Common gene expression patterns responsive to mild temperature hyperthermia in normal human fibroblastic cells

Abstract

Purpose: Heat stress induces complex cellular responses, and its detailed molecular mechanisms still remain to be clarified. The objective of this study was to investigate the molecular mechanisms underlying cellular responses to mild hyperthermia (MHT) in normal human fibroblastic (NHF) cells.

Materials and methods: Cells were treated with MHT (41°C, 30 min) and then cultured at 37°C. Gene expression was determined by the GeneChip® system and bioinformatics tools.

Results: Treatment of the NHF cell lines, Hs68 and OUMS-36, with MHT did not affect the cell viability or cell cycle. In contrast, many probe sets were differentially expressed by >1.5-fold in both cell lines after MHT treatment. Of the 1,196 commonly and differentially expressed probe sets analysed by k-means clustering, three gene clusters, Up-I, Down-I and Down-II, were observed. Interestingly, two gene networks were obtained from the up-regulated genes in cluster Up-I. The gene network E contained DDIT3 and HSPA5 and was mainly associated with the biological process of endoplasmic reticulum stress, while the network S contained HBEGF and LIF and was associated with the biological process of cell survival. Eighteen genes were validated by quantitative real-time polymerase chain reaction, consistent with the microarray data, in four kinds of NHF cells.

Conclusions: Common genes that were differentially expressed and/or acted within a gene network in response to MHT in NHF cells were identified. These findings provide the molecular basis for a further understanding of the mechanisms of the MHT response in NHF cells.

• Mild temperature hyperthermia
• microarray
• gene function
• gene network
• normal human fibroblastic cell

Introduction

Hyperthermia (HT) induced by heat stress in the range of 40–45°C has been used as a possible mode of cancer treatment and its pleiotropic effects argue for its combined use with already established strategies, especially radiotherapy and/or chemotherapy [1–6]. In general, cancer cells exposed to temperatures of more than 42.5°C, the breakpoint of HT, will undergo cell death with increasing temperature, whereas temperatures up to 42.5°C, in the mild hyperthermia (MHT) range, induce only slight or no cytotoxicity [7]. However, it is very difficult to restrict the application of heat to the cancer itself and avoid damaging the surrounding normal tissue [8]. It has also been suggested that HT monotherapy maintaining a cancer temperature above 42.5°C for direct cytotoxicity has been associated with higher toxicity and less efficacy against cancer. Moreover, in accordance with the close relationship established between cancer and immune reactions, recent findings have suggested that an anti-tumour immune system including heat shock protein (HSP) chaperone tumour antigens might be effective as an HT-based cancer immunotherapy [9], [10]. Therefore, MHT in combination with radiotherapy and/or chemotherapy have been used for various cancers, and the anticancer effects of such combinations have been verified in many clinical trials [1–6].

The heat shock response is an ancient and evolutionally conserved protective reaction to elevated temperatures. Heat induces a wide spectrum of stress responses, such as an induction of HSPs, protein aggregation, DNA and RNA damage, an imbalance of protein homeostasis, reactive oxygen species production, cell growth arrest and different types of cell death, including apoptosis [11], [12]. HSPs, which are especially induced by heat, behave as molecular chaperones for other cellular proteins [11]. From the point of view of cancer cell death, HSPs function as strong cytoprotective molecules preventing apoptosis and other types of cell death and participate in thermotolerance [13–15]. HSPs achieve these effects by interacting with components of the cell signalling pathway, such as caspase-dependent apoptosis, upstream and downstream of the mitochondrial level [16]. Furthermore, heat activates extracellular signal-regulated kinases, and this activation provides cytoprotection from cell death by heating [17]. On the other hand, HT has been reported to induce apoptosis via activation of the protein kinase c-Jun N-terminal kinase [18], [19]. More recently, MHT was reported to activate the endoplasmic reticulum (ER) stress pathway in mammalian cells [20], [21].

Recent microarray technology coupled with bioinformatics tools has provided a view of the genome-wide expression profiles, and the relevant biological functions and gene networks based on the gene-expression data. It has been reported that heat stresses including MHT and HT affect the expression pattern of genes and a variety of biological functions in normal cells [14], [22–26] and cancer cells [24], [27–34]. Because heat stress induces complex cellular responses and changes in signal transduction, the precise mechanisms of heat response have not been elucidated.

