Distinct Cellular Tools of Mild Hyperthermia-Induced Acquired Stress Tolerance in Chinese Hamster Ovary Cells

Abstract

Mild stress could help cells to survive more severe environmental or pathophysiological conditions. In the current study, we investigated the cellular mechanisms which contribute to the development of stress tolerance upon a prolonged (0-12 h) fever-like (40 °C) or a moderate (42.5 °C) hyperthermia in mammalian Chinese Hamster Ovary (CHO) cells. Our results indicate that mild heat triggers a distinct, dose-dependent remodeling of the cellular lipidome followed by the expression of heat shock proteins only at higher heat dosages. A significant elevation in the relative concentration of saturated membrane lipid species and specific lysophosphatidylinositol and sphingolipid species suggests prompt membrane microdomain reorganization and an overall membrane rigidification in response to the fluidizing heat in a time-dependent manner. RNAseq experiments reveal that mild heat initiates endoplasmic reticulum stress-related signaling cascades resulting in lipid rearrangement and ultimately in an elevated resistance against membrane fluidization by benzyl alcohol. To protect cells against lethal, protein-denaturing high temperatures, the classical heat shock protein response was required. The different layers of stress response elicited by different heat dosages highlight the capability of cells to utilize multiple tools to gain resistance against or to survive lethal stress conditions.

Keywords: Chinese hamster ovary cells; acquired stress tolerance; heat shock response; lipidomics; membrane; membrane lipid metabolism; stress; transcriptomics; unfolded protein response.

Figure 1

The effect of heat dosage on Hsp induction measured by Western blotting. (a,b) Time-dependent induction of Hsp25 upon mild heat treatment of CHO cells. Samples (40 °C and 42.5 °C denoted by black circles and red squares, respectively) were collected (a) right after (empty marks) HS or (b) following a 6 h recovery at 37 °C (solid marks). (c) Blot images of Hsp25, Hsp70 and GAPDH protein levels following a heat priming at 40 °C for 1 h or 6 h and 42.5 °C for 1 h without or with 6 h recovery (R) at 37 °C. (d) Relative quantification for Hsp25 induction in bar chart, in which recovery is indicated by checkerboard pattern. All data represent the means ± SD; n ≥ 5, p < 0.05 was considered statistically significant. Paired t-test was used for statistical comparisons, in which *, ° and + denote significant difference compared to 37 °C; 40 °C, 1 h with recovery; or 40 °C, 6 h with recovery, respectively. See experimental setup and blot images in Supplementary Figures S1 and S2.

Figure 1

The effect of heat dosage on Hsp induction measured by Western blotting. (a,b) Time-dependent induction of Hsp25 upon mild heat treatment of CHO cells. Samples (40 °C and 42.5 °C denoted by black circles and red squares, respectively) were collected (a) right after (empty marks) HS or (b) following a 6 h recovery at 37 °C (solid marks). (c) Blot images of Hsp25, Hsp70 and GAPDH protein levels following a heat priming at 40 °C for 1 h or 6 h and 42.5 °C for 1 h without or with 6 h recovery (R) at 37 °C. (d) Relative quantification for Hsp25 induction in bar chart, in which recovery is indicated by checkerboard pattern. All data represent the means ± SD; n ≥ 5, p < 0.05 was considered statistically significant. Paired t-test was used for statistical comparisons, in which *, ° and + denote significant difference compared to 37 °C; 40 °C, 1 h with recovery; or 40 °C, 6 h with recovery, respectively. See experimental setup and blot images in Supplementary Figures S1 and S2.

Figure 2

Acquired stress tolerance of heat-primed CHO cells. The quantitative analysis is based on colony formation assay. Primings were 1, 6 h at 40 °C or 42.5 °C for 1 h without or with 6 h recovery (R/checkerboard pattern) at 37 °C, which was followed by (a) a heat challenge with 46 °C for 20 min, (b) membrane fluidizing challenge by 95 mM BA for 20 min or (c) oxidative challenge by 250 µM TBHP for 3 h. Survival is represented as fold change compared to non-primed controls (37 °C). All data represent the means ± SD; n ≥ 3, p < 0.05 was considered statistically significant. Non-paired t-test was used for statistical comparisons, in which * denotes significant difference compared to 37 °C samples that received heat or BA challenge. See experimental setup and colony formation assay images in Supplementary Figure S4.

