scholarly journals Development of a Screening Assay for Surrogate Markers of Chk1 Inhibitor-Induced Cell Cycle Release

2006 ◽  
Vol 11 (7) ◽  
pp. 792-806 ◽  
Author(s):  
Christie P. Fanton ◽  
Michael W. Rowe ◽  
Edward J. Moler ◽  
Marjorie Ison-Dugenny ◽  
Susan K. De Long ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Differential Expression ◽  
Expression Analysis ◽  
Topoisomerase I ◽  
Marker Gene ◽  
Marker Genes ◽  
Surrogate Markers ◽  
Rt Pcr ◽  
Screening Assay

Chk1 is a key regulator of the S and G2/M checkpoints and is activated following DNA damage by agents such as the topoisomerase I inhibitor camptothecin (CPT). It has been proposed that Chk1 inhibitors used in combination with such a DNA damaging agent to treat tumors would potentiate cytotoxicity and increase the therapeutic index, particularly in tumors lacking functional p53. The aim of this study was to determine whether gene expression analysis could be used to inform lead optimization of a novel series of Chk1 inhibitors. The candidate small-molecule Chk1 inhibitors were used in combination with CPT to identify potential markers of functional Chk1 inhibition, as well as resulting cell cycle progression, using cDNA-based microarrays. Differential expression of several of these putative marker genes was further validated by RT-PCR for use as a medium-throughput assay. In the presence of DNA damage, Chk1 inhibitors altered CPT-dependent effects on the expression of cell cycle and DNA repair genes in a manner consistent with a Chk1-specific mechanism of action. Furthermore, differential expression of selected marker genes, cyclin E2, EGR1, and DDIT3, was dose dependent for Chk1 inhibition. RT-PCR results for these genes following treatment with a panel of Chk1 inhibitors showed a strong correlation between marker gene response and the ability of each compound to abrogate cell cycle arrest in situ following CPT-induced DNA damage. These results demonstrate the utility of global expression analysis to identify surrogate markers, providing an alternative method for rapid compound characterization to support advancement decisions in early drug discovery.

Biological Characteristics and Multilineage Differentiation of Kidney Mesenchymal Stem Cells Derived from the Tibetan Mastiff

2019 ◽  
Vol 9 (7) ◽  
pp. 904-913
Author(s):  
Bing Yan ◽  
Ruining Liang ◽  
Meng Ji ◽  
Qi-Qige Wuyun ◽  
Weijun Guan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Marker Gene ◽  
Cell Types ◽  
Immunofluorescence Staining ◽  
Marker Genes ◽  
Rt Pcr ◽  
Multipotent Stem Cells ◽  
Different Cell Types

Of all the significant researches that have taken place in isolation, culture and characterization of mesenchymal stem cells (MSCs), the field of kidney-derived mesenchymal stem cells (KMSCs) in Tibetan mastiff is still a blank. Therefore, the purpose of this study is to isolate, culture and characterize the Tibetan mastiff KMSCs. The KMSCs were successfully isolated from one-day year old Tibetan mastiff kidney, cultured for 16 passages and distinguished by two methods: immunofluorescence staining and RT-PCR. The Tibetan mastiff KMSCs expressed specific surface marker genes (VIM, CD44, FN1, CD90, CD109, CD73, FN1) and kidney marker gene PAX2. The proliferation ability of Tibetan mastiff KMSCs was measured through cell count and clonality. Furthermore, cells differentiated into different cell types (hepatocellular cells, osteogenic cells, adipogenic cells and chondrogenic cells) under special induced medium, and the marker genes of induced cells were identified with Immunofluorescence staining and RT-PCR. All of these results indicated that the Tibetan mastiff KMSCs were obtained successfully, which possessed certain characteristics of multipotent stem cells. Therefore, MSCs in Tibetan mastiff kidney hold potential for clinical applications for regenerative therapy and their further studies are waiting to be required to investigate their functions.

scholarly journals Signatures and Prognostic Values of Related Immune Targets in Tongue Cancer

2021 ◽  
Author(s):  
Xi Yu ◽  
Xiaofei Lv
Keyword(s):  
Differential Expression ◽  
Expression Analysis ◽  
Tongue Cancer ◽  
Differential Expression Analysis ◽  
Marker Genes ◽  
Data Sets ◽  
Expression Data ◽  
Oral Cancers ◽  
Limma Package ◽  
Cancer Bioinformatics

