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A microarray analysis of the effects of moderate hypothermia and rewarming on gene expression by human hepatocytes (HepG2).

ABSTRACT: The gene expression changes produced by moderate hypothermia are not fully known, but appear to differ in important ways from those produced by heat shock. We examined the gene expression changes produced by moderate hypothermia and tested the hypothesis that rewarming after hypothermia approximates a heat-shock response. Six sets of human HepG2 hepatocytes were subjected to moderate hypothermia (31 degrees C for 16 h), a conventional in vitro heat shock (43 degrees C for 30 min) or control conditions (37 degrees C), then harvested immediately or allowed to recover for 3 h at 37 degrees C. Expression analysis was performed with Affymetrix U133A gene chips, using analysis of variance-based techniques. Moderate hypothermia led to distinct time-dependent expression changes, as did heat shock. Hypothermia initially caused statistically significant, greater than or equal to twofold changes in expression (relative to controls) of 409 sequences (143 increased and 266 decreased), whereas heat shock affected 71 (35 increased and 36 decreased). After 3 h of recovery, 192 sequences (83 increased, 109 decreased) were affected by hypothermia and 231 (146 increased, 85 decreased) by heat shock. Expression of many heat shock proteins was decreased by hypothermia but significantly increased after rewarming. A comparison of sequences affected by thermal stress without regard to the magnitude of change revealed that the overlap between heat and cold stress was greater after 3 h of recovery than immediately following thermal stress. Thus, while some overlap occurs (particularly after rewarming), moderate hypothermia produces extensive, time-dependent gene expression changes in HepG2 cells that differ in important ways from those induced by heat shock.

SUBMITTER: Sonna LA

PROVIDER: S-EPMC3006613 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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A microarray analysis of the effects of moderate hypothermia and rewarming on gene expression by human hepatocytes (HepG2).

Sonna Larry A LA Kuhlmeier Matthew M MM Khatri Purvesh P Chen Dechang D Lilly Craig M CM

Cell stress & chaperones 20100606 5

The gene expression changes produced by moderate hypothermia are not fully known, but appear to differ in important ways from those produced by heat shock. We examined the gene expression changes produced by moderate hypothermia and tested the hypothesis that rewarming after hypothermia approximates a heat-shock response. Six sets of human HepG2 hepatocytes were subjected to moderate hypothermia (31 degrees C for 16 h), a conventional in vitro heat shock (43 degrees C for 30 min) or control cond ...[more]

PMID: 20526826

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Project description:Mercury is widely distributed in the biosphere, and its toxic effects have been associated with human death and several ailments that include cardiovascular diseases, anemia, kidney and liver damage, developmental abnormalities, neurobehavioral disorders, autoimmune diseases, and cancers in experimental animals. At the cellular level, mercury has been shown to interact with sulphydryl groups of proteins and enzymes, to damage DNA, and to modulate cell cycle progression and/or apoptosis. However, the underlying molecular mechanisms of mercury toxicity remain to be elucidated. Our laboratory has demonstrated that mercury exposure induces cytotoxicity and apoptosis, modulates cell cycle, and transcriptionally activates specific stress genes in human liver carcinoma cells. The liver is one of the few organs capable of regeneration from injury. Dormant genes in the liver are therefore capable of reactivation. In this research, we hypothesize that mercury-induced hepatotoxicity is associated with the modulation of specific gene expressions in liver cells that can lead to several disease states involving immune system dysfunctions. In testing this hypothesis, we used an Affymetrix oligonucleotide microarray with probe sets complementary to more than 20,000 genes to determine whether patterns of gene expressions differ between controls and mercury (1-3 microg/mL) treated cells. There was a clear separation in gene expression profiles between controls and mercury-treated cells. Hierarchical cluster analysis identified 2,211 target genes that were affected. One hundred and eighty-eight of these genes were up-regulated, among which forty eight were significant (p = 0.001) with greater than a two-fold change difference in the concentration range (1-3 microg/mL) of mercury-treated cells; twelve genes were moderately over-expressed with an increase of more than one fold (p = 0.004). 2,023 genes were down-regulated with only forty of them reaching statistically significant decline at p = 0.05 according to the Welch's ANOVA/Welch's t-test. Further analyses of affected genes identified genes located on all human chromosomes with higher than normal effects on genes found on chromosomes 1-14, 17-20 (sex-determining region Y)-box18SRY, 21 (splicing factor, arginine/serine-rich 15 and ATP-binding), and X (including BCL6-co-repressor). These genes are categorized as control and regulatory genes for metabolic pathways involving the cell cycle (cyclin-dependent kinases), apoptosis, cytokine expression, Na+/K+ ATPase, stress responses, G-protein signal transduction, transcription factors, DNA repair as well as metal-regulatory transcription factor 1, MTF1 HGNC, chondroitin sulfate proteoglycan 5 (neuroglycan C), ATP-binding cassette, sub-family G (WHITE), cytochrome b-561 family protein, CDC-like kinase 1 (CLK1 HGNC) (protein tyrosine kinase STY), Na+/H+ exchanger regulatory factor (NHERF HGNC), potassium voltage-gated channel subfamily H member 2 (KCNH2), putative MAPK activating protein (PM20, PM21), ras homolog gene family, polymerase (DNA directed), delta regulatory subunit (50 kDa), leptin receptor involved in hematopoietin/interferon-class (D200- domain) cytokine receptor activity and thymidine kinase 2, mitochondrial TK2 HGNC and related genes. Significant alterations in these specific genes provide new directions for deeper mechanistic investigations that would lead to a better understanding of the molecular basis of mercury-induced toxicity and human diseases that may result from disturbances in the immune system.

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Project description:Background: Moderate hypothermia (32oC for 12 – 72 hours) has therapeutic applications, but the mechanisms by which it affects cellular function are unclear. We tested the hypothesis that moderate hypothermia produces broad changes in gene expression by human cells at the level of mRNA. Methods: Acute monocytic leukemia THP-1 cells were incubated under control conditions (37oC) or moderate hypothermia (32oC) for 24 hours. Cellular mRNA was extracted and a cold stress response was confirmed by examining the expression of the cold-inducible gene CIRBP by reverse transcription PCR (RT-PCR). Gene expression analysis was performed on seven sets of samples with Affymetrix U133A chips, using established statistical methods. Sequences were considered to be affected by cold if they showed statistically significant changes in expression and also met published post-hoc filter criteria (changes in geometric mean expression of 2-fold or greater and detection calls of “present” or “marginal” in at least half of the experiments). The changes in expression of a select number of these sequences were further confirmed by PCR. Results: 167 sequences met our prospectively defined criteria for a change in expression; 67 showed increases in expression and 100 showed decreases in expression. Consistent with prior literature, the cold-inducible genes CIRBP and RBM3 showed increases in expression. Functional categories affected by cold stress included genes involved in immune responses, cell growth, proliferation, and differentiation, and metabolism and biosynthesis. Several heat shock proteins (HSPs) showed decreases in expression. Conclusions: Moderate hypothermia produces substantial changes in cellular gene expression, in categories potentially of importance to systemic function. Interestingly, cold exposure without re-warming decreased the expression of several HSPs. Our in vitro findings suggest that moderate hypothermia in vivo might produce physiologically important changes in gene expression by circulating leukocytes. Keywords: Stress response / hypothermia

Dataset Information

A microarray analysis of the effects of moderate hypothermia and rewarming on gene expression by human hepatocytes (HepG2).

Publications

A microarray analysis of the effects of moderate hypothermia and rewarming on gene expression by human hepatocytes (HepG2).

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