As described above, MHT-based cancer therapy has been regarded as an effective cancer treatment with relatively few side effects. However, the temperature of the surrounding normal tissue of the tumour is also elevated to within the MHT range. Therefore, it is very important to understand the heat responses to normal cells in addition to cancer cells. Previously, we reported differentially expressed genes and gene networks in MHT-treated human leukaemia U937 cells [32], [33] or human oral squamous cell carcinoma HSC-3 cells [34]. The objective of this study was to investigate the molecular mechanisms underlying cellular responses to MHT in normal human fibroblastic (NHF) cells. To assess the global-scale gene expression profiles we employed high-density oligonucleotide microarrays and computational gene expression analysis tools in two kinds of NHF cells, Hs68 and OUMS-36, responsive to MHT at 41°C. Moreover, a real-time quantitative polymerase chain reaction (qPCR) assay was performed to identify the common expression patterns responsive to MHT in two other NHF cell types, KD and NTI-4, as well as Hs68 and OUMS-36 cells.

Materials and methods

Cell culture

Human fibroblastic cell lines, Hs68 (derived from newborn foreskin), KD (lip), NTI-4 (foetus body) and OUMS-36 (embryo), were obtained from the Human Science Research Resources Bank, Japan Health Sciences Foundation (Tokyo, Japan). These cells were cultured in Dulbecco's modified Eagle medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (Equitech-Bio, Kerrville, TX, USA) at 37°C in humidified air with 5% CO₂ and 95% air.

Heat treatment

Heat treatment was performed by immersing plastic vessels containing the attached cells in a water bath at 41°C (±0.05°C) or 44°C (±0.05°C) for 30 min, respectively. The temperature was monitored with a digital thermometer (No. 7563, Yokogawa, Tokyo) during heating. After heat treatment, the cells were incubated for 0–24 h at 37°C.

Analyses of cell viability and cell cycle

For cell viability, the trypan blue dye exclusion test was performed. The number of cells excluding the dye was counted by using a haemocytometer. The cell cycle was determined by flow cytometry as described previously [35]. In brief, for detection of the cell cycle, cells were fixed with 70% ice-cold ethanol, and then treated with RNase A (Nacalai Tesque, Kyoto, Japan) and propidium iodide (Wako Pure Chemical Industries, Osaka, Japan). The samples were run on an Epics XL flow cytometer (Beckman Coulter, Fullerton, CA, USA).

RNA isolation

Total RNA was extracted from cells using an RNeasy Total RNA Extraction Kit (Qiagen, Valencia, CA) along with on-column DNase I treatment (RNase-free DNase kit, Qiagen). RNA quality was analysed using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). RNA samples that had RIN (RNA integrity number) values above 9.5 were considered acceptable.

Microarray gene expression analysis

Microarray gene expression analysis was performed using a GeneChip® system with a Human Genome U133-plus 2.0 array, which was spotted with 54,675 probe sets (Affymetrix, Santa Clara) according to the manufacturer's instructions. In short, 500 ng of total RNA was used to synthesise cRNA with a GeneChip® 3’ IVT Express Kit (Affymetrix). After fragmentation, biotin-labelled cRNA was hybridised to the array at 45°C for 16 h. The arrays were washed, stained with streptavidin-phycoerythrin, and scanned using a probe array scanner. The obtained hybridisation intensity data were analysed using GeneSpring® GX (Agilent) to extract the significant genes. To examine gene ontology, including biological processes, cellular components, molecular functions, and gene networks, the obtained data were analysed using Ingenuity® Pathway Analysis tools (Ingenuity Systems, Mountain View, CA), a web-delivered application that enables the identification, visualisation, and exploration of molecular interaction networks in gene expression data [36], [37].

Real-time qPCR assay

Real-time qPCR was performed on an Mx3005P real-time PCR system (Agilent) using SYBR PreMix ExTaq (Takara Bio, Shiga, Japan) or Premix Ex Taq (for the use of TaqMan probes, Takara Bio) according to the manufacturer's protocols. The reverse transcriptase reaction was carried out with total RNA by using a random 6 mers and an oligo dT primer (PrimeScript RT Reagent Kit; Takara Bio). Real-time qPCR was performed by using the specific primers and probes listed in Tables IS and IIS of the supplementary materials. Each mRNA expression level was normalised with respect to the mRNA expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Table I.  The top 40 genes in the cluster Up-I.