Abstract Tongue cancer, as one of the most malignant oral cancers, is highly invasive and has a high risk of recurrence. At present, tongue cancer in the advanced stage is not obvious, easy to miss the opportunity of early diagnosis. It is important to find markers that can predict the occurrence and progression of tongue cancer. Bioinformatics analysis plays an important role in the acquisition of marker genes. GEO and TCGA data are very important public databases. In addition to expression data, TCGA database also contains corresponding clinical data. In this study, we screened three GEO datasets included GSE13601, GSE34105 and GSE34106 that met the standard. These data sets were combined using the SVA package to prepare the data for differential expression analysis, and then the LIMMA package was used to set the standard to p<0.05 and |log2 (FC)| ≥1.5. We got 170 DEGs (104, raised 66 downgrade). Besides, the DEseq package was used for differential expression analysis using the same criteria for samples in TCGA database. It ended up with 1589 DEGs (644 up-regulated, 945 down-regulated). By merging these two sets of DEGs, 5 common up-regulated DEGs (CCL20, SCG5, SPP1, KRT75 and FOLR3) and 15 common down-regulated DEGs were obtained. Further functional analysis of the DEGs showed that CCL20, SCG5 and SPP1 is closely related to prognosis and may be a therapeutic target of TSCC.

scholarly journals A novel mutation in DNA topoisomerase I of yeast causes DNA damage and RAD9-dependent cell cycle arrest.

Genetics ◽  
1993 ◽  
Vol 133 (4) ◽  
pp. 799-814 ◽  
Author(s):  
N A Levin ◽  
M A Bjornsti ◽  
G R Fink
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Topoisomerase I ◽  
Novel Mutation ◽  
Constitutive Expression ◽  
Repair System ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cycle Arrest ◽  
Dependent Cell

Abstract DNA topoisomerases, enzymes that alter the superhelicity of DNA, have been implicated in such critical cellular functions as transcription, DNA replication, and recombination. In the yeast Saccharomyces cerevisiae, a null mutation in the gene encoding topoisomerase I (TOP1) causes elevated levels of mitotic recombination in the ribosomal DNA (rDNA), but has little effect on growth. We have isolated a missense mutation in TOP1 that causes mitotic hyper-recombination not only in the rDNA, but also at other loci, in addition to causing a number of other unexpected phenotypes. This topoisomerase I mutation (top1-103) causes slow growth, constitutive expression of DNA damage-inducible genes, and inviability in the absence of the double-strand break repair system. Overexpression of top1-103 causes RAD9-dependent cell cycle arrest in G2. We show that the Top1-103 enzyme nicks DNA in vitro, suggesting that it damages DNA directly. We propose that Top1-103 mimics the action of wild-type topoisomerase I in the presence of the anti-tumor drug, camptothecin.

scholarly journals genesorteR: Feature Ranking in Clustered Single Cell Data

2019 ◽  
Author(s):  
Mahmoud M Ibrahim ◽  
Rafael Kramann
Keyword(s):  
Single Cell ◽  
Differential Expression ◽  
Expression Analysis ◽  
Differential Expression Analysis ◽  
Large Cell ◽  
R Package ◽  
Marker Genes ◽  
Data Sets ◽  
Cell Type ◽  
Cell Data

ABSTRACTMarker genes identified in single cell experiments are expected to be highly specific to a certain cell type and highly expressed in that cell type. Detecting a gene by differential expression analysis does not necessarily satisfy those two conditions and is typically computationally expensive for large cell numbers.Here we present genesorteR, an R package that ranks features in single cell data in a manner consistent with the expected definition of marker genes in experimental biology research. We benchmark genesorteR using various data sets and show that it is distinctly more accurate in large single cell data sets compared to other methods. genesorteR is orders of magnitude faster than current implementations of differential expression analysis methods, can operate on data containing millions of cells and is applicable to both single cell RNA-Seq and single cell ATAC-Seq data.genesorteR is available at https://github.com/mahmoudibrahim/genesorteR.

scholarly journals Anterior-posterior gene expression differences in three Lake Malawi cichlid fishes with variation in body stripe orientation

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e4080 ◽  
Author(s):  
Ehsan Pashay Ahi ◽  
Kristina M. Sefc
Keyword(s):  
Differential Expression ◽  
Target Genes ◽  
Marker Gene ◽  
Horizontal Distribution ◽  
Marker Genes ◽  
Expression Levels ◽  
Cichlid Species ◽  
Expression Of Genes ◽  
Species Specific ◽  
Anterior Posterior

