scholarly journals NOTCH1 Mutation in CLL Enhances Cell Cycle G1/S Transition through Specific Cyclin Overexpression: Preclinical Ground for CDK4/6 Targeted Inhibition

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1544-1544
Author(s):  
Salvador Carrillo-Tornel ◽  
Tzu Hua Chen-Liang ◽  
María Zurdo ◽  
Anna Puiggros ◽  
Andrea Gómez-Llonín ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Flow Cytometry ◽  
Next Generation Sequencing ◽  
S Phase ◽  
Wild Type ◽  
Trisomy 12 ◽  
Anti Cd20 ◽  
Generation Sequencing

Abstract Introduction: The partially understood biological consequences of the NOTCH1 acquired lesion, seems to be distinctive enough among chronic lymphocytic leukemia (CLL) patients, as clinical studies have repeatedly found specific features: intermediate prognosis, anti-CD20 poorer responses, and a higher frequency of trisomy 12 and Richter transformation. Though located in a different domain, the activating nature of NOTCH1 mutation in T lymphoblastic leukemia relies on cell cycle regulators. In fact, pivotal studies, from the pre-next generation sequencing era, showed dysregulation of cyclins-gene expression, as driver of the unique CLL features. Thus, our goal was to revisit the cell cycle in CLL, but focusing now in the NOTCH1 mutated subset (NOTCH1MUT), hypothesizing that biological differences versus wild type cases (NOTCH1WT) would explain the clinical ones, and exploiting potential differences with targeted molecules in vitro. Methods: From 2010 to 2019, presentation bone marrow aspirates or blood samples DNA was collected during the diagnostic workout from 378 CLL patients, all of them annotated by next generation sequencing. G 0/early-G 1 effectors gene expression was measured by RT-qPCR in negatively immunoselected circulating CLL cells. A siRNA approach was selected for silencing by electroporation 7 NOTCH1WT and 2 NOTCH1MUT cases. Cell cycle and apoptosis flow cytometry assays were performed on cultured fresh primary cells from n? NOTCH1MUT and 4 NOTCH1WT cases, before and after exposure to different concentrations of palbociclib, a CDK4/6 inhibitor. Results: We found that 37/378 (9.8%) of patients harbored a NOTCH1 mutation. NOTCH1MUTcases presented with higher lymphocyte counts [NOTCH1MUT 17.2 x10 9/L vs. NOTCH1WT 9.7 x10 9/L; p=0.042], trisomy 12 (35.1% vs. 11.4%; p<0.001) and a higher frequency of an unmutated IGHV status (70% vs. 21%; p<0.001). Of note, NOTCH1MUT patients had poorer responses to anti-CD20 based schemes than NOTCH1WT patients (35.7 vs. 69.8% complete response; p). We found that NOTCH1MUT cases showed a relevant increase of 38-fold change (FC) for CCND3, 27-FC for CDK4 and CCND2, 11-FC for CCND1 and 9-FC for CDK6 gene expression in negatively immunoselected circulating CLL cells at diagnosis. In addition, NOTCH1MUT cases displayed a statistically significant higher percentage of cells in the S phase than the wild type cases (21% vs. 1%, p=0.004). Though significance was not met, NOTCH1MUT cases showed a higher percentage of events within G 2-M (28% vs. 26 %). Next, we incorporated the flow cytometry assay to in vitro palbociclib treated CLL cells from 3 NOTCH1MUT and 4 NOTCH1WT cases. Five days after culture stimulation, cells were exposed to 38 and 76 μM (dose range for reaching maximum CLL cells sensitivity plateau) of the drug for 48 hours. As stated above, NOTCH1MUT cases were characterized by a much higher proportion of cells in S phase at baseline (21%), which was reduced in a dose dependent manner to an 8% and a 6% after exposure to palbociclib, respectively. The standard 48-72 hours drug assay may not be the most suitable for slow growth tumors as CLL and, in particular, for testing cell cycle inhibitors. Thus, we designed an assay for two cell cycles based on the average population doubling time of the primary cell culture experiments (0.6 in 72 hours), and using the mean steady state plasma concentration of palbociclib achieved clinically: 1 μM. After 120 hours, the baseline 15% of cells in S phase was reduced to an 0.64% after exposure to palbociclib 1 μM in NOTCH1MUT cases and a 1.8x-increase in the percentage dead cells was noted, compared with NOTCH1WT cases. Conclusions: Compared with NOTCH1WT CLL cases, we describe an overexpression of effectors of early phase in NOTCH1MUT. This profile made NOTCH1MU cells more suited to enter and traverse through the cell cycle and could explain, in part, the proliferative clinical-biological features of this subset of patients and opening a window for exploiting therapeutically these differences. Ours experiments in vitro with palbociclib sets the ground for the clinical research. Figure 1 Figure 1. Disclosures Jerez: BMS: Consultancy; Novartis: Consultancy; GILEAD: Research Funding.

scholarly journals Regulation of Id3 cell cycle function by Cdk-2-dependent phosphorylation.