Gene symbol	Hs68 (fold change)			OUMS-36 (fold change)			Gene title
	0 h	1 h	3 h	0 h	1 h	3 h
HSPA6	12.4	20.3	1.4	1.5	2.9	0.3	Heat shock 70 kDa protein 6 (HSP70B')
LIF	1.5	16.1	3	0.9	1.5	1.7	Leukaemia inhibitory factor
BHLHE40	1.9	8	2.2	1	4	1.4	Basic helix-loop-helix family, member e40
DLX2	2.5	6.9	2	1	2.9	1.6	Distal-less homeobox 2
EDN1	0.7	6.4	1.4	0.9	4.6	1.4	Endothelin 1
HSPA1A/B	4.8	5.6	3.9	1.1	2.2	1	Heat shock 70 kDa protein 1 A/B
HES1	5.1	5.1	0.5	1	2.4	0.2	Hairy and enhancer of split 1
C6orf62	1.4	3.9	1	1.2	3.8	1.5	Chromosome 6 open reading frame 62
HSPA1L	2.7	3.6	0.9	1.2	2.5	0.4	Heat shock 70 kDa protein 1-like
GADD45B	1.6	3.5	1.3	1.1	1.8	0.8	Growth arrest and DNA damage inducible, beta
HBEGF	1.3	3.5	2.8	1	1.4	1.7	Heparin-binding EGF-like growth factor
ADSSL1	1.6	3.1	1.1	0.6	2.2	0.9	Adenylosuccinate synthase like 1
HMOX1	1.2	3.1	2.5	1	2.4	2.8	Haem oxygenase (decycling) 1
HSPA5	1.2	3	2.6	1.1	2.1	1.8	Heat shock 70 kDa protein 5
HERPUD1	1	2.9	1.1	1	2.8	1.4	Homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1
DNAJB1	2.7	2.9	1.3	1.2	2	1.4	DnaJ (Hsp40) homologue, subfamily B, member 1
DNAJB9	1.3	2.8	2.6	0.9	2	1.9	DnaJ (Hsp40) homologue, subfamily B, member 9
CSRNP1	1.6	2.8	0.9	1.1	2.4	0.9	Cysteine-serine-rich nuclear protein 1
SERTAD1	1.6	2.7	1.1	1.1	2	1	SERTA domain containing 1
TUFT1	1.1	2.7	1.7	1	2	1.8	Tuftelin 1
FAM83G	1.4	2.7	1.7	1	1.6	1.2	Family with sequence similarity 83, member G
JMJD6	2.5	2.7	1.6	0.9	2	1.4	Jumonji domain containing 6
DDIT3	0.5	2.6	0.7	0.9	4.7	3.2	DNA damage inducible transcript 3
ZFAND2A	1.4	2.5	1.5	1.1	1.8	1.3	Zinc finger, AN1-type domain 2 A
SDF2L1	0.9	2.5	1.8	1.1	2.2	1.7	Stromal cell-derived factor 2-like 1
ENC1	1.3	2.4	2.6	1	1.7	2.4	Ectodermal–neural cortex 1 (with BTB-like domain)
KLF10	1.4	2.4	1.3	0.9	1.6	0.9	Kruppel-like factor 10
SLC35C2	1.4	2.4	1.1	1.6	1.8	1.6	Solute carrier family 35, member C2
CALD1	0.9	2.4	1.1	1.8	4.6	1.1	Caldesmon 1
STMN1	3.9	2.3	1.5	0.6	2	0.7	Stathmin 1
HSPH1	2	2.3	2.3	1	1.5	1.4	Heat shock 105kDa/110kDa protein 1
NEDD9	0.7	2.3	1.5	1	2.7	1.4	Neural precursor cell expressed, developmentally down-regulated 9
C6orfl45	1.2	2.3	1.7	1.1	2.2	1.4	Chromosome 6 open reading frame 145
EFNB2	1.3	2.3	1.9	1	1.7	1.9	Ephrin-B2
PIP4K2A	0.9	2.2	1	1.6	3.2	1.7	Phosphatidylinositol-5-phosphate 4-kinase, type II, alpha
SMAD7	1.4	2.2	1.3	1	1.8	1	SMAD family member 7
DACT1	1	2.1	1.5	1.1	1.9	1	Dapper, antagonist of beta-catenin, homologue 1
SCHIP1	1.1	2.1	1.1	0.9	1.8	1.1	Schwannomin interacting protein 1
AKAP17A	1.5	2.1	1	0.9	1.8	1	Akinase (PRKA) anchor protein 17A
ANP32E	1.1	2.1	1	0.8	2.1	1.2	Acidic (leucine-rich) nuclear phosphoprotein 32 family member E

Table II.  The top 20 genes in the cluster Down-I.

