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Functional genomics analysis of low concentration of ethanol in human hepatocellular carcinoma (HepG2) cells. Role of genes involved in transcriptional and translational processes.

ABSTRACT: We previously found that ethanol at millimolar level (1 mM) activates the expression of transcription factors with subsequent regulation of apoptotic genes in human hepatocellular carcinoma (HCC) HepG2 cells. However, the role of ethanol on the expression of genes implicated in transcriptional and translational processes remains unknown. Therefore, the aim of this study was to characterize the effect of low concentration of ethanol on gene expression profiling in HepG2 cells using cDNA microarrays with especial interest in genes with transcriptional and translational function. The gene expression pattern observed in the ethanol-treated HepG2 cells revealed a relatively similar pattern to that found in the untreated control cells. The pairwise comparison analysis demonstrated four significantly up-regulated (COBRA1, ITGB4, STAU2, and HMGN3) genes and one down-regulated (ANK3) gene. All these genes exert their function on transcriptional and translational processes and until now none of these genes have been associated with ethanol. This functional genomic analysis demonstrates the reported interaction between ethanol and ethanol-regulated genes. Moreover, it confirms the relationship between ethanol-regulated genes and various signaling pathways associated with ethanol-induced apoptosis. The data presented in this study represents an important contribution toward the understanding of the molecular mechanisms of ethanol at low concentration in HepG2 cells, a HCC-derived cell line.

SUBMITTER: Castaneda F

PROVIDER: S-EPMC1752234 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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Functional genomics analysis of low concentration of ethanol in human hepatocellular carcinoma (HepG2) cells. Role of genes involved in transcriptional and translational processes.

Castaneda Francisco F Rosin-Steiner Sigrid S Jung Klaus K

International journal of medical sciences 20061221 1

We previously found that ethanol at millimolar level (1 mM) activates the expression of transcription factors with subsequent regulation of apoptotic genes in human hepatocellular carcinoma (HCC) HepG2 cells. However, the role of ethanol on the expression of genes implicated in transcriptional and translational processes remains unknown. Therefore, the aim of this study was to characterize the effect of low concentration of ethanol on gene expression profiling in HepG2 cells using cDNA microarra ...[more]

PMID: 17211498

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Project description:Background: Little is known about the impact of nutrients on cellular transcriptional responses, especially in face of environmental stressors such as oxygen deprivation. Hypoxia-inducible factors (HIF) coordinate the expression of genes essential for adaptation to oxygen-deprived environments. A second family of oxygen-sensing genes known as the alpha-ketoglutarate-dependent dioxygenases are also implicated in oxygen homeostasis and epigenetic regulation. The relationship between nutritional status and cellular response to hypoxia is understudied. In vitro cell culture systems frequently propagate cells in media that contains excess nutrients, and this may directly influence transcriptional response in hypoxia. Methods: We studied the effect of glucose and glutamine concentration on HepG2 hepatoma transcriptional response to low oxygen and expression of hypoxia inducible factor-1α (HIF-1α). Mass spectrometry confirmed low oxygen perturbation of dioxygenase transcripts resulted in changes in DNA methylation. Results: Under normoxic conditions, we observed a significant upregulation of both HIF-target genes and oxygen-dependent dioxygenases in HepG2 cells cultured with physiological levels of glucose or glutamine relative to regular DMEM media, demonstrating that excess glutamine/glucose can mask changes in gene expression. Under hypoxic conditions, CA9 was the most upregulated gene in physiological glutamine media while TETs and FTO dioxygenases were downregulated in physiological glucose. Hypoxic regulation of these transcripts did not associate with changes in HIF-1α protein expression. Downregulation of TETs suggests a potential for epigenetic modulation. Mass-spectrometry quantification of modified DNA bases confirmed our transcript data. Hypoxia resulted in decreased DNA hydroxymethylation, which correlated with TETs downregulation. Additionally, we observed that TET2 expression was significantly downregulated in patients with hepatocellular carcinoma, suggesting that tumour hypoxia may deregulate TET2 expression resulting in global changes in DNA hydroxymethylation. Conclusion: Given the dramatic effects of nutrient availability on gene expression, future in vitro experiments should be aware of how excess levels of glutamine and glucose may perturb transcriptional responses.

Project description:Listeria monocytogenes (Lm) is a foodborne pathogen that can cause severe human illness. Standard control measures for restricting bacterial growth, such as refrigeration, are often inadequate as Lm grows well at low temperatures. To identify genes involved in growth at low temperatures, a powerful functional genomics method Tn-seq was performed in this study. This genome-wide screening comprehensively identified the known and novel genetic determinants involved in low-temperature growth. A novel gene lmo1366, encoding rRNA methyltransferase, was identified to play an essential role in Lm growth at 16°C. In contrast, the inactivation of lmo2301, a gene encoding the terminase of phage A118, significantly enhanced the growth of Lm at 16°C. The deletion of lmo1366 or lmo2301 resulted in cell morphology alterations and impaired the growth rate in milk and other conditions at low temperatures. Transcriptomic analysis revealed that the Δlmo1366 and Δlmo2301 mutants exhibited altered transcriptional patterns compared to the wild-type strain at 16°C with significant differences in genes involved in ribosome structural stability and function, and membrane biogenesis, respectively. This work uncovered novel genetic determinants involved in Lm growth at 16°C, which could lead to a better understanding of how bacteria survive and multiply at low temperatures. Furthermore, these findings could potentially contribute to developing novel antibacterial strategies under low-temperature conditions. IMPORTANCE Listeria monocytogenes is a Gram-positive pathogen that contributes to foodborne outbreaks due to its ability to survive at low temperatures. However, the genetic determinants of Lm involved in growth at low temperatures have not been fully defined. Here, the genetic determinants involved in the low-temperature growth of Lm were comprehensively identified on a genome-wide scale by Tn-seq. The gene lmo1366, encoding rRNA methyltransferase, was identified essential for growth under low-temperature conditions. On the other hand, the gene lmo2301, encoding terminase of phage A118, plays a negative role in bacterial growth at low temperatures. The transcriptomic analysis revealed the potential mechanisms. These findings lead to a better understanding of how bacteria survive and multiply at low temperatures and could provide unique targets for novel antibacterial strategies under low-temperature conditions.

Dataset Information

Functional genomics analysis of low concentration of ethanol in human hepatocellular carcinoma (HepG2) cells. Role of genes involved in transcriptional and translational processes.

Publications

Functional genomics analysis of low concentration of ethanol in human hepatocellular carcinoma (HepG2) cells. Role of genes involved in transcriptional and translational processes.

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