Figure 3

The effect of heat priming on mitochondrial membrane potential and lipid peroxidation of CHO cells. (a) Change in mitochondrial membrane potential measured by fluorescent JC-1 dye. Cells were primed at 40 °C for 1 h and 42.5 °C for 1 h, then fluorescence was read before (empty bars) and after (solid bars) addition of a challenging dose of 95 mM BA. Fold change was calculated in comparison to non-treated 37 °C. (b) CHO cells were primed at 40 °C for 1 h and 6 h and at 42.5 °C for 1 h, followed by lipid peroxidation-sensitive DPPP fluorescence measurement (empty bars). After heat pretreatments a challenging dose of 95 mM BA was introduced to the cells (solid bars). In (a,b), all data represent the means ± SD; n ≥ 9, p < 0.05 was considered statistically significant. Paired t-test was used for statistical comparisons. In (a), * and ° denote statistically significant difference compared to 37 °C and 37 °C + BA, respectively, while in (b), significant difference was compared to 37 °C + BA and denoted by *.

Figure 4

RNA sequencing data reveal distinct stress transcriptome upon different stress conditions. (a) RNAseq samples of CHO cells distinguished by principal component analysis. (b) Heatmap representation of hierarchical clustering. (c) Expression of total of 920 genes had significantly changed by different doses of mild (40 °C, 1 h and 6 h) and moderate (42.5 °C, 1 h) heat treatments in pairwise comparison to controls (37 °C)—shown in Venn diagram.

Figure 4

RNA sequencing data reveal distinct stress transcriptome upon different stress conditions. (a) RNAseq samples of CHO cells distinguished by principal component analysis. (b) Heatmap representation of hierarchical clustering. (c) Expression of total of 920 genes had significantly changed by different doses of mild (40 °C, 1 h and 6 h) and moderate (42.5 °C, 1 h) heat treatments in pairwise comparison to controls (37 °C)—shown in Venn diagram.

Figure 5

The most significantly enriched canonical pathways (−log(B-H p-value) > 2.0) in heat-treated CHO cells derived from Ingenuity Pathway Analysis (IPA).

Figure 6

Heat-induced lipidomic changes. CHO cells were left untreated or subjected to 40 °C for 1 or 6 h or 42.5 °C for 1 h. (a) Partial least squares discriminant analysis score plots of lipidomic dataset based on relative concentration values. Circles display 95% confidence regions. The model was validated by a 2000-time permutation test (p = 0.0015). (b) Venn diagram displaying the number of statistically different components.

Figure 7

Heat-induced lipidomic changes. CHO cells were left untreated or subjected to 40 °C for 1 or 6 h or 42.5 °C for 1 h. (a–d) Changes in the relative concentration (a) of disaturated and monounsaturated membrane lipid species (db ≤ 1), (b) in polyunsaturated components (db ≥ 4), (c) in lysolipid species and (d) in sphingolipids. Data are expressed as mean ± SEM, n = 8; * denotes p < 0.025 compared to the untreated control. db, number of double bonds; PC, phosphatidylcholine; PI, phosphatidylinositol; LPC and LPI, the corresponding lyso species; Cer, ceramide; SM, sphingomyelin; HexCer, hexosylceramide.

Figure 7

Heat-induced lipidomic changes. CHO cells were left untreated or subjected to 40 °C for 1 or 6 h or 42.5 °C for 1 h. (a–d) Changes in the relative concentration (a) of disaturated and monounsaturated membrane lipid species (db ≤ 1), (b) in polyunsaturated components (db ≥ 4), (c) in lysolipid species and (d) in sphingolipids. Data are expressed as mean ± SEM, n = 8; * denotes p < 0.025 compared to the untreated control. db, number of double bonds; PC, phosphatidylcholine; PI, phosphatidylinositol; LPC and LPI, the corresponding lyso species; Cer, ceramide; SM, sphingomyelin; HexCer, hexosylceramide.