Effectiveness of Transcriptional CDK Inhibitors in Targeting Non-Proliferating CD34+CD38- AML Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3482-3482
Author(s):  
Monica Pallis ◽  
Francis Burrows ◽  
Abigail Whittall ◽  
Claire Seedhouse ◽  
Nicholas Boddy ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Rna Polymerase Ii ◽  
Rna Synthesis ◽  
Cdk Inhibitors ◽  
Proliferating Cells ◽  
Median Percentage ◽  
Homogeneous Population ◽  
Quiescent Cells ◽  
Species Production

Abstract Abstract 3482 Quiescent cells conserve energy and are characterised by low RNA synthesis. In contrast, cancer cells are thought to be addicted to high RNA synthesis, particularly synthesis of survival molecules. We hypothesised that quiescent cancer cells, already low in RNA, would be sensitive to apoptosis induced by transcriptional cyclin-dependent kinase (CDK) inhibitors that further deplete RNA. We cultured the CD34+CD38- KG1a cell line continuously in the presence of an mTOR inhibitor, which maintained excellent viability and enriched the cells for quiescent stem cell features including low RNA content, low metabolism, low reactive oxygen species production and decreased size. Sensitivity to mitochondrial pore transition was similar in proliferating and quiescence-enriched cells, indicating that the basal mitochondrial apoptotic machinery is neither impaired nor improved. We treated quiescence-enriched cells for 48 hours with the nucleoside analogues ara-C, 5-azacytidine and clofarabine, the topoisomerase targeting agents daunorubicin, etoposide and irinotecan and three multikinase inhibitors with activity against transcriptional CDKs - flavopiridol, roscovotine and TG02. All of the agents used showed increased kill in the unmanipulated compared to the quiescence-enriched cells, emphasising the chemoresistant nature of quiescent cells. To put a value on the difficulty of eradicating the leukamic clone, we used the parameter 2 X IC50 (which theoretically would kill 100% cells in a totally homogeneous population). In quiescence-enriched cells, the percentage of cells killed at 2 X IC50 was roscovotine - 80%, TG02 – 72%, flavopiridol - 62%, 5-azacydidine - 61%, daunorubicin, clofarabine and irinotecan - 60%, etoposide - 58%, and for araC no IC50 for quiescence-enriched cells was reached at 20 times the IC50 of proliferating cells. The data show conclusively that, in this model, transcriptional CDK inhibitors outperformed the agents conventionally used in AML chemotherapy. We also showed that transcriptional CDK inhibitors induced serine 2 dephosphorylation of RNA polymerase II in equal measure in proliferating and quiescence- enriched cells. We have already shown that TGO2 targets primary CD34+CD38- cells in vitro (ASH 2010, abstract 1823). Here we also show that in four primary AML samples sensitive to roscovotine in vitro, the median percentage kill is greater in the CD34+CD38- subset, (which consists largely of quiescent cells and tends to contain the leukaemic stem cells,) than in the bulk cells (40.5% versus 19.5% kill at 2 micromolar roscovotine). We suggest that transcriptional CDK inhibitors may be a useful class of agent for targetting the occult quiescent CD34+CD38- cells thought to contribute to relapse in AML. Disclosures: Burrows: Tragara Pharmaceuticals: Employment.

scholarly journals Origin of Hepatitis Delta Virus mRNA

2000 ◽  
Vol 74 (16) ◽  
pp. 7204-7210 ◽  
Author(s):  
Severin Gudima ◽  
Shwu-Yuan Wu ◽  
Cheng-Ming Chiang ◽  
Gloria Moraleda ◽  
John Taylor
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
Genome Replication ◽  
Rna Transcription ◽  
Hepatitis Delta ◽  
New Findings ◽  
Delta Virus

ABSTRACT Hepatitis delta virus (HDV) is unique relative to all known animal viruses, especially in terms of its ability to redirect host RNA polymerase(s) to transcribe its 1,679-nucleotide (nt) circular RNA genome. During replication there accumulates not only more molecules of the genome but also its exact complement, the antigenome. In addition, there are relatively smaller amounts of an 800-nt RNA of antigenomic polarity that is polyadenylated and considered to act as mRNA for translation of the single and essential HDV protein, the delta antigen. Characterization of this mRNA could provide insights into the in vivo mechanism of HDV RNA-directed RNA transcription and processing. Previously, we showed that the 5′ end of this RNA was located in the majority of species, at nt 1630. The present studies show that (i) at least some of this RNA, as extracted from the liver of an HDV-infected woodchuck, behaved as if it contained a 5′-cap structure; (ii) in the infected liver there were additional polyadenylated antigenomic HDV RNA species with 5′ ends located at least 202 nt and even 335 nt beyond the nt 1630 site, (iii) the 5′ end at nt 1630 was not detected in transfected cells, following DNA-directed HDV RNA transcription, in the absence of genome replication, and (iv) nevertheless, using in vitro transcription with purified human RNA polymerase II holoenzyme and genomic RNA template, we did not detect initiation of template-dependent RNA synthesis; we observed only low levels of 3′-end addition to the template. These new findings support the interpretation that the 5′ end detected at nt 1630 during HDV replication represents a specific site for the initiation of an RNA-directed RNA synthesis, which is then modified by capping.