Gene symbol	Hs68 (fold change)			OUMS-36 (fold change)			Gene title
	0 h	1 h	3 h	0 h	1 h	3 h
PNRC1	0.53	0.21	0.23	0.77	0.34	0.54	Proline-rich nuclear receptor coactivator 1
EGR1	0.87	0.22	0.11	0.88	0.51	0.25	Early growth response 1
NACAP1	0.73	0.25	0.32	0.66	0.51	0.75	Nascent polypeptide-associated complex alpha polypeptide pseudogene 1
KPNB1	0.46	0.3	0.42	0.77	0.61	0.63	Karyopherin (importin) beta 1
HERPUD2	0.77	0.32	0.61	0.87	0.44	0.67	HERPUD family member 2
GAS1	0.77	0.33	0.44	0.93	0.55	0.74	Growth arrest-specific 1
PLAG1	0.87	0.34	0.49	0.81	0.48	0.81	Pleiomorphic adenoma gene 1
TAF1B	0.77	0.37	0.62	0.89	0.54	0.62	TATA box binding protein (TBP)-associated factor, RNA polymerase I, B, 63 kDa
KDM4A	0.9	0.38	0.73	1.28	0.61	0.86	Lysine (K)-specific demethylase 4 A
MSRB3	0.82	0.38	0.83	0.79	0.38	0.69	Methionine sulphoxide reductase B3
PXK	0.86	0.38	0.73	0.78	0.5	0.9	PX domain containing serine/threonine kinase
KLF6	0.68	0.39	1.03	0.95	0.46	1.1	Kruppel-like factor 6
NR2F2	0.55	0.39	0.8	0.94	0.52	1.52	Nuclear receptor subfamily 2, group F, member 2
LMLN	0.87	0.39	0.79	0.8	0.4	0.73	Leishmanolysin-like (metallopeptidase M8 family)
PLDN	0.77	0.4	0.79	0.95	0.41	0.86	Pallidin homologue (mouse)
HEATR6	0.84	0.41	0.77	0.77	0.43	0.84	HEAT repeat containing 6
THSD4	0.96	0.41	0.78	0.84	0.44	0.7	Thrombospondin, type I, domain containing 4
AK3	0.91	0.41	0.79	0.9	0.44	0.83	Adenylate kinase 3
BTG1	0.82	0.42	0.96	1.06	0.61	1.27	B-cell translocation gene 1, anti-proliferative
PHACTR2	0.89	0.42	0.75	0.84	0.51	0.67	Phosphatase and actin regulator 2

Statistical analysis

Data are shown as the means ± SD. Student's t-test was used for statistical analysis and significance was assumed for p values <0.05.

Results

Effects of heat on cell viability and cell cycle in NHF Hs68 and OUMS-36 cells

We used a trypan blue dye exclusion test for the detection of cell viability. When Hs68 and OUMS-36 cells were treated with MHT at 41°C for 30 min followed by culture at 37°C for 24 h, the percentage of viable cells did not change compared to the control (37°C treatment) (Figure 1, A and B). On the other hand, HT at 44°C for 30 min significantly decreased cell viability, to levels of 78.4 ± 11.2% and 74.8 ± 11.3% (mean ± SD; each control = 100%) for Hs68 and OUMS-36 cells, respectively (Figure 1, A and B). In addition, the cell cycle distribution was monitored by propidium iodide staining with flow cytometry. As shown in Figure 1C and D, the percentages of control Hs68 or OUMS-36 cells in the G0/G1, S, and G2/M phases were 78.1 ± 0.6%, 9.3 ± 0.3%, and 12.6 ± 0.7% or 63.0 ± 8.3%, 19.3 ± 4.3%, and 17.7 ± 4.0% (mean ± SD), respectively. Although changes in the distribution of cell cycle phases were not observed in the Hs68 or OUMS-36 cells treated with MHT at 41°C for 30 min, HT at 44°C for 30 min could arrest cells of either type in the G2/M phase (Figure 1, C and D). These data indicated that no cell damage and no cell cycle arrest was present in the cells treated with MHT at 41°C for 30 min.

