Molecular radiobiologyThe hypoxic proteome is influenced by gene-specific changes in mRNA translation
Section snippets
Cell culture and hypoxic conditions
Exponentially growing cervical (HeLa) and prostate (DU145) carcinoma cells were seeded on glass dishes in DMEM or McCoy media with 10% foetal calf serum (FCS) (Sigma-Aldrich) and transferred to a hypoxic culture chamber (MACS VA500 microaerophilic workstation, Don Whitley Scientific, Shipley, UK). The atmosphere in the chamber consisted of 5% H2, 5% CO2, the desired concentration of O2 and residual N2. An anoxic atmosphere was ensured by the inclusion of a catalyst in the hypoxic chamber that
Results
We have reported that hypoxia results in a rapid inhibition of mRNA translation. In order to investigate the potential contribution of this inhibition on protein expression we analyzed the cellular proteome after short (1 h) and long (24 h) exposures to hypoxia. The proteome from aerobic or hypoxic HeLa cells was separated in two dimensions on the basis of charge and mass and proteins visualized by staining with SYPRO-Ruby. The early time point (1 h) was chosen to minimize the contribution of
Discussion
Tumor oxygenation varies dynamically in time at frequencies that would be difficult to compensate for by changes in transcription. Thus, it is not clear to what degree the cellular proteome, and ultimately cell behavior, changes during acute exposures to hypoxia. To address this question we assessed the effect of both acute (1 h) and chronic (24 h) hypoxia on the overall pool of expressed proteins. The results shown in Fig. 1 and Table 1 demonstrate that significant changes in the proteome take
Acknowledgements
This work was financially supported by Siemens, Netherlands Organization for Scientific Research (NWO) and the Dutch Cancer Society (KWF Kankerbestrijding). We thank Mieke Duysinx for technical assistance.
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2015, Biochimica et Biophysica Acta - Gene Regulatory MechanismsCitation Excerpt :Indeed, few tumor cells are refractory to the translation block induced under these conditions [64], whereas in most tumor cells examined the translation rate is inhibited (by 40% to 80%) by moderate hypoxia [64–66]. As discussed above, overall translation is primarily blocked at the initiation step under hypoxia, as assessed by polysome profiling and the inhibition of the eIF2α translation initiation factor (see Section 2.3) [60,62,67]. Translation elongation may also be inhibited in addition to initiation, based on the moderate retention of polysomes in tumor cells in response to hypoxia [60,62,67] and on the inhibition of the main translation elongation factor, eEF2, under these conditions (see Section 2.3).
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These authors contributed equally to this work.