance of the H/R-adapted cells. Finally, UCP-3 protein abundance of PT-derived clear cell renal cell carcinoma and normal renal tissue was compared in human specimens indicating upregulation of UCP-3 during tumor development. Combined, our data suggest functional significance of UCP-3 for H/R resistance.
Highlights
Surrounded by a microenvironment with varying oxygen pressure are, under continuous risk of mitochondria-derived oxidative insults
For H/R adaptation, immortalized primary cultures of mouse proximal convoluted tubule (PT) which are highly dependent on oxidative respiration and, highly hypoxia-sensitive were subjected to repeated cycles of hypoxia and reoxygenation[5]
To test for an acquired H/R resistance, sub-confluent H/R-adapted and control cultures were grown for 48 h under normoxia or hypoxia (0.1% oxygen) followed by 0.5, 24 or 48 h of reoxygenation
Summary
Surrounded by a microenvironment with varying oxygen pressure are, under continuous risk of mitochondria-derived oxidative insults. The present study aimed to define mechanisms of hypoxia/reoxygenation (H/R) adaptation in vitro by comparing H/R-adapted with highly hypoxia-sensitive parental cells. For H/R adaptation, immortalized primary cultures of mouse proximal convoluted tubule (PT) which are highly dependent on oxidative respiration and, highly hypoxia-sensitive were subjected to repeated cycles of hypoxia and reoxygenation[5]. That way H/R-adapted PT cultures were compared with the continuously normoxic-grown parental control cells in terms of H/R-induced impairment of mitochondrial function, formation of reactive oxygen species (ROS), cell death and gene expression. To estimate whether this in vitro finding might be translated to the in vivo situation, the present study analyzed UCP-3 expression in PT-derived clear cell renal cell carcinoma demonstrating marked up-regulation of UCP-3 by the tumor cells
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