Figure 1. The effects of heat on cell viability and the cell cycle in two NHF cell lines, Hs68 and OUMS-36. Cells were exposed to MHT at 41°C for 30 min or HT at 44°C for 30 min. After heat treatment, the cells were incubated for 24 h at 37°C. The cell viability (A, B) and cell cycle (C, D) were measured. (A, C) Hs68 cells. (B, D) OUMS-36 cells. Data indicate the means ± SD for four different experiments. *P < 0.05 versus the control (37°C treatment).

Global gene expression and cluster analyses

Global-scale gene expression analysis was carried out using a GeneChip® system with a Human Genome U133-plus 2.0 array, which was s potted with 54,675 probe sets. We detected 15,704 (28.7%) and 15,436 (28.2%) probe sets that were expressed in Hs68 and OUMS-36 cells subjected to MHT treatment, respectively. The complete lists of genes from Hs68 and OUMS-36 cell samples have been deposited in the Gene Expression Omnibus (GEO), a public database (accession numbers: GSE39177 and GSE39178). Expression analysis using GeneSpring® software of cells treated with MHT demonstrated many probe sets that were differentially regulated by a factor of 1.5 or greater. The Venn diagram in Figure 2A summarises the numbers of specifically and commonly expressed probe sets affected by MHT at 41°C for 30 min in Hs68 and OUMS-36 cells. In all, 2,567 and 2,169 probe sets were found to be differentially expressed in Hs68 and OUMS-36 cells, respectively, and 1,196 probe sets were differentially expressed in both cell lines (Figure 2A). Moreover, k-means clustering, a non-hierarchical gene clustering algorithm included in the GeneSpring® software package, was performed to generate the major patterns of gene expression during MHT treatment. In both cell lines, probe sets were grouped in three clusters with distinct expression profiles, designated as Up-I, Down-I and Down-II. The expression levels of probe sets in the Up-I cluster were gradually increased and this cluster also contained increased probe sets whose peak expressions occurred at 0 and 1 h; the expression levels of probe sets in the Down-I cluster were gradually decreased and this cluster also contained decreased probe sets whose lowest expression was at 1 h; the Down-II cluster contained transiently decreased probe sets whose lowest expression was at 1 h. The numbers of commonly expressed probe sets and genes in the Up-I, Down-I or Down-II cluster were 201 and 168, 493 and 443 or 283 and 238, respectively (Figure 2B). The top 40, 20 and 20 of genes in the Up-I, Down-I and Down-II clusters are shown in Tables I, II and III, respectively.

Figure 2. Gene expression analysis. Gene expression analysis of the probe sets that were differentially expressed by a factor of 1.5 or greater in Hs68 and OUMS-36 cells treated with MHT at 41°C for 30 min was performed using bioinformatics analysis tools. (A) Venn diagram of the probe sets that were differentially expressed in Hs68 and OUMS-36 cells. The numbers of probe sets are shown. (B) K-means clustering of commonly and differentially expressed probe sets (1,196 probe sets). Probe sets were grouped in three clusters with distinct expression profiles, Up-I, Down-I and Down-II. The numbers of commonly expressed probe sets and genes are shown. C, control.

Table III.  The top 20 of genes in the cluster Down-II.

