Microarray-based expression profiling in prostate tumors

High throughput gene expression profiling is increasingly becoming a desirable method for identifying genes differentially expressed in disease versus normal tissues. Microarrays and gene chips containing hundreds to thousands of genes of interest, both known and novel, can be used to establish the expression profile of numerous genes in a single experiment. In order to validate the hits emerging out of such an experiment it is necessary to use an appropriate panel of the cDNA repository. We investigated the usefulness of such a method to identify prostate cancer-specific genes. A microarray containing 588 known genes was analyzed using cDNA probes derived from normal and three independent prostate tumors. At least 19/588 genes were found to be differentially expressed in the tumors in comparison to the normal tissue. Among the nine test genes chosen, one gene, Glutathione-S-transferase theta 1 (GSTT1), showed a correlation with the microarray results when analyzed by RT-PCR. Using a comprehensive panel of normal and tumor tissues and cancer-derived cell lines, we have rapidly validated the expression relevance of GSTT1 in solid tumors. The microarray was also useful in the preliminary identification of androgen-regulated genes in the prostate tumor models. These results indicate that microarray in combination with a relevant cDNA repository can facilitate rapid identification of potential targets for therapy and diagnosis of prostate and other cancers.     

Monitoring therapy with gene expression profiling reveals physiological differences in drug action

Gene expression profiling has become a versatile tool for biomedical research, which allows the assessment of a wide variety of basic questions in cellular regulation, in particular when a large number of molecular parameters have changed. There are various applications in drug research for which gene expression profiling is a very suitable approach. This includes: target identification, target validation, validation of drug specificity and monitoring of drug action during therapy. The focus of this article is the therapy monitoring and the interpretation of the gene expression profiles in respect to physiological differences of drug action. As an example, we will discuss changes in gene expression in blood samples from CML patients treated with the tyrosine kinase inhibitor (imatinib mesylate) and compare the observed effects on gene expression with the effects of IFNalpha treatment. In comparison with other examples of therapy monitoring the potential of this application of gene expression profiling for optimizing individual therapy will be discussed.     

Expression profiling of neural cells reveals specific patterns of ethanol-responsive gene expression

Adaptive changes in gene expression are thought to contribute to dependence, addiction and other behavioral responses to chronic ethanol abuse. DNA array studies provide a nonbiased detection of networks of gene expression changes, allowing insight into functional consequences and mechanisms of such molecular responses. We used oligonucleotide arrays to study nearly 6000 genes in human SH-SY5Y neuroblastoma cells exposed to chronic ethanol. A set of 42 genes had consistently increased or decreased mRNA abundance after 3 days of ethanol treatment. Groups of genes related to norepinephrine production, glutathione metabolism, and protection against apoptosis were identified. Genes involved in catecholamine metabolism are of special interest because of the role of this pathway in mediating ethanol withdrawal symptoms (physical dependence). Ethanol treatment elevated dopamine beta-hydroxylase (DBH, EC 1.14.17.1) mRNA and protein levels and increased releasable norepinephrine in SH-SY5Y cultures. Acute ethanol also increased DBH mRNA levels in mouse adrenal gland, suggesting in vivo functional consequences for ethanol regulation of DBH. In SH-SY5Y cells, ethanol also decreased mRNA and secreted protein levels for monocyte chemotactic protein 1, an effect that could contribute to the protective role of moderate ethanol consumption in atherosclerotic vascular disease. Finally, we identified a subset of genes similarly regulated by both ethanol and dibutyryl-cAMP treatment in SH-SY5Y cells. This suggests that ethanol and cAMP signaling share mechanistic features in regulating a subset of ethanol-responsive genes. Our findings offer new insights regarding possible molecular mechanisms underlying behavioral responses or medical consequences of ethanol consumption and alcoholism.     