1997 ◽  
Vol 17 (12) ◽  
pp. 6815-6821 ◽  
Author(s):  
R W Deed ◽  
E Hara ◽  
G T Atherton ◽  
G Peters ◽  
J D Norton
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Complex Formation ◽  
S Phase ◽  
Regulatory Function ◽  
Specific Gene ◽  
Wild Type ◽  
E Box ◽  
Phase Entry

The functions of basic helix-loop-helix (bHLH) transcription factors in activating differentiation-linked gene expression and in inducing G1 cell cycle arrest are negatively regulated by members of the Id family of HLH proteins. These bHLH antagonists are induced during a mitogenic signalling response, and they function by sequestering their bHLH targets in inactive heterodimers that are unable to bind to specific gene regulatory (E box) sequences. Recently, cyclin E-Cdk2- and cyclin A-Cdk2-dependent phosphorylation of a single conserved serine residue (Ser5) in Id2 has been shown to occur during late G1-to-S phase transition of the cell cycle, and this neutralizes the function of Id2 in abrogating E-box-dependent bHLH homo- or heterodimer complex formation in vitro (E. Hara, M. Hall, and G. Peters, EMBO J. 16:332-342, 1997). We now show that an analogous cell-cycle-regulated phosphorylation of Id3 alters the specificity of Id3 for abrogating both E-box-dependent bHLH homo- or heterodimer complex formation in vitro and E-box-dependent reporter gene function in vivo. Furthermore, compared with wild-type Id3, an Id3 Asp5 mutant (mimicking phosphorylation) is unable to promote cell cycle S phase entry in transfected fibroblasts, whereas an Id3 Ala5 mutant (ablating phosphorylation) displays an activity significantly greater than that of wild-type Id3 protein. Cdk2-dependent phosphorylation therefore provides a switch during late G1-to-S phase that both nullifies an early G1 cell cycle regulatory function of Id3 and modulates its target bHLH specificity. These data also demonstrate that the ability of Id3 to promote cell cycle S phase entry is not simply a function of its ability to modulate bHLH heterodimer-dependent gene expression and establish a biologically important mechanism through which Cdk2 and Id-bHLH functions are integrated in the coordination of cell proliferation and differentiation.

scholarly journals Sequential Assessment of Cell Cycle S Phase in Flow Cytometry: A Non-Isotopic Method to Measure Lymphocyte ActivationIn Vitro

1997 ◽  
Vol 14 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Ch. Kohler ◽  
M. N. Kolopp‐Sarda ◽  
A. De March‐Kennel ◽  
A. Barbaud ◽  
M. C. Béné ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cellular Proliferation ◽  
Tritiated Thymidine ◽  
S Phase ◽  
Phase Cell ◽  
Isotopic Method ◽  
Isotopic Methods ◽  
Recall Antigen

Lymphocyte multiplication can be inducedin vitroby mitogens or specific antigens, and is usually measured using isotopic methods involving tritiated thymidine. Cellular proliferation can also be analyzed by flow cytometry techniques based on cell cycle analysis through the measurement of DNA content. We applied this method to lymphocytes from 113 individuals, to evaluate lymphocyte proliferation after stimulationin vitroby a mitogen (phytohaemagglutinin, PHA) or a recall antigen (tetanus toxoid), using a kinetic approach with four points sequential measurements of the S and G2 phases over six days of culture. The proportion of cells in S phase after PHA stimulation was significantly higher than in controls overall and as early as on day three of the culture. Activation with a recall antigen significantly induced increasing S phase cell proportions up to day six. These data suggest that flow cytometric assessment of the S phase could be a useful alternative to isotopic methods measuring lymphocyte reactivityin vitro.

scholarly journals Identification of potential genes in upper tract urothelial carcinoma using next-generation sequencing with bioinformatics and in vitro analyses

