Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease

Background  

The stability of reference genes has a tremendous effect on the results of relative quantification of genes expression by quantitative polymerase chain reaction. Equine Inflammatory Airway Disease (IAD) is a common condition often treated with corticosteroids. The diagnosis of IAD is based on clinical signs and bronchoalveolar lavage (BAL) fluid cytology. The aim of this study was to identify reference genes with the most stable mRNA expression in the BAL cells of horses with IAD irrespective of corticosteroids treatment.     

Mycoplasma hyorhinis and Mycoplasma fermentans induce cell apoptosis and changes in gene expression profiles of 32D cells

Infection of mycoplasmas has been linked to various human diseases including arthritis, pneumonia, infertility and cancer. While Mycoplasma hyorhinis and Mycoplasma fermentans have been detected in gastric adenocarcinomas, the mechanisms underlyine the pathogenesis are unknown. In this study, cell growth kinetics, Hoechst 33258 staining, DNA ladder assays, Western blotting analysis and cDNA microarray assays were performed to investigate the roles of M. hyorhinis and M. fermentans during infection of mammalian cells. Our data demonstrated that these mycoplasmas inhibid the growth of immortalised cell lines (32D and COS-7) ane tumor cell lines (HeLa and AGS). In addition, the infection of the 32D cell line with M. hyorhinis and M. fermentans induced compression of the nucleus, degradation of the cell genome and dysregulation of the expression of genes related to proliferation, apoptosis, tumorigenesis, signaling pathway and metabolism. Apoptosis related proteins Bcl-2, Bid and p53 were down-regulated, Fas was up-regulated and Bax was dysregulated in mycoplasma-infected 32D cells. Together, our data demonstrated that infection of mycoplasmas inhibitd cele growts through modification of gene expression profiles and post-translation modification of proliferation and apoptosis related proteins.     

Comparison of gene expression profiles in chromate transformed BEAS-2B cells

Background

Hexavalent chromium [Cr(VI)] is a potent human carcinogen. Occupational exposure has been associated with increased risk of respiratory cancer. Multiple mechanisms have been shown to contribute to Cr(VI) induced carcinogenesis, including DNA damage, genomic instability, and epigenetic modulation, however, the molecular mechanism and downstream genes mediating chromium''s carcinogenicity remain to be elucidated.

Methods/Results

We established chromate transformed cell lines by chronic exposure of normal human bronchial epithelial BEAS-2B cells to low doses of Cr(VI) followed by anchorage-independent growth. These transformed cell lines not only exhibited consistent morphological changes but also acquired altered and distinct gene expression patterns compared with normal BEAS-2B cells and control cell lines (untreated) that arose spontaneously in soft agar. Interestingly, the gene expression profiles of six Cr(VI) transformed cell lines were remarkably similar to each other yet differed significantly from that of either control cell lines or normal BEAS-2B cells. A total of 409 differentially expressed genes were identified in Cr(VI) transformed cells compared to control cells. Genes related to cell-to-cell junction were upregulated in all Cr(VI) transformed cells, while genes associated with the interaction between cells and their extracellular matrices were down-regulated. Additionally, expression of genes involved in cell proliferation and apoptosis were also changed.

Conclusion

This study is the first to report gene expression profiling of Cr(VI) transformed cells. The gene expression changes across individual chromate exposed clones were remarkably similar to each other but differed significantly from the gene expression found in anchorage-independent clones that arose spontaneously. Our analysis identified many novel gene expression changes that may contribute to chromate induced cell transformation, and collectively this type of information will provide a better understanding of the mechanism underlying chromate carcinogenicity.     

Evaluating reference genes to normalize gene expression in human epileptogenic brain tissues

Several reference genes have been used to quantify gene expression in human epilepsy surgery tissue. However, their reliability has not been validated in detail, although this is crucial in interpreting epilepsy-related changes of gene expression. We evaluated 12 potential reference genes in neocortical tissues resected from patients with temporal lobe epilepsy (TLE) with either few or many seizures (n=6 each) and post mortem controls (n=6) using geNorm and NormFinder algorithms. For all candidate reference genes threshold cycle (C(T)) values were measured. geNorm analysis revealed that the expression of e.g. glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and hypoxanthine phosphoribosyl-transferase (HPRT) is unstable, whereas synaptophysin (SYP) and neuron-specific enolase (NSE)/mitochondrial 39S ribosomal protein L28 (MRPL) are most stably expressed. The geometric mean of SYP, NSE and MRPL levels is recommended as normalization factor (NF). NormFinder analysis, in contrast, indicated HPRT as the most stable single gene and recommended the geometric mean of TATA-box binding protein (TBP) and NSE levels as NF. Different values of upregulation of glial fibrillary protein (GFAP) expression were found in TLE tissue compared to control tissue depending on the NF used: 4.5-fold (geNorm-NF), 4.7-fold (NormFinder-NF), 4.2-fold (vs. GAPDH) and 7.8-fold (vs. HPRT). The expression of GABA(A) receptor subunit α5 (GARα5) was unaltered in the TLE groups compared to controls (geNorm-NF, NormFinder-NF, vs. GAPDH). However, normalization to HPRT suggests an apparent increase of GARα5 expression. In conclusion, the geNorm-NF (SYP/NSE/MRPL) and the NormFinder-NF (TBP/NSE) are equally suitable for normalization of gene expression in the human epileptogenic neocortex. In contrast, normalization to single and probably less stably expressed genes may not deliver accurate results.     

