Project description:Experiment comparing Low-density, CCg derived, progenitors (LDP) with high-density progenitors (HDP), and neurons (LDN) and astrocytes (LDA) derived from the LDP condition Keywords: other
Project description:Analysis of serum starved PC-3 cells treated with CCG-1423, Latrunculin B, or the transcription elongation inhibitor DRB for 2 or 24 hours. Results provide insights to potential therapeutic approaches to cancer metastasis. Twenty one samples in triplicate were analyzed and compared to the DMSO-treated control. The primary condition tested was the effect of the Rho-transcription pathway inhibitor, CCG-1423 As a biologically related control, the actin polymerization inhibitor, Latrunculin B, that also blocks Rho-stimulated gene transcription was tested. As a control for non-specific transcription inhibition, DRB was used. All samples, 2-hr and 24-hr treated samples were compared to the 24-hr DMSO sample.
Project description:Myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF), and thereby regulate cytoskeletal gene expression in response to actin dynamics. MRTFs have also been implicated in heat shock protein (hsp) transcription in fly ovaries, but the mechanisms remain unclear. Here we demonstrate that in mammalian cells, MRTFs are dispensable for hsp gene induction. However, the widely used small molecule inhibitors of MRTF/SRF transcription pathway, derived from CCG-1423, efficiently inhibit hsp gene transcription in both fly and mammalian cells also in absence of MRTFs. Quantifying RNA synthesis and RNA polymerase distribution demonstrates that CCG-1423-derived compounds have a genome-wide effect on transcription. Indeed, tracking nascent transcription at nucleotide resolution reveals that CCG-1423-derived compounds reduce RNA polymerase II elongation, and severely dampen the transcriptional response to heat shock. The effects of CCG-1423-derived compounds therefore extend beyond the MRTF/SRF pathway into nascent transcription, opening novel opportunities for their use in transcription research.
Project description:Myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF), and thereby regulate cytoskeletal gene expression in response to actin dynamics. MRTFs have also been implicated in heat shock protein (hsp) transcription in fly ovaries, but the mechanisms remain unclear. Here we demonstrate that in mammalian cells, MRTFs are dispensable for hsp gene induction. However, the widely used small molecule inhibitors of MRTF/SRF transcription pathway, derived from CCG-1423, efficiently inhibit hsp gene transcription in both fly and mammalian cells also in absence of MRTFs. Quantifying RNA synthesis and RNA polymerase distribution demonstrates that CCG-1423-derived compounds have a genome-wide effect on transcription. Indeed, tracking nascent transcription at nucleotide resolution reveals that CCG-1423-derived compounds reduce RNA polymerase II elongation, and severely dampen the transcriptional response to heat shock. The effects of CCG-1423-derived compounds therefore extend beyond the MRTF/SRF pathway into nascent transcription, opening novel opportunities for their use in transcription research.
Project description:Analysis of serum starved PC-3 cells treated with CCG-1423, Latrunculin B, or the transcription elongation inhibitor DRB for 2 or 24 hours. Results provide insights to potential therapeutic approaches to cancer metastasis.
Project description:Stable expression of tRNA-Glu(UUC) and tRNA-Arg(CCG) followed by whole-genome transcript stability measurements using a-amanitin mediated inhibition of RNA Pol II.
Project description:The Ras/MEK/ERK pathway has been the primary focus of targeted therapies in melanoma, given that it is aberrantly activated in almost 80% of human cutaneous melanomas (~50% BRAFV600 mutations and ~30% NRAS mutations). While targeted therapies have yielded success in BRAFV600 mutant melanoma patients, such therapies have been ineffective in NRAS mutant melanomas in part due to their cytostatic effects and primary resistance in this patient population. Here, we demonstrate that increased Rho/MRTF-pathway activation correlates with high intrinsic resistance to trametinib, a MEK inhibitor, in a panel of NRAS mutant melanoma cell lines. Combination of trametinib with the Rho/MRTF-pathway inhibitor, CCG-222740, synergistically reduced cell viability in NRAS mutant melanoma cell lines in vitro. Furthermore, the combination of CCG-222740 with trametinib induced apoptosis and reduced clonogenicity in the highly trametinib-resistant SK-Mel-147 cells. These findings suggest a role of the Rho/MRTF-pathway in high intrinsic trametinib resistance to a subset of NRAS mutant melanoma cell lines and highlights the potential of concurrently targeting the Rho/MRTF-pathway and MEK in NRAS mutant melanomas.