72 research outputs found
Four-laser scanning confocal system for microarray analysis.
No full textWe have constructed a confocal scanner suitable for routine microarray analysis from commercially available parts. We have outlined the details that should be considered when designing such an instrument and listed some of the specific components comprising the system [the full list of system components is available on CD from the corresponding author (D.J.G.) at no charge]. Here, we describe the methods used to test the linearity and sensitivity of the instrument. Performance was evaluated with two commonly used dyes, fluorescein and Cy5. While the instrument had a linear correlation between the dye concentration and fluorescence intensity, the observed deviation from a slope of 1.0 underscores the importance of running multipoint calibration experiments to obtain accurate dye quantitation over the full dynamic range of the scanner. This method has utility in testing commercial instruments in addition to the scanner described here. An array with over 300 spots dyed with Cy3 was scanned with our instrument and a high-end commercial instrument. The agreement between the two instruments was very good over a 1000-fold intensity range. Our scanner is a cost-effective alternative to more costly commercial scanners with similar capabilities
NFκB activation and stimulation of chemokine production in normal human macrophages by the gadolinium-based magnetic resonance contrast agent Omniscan: possible role in the pathogenesis of nephrogenic systemic fibrosis.
No full textNephrogenic systemic fibrosis (NSF) is a generalised fibrotic disorder occurring in certain individuals with renal insufficiency exposed to gadolinium-based contrast agents (GdBCA) for MRI. Histopathological examination of affected tissues shows increased numbers of activated macrophages. To elucidate the mechanisms responsible for macrophage activation, the effects of the GdBCA Omniscan on normal human macrophage global gene expression, chemokine production and nuclear factor κB (NFκB) activation was examined. Normal human monocyte-derived macrophages were incubated with Omniscan (50 mM) and their gene expression analysed by microarrays and real-time PCR. Macrophage chemokine production was assayed by multiplex ELISA. NFκB activation was assessed by NFκB nuclear localisation and quantitation of intracellular levels of inducible nitric oxide synthase (iNOS) protein. A specific cell-permeable NFκB peptide inhibitor was used to abrogate NFκB stimulation of chemokine and iNOS protein levels. CCL8/MCP-2 in affected skin of patients with NSF was examined by indirect immunofluorescence. Omniscan caused a profound change in the transcriptome of differentiated human normal macrophages in vitro, including a large increase in the expression of genes encoding CC and CXC chemokines. It induced rapid nuclear localisation of NFκB and stimulation of iNOS protein levels and chemokine production which were blocked by an NFκB inhibitory peptide. CCL8/MCP-2, the most upregulated chemokine following in vitro macrophage exposure to Omniscan, was strongly increased in NSF-affected skin. The GdBCA Omniscan induces potent stimulation of macrophage gene expression, NFκB activation and increased NFκB-mediated production of CC and CXC chemokines and iNOS. These alterations may play a crucial role in the pathogenesis of NSF
Regulation of miR106b cluster through the RB pathway: mechanism and functional targets.
No full textThe RB pathway plays a critical role in proliferation control that is commonly subverted in tumor development. However, restoration of RB pathway function can be elicited in many tumor cells by the inhibition of CDK4/6 activity that leads to dephosphorylation of RB and subsequent repression of E2F-mediated transcription. In this context, active RB/E2F complexes inhibit the expression of a critical program of coding genes that promote cell cycle progression. However, the non-coding RNA target genes downstream from RB that could be relevant for tumor biology remain obscure. Here, miRNA gene expression profiling identified the miR106b cluster as being efficiently repressed with CDK4/6 inhibition in an E2F and RB-dependent manner. Importantly, the miR106B-cluster is intragenic of MCM7, and through a series of functional studies, the basis of MCM7 regulation and concordant expression of the miRNA species within the 106b cluster was determined. Importantly, RB-mediated repression of the 106b cluster enhances the transcript levels of p21Cip1 and PTEN. These data provide a mechanistic basis for cross-talk between the RB pathway and p21 and PTEN through the regulation of the MCM7/miR106b locus. © 2013 Landes Bioscience
Substrate uptake and metabolism are preserved in hypertrophic caveolin-3 knockout hearts.