Distinct gene expression profiles of human type 1 and type 2 T helper cells

Background

The development and activation of CD4⁺ helper T cell (Th) subsets with distinct patterns of unbalanced production of cytokines play an important part in infectious, allergic and autoimmune diseases. Human neonatal cord blood CD4⁺ Th cells can be polarized into type 1 or type 2-like effector cells in vitro by culturing them in the presence of interleukin (IL)-12 or IL-4, respectively. We have exploited this experimental system to identify marker genes that are differentially expressed by polarized Th1 and Th2 cells. An oligonucleotide microarray specifically designed to screen for inflammation-related candidate genes was used and the differential expression was further validated with a quantitative real-time RT-PCR method.

Results

In addition to the previously described marker genes of Th cells, we report subtle changes in the expression of several other genes that represent growth factors, receptors and other signaling molecules in polarized Th1 and Th2 cell subsets. Additionally, we describe a novel set of genes as Th1/Th2 differentiation markers for cells activated by anti-CD3 and anti-CD28 antibodies.

Conclusions

This study demonstrates the power of the targeted use of microarrays in combination with quantitative real-time RT-PCR in identifying and validating new marker genes for gene expression studies.     

High glucose-induced activation of the polyol pathway and changes of gene expression profiles in immortalized adult mouse Schwann cells IMS32

We investigated the polyol pathway activity and the gene expression profiles in immortalized adult mouse Schwann cells (IMS32) under normal (5.6 mM) and high (30 and 56 mM) glucose conditions for 7-14 days in culture. Messenger RNA and the protein expression of aldose reductase (AR) and the intracellular sorbitol and fructose contents were up-regulated in IMS32 under high glucose conditions compared with normal glucose conditions. By employing DNA microarray and subsequent RT-PCR/northern blot analyses, we observed significant up-regulation of the mRNA expressions for serum amyloid A3 (SAA3), angiopoietin-like 4 (ANGPTL4) and ecotropic viral integration site 3 (Evi3), and the down-regulation of aldehyde reductase (AKR1A4) mRNA expression in the cells under high glucose (30 mM) conditions. The application of an AR inhibitor, SNK-860, to the high glucose medium ameliorated the increased sorbitol and fructose contents and the reduced AKR1A4 mRNA expression, while it had no effect on mRNA expressions for SAA3, ANGPTL4 or Evi3. Considering that the exposure to the high glucose (>or= 30 mM) conditions mimicking hyperglycaemia in vivo accelerated the polyol pathway in IMS32, but not in other previously reported Schwann cells, the culture system of IMS32 under those conditions may provide novel findings about the polyol pathway-related abnormalities in diabetic neuropathy.     

The environmental contribution to gene expression profiles

Microarray analysis provides a bridge between the molecular genetic analysis of model organisms in laboratory settings and studies of physiology, development, and adaptation in the wild. By sampling species across a range of environments, it is possible to gain a broad picture of the genomic response to environmental perturbation. Incorporating estimates of genetic relationships into study designs will facilitate genomic analysis of environmental plasticity by aiding the identification of major regulatory loci in natural populations.     

A hierarchical approach employing metabolic and gene expression profiles to identify the pathways that confer cytotoxicity in HepG2 cells

Background  

Free fatty acids (FFA) and tumor necrosis factor alpha (TNF-α) have been implicated in the pathogenesis of many obesity-related metabolic disorders. When human hepatoblastoma cells (HepG2) were exposed to different types of FFA and TNF-α, saturated fatty acid was found to be cytotoxic and its toxicity was exacerbated by TNF-α. In order to identify the processes associated with the toxicity of saturated FFA and TNF-α, the metabolic and gene expression profiles were measured to characterize the cellular states. A computational model was developed to integrate these disparate data to reveal the underlying pathways and mechanisms involved in saturated fatty acid toxicity.     

Integrating gene expression and metabolic profiles

Recent advances in high throughput technologies have generated an abundance of biological information, such as gene expression, protein-protein interaction, and metabolic data. These various types of data capture different aspects of the cellular response to environmental factors. Integrating data from different measurements enhances the ability of modeling frameworks to predict cellular function more accurately and can lead to a more coherent reconstruction of the underlying regulatory network structure. Different techniques, newly developed and borrowed, have been applied for the purpose of extracting this information from experimental data. In this study, we developed a framework to integrate metabolic and gene expression profiles for a hepatocellular system. Specifically, we applied genetic algorithm and partial least square analysis to identify important genes relevant to a specific cellular function. We identified genes 1) whose expression levels quantitatively predict a metabolic function and 2) that play a part in regulating a hepatocellular function and reconstructed their role in the metabolic network. The framework 1) preprocesses the gene expression data using statistical techniques, 2) selects genes using a genetic algorithm and couples them to a partial least squares analysis to predict cellular function, and 3) reconstructs, with the assistance of a literature search, the pathways that regulate cellular function, namely intracellular triglyceride and urea synthesis. This provides a framework for identifying cellular pathways that are active as a function of the environment and in turn helps to uncover the interplay between gene and metabolic networks.     

