Early Gene Changes Research Articles

Early Changes in Structural and Stress-Like Genes after Radiation to the Submental Muscles

INTRODUCTION: Radiation treatments for head and neck cancer can lead to early alterations in swallowing kinematics that ultimately develop into dysphagia and require modifications in oral intake. The submental muscles (ie mylohyoid) are located inferior to the mandible and are crucial for swallowing function. Expression of collagen and heat shock proteins can be altered following muscle injury, which can impair muscle fiber regeneration leading to conditions of muscle atrophy or fibrosis. The aim of this study was to evaluate collagen and heat shock protein (HSP) 72 (HSPA1A/B) expression in a rat model of radiation-induced dysphagia. HSP72 is known to increase in response to stress and is needed to mediate muscle repair after injury. METHODS: We hypothesized that the submental muscles treated with 48 Gy of fractionated radiation would have increased expression of HSPA1A, HSPA1B, COL1, COL3, and COL4 genes at 2 and 4 weeks post treatment. Relative gene expression was measured using RT-PCR. RESULTS: The results showed that there was a sequential increase in expression of HSPA1A, HSPA1B, COL1, and COL3 at both the 2-week and 4-week post irradiation points. There was a greater increase in expression in COL4 at the 2-week compared to the 4-week post irradiation point. The highest change in gene expression was observed with HSPA1A and COL3. CONCLUSION: Results support the idea that irradiation treatment provokes early gene changes that may contribute to modifying the muscle structure and promoting dysphagia which can lead to decreased quality of life for head and neck cancer patients.

Mobile Ex Vivo Perfusion of Donor Lungs Alters Early Lung Gene Expression Profiles

<h3>Purpose</h3> Despite increasing use of mobile <i>ex vivo</i> lung perfusion (mEVLP) prior to lung transplant, very little is known about biological changes induced by lung perfusion and how long they may last. Use of mEVLP has been associated with improved early graft function, but the mechanisms of this protection remain unknown. We hypothesized that use of mEVLP induces early gene changes that alter early inflammatory responses. <h3>Methods</h3> We collected lung biopsies from 6 donor lungs with mEVLP (TransMedics OCS™) prior to lung transplant, and 6 patients whose donor lungs were preserved in Perfadex solution on ice prior to transplant. EVLP and non-EVLP donors were matched by donor gender, age and smoking status, as well as recipient age, gender and lung disease. Standard immunosuppression for each group was employed with Basiliximab induction. We compared biopsies obtained 1) prior to implant during back-table preparation, 2) at the end of the surgery, after reperfusion, but before chest closure, and 3) 1 month after transplant (Follow up). We measured and analyzed 785 genes using nCounter® human organ transplant panel with nSolver (version 4.0, NanoString) at each time point. <h3>Results</h3> Prior to implant, mEVLP lungs had higher levels of expression for genes associated with apoptosis and cell cycle regulation, autophagy, chemokine, and cytokine signaling. After surgical implantation and reperfusion, mEVLP lungs had increased expression of genes associated with adaptive immune system, apoptosis and cell cycle regulation, autophagy, cell- extracellular matrix interaction score, MAPK signaling, oxidative stress, TH1 differentiation, TGF-β signaling, and TH-17 differentiation. Tissue homeostasis was lower in mEVLP. One month after surgery, transbronchial biopsies from lungs that had undergone mEVLP demonstrated lower levels of angiogenesis, autophagy, inflammasomes, MAPK signaling, metabolism, mTOR signaling, and oxidative stress. <h3>Conclusion</h3> In general, our results demonstrate higher levels of inflammation in mEVLP lungs prior to and immediately after transplant. However, one month after transplant, mEVLP lungs have lower levels of inflammation and oxidative stress. It is possible that early robust gene responses in mEVLP lungs are followed by a more rapid recovery of inflammation.

Host response transcriptomic analysis of Crimean-Congo hemorrhagic fever pathogenesis in the cynomolgus macaque model

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne RNA virus prevalent in Asia, Europe, and Africa, and can cause a hemorrhagic disease (CCHF) in humans with mortality rates as high as 60%. A general lack of both effective medical countermeasures and a comprehensive understanding of disease pathogenesis is partly driven by an historical lack of viable CCHF animal models. Recently, a cynomolgous macaque model of CCHF disease was developed. Here, we document the targeted transcriptomic response of non-human primates (NHP) to two different CCHFV strains; Afghan09-2990 and Kosova Hoti that both yielded a mild CCHF disease state. We utilized a targeted gene panel to elucidate the transcriptomic changes occurring in NHP whole blood during CCHFV infection; a first for any primate species. We show numerous upregulated genes starting at 1 day post-challenge through 14 days post-challenge. Early gene changes fell predominantly in the interferon stimulated gene family with later gene changes coinciding with an adaptive immune response to the virus. There are subtle differences between viral strains, namely duration of the differentially expressed gene response and biological pathways enriched. After recovery, NHPs showed no lasting transcriptomic changes at the end of sample collection.

