Some chemotherapy drugs influence the formation of stress granules (SGs), cytoplasmic RNA foci involved in stress response pathways. The exact role of SGs in promoting cell survival or apoptosis needs further clarification. The chemotherapy drug lomustine induces SG formation by activating the eIF2α kinase HRI (EIF2AK1). We performed a DNA microarray transcriptome analysis to identify genes affected by lomustine-induced stress and to explore the role of SGs.
Our findings show that lomustine specifically regulates the expression of the pro-apoptotic EGR1 gene. The presence of EGR1 mRNA in SGs correlates with reduced translation of EGR1 mRNA. Lomustine likely sequesters EGR1 mRNA into SGs, preventing its ribosomal translation and thereby limiting apoptosis. This supports a model where SGs selectively sequester specific mRNAs in response to stress, modulate their translation, and thus determine the fate of stressed cells.
Overall design: The human-derived HAP1 (parental, S51A, ΔHRI, ΔGCN2, ΔPKR, ΔPERK, ΔEGR1 and ΔATF4) cell lines were purchased from Horizon Discovery, UK. The mutations in ΔHRI, ΔGCN2, ΔPKR and ΔPERK cell lines were confirmed by sequencing. Furthermore, parental, S51A, ΔHRI, ΔGCN2, ΔPKR and ΔPERK cell lines had already been used in another study, and their susceptibility for SG analysis was already confirmed (Aulas et al., 2018). HAP cell lines were cultured in Iscove’s modified Dulbecco’s medium (IMDM, Gibco #12440-053) supplemented with 10% FBS (Thermo Fisher Scientific #10270106) and 1% penicillin-streptomycin. All cell lines were maintained at 37°C with 5% CO2. All cells were frequently tested for mycoplasma contamination using a MycoAlert Mycoplasma Detection Kit (Lonza, LT07-418).
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