Gene symbol	Hs68 (fold change)			OUMS-36 (fold change)			Gene title
	0 h	1 h	3 h	0 h	1 h	3 h
CCDC88A	1.65	0.09	1. 01	0.69	0.06	0.39	Coiled coil domain containing 88 A
AGGF1	0.74	0.14	0.44	0.36	0.22	0.69	Angiogenic factor with G patch and FHA domains 1
ANKRD12	0.4	0.14	0.31	0.64	0.13	0.48	Ankyrin repeat domain 12
CHD9	1.74	0.15	1.11	1.02	0.12	0.55	Chromodomain helicase DNA binding protein 9
PHACTR2	1.28	0.15	1.18	0.93	0.15	0.59	Phosphatase and actin regulator 2
ATRX	1.06	0.16	0.86	0.81	0.14	0.63	Alpha thalassemia/mental retardation syndrome X-linked
C10orf118	1.26	0.16	1.05	1.02	0.13	0.88	Chromosome 10 open reading frame 118
SMC3	1.03	0.17	0.88	0.81	0.14	0.71	Structural maintenance of chromosomes 3
THRAP3	1.03	0.17	0.99	0.93	0.14	0.69	Thyroid hormone receptor associated protein 3
MALAT1	1.08	0.17	0.96	0.51	0.13	0.49	Metastasis associated lung adenocarcinoma transcript 1 (non-protein coding)
CEP290	0.99	0.18	0.74	0.56	0.15	0.46	Centrosomal protein 290 kDa
ASPM	1.52	0.19	1.03	0.66	0.14	0.51	Asp (abnormal spindle) homolog, microcephaly associated
SLFN5	1.05	0.19	0.77	0.9	0.19	0.76	Schlafen family member 5
SFRP1	1.22	0.2	1.04	0.86	0.18	0.98	Secreted frizzled-related protein 1
OSBPL8	1.88	0.2	2.3	0.7	0.08	0.73	Oxysterol binding protein-like 8
LUZP1	1.2	0.2	1.37	0.85	0.21	0.89	Leucine zipper protein 1
VEZF1	0.73	0.21	0.59	0.95	0.26	0.87	Vascular endothelial zinc finger 1
SPAG9	1.07	0.21	1.08	0.85	0.19	0.77	Sperm associated antigen 9
ABHD2	1.07	0.21	1.02	0.9	0.27	0.62	Abhydrolase domain containing 2
CASC5	1.16	0.21	0.84	0.9	0.16	0.54	Cancer susceptibility candidate 5

Identification of biological functions and gene networks

Functional category and gene network analyses of commonly and differentially expressed genes induced by MHT were conducted by using the Ingenuity® Pathways Knowledge Base. The top three biological functions at each cluster are summarised in Table IIIS of the supplementary materials. Three biological functions, i.e. cellular compromise, cellular function and maintenance, and cellular growth and proliferation, were observed in the up-regulated gene cluster Up-I. In the down-regulated gene cluster Down-I, the biological functions were determined to be gene expression, cell death, and cellular compromise. In addition, three biological functions, i.e. cell cycle, gene expression, and DNA replication, recombination and repair, were observed in gene cluster Down-II. In addition, two significant gene networks, E and S, that were obtained from up-regulated genes in the Up-I cluster were identified (Figure 3). The detailed time-course of changes in the expression levels of genes for each gene network in Hs68 and OUMS-36 cells is shown in Figures 1S and 2S of the supplementary materials. The gene network E contained many HSPs, DNA-damage-inducible transcript 3 (DDIT3), homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERPUD1), and Hsp70 kDa protein 5 (glucose-regulated protein, 78 kDa) (HSPA5), and was mainly associated with ER stress (functions annotation) in the category of biological functions (cellular compromise or cellular function and maintenance). The gene network S contained endothelin 1 (EDN1), heparin-binding EGF-like growth factor (HBEGF), and leukaemia inhibitory factor (LIF), and was mainly associated with cell survival (functions annotation) in the category of biological functions (cell death) (Figure 3).

Figure 3. Gene networks. Up-regulated genes in the cluster Up-I were analysed by Ingenuity® Pathways Analysis tools. Gene networks E (A) and S (B) associated with ER stress and cell survival, respectively, were identified. The network is displayed graphically as nodes (genes) and edges (the biological relationships between the nodes). The node colour of genes indicates the expression level of genes. Nodes and edges are displayed using various shapes and labels that present the functional class of genes and the nature of the relationship between the nodes, respectively.

