86 research outputs found
Supplementary materials - Analysis of gene expression during myc oncogene-induced lymphomagenesis in the bursa of Fabricius.
The transcriptional effects of deregulated myc gene overexpression are implicated in tumorigenesis in a spectrum of experimental and naturally occurring neoplasms. In follicles of the chicken bursa of Fabricius, myc induction of B-cell neoplasia requires a target cell population present during early bursal development and progresses through preneoplastic transformed follicles to metastatic lymphomas. We developed a chicken immune system cDNA microarray to analyze broad changes in gene expression that occur during normal embryonic B-cell development and during myc-induced neoplastic transformation in the bursa. The number of mRNAs showing at least 3-fold change was greater during myc-induced lymphomagenesis than during normal development, and hierarchical cluster analysis of expression patterns revealed that levels of several hundred mRNAs varied in concert with levels of myc overexpression. A set of 41 mRNAs were most consistently elevated in myc-overexpressing preneoplastic and neoplastic cells, most involved in processes thought to be subject to regulation by Myc. The mRNAs for another cluster of genes were overexpressed in neoplasia independent of myc expression level, including a small subset with the expression signature of embryonic bursal lymphocytes. Overexpression of myc, and some of the genes overexpressed with myc, may be important for generation of preneoplastic transformed follicles. However, expression profiles of late metastatic tumors showed a large variation in concert with myc expression levels, and some showed minimal myc overexpression. Therefore, high-level myc overexpression may be more important in the early induction of these lymphomas than in maintenance of late-stage metastases
Quality Assessment and Data Analysis for microRNA Expression Arrays
MicroRNAs are small (âŒ22 nt) RNAs that regulate gene expression and play important roles in both normal and disease physiology. The use of microarrays for global characterization of microRNA expression is becoming increasingly popular and has the potential to be a widely used and valuable research tool. However, microarray profiling of microRNA expression raises a number of data analytic challenges that must be addressed in order to obtain reliable results. We introduce here a universal reference microRNA reagent set as well as a series of nonhuman spiked-in synthetic microRNA controls, and demonstrate their use for quality control and between-array normalization of microRNA expression data. We also introduce diagnostic plots designed to assess and compare various normalization methods. We anticipate that the reagents and analytic approach presented here will be useful for improving the reliability of microRNA microarray experiments
Genomic alterations indicate tumor origin and varied metastatic potential of disseminated cells from prostate-cancer patients
Disseminated epithelial cells can be isolated from the bone marrow of a far greater fraction
of prostate-cancer patients than the fraction of patients who progress to metastatic disease.
To provide a better understanding of these cells, we have characterized their genomic alterations.
We first present an array comparative genomic hybridization method capable of detecting
genomic changes in the small number of disseminated cells (10-20) that can typically be obtained
from bone-marrow aspirates of prostate-cancer patients. We show multiple regions of
copy-number change, including alterations common in prostate cancer, such as 8p loss, 8q gain,
and gain encompassing the androgen-receptor gene on Xq, in the disseminated cell pools from
11 metastatic patients. We found fewer and less striking genomic alterations in the 48 pools of
disseminated cells from patients with organ-confined disease. However, we identify changes
shared by these samples with their corresponding primary tumors and prostate-cancer alterations
reported in the literature, evidence that these cells, like those in advanced disease, are
disseminated tumor cells (DTCs). We also demonstrate that DTCs from patients with advanced
and localized disease share several abnormalities, including losses containing cell-adhesion
genes and alterations reported to associate with progressive disease. These shared alterations
might confer the capability to disseminate or establish secondary disease. Overall, the spectrum
of genomic deviations is evidence for metastatic capacity in advanced-disease DTCs and variation
in that capacity in DTCs from localized disease. Our analysis lays the foundation for elucidation
of the relationship between DTC genomic alterations and progressive prostate cancer
Neural G0:a quiescent-like state found in neuroepithelial-derived cells and glioma
Singleâcell RNA sequencing has emerged as a powerful tool for resolving cellular states associated with normal and maligned developmental processes. Here, we used scRNAâseq to examine the cell cycle states of expanding human neural stem cells (hNSCs). From these data, we constructed a cell cycle classifier that identifies traditional cell cycle phases and a putative quiescentâlike state in neuroepithelialâderived cell types during mammalian neurogenesis and in gliomas. The Neural G0 markers are enriched with quiescent NSC genes and other neurodevelopmental markers found in nonâdividing neural progenitors. Putative glioblastoma stemâlike cells were significantly enriched in the Neural G0 cell population. Neural G0 cell populations and gene expression are significantly associated with less aggressive tumors and extended patient survival for gliomas. Genetic screens to identify modulators of Neural G0 revealed that knockout of genes associated with the Hippo/Yap and p53 pathways diminished Neural G0 in vitro, resulting in faster G1 transit, downâregulation of quiescenceâassociated markers, and loss of Neural G0 gene expression. Thus, Neural G0 represents a dynamic quiescentâlike state found in neuroepithelialâderived cells and gliomas