scholarly journals Development of a 3D Co-Culture System as a Cancer Model Using a Self-Assembling Peptide Scaffold

Gels ◽  
2018 ◽  
Vol 4 (3) ◽  
pp. 65 ◽  
Author(s):  
Nausika Betriu ◽  
Carlos Semino
Keyword(s):  
Cell Proliferation ◽  
Cancer Cells ◽  
Culture System ◽  
Critical Role ◽  
Tumor Stroma ◽  
Pharmaceutical Drugs ◽  
Cancer Model ◽  
Proliferating Cells ◽  
Self Assembling

Cancer research has traditionally relied on two-dimensional (2D) cell culture, focusing mainly on cancer cells and their abnormal genetics. However, over the past decade, tumors have been accepted as complex tissues rather than a homogenous mass of proliferating cells. Consequently, cancer cells’ behavior can only be deciphered considering the contribution of the cells existing in the tumor stroma as well as its complex microenvironment. Since the tumor microenvironment plays a critical role in tumorigenesis, it is widely accepted that culturing cells in three-dimensional (3D) scaffolds, which mimic the extracellular matrix, represents a more realistic scenario. In the present work, an in vitro 3D co-culture system based on the self-assembling peptide scaffold RAD16-I (SAPS RAD16-I) was developed as a cancer model. For that, PANC-1 cells were injected into a RAD16-I peptide scaffold containing fibroblasts, resulting in a 3D system where cancer cells were localized in a defined area within a stromal cells matrix. With this system, we were able to study the effect of three well-known pharmaceutical drugs (Gemcitabine, 5-Fluorouracil (5-FU), and 4-Methylumbelliferone (4-MU)) in a 3D context in terms of cell proliferation and survival. Moreover, we have demonstrated that the anti-cancer effect of the tested compounds can be qualitatively and quantitatively evaluated on the developed 3D co-culture system. Experimental results showed that Gemcitabine and 5-FU prevented PANC-1 cell proliferation but had a high cytotoxic effect on fibroblasts as well. 4-MU had a subtle effect on PANC-1 cells but caused high cell death on fibroblasts.

scholarly journals The Selectivity for Tumor Cells of Nuclear-Directed Cytotoxic RNases Is Mediated by the Nuclear/Cytoplasmic Distribution of p27KIP1

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1319
Author(s):  
Glòria García-Galindo ◽  
Jessica Castro ◽  
Jesús Matés ◽  
Marlon Bravo ◽  
Marc Ribó ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Tumor Cells ◽  
Complex Disease ◽  
Antitumor Action ◽  
Ovarian Cancer Cells ◽  
Proliferating Cells ◽  
New Strategy ◽  
Localization Signal ◽  
Multiple Species

Although single targeted anti-cancer drugs are envisaged as safer treatments because they do not affect normal cells, cancer is a very complex disease to be eradicated with a single targeted drug. Alternatively, multi-targeted drugs may be more effective and the tumor cells may be less prone to develop drug resistance although these drugs may be less specific for cancer cells. We have previously developed a new strategy to endow human pancreatic ribonuclease with antitumor action by introducing in its sequence a non-classical nuclear localization signal. These engineered proteins cleave multiple species of nuclear RNA promoting apoptosis of tumor cells. Interestingly, these enzymes, on ovarian cancer cells, affect the expression of multiple genes implicated in metabolic and signaling pathways that are critic for the development of cancer. Since most of these targeted pathways are not highly relevant for non-proliferating cells, we envisioned the possibility that nuclear directed-ribonucleases were specific for tumor cells. Here, we show that these enzymes are much more cytotoxic for tumor cells in vitro. Although the mechanism of selectivity of NLSPE5 is not fully understood, herein we show that p27KIP1 displays an important role on the higher resistance of non-tumor cells to these ribonucleases.

scholarly journals FSMP-09. FORMATE PROMOTES CANCER CELL INVASION AND METASTASIS VIA CALCIUM SIGNALING

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i18-i18
Author(s):  
Catherine Delbrouck ◽  
Vitaly I Pozdeev ◽  
Anais Oudin ◽  
Kamil Grzyb ◽  
Laura Neises ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Ex Vivo ◽  
Tumor Model ◽  
Underlying Mechanism ◽  
Tumor Escape ◽  
Proliferating Cells ◽  
Nucleotide Synthesis ◽  
Formate Production

