Molecular radiobiology
Phosphorylation of eIF2α is required for mRNA translation inhibition and survival during moderate hypoxia

https://doi.org/10.1016/j.radonc.2007.04.031Get rights and content

Abstracts

Background and purpose

Human tumors are characterized by temporal fluctuations in oxygen tension. The biological pathways that respond to the dynamic tumor microenvironment represent potential molecular targets for cancer therapy. Anoxic conditions result in eIF2α dependent inhibition of overall mRNA translation, differential gene expression, hypoxia tolerance and tumor growth. The signaling pathway which governs eIF2α phosphorylation has therefore emerged as a potential molecular target. In this study, we investigated the role of eIF2α in regulating mRNA translation and hypoxia tolerance during moderate hypoxia. Since other molecular pathways that regulate protein synthesis are frequently mutated in cancer, we also assessed mRNA translation in a panel of cell lines from different origins.

Materials and methods

Immortalized human fibroblast, transformed mouse embryo fibroblasts (MEFs) and cells from six cancer cell lines were exposed to 0.2% or 0.0% oxygen. We assayed global mRNA translation efficiency by polysome analysis, as well as proliferation and clonogenic survival. The role of eIF2α was assessed in MEFs harboring a homozygous inactivating mutation (S51A) as well as in U373-MG cells overexpressing GADD34 (C-term) under a tetracycline-dependent promoter. The involvement of eIF4E regulation was investigated in HeLa cells stably expressing a short hairpin RNA (shRNA) targeting 4E-BP1.

Results

All cells investigated inhibited mRNA translation severely in response to anoxia and modestly in response to hypoxia. Two independent genetic cell models demonstrated that inhibition of mRNA translation in response to moderate hypoxia was dependent on eIF2α phosphorylation. Disruption of eIF2α phosphorylation caused sensitivity to hypoxia and anoxia.

Conclusions

Disruption of eIF2α phosphorylation is a potential target for hypoxia-directed molecular cancer therapy.

Section snippets

Cell models

Our studies included the following human cell lines: HeLa (cervix carcinoma), DU145 (prostate carcinoma derived from brain metastasis), HT-29 (colorectal adenocarcinoma), MCF7 (mammary adenocarcinoma derived from pleural effusion), U373 MG (glioblastoma–astrocytoma), BJ-hTERT (normal skin fibroblasts transformed with human telomerase). Mouse embryo fibroblasts (MEF) wild-type (wt) or with a knock-in mutation for eIF2α (S51A) were transformed with SV40 Large T antigen and previously described

Results

We have previously shown that acute exposure to anoxia severely inhibits mRNA translation in HeLa cells [9]. However, since tumor cells are known to be exposed to a wide range of oxygen tensions, we investigated whether this inhibition also occurred at higher oxygen concentrations. To this end, we exposed HeLa cells to 0.0%, 0.2% and 2.0% oxygen and quantified global mRNA translation efficiency using the polysome assay. In this assay, RNA complexes are separated by centrifugation through a

Discussion

It can be argued that the molecular pathways which are activated early in response to hypoxia may be most attractive to target because they potentially affect all hypoxic cells. The data presented here show that a large panel of cells, including normal cells as well as metastatic breast and prostate cancer cells, inhibit mRNA translation to a very similar extent in response to acute hypoxia or anoxia. The molecular pathways that regulate translation during acute hypoxia do not appear to be

Acknowledgements

We acknowledge financial support from the Dutch Science Organization (ZonMW-NWO Top Grant 912-03-047 to BW, and ZonMW-VENI Grant 016.056.015 to MK), the Dutch Cancer Society (KWF Grant UM 2003-2821 to BW), and the EU 6th framework program (Euroxy program to BW).

The technical assistance of Mieke Duysinx is highly appreciated.

References (22)

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