Gene expression profiling analysis reveals arsenic-induced cell cycle arrest and apoptosis in p53-proficient and p53-deficient cells through differential gene pathways

Arsenic (As) is a well-known environmental toxicant and carcinogen as well as an effective chemotherapeutic agent. The underlying mechanism of this dual capability, however, is not fully understood. Tumor suppressor gene p53, a pivotal cell cycle checkpoint signaling protein, has been hypothesized to play a possible role in mediating As-induced toxicity and therapeutic efficiency. In this study, we found that arsenite (As³⁺) induced apoptosis and cell cycle arrest in a dose-dependent manner in both p53^+/+ and p53^−/− mouse embryonic fibroblasts (MEFs). There was, however, a distinction between genotypes in the apoptotic response, with a more prominent induction of caspase-3 in the p53^−/− cells than in the p53^+/+ cells. To examine this difference further, a systems-based genomic analysis was conducted comparing the critical molecular mechanisms between the p53 genotypes in response to As³⁺. A significant alteration in the Nrf2-mediated oxidative stress response pathway was found in both genotypes. In p53^+/+ MEFs, As³⁺ induced p53-dependent gene expression alterations in DNA damage and cell cycle regulation genes. However, in the p53^−/− MEFs, As³⁺ induced a significant up-regulation of pro-apoptotic genes (Noxa) and down-regulation of genes in immune modulation. Our findings demonstrate that As-induced cell death occurs through a p53-independent pathway in p53 deficient cells while apoptosis induction occurs through p53-dependent pathway in normal tissue. This difference in the mechanism of apoptotic responses between the genotypes provides important information regarding the apparent dichotomy of arsenic's dual mechanisms, and potentially leads to further advancement of its utility as a chemotherapeutic agent.     

Microarray gene expression profiling in meningiomas and schwannomas

Microarray gene expression profiling is a high throughput system recently used in basic and applied research. It provides a large amount of data -at molecular level- that once acquired, must be functionally integrated in order to find common patterns within a defined group of biological samples. In addition to identification of differentially expressed genes and the establishment of gene regulation patterns, microarrays may also allow us to discover new tumor markers that could have a great impact on the improvement of clinical practice and therapeutics for cancer. The classification method used for cancer is currently based on the morphological characteristics of the biological samples. The information obtained with this method is limited, omitting many important tumor characteristics like the proliferation rate, the capacity of invasion and metastases, as well as the possible development of mechanisms of cellular resistance to treatment. Microarrays can be used in combination with conventional diagnostics as a helpful complement. In this review we focus on how this technology has contributed to our knowledge of the molecular pathogenesis of meningiomas and schwannomas, its potential role as a useful tool for tumor classification and its application in clinical practice.     

雷公藤红素纳米混悬剂的制备及其抗肿瘤作用研究

目的制备雷公藤红素纳米混悬剂,并进行体内外抗肿瘤作用研究。方法采用超声注入联合旋转蒸发法制备雷公藤红素纳米混悬剂,以粒径大小为指标,筛选合适的稳定剂;采用动态光散射法、透射电镜考察粒径和形态,并对其不同介质稳定性、体外释放、溶血性、体外抗肿瘤活性进行研究;建立H22荷瘤小鼠模型,以雷公藤红素注射液为对照组,2 mg/kg iv给药,考察体内抗肿瘤作用。结果聚乙二醇-聚己内酯(m PEG2000-PCL2000)为雷公藤纳米混悬剂的优良的稳定剂,所制备的纳米混悬剂粒径为(67.1±3.0)nm,Zeta电位为(-10.4±1.45)m V,多分散性指数为0.232±0.08,近乎为球形,分布比较均匀。在磷酸缓冲液(PBS)、血浆、生理盐水、5%葡萄糖中均稳定;体外缓慢释放,在144 h累积释放率达到74.04%;MTT结果显示雷公藤红素纳米混悬剂对HepG2细胞的毒性强于溶液(IC50,1.179μg/m L vs 2.377μg/m L,P0.05)。体内研究中雷公藤红素纳米混悬剂对H22荷瘤小鼠的的抑瘤率显著高于注射液组(70.36%vs 51.1%,P0.05)。结论制备的雷公藤红素纳米混悬剂粒径小、载药量高、稳定性好,显著提高了雷公藤红素的抗肿瘤效果,可以作为雷公藤红素作为抗肿瘤药物应用的合适剂型。     

Emerging treatments and gene expression profiling in high-risk medulloblastoma

The past decades have seen an increase in the survival rates of patients with standard-risk medulloblastoma. Efforts have, therefore, been focused on obtaining better results in the treatment of patients with high-risk tumors. In addition to consolidated therapies, novel approaches such as small molecules, monoclonal antibodies, and antiangiogenic therapies that aim to improve outcomes and quality of life are now available through new breakthroughs in the molecular biology of medulloblastoma. The advent of innovative anticancer drugs tested in brain tumors has important consequences for personalized therapy. Gene expression profiling of medulloblastoma can be used to identify the genes and signaling transduction pathways that are crucial for the tumorigenesis process, thereby revealing both new targets for therapy and sensitive/resistance phenotypes. The interpretation of microarray data for new treatments of patients with high-risk medulloblastoma, as well as other poor prognosis tumors, should be developed through a consensus multidisciplinary approach involving oncologists, neurosurgeons, radiotherapists, biotechnologists, bioinformaticists, and other professionals.     