Effective interaction between helical bio-molecules
The effective interaction between two parallel strands of helical
bio-molecules, such as deoxyribose nucleic acids (DNA), is calculated using
computer simulations of the "primitive" model of electrolytes. In particular we
study a simple model for B-DNA incorporating explicitly its charge pattern as a
double-helix structure. The effective force and the effective torque exerted
onto the molecules depend on the central distance and on the relative
orientation. The contributions of nonlinear screening by monovalent counterions
to these forces and torques are analyzed and calculated for different salt
concentrations. As a result, we find that the sign of the force depends
sensitively on the relative orientation. For intermolecular distances smaller
than it can be both attractive and repulsive. Furthermore we report a
nonmonotonic behaviour of the effective force for increasing salt
concentration. Both features cannot be described within linear screening
theories. For large distances, on the other hand, the results agree with linear
screening theories provided the charge of the bio-molecules is suitably
renormalized.Comment: 18 pages, 18 figures included in text, 100 bibliog
Non-monotonic variation with salt concentration of the second virial coefficient in protein solutions
The osmotic virial coefficient of globular protein solutions is
calculated as a function of added salt concentration at fixed pH by computer
simulations of the ``primitive model''. The salt and counter-ions as well as a
discrete charge pattern on the protein surface are explicitly incorporated. For
parameters roughly corresponding to lysozyme, we find that first
decreases with added salt concentration up to a threshold concentration, then
increases to a maximum, and then decreases again upon further raising the ionic
strength. Our studies demonstrate that the existence of a discrete charge
pattern on the protein surface profoundly influences the effective interactions
and that non-linear Poisson Boltzmann and Derjaguin-Landau-Verwey-Overbeek
(DLVO) theory fail for large ionic strength. The observed non-monotonicity of
is compared to experiments. Implications for protein crystallization are
discussed.Comment: 43 pages, including 17 figure
Genome-wide RNAi screens in human brain tumor isolates reveal a novel viability requirement for PHF5A
To identify key regulators of human brain tumor maintenance and initiation, we performed multiple genome-wide RNAi screens in patient-derived glioblastoma multiforme (GBM) stem cells (GSCs). These screens identified the plant homeodomain (PHD)-finger domain protein PHF5A as differentially required for GSC expansion, as compared with untransformed neural stem cells (NSCs) and fibroblasts. Given PHF5A's known involvement in facilitating interactions between the U2 snRNP complex and ATP-dependent helicases, we examined cancer-specific roles in RNA splicing. We found that in GSCs, but not untransformed controls, PHF5A facilitates recognition of exons with unusual C-rich 3âČ splice sites in thousands of essential genes. PHF5A knockdown in GSCs, but not untransformed NSCs, astrocytes, or fibroblasts, inhibited splicing of these genes, leading to cell cycle arrest and loss of viability. Notably, pharmacologic inhibition of U2 snRNP activity phenocopied PHF5A knockdown in GSCs and also in NSCs or fibroblasts overexpressing MYC. Furthermore, PHF5A inhibition compromised GSC tumor formation in vivo and inhibited growth of established GBM patient-derived xenograft tumors. Our results demonstrate a novel viability requirement for PHF5A to maintain proper exon recognition in brain tumor-initiating cells and may provide new inroads for novel anti-GBM therapeutic strategies
Supplementary Data Only: Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network.
The Myc/Max/Mad transcription factor network is critically involved in cell behavior; however, there is relatively little information on its genomic binding sites. We have employed the DamID method to carry out global genomic mapping of the Drosophila Myc, Max, and Mad/Mnt proteins. Each protein was tethered to Escherichia coli DNA adenine-methyltransferase (Dam) permitting methylation proximal to in vivo binding sites in Kc cells. Microarray analyses of methylated DNA fragments reveals binding to multiple loci on all major Drosophila chromosomes. This approach also reveals dynamic interactions among network members as we find that increased levels of dMax influence the extent of dMyc, but not dMnt, binding. Computer analysis using the REDUCE algorithm demonstrates that binding regions correlate with the presence of E-boxes, CG repeats, and other sequence motifs. The surprisingly large number of directly bound loci ( approximately 15% of coding regions) suggests that the network interacts widely with the genome. Furthermore, we employ microarray expression analysis to demonstrate that hundreds of DamID-binding loci correspond to genes whose expression is directly regulated by dMyc in larvae. These results suggest that a fundamental aspect of Max network function involves widespread binding and regulation of gene expression
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