No full textCaveolin-3
(Cav3), the primary protein component of caveolae in muscle cells,
regulates numerous signaling pathways including insulin receptor signaling
and facilitates free fatty acid (FA) uptake by interacting with several
FA transport proteins. We previously reported that Cav3 knockout mice
(Cav3KO) develop cardiac hypertrophy with diminished contractile function;
however, the effects of Cav3 gene ablation on cardiac substrate
utilization are unknown. The present study revealed that the uptake
and oxidation of FAs and glucose were normal in hypertrophic
Cav3KO hearts. Real-time PCR analysis revealed normal expression
of lipid metabolism genes including FA translocase (CD36) and
carnitine palmitoyl transferase-1 in Cav3KO hearts. Interestingly,
myocardial cAMP content was significantly increased by 42%; however,
this had no effect on PKA activity in Cav3KO hearts. Microarray
expression analysis revealed a marked increase in the expression of genes
involved in receptor trafficking to the plasma membrane, including
Rab4a and the expression of WD repeat/FYVE domain containing
proteins. We observed a fourfold increase in the expression of cellular
retinol binding protein-III and a 3.5-fold increase in 17-hydroxysteroid
dehydrogenase type 11, a member of the short-chain dehydrogenase/
reductase family involved in the biosynthesis and inactivation of steroid
hormones. In summary, a loss of Cav3 in the heart leads to cardiac
hypertrophy with normal substrate utilization. Moreover, a loss of Cav3
mRNA altered the expression of several genes not previously linked to
cardiac growth and function. Thus we have identified a number of new
target genes associated with the pathogenesis of cardiac hypertrophy
Gene expression profiling during the transition to failure in TNF-a over-expressing mice demonstrates the development of autoimmune myocarditis.
No full textTransgenic mice with cardiac-specific over-expression of tumor necrosis factor-a (TNF1.6) progress to dilated heart failure. A significant
inflammatory response precedes functional deterioration, and may contribute to cardiac damage in this model. To evaluate the underlying
molecular mechanisms, we assessed the gene expression in six groups of mouse hearts defined by age, gender, and phenotype (n = 3/group)
using Affymetrix microarray analysis. Phenotype was defined as compensated (in young TNF1.6) or decompensated (in older TNF1.6) via
echocardiogram. Of the >1000 transcripts altered in the compensated hearts (fold change > 2, P < 0.05 vs. wild-type (WT)), 102 were
identified as immune response genes, 20 of which function in antigen presentation and processing. When comparing the compensated and
decompensated hearts, >50 genes were differentially regulated, including seven immunoglobulin genes. Real-time reverse transcriptasepolymerase
chain reaction and cDNA microarray confirmed the Affymetrix data. Mac3+ macrophages, CD4+ T and CD45/B220+ B-cells
were identified in both compensated and decompensated hearts. However, a large amount of IgG was found deposited in areas devoid of
B-lymphocytes in the myocardium of decompensated TNF1.6 mice; no such accumulation was seen in the compensated or age-matched
controls. Furthermore, nuclei density analyses showed a two-fold increase in the myocardium of both compensated and decompensated
TNF1.6 mice (vs. WT). This study suggests that TNF-a over-expression activates not only the inflammatory response, but also humoral
immune responses within the transgenic hearts. The autoimmune response occurs concomitantly with cardiac decompensation and may
participate in triggering the transition to failure in TNF1.6 mice
Mechanisms of Endothelial Cell Attachment, Proliferation, and Differentiation on 4 Types of Platinum-Based Endovascular Coils
No full textObjective: A subarachnoid hemorrhage is neurologically devastating, with 50% of patients becoming disabled or deceased. Advent of Guglielmi detachable coils in 1995 permitted endovascular treatment of cerebral aneurysms. Coiling is efficacious and safe, but durability needs improvement, as nearly 20% of patients require further invasive intervention secondary to aneurysm recurrence. The aim of this study is to develop an in vitro model of endothelial cell (EC) proliferation and differentiation on four types of platinum-based coils, using gene expression profiling to understand EC biology as they colonize and differentiate on coils. Methods: Human umbilical vein ECs were grown in vitro on platinum coil segments. Growth patterns were assessed as a function of coil type. Gene expression profiles for coil attached versus coil unattached ECs were determined using immunohistochemistry and gene array analysis. Results: ECs showed rapid, robust attachment to all coil types. Some detachment occurred within 24-48 hours. Significant growth of remaining attached cells occurred during the next week, creating a confluence on coils and within coil grooves. Similar growth curve results were obtained with human brain ECs on platinum-based coil surfaces. Differentiation markers in attached cells (α1, α2, β1 integrins) were expressed on immunostaining, whereas microarray gene expression revealed 48 up-regulated and 68 down-regulated genes after 24-hour growth on coils. Major pathways affected as a function of time of colonization on coils and coil type included those involved in regulation of cell cycle and cell signaling. Conclusions: We developed an in vitro model for evaluating endothelialization of platinum coils to optimize coil design to support robust EC colonization and differentiation. © 2014 Elsevier Inc. All rights reserved
Gfi-1 inhibits proliferation and colony formation of p210BCR/ABL-expressing cells via transcriptional repression of STAT 5 and Mcl-1
No full textExpression of the transcription repressor Gfi-1 is required for the maintenance of murine hematopoietic stem cells. In human cells, ectopic expression of Gfi-1 inhibits and RNA interference-mediated Gfi-1 downregulation enhances proliferation and colony formation of p210BCR/ABL expressing cells. To investigate the molecular mechanisms that may explain the effects of perturbing Gfi-1 expression in human cells, Gfi-1-regulated genes were identified by microarray analysis in K562 cells expressing the tamoxifen-regulated Gfi-1-ER protein. STAT 5B and Mcl-1, two genes important for the proliferation and survival of hematopoietic stem cells, were identified as direct and functionally relevant Gfi-1 targets in p210BCR/ABL-transformed cells because: (i) their expression and promoter activity was repressed by Gfi-1 and (ii) when constitutively expressed blocked the proliferation and colony formation inhibitory effects of Gfi-1. Consistent with these findings, genetic or pharmacological inhibition of STAT 5 and/or Mcl-1 markedly suppressed proliferation and colony formation of K562 and CD34+ chronic myelogenous leukemia (CML) cells. Together, these studies suggest that the Gfi-1STAT 5B/Mcl-1 regulatory pathway identified here can be modulated to suppress the proliferation and survival of p210BCR/ABL-transformed cells including CD34+ CML cells
Mammary gland selective excision of c-jun identifies its role in mRNA splicing.