Regulatory RNAs as mediators of virulence gene expression in bacteria

Bacteria exploit functional diversity of RNAs in a wide range of regulatory mechanisms to control gene expression. In last few years, small RNA molecules have been discovered at a staggering rate in bacteria, mainly in Escherichia coli. While functions of many of these RNA molecules are still not known, several of them behave as key effectors of adaptive responses, such as environmental cue recognition, stress response, and virulence control. Most fascinating, perhaps, is the discovery that mRNAs behave as direct sensors of small molecules or of environmental cues. The astonishing diversity of RNA-dependent regulatory mechanisms is linked to the dynamic properties and versatility of the RNA structure. In this review, we relate several recent studies in different bacterial pathogens that illustrate the diverse roles of RNA to control virulence gene expression.     

Aspects of lung cancer gene expression profiling

Lung cancer is the most common cancer in the world. In the majority of cases, a tumor will have already become disseminated at the time of diagnosis, and extensive efforts to improve diagnosis and therapy have had no major success so far. Lung cancer gene expression profiling provides novel diagnostic and prognostic markers and a basis for targeted therapies involving small-molecule and antibody-based approaches. To enhance further research and to translate knowledge gained at the molecular level into novel therapeutic concepts, this review summarizes gene expression analyses of lung cancer performed at the RNA level using suppression subtractive hybridization, serial analysis of gene expression and cDNA and oligonucleotide microarrays, and discusses major findings.     

MicroRNA expression profiling involved in MC-LR-induced hepatotoxicity using high-throughput sequencing analysis

Microcystin-LR (MC-LR), the most common microcystin (MC) present in water is known to pose a significant threat to human health especially hepatotoxicity. However, the specific molecular mechanisms underlying MC-LR-induced hepatic cellular damage still remain to be determined. MicroRNAs (miRNAs) are known to play key roles in cellular processes including development, cell proliferation and responsiveness to stress. Thus, this study aimed to examine, whether miRNAs were involved in the observed MC-LR-mediated liver damage using miRNA profiling of a human normal liver cell line HL7702 using high-throughput sequencing techniques. Protein phosphatase 2A (PP2A) activity, an established biomarker of microcystin toxicity, was determined 24 hr following treatment with the algal toxin to confirm responsiveness. Data demonstrated that MC-LR significantly inhibited PP2A activity in a concentration-dependent manner with inhibitory concentration (IC₅₀) value of 4.6 μM. Compared with control cells, treatment with MC-LR at concentrations of 1, 2.5, 5 or 10 μM significantly modified expression of levels of 3, 10, 9, and 99 miRNAs, respectively. Expression levels of miR-15b-3p were significantly increased in all 4 treatment groups, while miR-4521 expression levels were markedly reduced. In the case of miR-451a, 1, 5 or 10 μM also significantly lowered expression levels. However, a significant rise in miR-451a was noted in cells exposed to 2.5 μM toxin. The results obtained from miRNA differential expression levels were confirmed by real-time fluorescent quantitative PCR (qPCR). Gene Ontology (GO) enrichment analysis of hepatic cells demonstrated that miRNAs significantly altered were involved in systems development, metabolism, and protein binding. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis data showed that target genes of differentially expressed miRNAs in liver cells predominantly participated in mechanistic target of rapamycin kinase (mTOR), Ras, Ras-related protein 1 (Rap1), hypoxia inducible factor 1 (HIF-1), and cancer development. In summary, evidence indicates that MC-LR-induced hepatotoxicity may be associated with alterations in miRNAs. Evidence indicates that alterations in miR-451a, miR-4521 and miR-15b-3p may be involved in the observed MC-LR- induced hepatotoxicity     