Inhibition of Activin A Ameliorates Skeletal Muscle Injury and Rescues Contractile Properties by Inducing Efficient Remodeling in Female Mice

Activin A, a member of the transforming growth factor-β superfamily, provides pleiotropic regulation of fibrosis and inflammation. We aimed at determining whether selective inhibition of activin A would provide a regenerative benefit. The introduction of activin A into normal muscle increased the expression of inflammatory and muscle atrophy genes Tnf, Tnfrsf12a, Trim63, and Fbxo32 by 3.5-, 10-, 2-, and 4-fold, respectively. The data indicate a sensitive response of muscle to activin A. Two hours after cardiotoxin-induced muscle damage, local activin A protein expression increased by threefold to ninefold. Neutralization of activin A with a specific monoclonal antibody in this muscle injury model decreased the muscle protein levels of lymphotoxin α and Il17a by 32% and 42%, respectively. Muscle histopathological features showed that activin A antibody-treated mice displayed an increase in muscle degradation, with the concomitant 9.2-fold elevation in F4/80-positive cells 3 days after injury. At the same time, the number of Pax7/Myod1-positive cells also increased, indicative of potentiated muscle precursor activation. Ultimately, activin A inhibition resulted in rapid recovery of muscle contractile properties indicated by a restoration of maximum and specific force. In summary, selective inhibition of activin A with a monoclonal antibody in muscle injury leads to the early onset of tissue degradation and subsequent enhanced myogenesis, thereby accelerating muscle repair and functional recovery.

Identification of early target genes of aflatoxin B1 in human hepatocytes, inter-individual variability and comparison with other genotoxic compounds

Gene expression profiling has recently emerged as a promising approach to identify early target genes and discriminate genotoxic carcinogens from non-genotoxic carcinogens and non-carcinogens. However, early gene changes induced by genotoxic compounds in human liver remain largely unknown. Primary human hepatocytes and differentiated HepaRG cells were exposed to aflatoxin B1 (AFB1) that induces DNA damage following enzyme-mediated bioactivation. Gene expression profile changes induced by a 24 h exposure of these hepatocyte models to 0.05 and 0.25 μM AFB1 were analyzed by using oligonucleotide pangenomic microarrays. The main altered signaling pathway was the p53 pathway and related functions such as cell cycle, apoptosis and DNA repair. Direct involvement of the p53 protein in response to AFB1 was verified by using siRNA directed against p53. Among the 83 well-annotated genes commonly modulated in two pools of three human hepatocyte populations and HepaRG cells, several genes were identified as altered by AFB1 for the first time. In addition, a subset of 10 AFB1-altered genes, selected upon basis of their function or tumor suppressor role, was tested in four human hepatocyte populations and in response to other chemicals. Although they exhibited large variable inter-donor fold-changes, several of these genes, particularly FHIT, BCAS3 and SMYD3, were found to be altered by various direct and other indirect genotoxic compounds and unaffected by non-genotoxic compounds. Overall, this comprehensive analysis of early gene expression changes induced by AFB1 in human hepatocytes identified a gene subset that included several genes representing potential biomarkers of genotoxic compounds.

Exposure to atrazine affects the expression of key genes in metabolic pathways integral to energy homeostasis in Xenopus laevis tadpoles

