Molecular radiobiology
The hypoxic proteome is influenced by gene-specific changes in mRNA translation

https://doi.org/10.1016/j.radonc.2005.06.036Get rights and content

Abstract

Background and purpose

Hypoxia causes a rapid reduction in mRNA translation efficiency. This inhibition does not affect all mRNA species to the same extent and can therefore contribute significantly to hypoxia-induced differential protein expression. Our aim in this study was to characterize changes in gene expression during acute hypoxia and evaluate the contribution of regulation via mRNA translation on these changes. For each gene, the contribution of changes in mRNA abundance versus mRNA translation was determined.

Materials and methods

DU145 prostate carcinoma cells were exposed to 4 h of hypoxia (<0.02% O2). Efficiently translated mRNAs were isolated by sedimentation through a sucrose gradient. Affymetrix microarray technology was used to evaluate both the transcriptional and translational contribution to gene expression. Results were validated by quantitative PCR.

Results

One hundred and twenty genes were more than 4-fold upregulated by hypoxia in the efficiently translated fraction of mRNA, in comparison to only 76 genes at the level of transcription. Of the 50 genes demonstrating the largest changes in translation, 11 were found to be more than 2-fold over represented in the translated fraction in comparison to their overall transcriptional level. The gene with the highest translational contribution to its induction was CITED-2, which is a negative regulator of HIF-1 transcriptional activity.

Conclusions

Gene-specific regulation of mRNA translation contributes significantly to differential gene expression during hypoxia.

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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