Morphological differentiation among closely related species provides opportunities to study mechanisms shaping natural phenotypic variation. Here, we address variation in the orientation of melanin-colored body stripes in three cichlid species of the tribe Haplochromini. Melanochromis auratus displays a common pattern of dark, straight horizontal body stripes, whereas in Aristochromis christyi and Buccochromis rhoadesii, oblique stripes extend from the anterior dorsal to the posterior mid-lateral trunk. We first validated a stably reference gene, and then, investigated the chromatophore distribution in the skin by assessing the expression levels of the iridophore and melanophore marker genes, ltk and slc24a5, respectively, as well as pmel, a melanophore pigmentation marker gene. We found anterior-posterior differences in the expression levels of the three genes in the oblique-striped species. The higher anterior expression of ltk, indicates increased iridophore density in the anterior region, i.e., uneven horizontal distribution of iridophores, which coincides with the anterior dorsalization of melanophore stripe in these species. The obliqueness of the horizontal body stripes might be a result of distinct migratory or patterning abilities of melanophores in anterior and posterior stripe regions which could be reflected by variation in the expression of genes involved in melanophore patterning. To address this, we investigated anterior-posterior expression levels of a primary set of candidate target genes with known functions in melanophore migration and stripe patterning in the adult zebrafish, and their related gene regulatory network. Among these genes, those with differences in anterior-posterior expression showed only species-specific differential expression, e.g., sdf1a, col14a1a, ifitm5, and agpat3, with the exception of fbxw4/hagoromo (differentially expressed in an oblique-and the straight-striped species). In summary, distinct anterior-posterior gradients in iridophore density found to be more similar characteristic between the two oblique-striped species. Furthermore, the species-specific differential expression of genes involved in stripe patterning might also implicate distinct molecular processes underlying the obliqueness of body stripe in two closely related cichlid species.

EphB1 Downregulation In Acute Myeloid Leukemia: Suppressing p53-Dependent DNA Damage Control System

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2484-2484
Author(s):  
Kim R Kampen ◽  
Frank JG Scherpen ◽  
Guillermo Garcia-Manero ◽  
Hui Yang ◽  
Steven M. Kornblau ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Control System ◽  
Cell Survival ◽  
Cell Lines ◽  
Damage Control ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Pediatric Aml ◽  
Stimulation Of