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11343
Author(s):  
Hsiang-Ying Lee ◽  
Ching-Chia Li ◽  
Wei-Ming Li ◽  
Ya-Ling Hsu ◽  
Hsin-Chih Yeh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Next Generation Sequencing ◽  
Candidate Genes ◽  
Venn Diagram ◽  
Next Generation ◽  
Upper Tract ◽  
Urothelial Carcinomas ◽  
Gene And Protein Expression ◽  
Generation Sequencing

Background We aimed to identify prognostic biomarkers of upper tract urothelial carcinomas (UTUCs), including microRNAs (miRNAs) and genes which account for only 5% to 10% of all urothelial carcinomas (UCs). In Taiwan, this figure is markedly higher, where it can reach up to 30% of UC cases. Materials and Methods Using next-generation sequencing (NGS), we analyzed two pairs of renal pelvis tumors and adjacent normal urothelial tissues to screen miRNAs and messenger RNAs. By combining bioinformatics analysis from miRmap, Gene Expression Omnibus (GEO), and Oncomine and Ingenuity® Pathway Analysis databases, we identified candidate genes. To search for upstream miRNAs with exact target binding sites, we used miRmap, TargetScan, and miRDB to enforce evidence. Then, we clarified gene and protein expression through an in vitro study using western blot analysis and quantitative real-time reverse transcriptase-PCR. Results Interactions between selected target genes obtained using the NGS and miRmap methods were assessed through a Venn diagram analysis. Six potential genes, namely, PDE5A, RECK, ZEB2, NCALD, PLCXD3 and CYBRD1 showed significant differences. Further analysis of gene expression from the GEO dataset indicated lower expression of PDE5A, RECK, ZEB2, and CYBRD1 in bladder cancer tissue than in normal bladder mucosa, which indicated that PDE5A, RECK, ZEB2, and CYBRD1 may act as tumor suppressors in UTUC. In addition, we compared the expression of these genes in various UC cell lines (RT4, BFTC905, J82, T24, UMUC3, 5637, BFTC 909, UMUC14) and found decreased expression of PDE5A in muscle-invasive UC cells compared with the RT4 cell line. Furthermore, by using paired UTUC and normal tissues from 20 patients, lower PDE5A expression was also demonstrated in tumor specimens. Conclusions Our findings suggest these candidate genes may play some roles in UTUC progression. We propose that these markers may be potential targets clarified by in vitro and in vivo experiments. PDE5A also potentially presents tumor suppressor genes, as identified by comparing the expression between normal and tumor specimens.

scholarly journals Endothelial cells isolated from caveolin-2 knockout mice display higher proliferation rate and cell cycle progression relative to their wild-type counterparts

2010 ◽  
Vol 298 (3) ◽  
pp. C693-C701 ◽  
Author(s):  
Leike Xie ◽  
Philippe G. Frank ◽  
Michael P. Lisanti ◽  
Grzegorz Sowa
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Inhibitors ◽  
Fold Increase ◽  
S Phase ◽  
Lower Percentage ◽  
Wild Type ◽  
Cycle Progression ◽  
Molecular Events

The goal of this study was to determine whether caveolin-2 (Cav-2) is capable of controlling endothelial cell (EC) proliferation in vitro. To realize this goal, we have directly compared proliferation rates and cell cycle-associated signaling proteins between lung ECs isolated from wild-type (WT) and Cav-2 knockout (KO) mice. Using three independent proliferation assays, we have determined that Cav-2 KO ECs proliferate by ca. 2-fold faster than their WT counterparts. Cell cycle analysis by flow cytometry of propidium iodide-stained cells showed a relatively higher percentage of Cav-2 KO ECs in S and G2/M and lower percentage in Go/G1 phases of cell cycle relative to their WT counterparts. Furthermore, an over 2-fold increase in the percentage of S phase-associated Cav-2 KO relative to WT ECs was independently determined with bromodeoxyuridine incorporation assay. Mechanistically, the increase in proliferation/cell cycle progression of Cav-2 KO ECs correlated well with elevated expression levels of predominantly S phase- and G2/M phase-associated cyclin A and B1, respectively. Further mechanistic analysis of molecular events controlling cell cycle progression revealed increased level of hyperphosphorylated (inactive) form of G1 to S phase transition inhibitor, the retinoblastoma protein in hyperproliferating Cav-2 KO ECs. Conversely, the expression level of the two cyclin-dependent kinase inhibitors p16INK4 and p27Kip1 was reduced in Cav-2 KO ECs. Finally, increased phosphorylation (activation) of proproliferative extracellular signal-regulated kinase 1/2 was observed in hyperproliferating Cav-2 KO ECs. Overall, our data suggest that Cav-2 negatively regulates lung EC proliferation and cell cycle progression.