Abstract Serine catabolism via the folate cycle provides formate that is essential for nucleotide synthesis in proliferating cells. In addition to this canonical function to support biomass production in anabolic cells, we have recently demonstrated in vitro and in vivo that formate production in cancer cells is often in excess of the anabolic demand. This excess formate production is characterized by formate overflow and thus, net formate excretion into the tumor microenvironment. Interestingly, we observe increased rates of formate overflow upon different chemical perturbations that induce growth arrest. Thus, stressed cancer cells that encounter growth restriction such as upon chemotherapy, are often characterized by increased formate release rates. We demonstrated that such high formate levels in the extracellular space promote invasion of glioblastoma cells. Using ex vivo brain slice cultures and an orthotopic brain tumor model, we demonstrate that silencing MTHFD1L, the essential enzyme to enable formate overflow, results in decreased invasiveness of the tumor. Embarking from this observation, we investigated the underlying mechanism and now provide evidence that the formate-dependent increase of cell motility is mediated by an activation of Ca2+ signaling. Activation of Ca2+ signaling triggers integrin and matrix metallopeptidase (MMP) responses enabling the invasion process. Targeting either the Ca2+ response or MMP release can suppress the formate dependent increase in invasion. Finally, we tested the effect of formate also in context of breast cancer where we were able to recapitulate our observation of increased invasiveness and, in this case, formate also promoted the metastatic potential. We conclude that excreted formate might serve as a cellular stress signal that represents a promotive trigger to support tumor escape mechanisms.

scholarly journals Swift Formation of Optimal Single Spheroids towards In-Vitro 3-Dimensional Tumour Models

2021 ◽  
Author(s):  
Stephanie Eugenie Brigitte McArdle ◽  
Kinana Habra ◽  
Joshua R D Pearson
Keyword(s):  
Cancer Cells ◽  
3D Structure ◽  
Proliferating Cells ◽  
Monolayer Cell Culture ◽  
3 Dimensional ◽  
Rapid Generation ◽  
The Cost ◽  
High Throughput Experiments

Monolayer cell culture, while useful for basic in vitro studies, are not physiologically relevant. Spheroids, on the other hand provide a more complex 3-dimensional (3D) structure which more resemble the in vivo tumour growth thereby allowing results obtained with those on proliferation, cell death, differentiation, metabolism, and various anti-tumour therapies to be more predictive of in vivo outcomes. However, the cost associated with their generation often involve expensive, plate, media, and growth supplements, which have limited their use for high throughput experiments. The protocol herein presents a novel and rapid generation for single spheroids of various cancer cell lines, U87 MG; SEBTA-027; SF188, brain cancer cells, DU-145, TRAMP-C1, prostate cancer cells, in 96-round bottom well plates. Cells are washed with anti-adherent solution, and the homogeneous compact spheroid morphology was evidenced as early as 24 hours after 10 minutes centrifugation for the seeded cells. By using confocal microscopy, the proliferating cells were traced in the rim and the dead cells were found inside the core region of the spheroid. The H&E stain of spheroid slices and the western blotting were utilised to investigate the tightness of the cell packaging by adhesion proteins. Carnosine was used as an example of treatment for U87 single spheroids. The protocol allows the rapid generation of spheroids, which will help towards reducing the number of tests performed on animals.

RNA polymerase II subunit composition, stoichiometry, and phosphorylation

1990 ◽  
Vol 10 (5) ◽  
pp. 1915-1920 ◽  
Author(s):  
P A Kolodziej ◽  
N Woychik ◽  
S M Liao ◽  
R A Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Synthesis ◽  
Subunit Composition ◽  
Subunit Gene ◽  
Cell Extracts ◽  
Alpha Amanitin

RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.

scholarly journals H3K27me3 Depletion during Differentiation Promotes Myogenic Transcription in Porcine Satellite Cells

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 231
Author(s):  
Sheng Wang ◽  
Yan Sun ◽  
Ruimin Ren ◽  
Junhui Xie ◽  
Xiaohuan Tian ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Rna Polymerase Ii ◽  
Satellite Cells ◽  
Myogenic Differentiation ◽  
Integrated Analysis ◽  
Proliferating Cells ◽  
A Genome ◽  
Chromatin Immunoprecipitation Sequencing ◽  
Wide Scale