Abstract Background Ephrin receptors (Eph) have been implicated to contribute to cancer pathogenesis in a way by which receptors can both promote or suppress cancer progression depending on their cellular contexts. Mechanism for Eph receptor tumor suppressor functions are unknown. In a previous study, we found that pediatric acute myeloid leukemia (AML) sample lysates showed a significant decreased EphB1 peptide activity in comparison to normal bone marrow (NBM). In this study, we challenged to investigate EphB1 receptor transcriptional regulation and biological insights in AML. Materials and Methods Quantitative RT-PCR analysis and bisulfate pyrosequencing was used to examine mRNA expression and methylation in AML cell lines and AML patient samples. AML cell proliferation (cell counts) and apoptosis (annexin V/PI) were analyzed after EfnB1 ligand stimulation. We generated constitutive EphB1 overexpression constructs in AML cell lines. Downstream in vitro effects were studied using quantitative RT-PCR, flowcytometry, phospho-proteome arrays, chromatin immunoprecipitation (ChIP), and immunoblot analysis. Results First, we examined EphB1 mRNA expression and methylation in primary pediatric AML samples, which revealed that EphB1 mRNA expression was significantly lower in unfavorable AML cytogenetic subgroups as compared to favorable AML patient samples (Fig. A, P = 0.013). The EphB1 promoter region was found increasingly methylated in approximately 20% of the pediatric AML patient samples (n=21), but not in NBM (n=5, Fig. B). In AML cell lines, we confirmed that EphB1 promoter methylation resulted in epigenetic gene silencing (P = 0.010). As a next step we challenged to study the biological relevance of EphB1 suppression. EfnB1 stimulation of EphB1high THP-1 cells inhibited the cell proliferation and induced apoptosis, while EphB1low HL60 and MOLM13 cells remained unaffected upon EfnB1 stimulation. Cell cycle analysis revealed that EfnB1 stimulation mediated phenotypic effects in EphB1high THP-1 cells could be assigned to a G2/M cell cycle arrest. The cell cycle arrest and the induction of apoptosis as a common consequence was confirmed by phospho-proteome, immunoblot, and qRT-PCR analysis. We found an induction of DNA damage control protein activation and cell cycle inhibitors Chk-2, p21, p27, p53, and CDC2tyr15, and a reduction of cell survival proteins HSP27 and BCL-2 accompanied by an increase of Bax. Next, we investigated whether EfnB1 stimulation of THP-1 could have restored the DNA damage control system by increasing DNA binding of mutant and wildtype p53. Performing ChIP assays using p53 antibodies recognizing wildtype or both mutant and wildtype p53 followed by p21 and MDM2 quantitative RT-PCR analysis revealed that EfnB1 stimulation of THP-1 cells significantly increased p53 DNA binding (Fig. C). Introducing a constitutive EphB1 overexpression into EphB1 methylated HL60 and MOLM13 cell lines resulted in an instant increase in apoptosis relatively to empty vector control cells. As expected, G2/M cell cycle inhibiting phosphorylation of CDC2tyr15 was upregulated. EfnB1 stimulation of the GFP sorted remaining viable cells showed to decrease the AML cell survival in EphB1 overexpressed AML cells as compared to empty vector controls. Additionally, we found that EphB1 demethylation by 5-Aza-2'-deoxycytidine was able to restore EphB1 membrane protein expression in EphB1 methylated AML cells and simultaneously reduced the AML cell survival. Conclusions In this study, we found a common downregulation of EphB1 mRNA that was significantly associated with an unfavorable prognosis in pediatric AML. The downregulation of EphB1 could be explained by EphB1 promoter hypermethylation in a subset of AML samples. Downregulation of EphB1 was found to be one of the mechanisms through which AML cells suppress p53 DNA binding that impairs the DNA damage control system to support a proliferative and anti-apoptotic AML cell survival advantage. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A novel topoisomerase I inhibitor DIA-001 induces DNA damage mediated cell cycle arrest and apoptosis in cancer cell

2020 ◽  
Vol 8 (4) ◽  
pp. 89-89
Author(s):  
Jiaqi Liu ◽  
Guohe Geng ◽  
Guang Liang ◽  
Ling Wang ◽  
Kuntian Luo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Cancer Cell ◽  
Topoisomerase I ◽  
Topoisomerase I Inhibitor ◽  
Cycle Arrest

scholarly journals Invariant Differential Expression Analysis Reveals Mechanism of Cancer Resistance to Cell Cycle Inhibitors

2021 ◽  
Author(s):  
Amit Chatterjee ◽  
Sonalisa Pandey ◽  
Ravikanth Danda ◽  
R Ranjith Kumar ◽  
S Maheswari ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Resistance ◽  
Differential Expression ◽  
Differentially Expressed Genes ◽  
Expression Analysis ◽  
Drug Sensitivity ◽  
Differential Expression Analysis ◽  
Mouse Xenograft ◽  
Invariant Differential ◽  
Cell Cycle Inhibitors

AbstractRetinoblastoma (RB) is a good model to study drug resistance to cell-cycle inhibitors because it is driven by mutations in the core components of cell-cycle, i.e, Rb gene. However, there is limited gene expression dataset in RB which has major reproducibility issues. We have developed invariant differential expression analysis (iDEA) that improves the state of the art in differential expression analysis (DEA). iDEA uses strong Boolean implication relationships in a large diverse human dataset GSE119087 (n = 25,955) to filter the noisy differentially expressed genes (DEGs). iDEA was applied to RB datasets and a gene signature was computed that led to prediction and mechanism of drug sensitivity. The prediction was confirmed using drugs-sensitive/resistant RB cell-lines and mouse xenograft models using CDC25 inhibitor NSC663284. iDEA improved reproducibility of differential expression across diverse retina/RB cohorts and RB cell-lines with different drug sensitivity (Y79/Weri vs NCC). Pathway analysis revealed WNT/β-catenin involved in distinguishing drug sensitivity to CDC25 inhibitor NSC663284. NSC663284 inhibited tumour cell proliferation in mouse xenograft model containing Y79 cells indicating novel therapeutic option in RB. Invariant differentially expressed genes (iDEGs) are robustly associated with outcome in diverse cancer datasets and supports for a fundamental mechanism of drug resistance.

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