Hypoxia Strongly Affects Mitochondrial Ribosomal Proteins and Translocases, as Shown by Quantitative Proteomics of HeLa Cells

Abstract

Hypoxia is an important and common characteristic of many human tumors. It is a challenge clinically due to the correlation with poor prognosis and resistance to radiation and chemotherapy. Understanding the biochemical response to hypoxia would facilitate the development of novel therapeutics for cancer treatment. Here, we investigate alterations in gene expression in response to hypoxia by quantitative proteome analysis using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with LCMS/MS. Human HeLa cells were kept either in a hypoxic environment or under normoxic conditions. 125 proteins were found to be regulated, with maximum alteration of 18-fold. In particular, three clusters of differentially regulated proteins were identified, showing significant upregulation of glycolysis and downregulation of mitochondrial ribosomal proteins and translocases. This interaction is likely orchestrated by HIF-1. We also investigated the effect of hypoxia on the cell cycle, which shows accumulation in G1 and a prolonged S phase under these conditions. Implications. This work not only improves our understanding of the response to hypoxia, but also reveals proteins important for malignant progression, which may be targeted in future therapies.

Figure 1

HeLa cells under normoxic (a–d) and hypoxic (e–h) conditions. Panels (a) and (e) represent time point 0, and (b) and (f) show cells after 72 h in normoxic or hypoxic environments, respectively. Panels (c) and (g) show the oxygen profile over a period of 72 h, where the pericellular oxygen concentration was measured with an automated microsensor. The calculated usage of oxygen per cell per hour is depicted in panels 2(d) and (h).

Figure 2

Protein-protein interaction analysis using STRING. Nodes and edges are colored according to type of evidence; protein structures are sketched in the circles. Dark green: neighborhood; red: gene fusion; dark blue: cooccurrence; dark purple: coexpression; light purple: experiments; light blue/green: databases; light green: text-mining; light blue: homology. The gene names are matched to Uniprot accession numbers in Table S1. The backgrounds of up- and downregulated clusters are the shaded backgrounds in red and green, respectively (right: glycolysis, upregulated; bottom: mitochondrial translocases and left: mitochondrial ribosomal proteins, both downregulated).

Figure 3

Glycolysis/gluconeogenesis and citric acid cycle. H/L ratios and corrected p values are given in parentheses. Proteins that are up- and downregulated with statistical significance are depicted in green and red, respectively, while proteins in black do not satisfy the statistical criteria applied.

Figure 4

Cell cycle analysis. HeLa cells grown under (a) normoxic and (b) hypoxic conditions (1% O₂), stained with propidium iodide. The premitotic phases G1, S, and G2 are represented in the figure (calculated from the fluorescence intensity values; grey line). Cells grown under hypoxic conditions differ from those exposed to normoxia by showing an accumulation in G1 and a prolonged S phase.