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Updated: Jul 18, 2025
Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules
Published on: May 12, 2017
Differential Formation of Stress Granules in Radiosensitive and Radioresistant Head and Neck Squamous Cell Carcinoma
Safa Louati1, Anne-Sophie Wozny2, Céline Malesys3
1Cellular and Molecular Radiobiology Laboratory, Lyon-Sud Medical School, UMR CNRS 5822/IP2I, Université de Lyon, Lyon 1 University, Oullins, France; Department of Research and Teaching in Oncology, Hôpital Nord, Saint-Priest en Jarez, France.
Purpose:
Stress granules (SGs) are cytoplasmic aggregates in which mRNAs and specific proteins are trapped in response to a variety of damaging agents. They participate in the cellular defense mechanisms. Currently, their mechanism of formation in response to ionizing radiation and their role in tumor-cell radiosensitivity remain elusive.
Methods And Materials:
The kinetics of SG formation was investigated after the delivery of photon irradiation at different doses to head and neck squamous cell carcinoma cell lines with different radiosensitivities and the HeLa cervical cancer cell line (used as reference). In parallel, the response to a canonical inducer of SGs, sodium arsenite, was also studied. Immunolabeling of SG-specific proteins and mRNA fluorescence in situ hybridization enabled SG detection and quantification. Furthermore, a ribopuromycylation assay was used to assess the cell translational status. To determine whether reactive oxygen species were involved in SG formation, their scavenging or production was induced by pharmacologic pretreatment in both SCC61 and SQ20B cells.
Results:
Photon irradiation at different doses led to the formation of cytoplasmic foci that were positive for different SG markers. The presence of SGs gradually increased from 30 minutes to 2 hours postexposure in HeLa, SCC61, and Cal60 radiosensitive cells. In turn, the SQ20B and FaDu radioresistant cells did not form SGs. These results indicated a correlation between sensitivity to photon irradiation and SG formation. Moreover, SG formation was significantly reduced by reactive oxygen species scavenging using dimethyl sulfoxide in SCC61 cells, which supported their role in SG formation. However, a reciprocal experiment in SQ20B cells that depleted glutathione using buthionine sulfoximide did not restore SG formation in these cells.
Conclusions:
SGs are formed in response to irradiation in radiosensitive, but not in radioresistant, head and neck squamous cell carcinoma cells. Interestingly, compared with sodium arsenite-induced SGs, photon-induced SGs exhibited a different morphology and cellular localization. Moreover, photon-induced SGs were not associated with the inhibition of translation; rather, they depended on oxidative stress.
Insights
Stress granules (SGs) form in response to irradiation in radiosensitive cancer cells but not radioresistant ones. This suggests SGs play a role in cellular defense against radiation damage.
Area of Science:
- Cell Biology
- Cancer Research
- Radiation Oncology
Background:
- Stress granules (SGs) are dynamic cytoplasmic foci involved in cellular defense mechanisms against various stressors.
- The formation and role of SGs in response to ionizing radiation and their impact on tumor cell radiosensitivity are not well understood.
Purpose of the Study:
- To investigate the mechanism of stress granule formation induced by ionizing radiation.
- To determine the role of stress granules in the radiosensitivity of head and neck squamous cell carcinoma cells.
Main Methods:
- Investigated SG formation kinetics in response to photon irradiation in different head and neck squamous cell carcinoma cell lines and HeLa cells.
- Utilized immunolabeling and mRNA fluorescence in situ hybridization for SG detection and quantification.
- Assessed cellular translational status and the involvement of reactive oxygen species (ROS) in SG formation.
Main Results:
- Photon irradiation induced SG formation in radiosensitive cell lines (HeLa, SCC61, Cal60) but not in radioresistant ones (SQ20B, FaDu).
- SG formation correlated with sensitivity to photon irradiation and was dependent on oxidative stress, as evidenced by ROS scavenging experiments.
- Photon-induced SGs differed in morphology and localization compared to those induced by sodium arsenite and were not associated with translation inhibition.
Conclusions:
- Stress granule formation in response to irradiation is specific to radiosensitive head and neck squamous cell carcinoma cells.
- Oxidative stress is a key factor in the formation of photon-induced stress granules.
- Photon-induced SGs may represent a distinct cellular response pathway compared to stress granule formation induced by other agents.