Global gene expression profiling of the polyamine system in suicide completers

In recent years, gene expression, genetic association, and metabolic studies have implicated the polyamine system in psychiatric conditions, including suicide. Given the extensive regulation of genes involved in polyamine metabolism, as well as their interconnections with the metabolism of other amino acids, we were interested in further investigating the expression of polyamine-related genes across the brain in order to obtain a more comprehensive view of the dysregulation of this system in suicide. To this end, we examined the expression of genes related to polyamine metabolism across 22 brain regions in a sample of 29 mood-disordered suicide completers and 16 controls, and identified 14 genes displaying differential expression. Among these, altered expression of spermidine/spermine N1-acetyltransferase, spermine oxidase, and spermine synthase, has previously been observed in brains of suicide completers, while the remainder of the genes represent novel findings. In addition to genes with direct involvement in polyamine metabolism, including S-adenosylmethionine decarboxylase, ornithine decarboxylase antizymes 1 and 2, and arginase II, we identified altered expression of several more distally related genes, including aldehyde dehydrogenase 3 family, member A2, brain creatine kinase, mitochondrial creatine kinase 1, glycine amidinotransferase, glutamic-oxaloacetic transaminase 1, and arginyl-tRNA synthetase-like. Many of these genes displayed altered expression across several brain regions, strongly implying that dysregulated polyamine metabolism is a widespread phenomenon in the brains of suicide completers. This study provides a broader view of the nature and extent of the dysregulation of the polyamine system in suicide, and highlights the importance of this system in the neurobiology of suicide.     

Molecular profiling of hepatotoxicity induced by a aminoguanidine carboxylate in the rat: gene expression profiling

The hepatotoxicity of the aminoguanidine carboxylate 2-[1-[hydrazino(imino)methyl]hydrazino]acetic acid was characterized using oligonucleotide micro arrays, with the goal to select compounds from the same class with lower toxicity potential. The approach included a 14-day repeated- and a single-dose study in the rat as well as in vitro studies. Common gene expression changes could be followed from in vivo to in vitro studies. Anyhow, comparing the in vivo and in vitro response of the compound on gene expression, significant discrepancies were detected. Many of the genes whose mRNA levels were increased/decreased in the livers of the animals treated with toxic doses of the compound, were expressed at higher/lower levels in control hepatocytes than in control liver. The expression of the majority of these genes was not affected by in vitro treatment. These data question the use of gene expression analysis as a marker for drug response in vitro and illustrate the need of a careful characterization of in vitro systems. The results presented show that array-based gene expression analysis can lead to a better understanding of the molecular basis of drug-induced liver injury and, potentially, be used in the selection process for compounds and in the design of safer drugs.     

Gene expression profiling of DEHP-treated cardiomyocytes reveals potential causes of phthalate arrhythmogenicity

BackgroundDi-(2-ethylhexyl)-phthalate (DEHP) is a widely used plasticizer that imparts flexibility to polyvinyl chloride. We have recently reported that clinically relevant concentrations of DEHP can affect electrical coupling between cardiac myocytes causing significant rhythm disturbances. The underlying causes for this effect are currently unknown.ObjectivesTo use data on global mRNA expression as a tool to reveal possible pathways leading to arrhythmogenic effects of DEHP.MethodsRat neonatal cardiomyocytes were treated with 50 μg/mL DEHP for 72 h. Extracted RNA samples were hybridized onto Affymetrix Rat Gene 1.0 ST arrays. The mRNA expression of a subset of genes was validated by qRT-PCR. In a second set of experiments, cells were treated in a concentration dependent manner to identify genes affected by low DEHP concentrations.ResultsDEHP exposure is associated with global changes in mRNA expression, with differentially expressed genes overrepresented in 47 Gene Ontology categories. Modified expression was detected for genes associated with cell electrical activity, calcium handling, adhesion and microtubular transport. For a number of key proteins, including kinesin, TGFβ2, α-tubulin, and α1 &; β1 integrins, changes in mRNA levels were confirmed on the level of the protein expression. A number of genes associated with cell adhesion and electrical activity were identified as early DEHP targets as they were affected by concentrations as low as 1 μg/mL.ConclusionsExposure of neonatal rat cardiomyocytes to clinically relevant DEHP concentrations leads to global changes in mRNA expression. These changes help to explain the arrhythmogenic effects of phthalates on these cells.