Quantitative analysis of differentially expressed genes

To verify the alterations observed by the microarray analysis, we used two NHF cell lines, KD and NTI-4, in addition to Hs68 and OUMS-36 cells. Time-dependent changes in the gene expression levels of selected genes that belonged to the gene networks E and S were monitored by using a real-time qPCR. The expression levels of all 10 genes in the gene network E, DDIT3, DnaJ (Hsp40) homolog, subfamily B, member 1 (DNAJB1), HERPUD1, haem oxygenase (decycling) 1 (HMOX1), Hsp70 kDa protein 1 A/B (HSPA1A/B), Hsp70 kDa protein 1-like (HSPA1L), HSPA5, Hsp70 kDa protein 6 (Hsp70B') (HSPA6), heat shock 105 kDa/110 kDa protein 1 (HSPH1), and protein phosphatase 1, regulatory subunit 15 A (PPP1R15A), were significantly increased in the Hs68 and OUMS-36 cells treated with MHT. These elevations were observed in the KD and NTI-4 cells treated with MHT (Figure 4). The expression levels of all 11 genes in the gene network S, basic helix-loop-helix family, member e40 (BHLHE40), EDN1, growth arrest and DNA-damage-inducible, alpha (GADD45A), growth arrest and DNA-damage-inducible, beta (GADD45B), HBEGF, hairy and enhancer of split 1, (Drosophila) (HES1), inhibitor of DNA binding 2, dominant negative helix-loop-helix protein (ID2), Kruppel-like factor 10 (KLF10), LIF, neural precursor cell expressed, developmentally down-regulated 9 (NEDD9), and SMAD family member 7 (SMAD7), were significantly increased in the Hs68 and OUMS-36 cells treated with MHT. The expression levels of eight of the 11 genes, with the exception being HES1, ID2 and KLF10, were significantly elevated in both KD and NTI-4 cells undergoing MHT treatment. On the other hand, increases in the expression of ID2 and KLF10 were detected in the NTI-4 and KD cells, respectively. A significant elevation of the expression of HES1 did not observed in the KD and NTI-4 cells (Figure 5). Taken together, the microarray results for all 21 genes in these networks were comparable to those from real-time qPCR analysis.

Figure 4. Effects of MHT on the time-course of changes in the expression levels of genes in the gene network E. Cells of the NHF lines Hs68 (Hs), KD, NTI-4 (NTI) and OUMS-36 (OUMS) were incubated at 41°C for 30 min and then cultured at 37°C for 0, 1 and 3 h. Real-time qPCR was carried out. DDIT3 (A), DNAJB1 (B), HERPUD1 (C), HMOX1 (D), HSPA1A/B (E), HSPA1L (F), HSPA5 (G), HSPA6 (H), HSPH1 (I) and PPP1R15A (J) were included in the gene network E. The expression level of each mRNA was normalised to the GAPDH expression level. Data are presented as the mean ± SD (n = 4). *P < 0.05 versus the control.

Figure 5. Effects of MHT on the time-course of changes in the expression levels of genes in the gene network S. Cells of the NHF cells Hs68 (Hs), KD, NTI-4 (NTI) and OUMS-36 (OUMS) were incubated at 41°C for 30 min and then cultured at 37°C for 0, 1 and 3 h. Real-time qPCR was carried out. BHLHE40 (A), EDN1 (B), GADD45A (C), GADD45B (D), HBEGF (E), HES1 (F), ID2 (G), KLF10 (H), LIF (I), NEDD9 (J) and SMAD7 (K) were included in the gene network S. The expression level of each mRNA was normalised to the GAPDH expression level. Data are presented as the mean ± SD (n = 4). *P < 0.05 versus the control.

Discussion

The heat shock response, which is a universal cellular response to heat, is a very important cellular event for cell adaptation. Because of the complex cellular response elicited by heat, we hypothesised that the most powerful technologies currently available for elucidating heat responses were global-scale microarrays and bioinformatics tools. Our approach with these technologies is the first to demonstrate the common genes and gene networks in response to MHT in NHF cells.

In the present study, MHT at 41°C did not affect cell viability or the cell cycle. However, a global-scale microarray clearly showed that many genes were differentially expressed by >1.5-fold in both cell lines, Hs68 and OUMS-36, after MHT treatment. This finding was in good agreement with previous results in MHT conditions [24], [31–34] and HT at relatively high temperature [14], [22], [23], [25–31], [33], [34] in normal cells [14], [22–26] and cancer cells [24], [27–34]. Here, computational bioinformatics tools were used to explore common responses to MHT. Approximately 50% of the probe sets were found to be commonly and differentially expressed probe sets between Hs68 and OUMS-36 cells, and these could be grouped in three clusters with distinct expression profiles, Up-I, Down-I and Down-II. Numerous genes in the clusters of Down-I and Down-II were transiently decreased at 1 h post-MHT treatment, but their expression levels were recovered immediately (Figure 2). In mammalian cells, heat activates transcription of heat responsive genes, including HSPs, coincident with a bulk decrease in mRNA and protein syntheses, and this overall reprogramming of gene expression permits the selective synthesis of HSPs [38–40].