In our laboratory, Xenopus laevis tadpoles exposed throughout development to 200 or 400 μg/L atrazine, concentrations reported to periodically occur in puddles, vernal ponds and runoff soon after application, were smaller and had smaller fat bodies (the tadpole's lipid storage organ) than controls. It was hypothesized that these changes were due to atrazine-related perturbations of energy homeostasis. To investigate this hypothesis, selected metabolic responses to exposure at the transcriptional and biochemical levels in atrazine-exposed tadpoles were measured. DNA microarray technology was used to determine which metabolic pathways were affected after developmental exposure to 400 μg/L atrazine. From these data, genes representative of the affected pathways were selected for assay using quantitative real time polymerase chain reaction (qRT-PCR) to measure changes in expression during a 2-week exposure to 400 μg/L. Finally, ATP levels were measured from tadpoles both early in and at termination of exposure to 200 and 400 μg/L. Microarray analysis revealed significant differential gene expression in metabolic pathways involved with energy homeostasis. Pathways with increased transcription were associated with the conversion of lipids and proteins into energy. Pathways with decreased transcription were associated with carbohydrate metabolism, fat storage, and protein synthesis. Using qRT-PCR, changes in gene expression indicative of an early stress response to atrazine were noted. Exposed tadpoles had significant decreases in acyl-CoA dehydrogenase (AD) and glucocorticoid receptor protein (GR) mRNA after 24 h of exposure, and near-significant ( p = 0.07) increases in peroxisome proliferator-activated receptor β (PPAR-β) mRNA by 72 h. Decreases in AD suggested decreases in fatty acid β-oxidation while decreases in GR may have been a receptor desensitization response to a glucocorticoid surge. Involvement of PPAR-β, an energy homeostasis regulatory molecule, also suggested changes in energy status. Despite, or possibly because of, these early gene changes, there were no differences in either absolute ATP levels or ADP:ATP ratios early in the exposure. However, livers from animals exposed to 200 μg/L atrazine had near-significant ( p = 0.06) increases in ADP:ATP ratios at the end of exposure suggesting tadpoles may have had difficulty maintaining energy homeostasis. Perturbations in the expression of genes regulating energy metabolism by 24 h into exposure to 400 μg/L atrazine was noteworthy, especially since these tadpoles were significantly smaller than controls by 72 h of exposure.

Differences in Lung and Right Ventricle Responses to Sustained and Intermittent Hypoxia

Sustained hypoxia (SH) induces pulmonary arterial hypertension, a model of right heart failure. Intermittent Hypoxia (IH) induces both pulmonary and systemic arterial hypertension and is a model of arterial hypoxemia accompanying occlusive sleep apnea. Our long‐term goal is to capitalize on differences between these two models to dissect out the pathophysiological mechanisms that regulate lung and heart function. We hypothesized that sustained and intermittent hypoxia would differentially alter lung and heart structure and function. We collected left ventricle (LV), right ventricle (RV), and lung tissue from three groups of rats subjected to 7 days of a) SH (n=12); b) IH (n=12); and c) normoxic controls (n=18). The lung to body weight ratios were increased in the SH group compared to the normoxic and IH groups (7.03±0.31 mg/g vs 4.72±0.14 mg/g and 4.74±0.16 mg/g, respectively (p&lt;0.05 for SH compared to both normoxic and IH groups). The RV to body ratios were also increased in the SH group compared to the normoxic and IH groups (0.47±0.01 mg/g vs 0.32±0.01 mg/g and 0.36±0.01 mg/g, respectively (p &lt;0.05 for SH compared to both normoxic and IH groups). The increase in the lung and RV masses indicates fluid accumulation in these organs, consistent with pulmonary hypertension and right heart dysfunction. The LV to body weight ratios were similar among the groups (2.35±0.07 mg/g for the SH; 2.29±0.03 mg/g for the normoxia, and 2.22±0.04 mg/g for the IH; p=ns). Our results show that the two models display different responses in terms of increases in organ weights. We can now use these tissues to explore changes in extracellular matrix composition to determine if early gene changes can be delineated in the two types of hypoxia.

Early gene changes induced by isotretinoin in the skin provide clues to its mechanism of action

Gene expression analysis of patient skin at one-week of oral isotretinoin therapy for severe acne illustrates the initial changes caused by 13-cis RA. Focus on the function of these significantly changed genes provides clues to its mechanism of action in skin. Significant gene changes included the up-regulation of lipocalin 2, which encodes the protein neutrophil gelatinase associated lipocalin (NGAL). Detailed studies of NGAL’s localization and function within the sebaceous gland demonstrated that NGAL mediates the apoptotic action of 13-cis RA on sebaceous glands, thereby suggesting that agents which selectively induce NGAL expression in sebaceous glands might represent therapeutic alternatives to the use of 13-cis RA for the treatment of acne

Transcriptional Profiling Identifies Cyclin D1 as a Critical Downstream Effector of Mutant Epidermal Growth Factor Receptor Signaling