Background: Porcine skeletal muscle satellite cells play important roles in myogenesis and muscle regeneration. Integrated analysis of transcriptome and histone modifications would reveal epigenomic roles in promoting myogenic differentiation in swine. Methods: Porcine satellite cells (PSCs) were isolated and in-vitro cultured from newborn piglets. RNA Sequencing (RNA-Seq) and Chromatin Immunoprecipitation Sequencing (ChIP-Seq) experiments were performed using proliferating cells and terminal myotubes in order to interrogate the transcriptomic profiles, as well as the distribution of histone markers—H3K4me3, H3K27me3, and H3K27ac—and RNA polymerase II. Results: The study identified 917 differentially expressed genes during cell differentiation. The landscape of epigenetic marks was displayed on a genome-wide scale, which had globally shrunken. H3K27me3 reinforcement participated in obstructing the transcription of proliferation-related genes, while its depletion was closely related to the up-regulation of myogenic genes. Furthermore, the degree of H3K27me3 modification was dramatically reduced by 50%, and 139 myogenic genes were upregulated to promote cell differentiation. Conclusions: The depletion of H3K27me3 was shown to promote porcine satellite cell differentiation through upregulating the transcription level of myogenic genes. Our findings in this study provide new insights of the epigenomic mechanisms occurring during myogenic differentiation, and shed light on chromatin states and the dynamics underlying myogenesis.

scholarly journals Enhanced Serine Palmitoyltransferase Expression in Proliferating Fibroblasts, Transformed Cell Lines, and Human Tumors

2003 ◽  
Vol 51 (6) ◽  
pp. 715-726 ◽  
Author(s):  
Jill M. Carton ◽  
David J. Uhlinger ◽  
Ameesha D. Batheja ◽  
Claudia Derian ◽  
George Ho ◽  
...  
Keyword(s):  
Wound Repair ◽  
Cell Activity ◽  
Serine Palmitoyltransferase ◽  
Proliferating Cells ◽  
Stromal Fibroblasts ◽  
Quiescent Cells ◽  
Cell Metastasis ◽  
Vitro Model ◽  
Transformed Cell Lines

Metastatic processes, including cell invasion, extracellular matrix degradation, and tissue remodeling, require cellular reorganization and proliferation. The cell signaling molecules required and the proteins involved in cell restructuring have not been completely elucidated. We have been studying the role of sphingolipids in normal cell activity and in several pathophysiological states. In this study we used immunohistochemistry to observe the presence of the two known subunits of serine palmitoyltransferase (SPT) in proliferating cells, in an in vitro model of wound repair, and in human malignant tissue. We report increased expression of the two subunits, SPT1 and SPT2, in the proliferating cells in these models. We also demonstrate a change in subcellular localization of the SPT subunits from predominantly cytosolic in quiescent cells to nuclear in proliferating cells. In addition, we observed SPT1 and SPT2 immunoreactivity in reactive stromal fibroblasts surrounding the carcinoma cells of some of the tumors. This enhanced SPT expression was absent in the stromal fibroblasts surrounding normal epithelial cells. Our results suggest a potential role for overexpression of SPT in the processes of cell metastasis.

scholarly journals RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

1990 ◽  
Vol 10 (5) ◽  
pp. 1915-1920 ◽  
Author(s):  
P A Kolodziej ◽  
N Woychik ◽  
S M Liao ◽  
R A Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Synthesis ◽  
Subunit Composition ◽  
Subunit Gene ◽  
Cell Extracts ◽  
Alpha Amanitin

RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.

scholarly journals Magnetic Compression of Tumor Spheroids Increases Cell Proliferation In Vitro and Cancer Progression In Vivo

Cancers ◽  
2022 ◽  
Vol 14 (2) ◽  
pp. 366
Author(s):  
Gaëtan Mary ◽  
Brice Malgras ◽  
Jose Efrain Perez ◽  
Irène Nagle ◽  
Nathalie Luciani ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Peritoneal Cancer Index ◽  
Colon Cancer Cells ◽  
Tumor Spheroids ◽  
Proliferating Cells ◽  
The Impact

A growing tumor is submitted to ever-evolving mechanical stress. Endoscopic procedures add additional constraints. However, the impact of mechanical forces on cancer progression is still debated. Herein, a set of magnetic methods is proposed to form tumor spheroids and to subject them to remote deformation, mimicking stent-imposed compression. Upon application of a permanent magnet, the magnetic tumor spheroids (formed from colon cancer cells or from glioblastoma cells) are compressed by 50% of their initial diameters. Such significant deformation triggers an increase in the spheroid proliferation for both cell lines, correlated with an increase in the number of proliferating cells toward its center and associated with an overexpression of the matrix metalloproteinase−9 (MMP−9). In vivo peritoneal injection of the spheroids made from colon cancer cells confirmed the increased aggressiveness of the compressed spheroids, with almost a doubling of the peritoneal cancer index (PCI), as compared with non-stimulated spheroids. Moreover, liver metastasis of labeled cells was observed only in animals grafted with stimulated spheroids. Altogether, these results demonstrate that a large compression of tumor spheroids enhances cancer proliferation and metastatic process and could have implications in clinical procedures where tumor compression plays a role.

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