scholarly journals Gene Expression Changes Associated with Nintedanib Treatment in Idiopathic Pulmonary Fibrosis Fibroblasts: A Next-Generation Sequencing and Bioinformatics Study

2019 ◽  
Vol 8 (3) ◽  
pp. 308 ◽  
Author(s):  
Chau-Chyun Sheu ◽  
Wei-An Chang ◽  
Ming-Ju Tsai ◽  
Ssu-Hui Liao ◽  
Inn-Wen Chong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Next Generation Sequencing ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Protein Interactions ◽  
Next Generation ◽  
Protein Protein Interactions ◽  
Bioinformatics Study ◽  
Generation Sequencing

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal interstitial lung disease. Therapeutic options for IPF remain limited. Nintedanib, a tyrosine kinase inhibitor approved for IPF treatment, is known to inhibit fibroblasts proliferation, migration and transformation to myofibroblasts. However, how nintedanib changes gene regulations in IPF has never been systematically investigated. We conducted a next-generation sequencing and bioinformatics study to evaluate the changes of mRNA and miRNA profiles in IPF fibroblasts treated with 2 µM and 4 µM nintedanib, compared to those without treatment. We identified 157 upregulated and 151 downregulated genes and used STRING and DAVID databases for analysis of protein–protein interactions, biological pathways, and molecular functions. We found strong protein–protein interactions within these dysregulated genes, mostly involved in the pathways of cell cycle and mitotic cell cycle. We also discovered 13 potential miRNA–mRNA interactions associated with nintedanib treatment. After validation using miRDB, TargetScan, and RT-qPCR, we identified 4 downregulated genes (DDX11, E2F1, NPTX1, and PLXNA4) which might be repressed by the upregulated hsa-miR-486-3p. According to the proposed functions of DDX11, E2F1, and PLXNA4 reported in previous studies, these gene expression changes together might contribute to decreased proliferation of fibroblasts and decreased angiogenesis in the microenvironment of IPF. Our findings need further studies to confirm.

Silencing of TEL/AML1 In Definitive Leukemic Cells Does Not Impair Cell Survival

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3229-3229
Author(s):  
Marketa Zaliova ◽  
Jozef Madzo ◽  
Gunnar Cario ◽  
Jan Trka
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Viability ◽  
Fusion Gene ◽  
Global Gene Expression ◽  
Leukemic Cells ◽  
Cell Cycle Distribution ◽  
Wild Type ◽  
Egfp Reporter

Abstract Abstract 3229 The most frequent structural chromosomal aberration in childhood acute lymphoblastic leukemia t(12;21) generates TEL/AML1 fusion gene. Resulting TEL/AML1 protein probably acts as an aberrant transcription factor that deregulates AML1-dependent transcription but its target genes and thus also the exact role in leukemic cells remain unknown. In vivo studies showed that TEL/AML1 itself is not sufficient to cause leukemia but may induce a preleukemic state characterized by the increased numbers of multipotent or B-cell progenitors with an incomplete block of differentiation. Despite its role for leukemia establishment the relevance of TEL/AML1 fusion gene for leukemia persistance has not been studied enough.To address this question and to explore the possibility of TEL/AML1-targeted therapy, we studied the effects of RNAi-mediated TEL/AML1 silencing on leukemic cells. As the only siRNA used for TEL/AML1 silencing published so far (Diakos et al, Blood, 2007) targets also the wild type AML1 (46% transcript reduction, our data), our first goal was to identify efficient and TEL/AML1-specific siRNA. We designed eleven different siRNAs spanning the fusion point of TEL/AML1 lacking the total sequence homology to wild type TEL and AML1 alleles to avoid their silencing. These 11 siRNAs were tested in HeLa cells transgenic for TEL/AML1-ires2-EGFP reporter. After lipofection into HeLa cells the efficiency of individual siRNAs was measured as a decrease of EGFP reporter fluorescence by flow cytometry. The best five siRNAs, that induced 50–58% silencing of the EGFP reporter, were tested at the mRNA level in TEL/AML-positive leukemic cell line. 24h after electroporation of siRNAs, when the silencing reached its maximum, two most efficient siRNAs induced 58% and 57% TEL/AML1 transcript reduction, respectively. We achieved 61% TEL/AML1 transcript reduction with the pool of both siRNAs while there was only slight reduction (14%) of wild type AML1 transcript. We used this efficient and specific siRNA pool to silence TEL/AML1 in REH and UOC-B6 TEL-AML1 positive cell lines and studied its effect on cell viability, proliferation and global gene expression. Applying two rounds of siRNA electroporation within 48 hours interval we achieved 74% and 86% TEL/AML1 protein knockdown in REH and UOC-B6 cells, respectively. We used trypan blue staining followed by optical microscopy to monitor cell viability and staining with annexin V and propidium idode to assess apoptosis rate by flow cytometry. Analysis of DNA content using staining with propidium iodide was performed to assess cell-cycle distribution. Incorporation of nucleoside analog was measured by flow cytometry to analyse de novo DNA synthesis as an indicator of proliferation rate. Despite the common expectation derived from studies on other fusion oncogenes (BCR/ABL, AML1/ETO, E2A/PBX), TEL/AML1 silencing neither decreased cell viability, nor induced apoptosis. On the contrary, TEL/AML1 depletion was accompanied by the slight but significant increase in the fraction of S-phase cells and corresponding rise in proliferation rate. Opposite effects on cell cycle distribution and proliferation were induced when we silenced wild type AML1. These findings support our hypothesis that TEL/AML1 may block previously established AML1 function in G1/S progression through the cell cycle. In line with the lack of effect on cell viability and discreet effect on cell-cycle distribution and proliferation we found no significant changes in global gene expression pattern upon TEL/AML1 depletion. Our data indicate, that TEL/AML1 is dispensable for the survival of definitive leukemic cells. This work was supported by grants MSM0021620813 and MZOFNM2005. Disclosures: No relevant conflicts of interest to declare.