Activating mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain determine responsiveness to EGFR tyrosine kinase inhibitors in patients with advanced non-small cell lung cancer (NSCLC). The modulation of transcriptional pathways by mutant EGFR signaling is not fully understood. Previously, we and others identified a single base pair change leading to a threonine to methionine (T790M) amino acid alteration in the ATP-binding pocket of the EGFR as a common mechanism of acquired resistance. The gefitinib-resistant, T790M-mutant H1975 NSCLC cell line undergoes prominent growth arrest and apoptosis when treated with the irreversible EGFR inhibitor, CL-387,785. We did a transcriptional profiling study of mutant EGFR target genes that are differentially expressed in the "resistant" gefitinib-treated and the "sensitive" CL387,785-treated H1975 cells to identify the pivotal transcriptional changes in NSCLC with EGFR-activating mutations. We identified a small subset of early gene changes, including significant reduction of cyclin D1 as a result of EGFR inhibition by CL-387,785 but not by gefitinib. The reduction in cyclin D1 transcription was associated with subsequent suppression of E2F-responsive genes, consistent with proliferation arrest. Furthermore, cyclin D1 expression was higher in EGFR-mutant lung cancer cells compared with cells with wild-type EGFR. EGFR-mutant cells were routinely sensitive to the cyclin-dependent kinase inhibitor flavopiridol, confirming the functional relevance of the cyclin D axis. These studies suggest that cyclin D1 may contribute to the emergence of EGFR-driven tumorigenesis and can be an alternative target of therapy.

Doxazosin Induces Apoptosis of Benign and Malignant Prostate Cells via a Death Receptor–Mediated Pathway

Quinazoline-based alpha1-adrenoceptor antagonists such as doxazosin and terazosin have been previously shown to induce apoptosis in prostate cancer cells via an alpha1-adrenoceptor-independent pathway, involving activation of transforming growth factor-beta1 (TGF-beta1) signaling. In this study, the molecular events initiating this apoptotic effect were further investigated in vitro using the human androgen-independent prostate cancer cells PC-3 and the human benign prostate epithelial cells BPH-1. Quantitative microarray assays were done in PC-3 and BPH-1 cells after treatment with doxazosin (25 micromol/L, 6 and 24 hours) to identify the early gene changes. Transient changes in the expression of several apoptosis regulators were identified, including up-regulation of Bax and Fas/CD95 and down-regulation of Bcl-xL and TRAMP/Apo3. Moreover, there were significant changes in the expression pattern of signaling components of the extracellular matrix such as integrins alpha2, alphaV, beta1, and beta8. Western blot analysis revealed activation of caspase-8 and caspase-3 within the first 6 to 12 hours of treatment with doxazosin in both PC-3 and BPH-1 cells. Doxazosin-induced apoptosis was blocked by specific caspase-8 inhibitors, supporting the functional involvement of caspase-8 in doxazosin-induced apoptosis. The effect of doxazosin on recruitment of Fas-associated death domain (FADD) and procaspase-8 to the Fas receptor was examined via analysis of death-inducing signaling complex formation. Doxazosin increased FADD recruitment and subsequent caspase-8 activation, implicating Fas-mediated apoptosis as the underlying mechanism of the effect of doxazosin in prostate cells. These results show that doxazosin exerts its apoptotic effects against benign and malignant prostate cells via a death receptor-mediated mechanism with a potential integrin contribution towards cell survival outcomes.

Changes in immediate early gene expression in the rat brain after unilateral lesions of the hippocampus

Activity of the immediate early genes c- fos and zif268 was compared across hemispheres in rats with unilateral, excitotoxic lesions of the hippocampus (dentate gyrus and CA fields 1–4). Counts of the protein products of these genes were made shortly after rats performed a test of spatial working memory in the radial-arm maze, a task that is sensitive to bilateral lesions of the hippocampus. Unilateral hippocampal lesions produced evidence of widespread hypoactivity. Significant reductions in immediate early gene counts were observed within all three anterior thalamic nuclei, as well as the entorhinal, perirhinal, and postrhinal cortices, and much of the subicular complex. In contrast, no observable changes were detected in the anterior cingulate, infralimbic or prelimbic cortices, as well as several amygdala nuclei, even though many of these regions receive projections from the subiculum. Instead, the immediate early gene changes were closely linked to sites that are thought to be required for successful task performance, with both immediate early genes giving similar patterns of results. The findings support the notion that the anterior thalamic nuclei, hippocampus, and parahippocampal cortices form the key components of an interdependent neuronal network involved in spatial mnemonic processing.