Next, we focused on the common and up-regulated genes induced by MHT. In this study it was of particular interest that the significant gene networks E and S derived from up-regulated genes in the cluster Up-I were mainly associated with the biological processes of ER stress and cell survival, respectively. Moreover, real-time qPCR demonstrated that the expression of almost all the genes (18 out of 21 genes) selected was induced by MHT in four kinds of NHF cells, indicating that these genes would be common MHT-responsive molecules in NHF cells. In network E, four genes, HSPA5 [41], [42], DDIT3 [41], [42], HERPUD1 [43], and PPP1R15A [42], which are very closely associated with the ER stress response, were present, and relationships between DDIT3 and HERPUD1 [44], HSPA5 [45] or PPP1R15A [46] were identified. More recently, Xu et al. [20] demonstrated that MHT (40°C) but not HT (43°C) induced a partial or full ER stress pathway in parallel with the induction of Hsp70 protein in five mammalian cell types. Pallepati and Averill-Bates [21] reported that MHT (40°C) enhanced the pro-survival effects of the PERK (prot ein kinase RNA-like endoplasmic reticulum kinase)/eIF2α (alpha subunit of eukaryotic initiation factor 2) branch of ER stress. In addition, ER stress-related genes including HERPUD1 and DNAJB9 were up-regulated in normal human fibroblastic cells treated with MHT at 42°C using cDNA microarrays [24]. Based on the present results and the previous reports [20], [21], [24], we suggested that heat stresses within an MHT range can activate ER stress in our NHF cell systems. Although DDIT3 [47], [48] and PPP1R15A [47], which are reported to be inducers of cell death during ER stress, were elevated in normal fibroblastic cells treated with MHT, no cell death was observed (Figure 1). It is presumed that HSPA5 [49], HERPUD1 [44] and many HSPs [16], which have cytoprotective activity, participate in the prevention of cell death induced by MHT. In the present study we uncovered a unique cell survival associated gene network S. In network S, most of the genes, including LIF [50], HBEGF [51], GADD45A [52] and GADD45B [52], are related to cell survival. In this network, HBEGF was reported to be positively regulated by SMAD7 [53] and functionally activated by EDN1 [54]. These genes in network S seemed to behave as anti-cell death molecules under the present experimental conditions.

Our previous study with the GeneChip® microarray system showed that heat stresses affect the expression of a large number of genes in human lymphoma U937 cells [32], [33] and human oral squamous cell carcinoma HSC-3 cells [34]. Heat maps of gene expression patterns in the gene networks E and S among NHF cells in the present study and in human cancer cells [32–34] are demonstrated in Figures 3S and 4S of the supplementary materials. Although up-regulations of HSPA1L and DDIT3 were not detected in cancer cells, the majority of genes in network E, including many HSPs, were observed in both the HNF cells and cancer cells (Figure 3S). On the other hand, a remarkable difference in the gene expression patterns of genes in network S was observed among NHF cells and cancer cells (Figures 4S). It has also been indicated that there are differences between cancer cells and normal fibroblast cells in terms of their response to heat stress [24]. These findings demonstrated that induction of HSPs is a common response to heat stresses even under different experimental conditions, including differences in heating temperature, duration of heat treatment and cell origin [14], [22–34]. However, at present, the specific gene expression patterns affected by heat stresses in normal cells and cancer cells are not fully understand. Further studies are required to clarify this issue.

In conclusion, common genes that were differentially expressed and/or acted within a gene network in response to MHT in NHF cells were identified. These findings provide the molecular basis for a further understanding of the mechanisms of the biological changes that are responsive to MHT in NHF cells. MHT has the advantage of being associated with fewer side effects than HT at a relatively high temperature range, and the combination of MHT with radiotherapy and/or chemotherapy is recognised as a more promising therapeutic approach than HT monotherapy in many cancers [1–6]. It is thus expected that the findings obtained herein will facilitate the design of a more effective strategy for combination therapy with MHT.

Declaration of interest: This study was supported in part by a Grant-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology. The authors alone are responsible for the content and writing of the paper.