ID3 Is a Novel Tumor Suppressor Gene in Burkitt Lymphom

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 898-898
Author(s):  
Cassandra L Love ◽  
Dereje Jima ◽  
Zhen Sun ◽  
Rodney R. Miles ◽  
Cherie H. Dunphy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Tumor Suppressor ◽  
Burkitt Lymphoma ◽  
Cell Cycle Progression ◽  
Suppressor Gene ◽  
S Phase ◽  
Wild Type ◽  
Cycle Progression ◽  
Helix Loop Helix

Abstract Abstract 898 Burkitt Lymphoma (BL) is a highly proliferative form of non-Hodgkin lymphoma and is characterized by translocation of the C-MYC gene to the immunoglobulin gene loci resulting in deregulation. The role of collaborating gene mutations in BL is largely unknown. We performed whole exome sequencing and gene expression profiling of 57 Burkitt lymphoma and 94 DLBCL exomes. Mutational analysis revealed that ID3 is recurrently mutated in 38% of Burkitt lymphoma samples. ID3 mutations did not occur in any of the 94 DLBCL cases. ID3 gene expression was also found to be a distinguishing feature of Burkitt lymphomas (P<10−6), compared to DLBCL. We found a total of 27 distinct mutations in the ID3 genes among the 22 BL cases. These included five frameshift, four nonsense, and 18 missense mutations. We validated 16 of these events with Sanger sequencing with over 90% concordance. All of these mutations were located in the highly conserved helix-loop-helix region located on Exon 1. We explored the biological significance of ID3 mutations by initially comparing the gene expression profiles of BL cases that had mutated and wild-type ID3. Gene set enrichment analysis showed that those samples with mutated ID3 had higher expression of genes that were involved in cell cycle regulation, specifically those involved in the G1-S transition (P=0.01). In order to experimentally investigate the functional consequences of ID3 mutation, we generated mutant constructs corresponding to six different ID3 mutations observed in BLs. These mutant constructs were cloned into lentiviral vectors and overexpressed in BL cells that were wild type for ID3. We then performed cell cycle analysis for these wild type cells expressing GFP controls or the mutant constructs. We found that BL cells expressing each of the six mutant constructs demonstrated significant cell cycle progression from G1 to S phase compared to wild-type (P=0.01). Separately, we tested the effects of expressing mutant ID3 in cell proliferation assays and found that cells expressing mutant ID3 were considerably more proliferative than those expressing wild type (P=0.03). Conversely, we over-expressed the wild type form of ID3 in BL cells that had mutated ID3. These experiments completely rescued the observed phenotypes of the mutant ID3 constructs, with reduced cell cycle progression through increased G1 phase and decreased S-phase (P=0.04). We also noted decreased cell proliferation in these cells (P=0.03). These experiments support a role for ID3 as a novel tumor suppressor gene in Burkitt lymphoma. ID3 is a basic helix loop helix (bHLH) protein that binds to other E-proteins, blocking their ability to bind DNA. ID3 has been shown to be involved in a variety of biological processes including development and T and B cell differentiation. ID3 knockout mice have been shown to develop T cell as well as B cell lymphomas. Our data implicates this gene for the first time as a tumor suppressor in human cancer. Disclosures: No relevant conflicts of interest to declare.