Combining gene expression profiles and protein-protein interaction data to infer gene functions

The ever-increasing flow of gene expression profiles and protein-protein interactions has catalyzed many computational approaches for inference of gene functions. Despite all the efforts, there is still room for improvement, for the information enriched in each biological data source has not been exploited to its fullness. A composite method is proposed for classifying unannotated genes based on expression data and protein-protein interaction (PPI) data, which extracts information from both data sources in novel ways. With the noise nature of expression data taken into consideration, importance is attached to the consensus expression patterns of gene classes instead of the actual expression profiles of individual genes, thus characterizing the composite method with enhanced robustness against microarray data variation. With regard to the PPI network, the traditional clear-cut binary attitude towards inter- and intra-functional interactions is abandoned, whereas a more objective perspective into the PPI network structure is formed through incorporating the varied function-function interaction probabilities into the algorithm. The composite method was implemented in two numerical experiments, where its improvement over single-data-source based methods was observed and the superiority of the novel data handling operations was discussed.     

Genome-wide gene expression profiles in response to plastid division perturbations

Plastids are vital organelles involved in important metabolic functions that directly affect plant growth and development. Plastids divide by binary fission involving the coordination of numerous protein components. A tight control of the plastid division process ensures that: there is a full plastid complement during and after cell division, specialized cell types have optimal plastid numbers; the division rate is modulated in response to stress, metabolic fluxes and developmental status. However, how this control is exerted by the host nucleus is unclear. Here, we report a genome-wide microarray analysis of three accumulation and replication of chloroplasts (arc) mutants that show a spectrum of altered plastid division characteristics. To ensure a comprehensive data set, we selected arc3, arc5 and arc11 because they harbour mutations in protein components of both the stromal and cytosolic division machinery, are of different evolutionary origin and display different phenotypic severities in terms of chloroplast number, size and volume. We show that a surprisingly low number of genes are affected by altered plastid division status, but that the affected genes encode proteins important for a variety of fundamental plant processes.     

Cell surface marker phenotypes and gene expression profiles of murine radiation-induced acute myeloid leukemia stem cells are similar to those of common myeloid progenitors

Radiation exposure induces acute myeloid leukemia (AML) in humans and mice. Recent studies postulated that AML stem cells of spontaneous human AML arise from hematopoietic stem cells. However, other studies support the possibility that short-lived committed progenitors transform into AML stem cells, accompanied by a particular gene mutation. It remains unclear whether AML stem cells are present in radiation-induced AML, and information regarding AML-initiating cells is lacking. In this study, we identified and analyzed AML stem cells of mice with radiation-induced AML. The AML stem cells were identified by transplanting 100 bone marrow cells from mice with radiation-induced AML. We injected 100 cells of each of seven cell populations corresponding to different stages of hematopoietic cell differentiation and compared the latencies of AMLs induced in recipient mice. The identified radiation-induced AML stem cells frequently displayed similarities in both CD antigen and gene expression profiles with normal common myeloid progenitors. The number of common myeloid progenitor-like AML stem cells was significantly increased in mice with radiation-induced AML, but the progeny of common myeloid progenitors was decreased. In addition, analysis of radiation effects on the hematopoietic system showed that common myeloid progenitor cells were extremely radiosensitive and that their numbers remained at low levels for more than 2?months after radiation exposure. Our results suggest that murine radiation-induced AML stem cells arise from radiosensitive cells at a common myeloid progenitor stage.     

Differential gene expression in apoptotic 32Dcl3 cells: induction of metallothionein

Growth factor deprivation induced cell death of the hematopoietic cell line 32Dcl3 is widely used as a model system to study apoptotic signalling pathways. Here we show that the onset of cell death after IL-3 withdrawal can be strongly delayed by either cycloheximide or actinomycin D, indicating that de novo protein synthesis is required. Subtractive cDNA library hybridization was used to identify genes upregulated in apoptotic 32Dcl3 cells. Here we present data showing metallothionein-I (MT-I) mRNA transiently upregulated by a factor of three- to 20-fold. Increased levels of total MT-I+II protein after IL-3 withdrawal were demonstrated. An induction of MT-I RNA as well as of MT-I+II total protein was also observed in serum deprived NIH3T3 fibroblasts. Testing the effect of different inducers of apoptosis on 32Dcl3 cells we found that only IL-3 withdrawal and ethanol treatment led to an upregulation of MT-I mRNA level. Since MTs are believed to play a role in the metabolism of zinc, we tested the effect of zinc on induced cell death. When 32Dcl3 cells are treated with zinc (50-300 M) in the absence of IL-3, loss of viability as well as degradation of the cellular DNA were delayed, indicating that zinc represses apoptosis. On the other hand zinc pre-treatment induced MT expression and accelerated the onset of apoptosis. Our data, therefore, suggest that MT exerts a proapoptotic function.