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. 2019 Jan:71:156-171.
doi: 10.1016/j.jbior.2018.09.007. Epub 2018 Sep 18.

Gle1 mediates stress granule-dependent survival during chemotoxic stress

Laura Glass  1 , Susan R Wente  2
Affiliations

Gle1 mediates stress granule-dependent survival during chemotoxic stress

Laura Glass et al. Adv Biol Regul. 2019 Jan.

Abstract

Stress granules (SGs) are non-membrane bound organelles that form in response to multiple different stress stimuli, including exposure to sodium arsenite. SGs are postulated to support cells during periods of stress and provide a protective effect, allowing survival. Gle1 is a highly conserved, essential modulator of RNA-dependent DEAD-box proteins that exists as at least two distinct isoforms in human cells. Gle1A is required for proper SG formation, whereas Gle1B functions in mRNA export at the nuclear pore complex. Since Gle1A is required for SG function, we hypothesized that SG-dependent survival responses would also be Gle1-dependent. We describe here an experimental system for quantifying and testing the SG-associated survival response to sodium arsenite stress in HeLa cells. Gle1A was required for the sodium arsenite survival response, and overexpression of Gle1A supported the survival response. Overexpression of the SG-component G3BP also enabled the response. Next, we analyzed whether cells undergoing multiple rounds of stress yield a subpopulation with a higher propensity for SG formation and an increased resistance to undergoing apoptosis. After ten doses of sodium arsenite treatment, cells became resistant to sodium arsenite and to diclofenac sodium (another SG-inducing drug). The sodium arsenite-resistant cells exhibited changes in SG biology and had an increased survival response that was conferred in a paracrine manner. Changes in secreted factors occurred including a significantly lower level of MCP-1, a known regulator of stress granules and stress-induced apoptosis. This study supports models wherein SGs play a role in cell evasion of apoptosis and further reveal Gle1A and SG functions as targets for clinical approaches directed at chemoresistant/refractory cells.

Keywords: Cancer; Evasion of apoptosis; Gle1; MCP-1; Stress granules; Survival.