Steady State Differences In Metabolic Properties Of Bone Marrow Versus Spleen Erythroid Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 943-943 ◽  
Author(s):  
Genís Campreciós ◽  
Jeffrey Barminko ◽  
Jeffrey Bernitz ◽  
Saghi Ghaffari
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
S Phase ◽  
Erythroid Cells ◽  
Wild Type ◽  
Mitochondrial Mass ◽  
Stress Erythropoiesis ◽  
Erythroid Precursors ◽  
Metabolic Conditions

Abstract Erythropoiesis in adult mice occurs principally in the bone marrow, while stress erythropoiesis is mostly localized in the spleen. Although both bone marrow and spleen produce fully functional mature red blood cells, we noticed that the rate of maturation of erythroid precursors (based on the ratio of mature/immature erythroid cells) is over 10 times higher in the spleen as compared to the bone marrow in C57BL6 mice (3.7 ± 0.6 in the spleen as compared to 0.4 ± 0.1 in the bone marrow, n ≥ 10 mice). Cell cycle analysis of erythroid precursors revealed that bone marrow erythroid cells cycle up to 3 times more than their spleen counterparts (88 ± 1% vs 25 ± 7% of cells in the S phase, n=3). As reactive oxygen species (ROS) influence cell cycle, we measured ROS levels by flow cytometry using the CM-H2DCFDA probe. To our surprise, we found ROS levels to decrease (rather than increase) progressively in the bone marrow erythroid cells as they mature and accumulate hemoglobin. Interestingly, the levels of ROS were twice as high in the spleen erythroid cells as compared to erythroid cells in the bone marrow. As mitochondria are a major site of ROS production we measured mitochondrial mass by flow cytometry using Mitotracker Green. Mitochondrial mass was found to be two fold lower in the spleen erythroid cells as compared to the bone marrow. In agreement with these findings, qRT-PCR expression analysis of different antioxidant enzymes such as gluthathione peroxidases Gpx1 and Gpx4 showed higher levels in the bone marrow as compared to the spleen erythroid precursors. In particular, Gpx1 expression increased ten fold during erythroid maturation in the bone marrow while in the spleen the expression of Gpx1 did not change significantly. Together these results suggest erythroid metabolic profile is distinct in the spleen as compared to the bone marrow at the steady state. In order to compare homeostatic versus stress erythropoiesis we analyzed bone marrow and spleen from Foxo3-/- and Th3/+ thalassemic mice, two models of ineffective erythropoiesis with different degrees of severity and splenomegaly. As anticipated Foxo3-/- and Th3/+ erythroid precursors displayed decreased rate of maturation as compared to wild type cells in both bone marrow (0.3 ± 0.02 and 0.12 ± 0.01 for Foxo3-/- and Th3/+ respectively, n ≥ 10) and spleen (2.1 ± 0.3 and 0.42 ± 0.1 for Foxo3-/- and Th3/+ respectively, n ≥ 10 mice per group) and cell cycle analysis showed an increased number of cells in the S phase in both Foxo3-/- and Th3/+ spleen erythroid cells as compared to wild type (63 ± 2% and 79 ± 2% respectively, n=3). Unexpectedly however, ROS levels in both Foxo3-/- and Th3/+ spleen erythroid cells were decreased as compared to their wild type counterparts. The observed inverse correlation between cell cycling and ROS levels was further supported by expression analysis of Gpx1 and Gpx4, the levels of which were increased in Foxo3-/- as compared to wild type spleen erythroid cells. Collectively our results highlight the different erythroid metabolic conditions in the bone marrow versus spleen under both homeostatic and disease states. Further investigations elucidating differences in metabolic conditions and properties of erythroid cells in bone marrow versus spleen should improve our understanding of generation of erythroid cells under stress and the production of erythroid cells in vitro. Disclosures: No relevant conflicts of interest to declare.

A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

2019 ◽  
Vol 19 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Yang Liu ◽  
Jingyin Zhang ◽  
Shuyun Feng ◽  
Tingli Zhao ◽  
Zhengzheng Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Cyclin D ◽  
Dna Relaxation ◽  
Cycle Arrest ◽  
H460 Cell ◽  
H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

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