Gene expression profiling in the inductive human hematopoietic microenvironment

Human hematopoietic stem cells (HSCs) and their progenitors can be maintained in vitro in long-term bone marrow cultures (LTBMCs) in which constituent HSCs can persist within the adherent layers for up to 2 months. Media replenishment of LTBMCs has been shown to induce transition of HSCs from a quiescent state to an active cycling state. We hypothesize that the media replenishment of the LTBMCs leads to the activation of important regulatory genes uniquely involved in HSC proliferation and differentiation. To profile the gene expression changes associated with HSC activation, we performed suppression subtractive hybridization (SSH) on day 14 human LTBMCs following 1-h media replenishment and on unmanipulated controls. The generated SSH library contained 191 differentially up-regulated expressed sequence tags (ESTs), the majority corresponding to known genes related to various intracellular processes, including signal transduction pathways, protein synthesis, and cell cycle regulation. Nineteen ESTs represented previously undescribed sequences encoding proteins of unknown function. Differential up-regulation of representative genes, including IL-8, IL-1, putative cytokine 21/HC21, MAD3, and a novel EST was confirmed by semi-quantitative RT-PCR. Levels of fibronectin, G-CSF, and stem cell factor also increased in the conditioned media of LTBMCs as assessed by ELISA, indicating increased synthesis and secretion of these factors. Analysis of our library provides insights into some of the immediate early gene changes underlying the mechanisms by which the stromal elements within the LTBMCs contribute to the induction of HSC activation and provides the opportunity to identify as yet unrecognized factors regulating HSC activation in the LTBMC milieu.

Early and late molecular events in neurodegeneration and neuroprotection in Parkinson's disease MPTP model as assessed by cDNA microarray; the role of iron.

Possible cell death mechanisms for pars compacta nigro-striatal dopamine neurons in Parkinson's disease include oxidative stress, inflammatory processes, nitric oxide iron accumulation, glutamate toxicity and diminished neurotrophic factor responses. There is a notion that Parkinson's disease is not a single disorder but a syndrome that can be initiated by several factors. Because of limitations of biochemical methods in the global analysis of neuronal death, a full picture of events has not been established. However, recently developed cDNA microarray or microchips, in which the global expression of thousands of genes can be assessed simultaneously, is changing the prospect for understanding the disease process, its progression, response to drugs, etc. The neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is considered the most valid model of Parkinson's disease. We employed the technique of cDNA microarray gene expression to determine the mechanism of action of MPTP in mouse substantia nigra. Also, we studied neuroprotective processes induced by several compounds, including R-apomorphine and the green tea polyphenol epigallo-catechin-3-gallate (EGCG). This was done in two ways: (1) the time-dependent acute effect of MPTP, for determining which of the initial genes might lead to dopamine neuron death and (2) gene expression at the time of MPTP-induced dopamine neuron death. We observed that early (acute MPTP) gene expression differs from effects seen at the time of death (chronic MPTP), and that early gene changes are crucial for setting into action genes that eventually cause dopamine neuron death. Furthermore, this process is a cascade of "domino" effects, some of which were previously established by biochemical means. However, our findings show an additional large number of events previously unknown. The neuroprotective drugs reversed some but not all of the gene expression, suggesting involvement of these genes in the neurodegenerative process. Because of the profound complexity of "domino" effect it is now reasonable to understand why a single neuroprotective drug has not shown clinical neuroprotective efficacy. Future multi neuroprotective drugs may be necessary for treatment of not only Parkinson's disease, but other neurodegenerative diseases (e.g. Alzheimer's disease) and detrimental states (e.g. ischaemia).

Effects of Cadmium on Bone: Anin VivoModel for the Early Response

Cadmium (Cd) exposure induces bone resorptionin vitroandin vivothat can lead to low bone mass and increased incidence of fracture. We have developed an animal model for following the early skeletal response to Cd. A low-calcium (but not calcium-deficient) diet is used to increase gastrointestinal absorption of calcium so that the endogenous fecal calcium excretion is essentially the total fecal calcium excretion. The bone response is followed by quantitation of stable fecal calcium and does not require a radioactive label. After mice were adjusted to a low-calcium diet, Cd was administered by a single gavage and fecal calcium was monitored to determine the magnitude of the calcium release from bone. Fecal calcium excretion (μg Ca/hr; mean ± SE) remained at the background level for 8 hr (13.6 ± 1.8,n= 18) but increased during the 8- to 24-hr and 24- to 56-hr collection periods (43.8 ± 6.8,n= 12; 50.75 ± 3.7,n= 6, respectively). The bone response was transient and dropped to nearly background levels during the 56- to 104-hr collection period. Blood calcium levels were normal throughout the time course. Bone resorption occurred at Cd levels of 7.9 ± 0.7 μg/liter blood (mean ± SE,n= 6), which is in the range of occupational exposure levels. The transient nature of the bone response contrasted to the slow but continuing rise observed in blood Cd levels. These results suggest that a threshold level of Cd is required for a bone response but that chronic levels of Cd in blood do not necessarily indicate the occurrence of continuous active bone resorption. This model can be used to probe early gene changes (prior to the bone response) that may be occurring in response to Cd exposure.