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Figures

Figure 1.
Figure 1.. High doses of sodium arsenite induce SG formation and a survival response in HeLa cells.
(A) HeLa cells (seeded at 1×105 cells/mL) were treated with sodium arsenite at concentrations ranging from 6 nM to 25 mM for 72 hours and viability was measured. A representative viability curve is shown, where each data point represents the average of 2 technical replicates normalized to duplicate untreated wells. Error bars denote range. Survival response is defined as the biphasic effect on cell viability induced at sodium arsenite concentrations > 100 μM. (B) SG formation was assessed by anti-G3BP indirect immunofluorescence, with Nuc Red® stain marking nuclear material. Scale bar represents 15 μm. (C) Viability and survival response were assessed at different seeding densities (1×104, 5×104, 1×105 or 2×105 cells/mL) Representative viability curves are shown. (D) The impact of seeding density on survival response (C) was quantified as area under the curve (AUC) from 100 μM-25 mM sodium arsenite, and shown for three independent trials. Data shown in (C) is indicated in (D) as “Trial I”.
Figure 2.
Figure 2.. Gle1 is required for survival response.
(A) Dose response curves of sodium arsenite on cell viability were assessed for nontargeting (CTRL) or GLE1 siRNA-treated HeLa cells exposed to 6 nM to 25 mM arsenite for 72 hours. Representative viability curves are shown, where each data point represents the average of 2 technical replicates normalized to duplicate untreated wells. Error bars denote range. (B) The sodium arsenite survival response was quantified as described, and plotted for four independent trials. Data shown in (A) is indicated in (B) as “Trial I”. (C) SG formation and mRNA export were evaluated in CTRL or GLE1 siRNA-treated HeLa cells exposed to 500 μM sodium arsenite for 1 hour. SGs were detected by anti-G3BP indirect immunofluorescence, and poly(A)+ localization was determined by in situ hybridization to Cy3-oligo dT. Scale bar represents 10 μm. (D, E) Size and number of SGs were determined for CTRL and GLE1 siRNA-treated HeLa cells after 500 μM sodium arsenite treatment 15 to 60 minutes. G3BP immunofluorescence was quantified using ImageJ 3D objects counter, where surface areas of 3D objects was reported. Data was collected at each time point for three independent experiments, and plotted onto a box and whiskers graph where whiskers denote 10th-90th percentile of data. (F) The percentage of cells exhibiting SGs was monitored over recovery time following 500 μM sodium arsenite treatment for 1 hour. SG-positive cells were quantified at each time point for three independent experiments, and plotted onto a box and whiskers graph. (G) Survival response to 1500 μM sodium arsenite for 72 hours was assessed in CTRL or GLE1 siRNA-treated HeLa cells exogenously expressing GFP, GFP-Gle1A or GFP-Gle1B. *p<0.05; **p<0.01; ***p<0.001; statistical significance was determined by Student’s paired t-test.
Figure 3.
Figure 3.. Sodium arsenite survival response is mediated by stimulation of SG assembly, not mRNA export inhibition
(A) Nxf1 localization and mRNA export were evaluated in control (CTRL) or NXF1 siRNA-treated HeLa cells by anti-Nxf1 indirect immunofluorescence and in situ hybridization to Cy3-oligo d(T), respectively. Scale bar represents 10 μm. (B, C, D, E) Dose response curves of sodium arsenite on cell viability and the sodium arsenite survival response were assessed as described for (B, C) CTRL or NXF1 siRNA-treated HeLa cells, or (D, E) HeLa cells exogenously expressing mCherry or mCherry-G3BP. Data shown in (B) and (D) are indicated in (C) and (D) as “Trial I”. *p<0.05; statistical significance was determined by Student’s paired t-test.
Figure 4.
Figure 4.. Sodium arsenite-resistant cells exhibit altered cell morphology and SG biology.
(A) Schematic depicting the process taken to generate arsenite-resistant HeLa cells. (B) Following 10 rounds of sodium arsenite treatment, cell morphology was evaluated by bright field microscopy. Scale bar represents 20 μM. (C) Cell viability was measured for non-resistant and arsenite-resistant HeLa cells seeded at increasing cell densities. (D, E) Dose response curves of sodium arsenite on cell viability were generated as described for non-resistant and arsenite-resistant HeLa cells. Survival responses and IC50 values were calculated from 3 independent experiments. (F, G) Non-resistant and arsenite-resistant HeLa cells were subjected to 0, 250, 500 or 1000 μM sodium arsenite treatment for 60 minutes and SG phenotypes were analyzed. Size (F) and number of SGs (G) were calculated as previously described. Data was collected for three independent experiments and plotted onto a box and whiskers. *p<0.05 using Student’s unpaired t-test.
Figure 5.
Figure 5.. Sodium arsenite resistance confers differential sensitivity to SG-inducing chemotoxic agents.
IC50s of non-resistant and arsenite resistant cell viability were determined after exposure to the indicated chemotoxic agents for 72 hours. Cells were treated with (A) 0.2 pM to 1 μM bortezomib (BTZ); (B) 9.5 nM to 40 mM diclofenac sodium; (C) 1 pM to 5 mM emetine; or (D) 2 pM to 8 mM paclitaxel (PCX). IC50s were calculated from 3 independent experiments. *p<0.05 using Student’s unpaired t-test.
Figure 6.
Figure 6.. Arsenite-resistance is mediated by MCP-1 and Serpin E1
(A) The conditioned media of non-resistant and arsenite-resistant HeLa cells was swapped and dose responses of sodium arsenite on cell viability were measured and plotted as described. (B) Cell viability was compared for each experimental group at 900 μM sodium arsenite, denoted by an arrow on the viability curves in (A). (C) Dose responses of sodium arsenite on cell viability were determined and plotted as described for untreated HeLa cells or HeLa cells pre-incubated with 50 μM 15-d-PGJ2 for 1 hour. (D) The arsenite survival response was quantified as described, and plotted for six independent trials. Data from (C) is represented in (D) as “Trial I”. *p<0.05 using Student’s paired t-test. (E, F) Conditioned media samples from non-resistant and arsenite-resistant cells were probed for 105 human cytokines using a membrane-based sandwich immunoassay. Densitometry was performed for each cytokine and normalized to a loading control. Two-way ANOVA with multiple comparisons was performed to identify differentially secreted factors; *p=0.0255, ****p<0.0001.
Figure 7.
Figure 7.. Model for the role of MCP-1 in differential response of non-resistant vs arsenite-resistant cell lines
In non-resistant cells (left), MCP-1 is secreted, which in turn induces MCPIP-1; a known suppressor of SG formation. When cells cannot form SGs, apoptosis is more likely to be induced. However, in arsenite-resistant cells (right), MCP-1 secretion is suppressed. This prevents stimulation of MCPIP-1 and therefore relieves the blockage of SG formation. This leaves the cells with an increased propensity to form SGs and allows the cell to survive the stress and recover.
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