GILA, a Replacement for the Soft‐Agar Assay that Permits High‐Throughput Drug and Genetic Screens for Cellular Transformation

2016 ◽  
Vol 116 (1) ◽  
Author(s):  
Benjamin Izar ◽  
Asaf Rotem
Keyword(s):  
High Throughput ◽  
Cellular Transformation ◽  
Soft Agar ◽  
Genetic Screens ◽  
Soft Agar Assay

scholarly journals Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation

2015 ◽  
Vol 112 (18) ◽  
pp. 5708-5713 ◽  
Author(s):  
Asaf Rotem ◽  
Andreas Janzer ◽  
Benjamin Izar ◽  
Zhe Ji ◽  
John G. Doench ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Cellular Transformation ◽  
Soft Agar ◽  
Ovarian Cancer Cells ◽  
Soft Agar Assay ◽  
Resistant Disease ◽  
Personalized Cancer

Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening, and they include kinase inhibitors and drugs for noncancer diseases. In addition to known oncogenes, the genetic screen identifies genes that contribute to cellular transformation. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment.

The Src family kinase Fgr is a transforming oncoprotein that functions independently of SH3-SH2 domain regulation

2018 ◽  
Vol 11 (553) ◽  
pp. eaat5916 ◽  
Author(s):  
Kexin Shen ◽  
Jamie A. Moroco ◽  
Ravi K. Patel ◽  
Haibin Shi ◽  
John R. Engen ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Colony Formation ◽  
Family Members ◽  
Cellular Transformation ◽  
Soft Agar ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Mass Spectrometry Data ◽  
Activation Loop ◽  
Exchange Mass Spectrometry

Fgr is a member of the Src family of nonreceptor tyrosine kinases, which are overexpressed and constitutively active in many human cancers. Fgr expression is restricted to myeloid hematopoietic cells and is markedly increased in a subset of bone marrow samples from patients with acute myeloid leukemia (AML). Here, we investigated the oncogenic potential of Fgr using Rat-2 fibroblasts that do not express the kinase. Expression of either wild-type or regulatory tail-mutant constructs of Fgr promoted cellular transformation (inferred from colony formation in soft agar), which was accompanied by phosphorylation of the Fgr activation loop, suggesting that the kinase domain of Fgr functions independently of regulation by its noncatalytic SH3-SH2 region. Unlike other family members, recombinant Fgr was not activated by SH3-SH2 domain ligands. However, hydrogen-deuterium exchange mass spectrometry data suggested that the regulatory SH3 and SH2 domains packed against the back of the kinase domain in a Src-like manner. Sequence alignment showed that the activation loop of Fgr was distinct from that of all other Src family members, with proline rather than alanine at the +2 position relative to the activation loop tyrosine. Substitution of the activation loop of Fgr with the sequence from Src partially inhibited kinase activity and suppressed colony formation. Last, Fgr expression enhanced the sensitivity of human myeloid progenitor cells to the cytokine GM-CSF. Because its kinase domain is not sensitive to SH3-SH2–mediated control, simple overexpression of Fgr without mutation may contribute to oncogenic transformation in AML and other blood cancers.

scholarly journals USP13 Regulates the Breast Tumor Proliferation and Migration through the Stabilization of PDCD4 Protein

2021 ◽  
Author(s):  
Jun Tian ◽  
Bei Li ◽  
Jing Qiao ◽  
Xinfeng Pang ◽  
Xiaojing Yue
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Breast Tumor ◽  
Soft Agar ◽  
Tumor Proliferation ◽  
Soft Agar Assay ◽  
And Migration ◽  
Cancer Tissues ◽  
Proliferation And Migration

Abstract Background: Programmed cell death protein 4 (PDCD4), which serves as a tumor suppressor protein, plays a important role in cell proliferation,apoptosis, cell migration and DNA-damage response.However, the exact mechanism for the deubiquitination of PDCD4 remain unclear.Methods: Western blotting was used to detect the expression of PDCD4 in the breast cancer tissues and BC cell lines. We identified the potential PDCD4 associated deubiquitinase by RNAi screening. GST-Pull down and domain-mapping analysis were used to reveal that USP13 and PDCD4 directly interact with each other.Flow cytometry was used to detect the changes of G1 to S phase. Soft agar assay was used to measure the changes of the cell proliferation efficiency.Results: The expression of PDCD4 was decreased in the breast cancer tissues and BC cell lines. USP13 as a potential PDCD4 associated deubiquitinase. USP13 physically interacted with PDCD4 and greatly increased the steady state of PDCD4 through the ubiquitin-proteasome pathway.Importantly, silencing of the USP13 facilitated cell cycle from G1 to Sphase, promoted breast tumor cells proliferation and migration through downregulation of PDCD4. Conclusions: Together, these results suggest that USP13 plays an important role in the breast tumor proliferation and migration through modulating PDCD4 stability.

scholarly journals Targeting Epigenetic Modifiers Can Reduce the Clonogenic Capacities of Sézary Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Alain Chebly ◽  
Martina Prochazkova-Carlotti ◽  
Yamina Idrissi ◽  
Laurence Bresson-Bepoldin ◽  
Sandrine Poglio ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Histone Deacetylase Inhibitors ◽  
Soft Agar ◽  
Tumor Formation ◽  
Human Telomerase Reverse Transcriptase ◽  
Hematological Disorders ◽  
Soft Agar Assay ◽  
Human Telomerase ◽  
Sézary Cells