No full textThe c-jun gene regulates cellular proliferation and apoptosis via direct regulation of cellular gene expression. Alternative splicing of pre-mRNA increases the diversity of protein functions, and alternate splicing events occur in tumors. Here, by targeting the excision of the endogenous c-jun gene within the mouse mammary epithelium, we have identified its selective role as an inhibitor of RNA splicing. Microarray-based assessment of gene expression, on laser capture microdissected c-jun(-/-) mammary epithelium, showed that endogenous c-jun regulates the expression of approximately 50 genes governing RNA splicing. In addition, genome-wide splicing arrays showed that endogenous c-jun regulated the alternate exon of approximately 147 genes, and 18% of these were either alternatively spliced in human tumors or involved in apoptosis. Endogenous c-jun also was shown to reduce splicing activity, which required the c-jun dimerization domain. Together, our findings suggest that c-jun directly attenuates RNA splicing efficiency, which may be of broad biologic importance as alternative splicing plays an important role in both cancer development and therapy resistance
Targeting CDK6 and BCL2 Exploits the MYB Addiction of Ph+ Acute Lymphoblastic Leukemia
No full textPhiladelphia chromosome–positive acute lymphoblastic leukemia (Phþ ALL) is currently treated with BCR-ABL1 tyrosine kinase inhibitors (TKI) in combination with chemotherapy. However, most patients develop resistance to TKI through BCR-ABL1–dependent and –independent mechanisms. Newly developed TKI can target Phþ ALL cells with BCR-ABL1–dependent resistance; however, overcoming BCR-ABL1–independent mechanisms of resistance remains challenging because transcription factors, which are difficult to inhibit, are often involved. We show here that (i) the growth of Phþ ALL cell lines and primary cells is highly dependent on MYB-mediated transcriptional upregulation of CDK6, cyclin D3, and BCL2, and (ii) restoring their expression in MYB-silenced Phþ ALL cells rescues their impaired proliferation and survival. Levels of MYB and CDK6 were highly correlated in adult Phþ ALL (P ¼ 0.00008). Moreover, Phþ ALL cells exhibited a specific requirement for CDK6 but not CDK4 expression, most likely because, in these cells, CDK6 was predominantly localized in the nucleus, whereas CDK4 was almost exclusively cytoplasmic. Consistent with their essential role in Phþ ALL, pharmacologic inhibition of CDK6 and BCL2 markedly suppressed proliferation, colony formation, and survival of Phþ ALL cells ex vivo and in mice. In summary, these findings provide a proof-of-principle, rational strategy to target the MYB addiction of Phþ ALL. © 2017 American Association for Cancer Research
Sighting acute myocardial infarction through platelet gene expression
No full text© 2019, The Author(s). Acute myocardial infarction is primarily due to coronary atherosclerotic plaque rupture and subsequent thrombus formation. Platelets play a key role in the genesis and progression of both atherosclerosis and thrombosis. Since platelets are anuclear cells that inherit their mRNA from megakaryocyte precursors and maintain it unchanged during their life span, gene expression profiling at the time of an acute myocardial infarction provides information concerning the platelet gene expression preceding the coronary event. In ST-segment elevation myocardial infarction (STEMI), a gene-by-gene analysis of the platelet gene expression identified five differentially expressed genes: FKBP5, S100P, SAMSN1, CLEC4E and S100A12. The logistic regression model used to combine the gene expression in a STEMI vs healthy donors score showed an AUC of 0.95. The same five differentially expressed genes were externally validated using platelet gene expression data from patients with coronary atherosclerosis but without thrombosis. Platelet gene expression profile highlights five genes able to identify STEMI patients and to discriminate them in the background of atherosclerosis. Consequently, early signals of an imminent acute myocardial infarction are likely to be found by platelet gene expression profiling before the infarction occurs
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