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL) in which the human Telomerase Reverse Transcriptase (hTERT) gene is re-expressed. Current available treatments do not provide long-term response. We previously reported that Histone deacetylase inhibitors (HDACi, romidespin and vorinostat) and a DNA methyltransferase inhibitor (DNMTi, 5-azacytidine) can reduce hTERT expression without altering the methylation level of hTERT promoter. Romidepsin and vorinostat are approved for CTCL treatment, while 5-azacytidine is approved for the treatment of several hematological disorders, but not for CTCL. Here, using the soft agar assay, we analyzed the functional effect of the aforementioned epidrugs on the clonogenic capacities of Sézary cells. Our data revealed that, besides hTERT downregulation, epidrugs’ pressure reduced the proliferative and the tumor formation capacities in Sézary cells in vitro.

scholarly journals MicroRNA-384 Inhibits the Progression of Papillary Thyroid Cancer by Targeting PRKACB

2020 ◽  
Vol 2020 ◽  
pp. 1-11 ◽  
Author(s):  
Yongxia Wang ◽  
Beixi Wang ◽  
Hong Zhou ◽  
Xiangnan Zhang ◽  
Xinlai Qian ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Papillary Thyroid Cancer ◽  
Target Gene ◽  
Underlying Mechanism ◽  
Soft Agar ◽  
Luciferase Reporter ◽  
Papillary Thyroid ◽  
Soft Agar Assay ◽  
Spearman’S Correlation ◽  
Dual Luciferase Reporter Assay

Background. Growing evidence shows that dysregulation of miRNAs plays a significant role in papillary thyroid cancer (PTC) tumorigenesis and development. The abnormal expression of miR-384 has been acknowledged in the proliferation or metastasis of some cancers. However, the function and the underlying mechanism of miR-384 in PTC progression remain largely unknown. Methods. Real-time PCR was conducted to detect miR-384 expression in 58 cases of PTC and their adjacent noncancerous tissues. MTT, soft agar assay Transwell assay, and wound-healing assay were carried out to explore the biological function of miR-384 in PTC cell lines of BCPAP and K1. Bioinformatics analysis, dual-luciferase reporter assay, western blot, and functional complementation analysis were conducted to explore the target gene of miR-384. Moreover, Spearman’s correlation analysis was conducted to reveal the correlation between miR-384 and PRKACB mRNA in PTC. Results. The expression of miR-384 decreased obviously in PTC, especially in the tumors with lymph node metastasis or larger tumor size. The ectopic upregulation of miR-384 significantly suppressed PTC progression, and the inhibition of miR-384 had the opposite effects. Moreover, PRKACB gene was confirmed as the target of miR-384. Conclusion. The study suggests that miR-384 serves as a tumor suppressor in PTC progression by directly targeting the 3′-UTR of PRKACB gene.

scholarly journals High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding

2019 ◽  
Vol 218 (8) ◽  
pp. 2797-2811 ◽  
Author(s):  
Yury S. Bykov ◽  
Nir Cohen ◽  
Natalia Gabrielli ◽  
Hetty Manenschijn ◽  
Sonja Welsch ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Sample Preparation ◽  
High Throughput ◽  
Fluorescent Microscopy ◽  
Large Datasets ◽  
Genetic Screens ◽  
Cell Type ◽  
Resolution Limit ◽  
Computer Image Analysis ◽  
Multiple Cell

Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called “MultiCLEM,” allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique.

Metformin Treatment Sensitizes Human Laryngeal Cancer Cell Line Hep- 2 to 5-Fluorouracil

2020 ◽  
Vol 7 (1) ◽  
pp. 16-24
Author(s):  
Neslisah Barlak ◽  
Fatma Sanli ◽  
Ozel Capik ◽  
Elanur Tuysuz ◽  
Elanur Aydın Karatas ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cancer Cell Line ◽  
Inhibitory Effect ◽  
Soft Agar ◽  
Invasive Potential ◽  
Matrigel Invasion ◽  
Soft Agar Assay ◽  
Anti Cancer ◽  
Apoptosis Assay ◽  
Cancer Types

Background: Larynx cancer (LCa) is the most common head and neck cancer and accounts for 1-2.5% of all human cancers worldwide. Metformin, an oral anti-diabetic drug, has been recently shown to have anti-cancer activity in various cancer types, and there are several studies in the literature pointing to its potential to sensitize cancer cells to chemotherapeutic drugs. Objective: This study was aimed at exploring the anti-cancer effects of metformin alone or in combination with 5-fluorouracil (5-FU) on Hep-2 cells. Methods: The effects of metformin and/or 5-FU on the proliferative, clonogenic, and apoptotic potential of Hep-2 cells were evaluated with Cell Viability Detection Kit-8, soft agar assay and Annexin VFITC Apoptosis assay, respectively. Migratory and invasive potential of cells was tested using scratch, transwell migration and Matrigel invasion assays. Gene expression of cells exposed to metformin and/or 5-FU was profiled using RT2 mRNA PCR Array plates. Results: Treatment of Hep-2 cells with metformin inhibited cell proliferation by inducing apoptosis, and suppressed cell migration. Besides, treatment of metformin along with 5-FU improved the antiproliferative and anti-migratory effects of 5-FU. However, unexpectedly, metformin was found to enhance cellular invasion and reverse the inhibitory effect of 5-FU on the invasive potential of Hep-2 cells. Conclusion: Our findings suggest that metformin might be used as an adjuvant agent in the treatment of LCa. However, the potential of metformin to promote the invasion of cancer cells should not be neglected.

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