scholarly journals Cell Cycle Regulates Phosphorylation of RUNX1 to Modulate Megakaryocyte-Erythroid Progenitor Fate Specification

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 15-15
Author(s):  
Yi-Chien Lu ◽  
Vanessa M Scanlon ◽  
Juliana Xavier-Ferrucio ◽  
Lin Wang ◽  
Alan Friedman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Tyrosine Phosphorylation ◽  
Mrna Levels ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Fate Specification ◽  
Protein Levels ◽  
Differential Phosphorylation ◽  
Cell Cycle Machinery

Human Megakaryocytic-Erythroid Progenitors (MEP) produce megakaryocytic progenitors (MkP) and erythroid progenitors (ErP). Though some of the players have been identified, the molecular mechanisms underlying the MEP fate decision have not yet been determined. Using a functional single cell CFU-Mk/E assay, and single-cell RNA sequencing (scRNAseq), we have revealed that MEP cell cycling regulates MEP fate decisions with decreased cycling promoting Mk fate commitment and increased cycling promoting E fate commitment (Lu et al, Cell Reports, 2018). Our data point to RUNX1 (aka AML1), already known to be important for both Mk and E maturation, as playing a key role in MEP fate determination. RUNX1 target genes vary significantly between Common Myeloid Progenitors (CMP), MEP, MkP, and ErP. For example, the RUNX1 targets MPL, FLI1, and THBS1 are higher in MkP than in MEP and lowest in CMP and ErP. Analysis of scRNAseq data indicates that 11.8% and 9.3% of differentially (adj.p < 0.05, fold change > 2) expressed genes are predicted RUNX1 targets when comparing MEP to MkP and MEP to ErP, respectively (p=2.9e-8 and 5.6e-16). However, RUNX1 mRNA levels do not change significantly between CMP, MEP, MkP and ErP. We therefore assessed whether total RUNX1 protein levels and post-translational modifications change with Mk and E lineage commitment. Intracellular staining normalized to levels in CMP revealed that total RUNX1 protein is elevated 1.34-fold from CMP to MEP, is highest (1.65-fold) in MkP, and is intermediate in ErP (1.5-fold) consistent with RUNX1 perhaps promoting Mk over E fate. RUNX1 can be phosphorylated by cyclin dependent serine/threonine kinases (CDKs) as well as Src tyrosine kinases. Significant differences in RUNX1 phosphoprotein levels were revealed by intracellular FACS assays for differential phosphorylation of RUNX1. Phosphoserine modified RUNX1 (Ser21, Ser276, and Ser 397) levels, which likely reflect active RUNX1, are highest in MkP (1.28-fold over MEP), and diminished in ErP (-1.32-fold over MEP). To determine whether RUNX1 and phosphor-RUNX1 variants affect MEP commitment, we overexpressed wildtype (WT) RUNX1 or RUNX1-S4D (phospho-mimetic on S249, S277, T273, S276 residues). WT RUNX1 overexpression affected fate specification of primary MEP (p<0.05) with an increase in the Mk-only colony percentage from 24% to 43% at the expense of E-only colonies (from 26% to 16%) with little change in bipotent CFU-Mk/E. RUNX1-S4D more dramatically (p<0.04) increased Mk-only colonies (from 25% to 75%) and decreased E-only (from 25% to < 4%) and Mk/E (from 50% to 21%) colonies. Surprisingly, overexpression of RUNX1-S4D in FACSorted primary human ErP caused them to develop more (p<0.01) Mk/E-colonies (from 12% to 60%) with a dramatic decrease in E-only colonies (from 85% to 28%) suggesting that the E commitment of ErP is reversed (or reprogrammed) by activated RUNX1. In contrast, RUNX1 variants mimicking tyrosine phosphorylation (RUNX1-Y6D) promote E fate specification. Tyrosine phosphorylation on RUNX1 is mediated by Src family kinases (Huang te al, Genes Dev., 2012). We found that Src kinase inhibition results in increasing Mk lineage bias (from 20% to 71%) suggest differential phosphorylation of RUNX1 plays a pivotal role in MEP fate specification. Epigenetic modifications induced by phospho-serine vs phospho-tyrosine mimetics are currently under evaluation. Combining our data on cell cycle control of MEP with RUNX1 reveals that RUNX1 inhibition prevents the Mk bias induced by slowing the cell cycle. For example, the RUNX1 inhibitor Ro5-3335 causes MEP to have an Er bias from 27% to 61% while the mTOR inhibitor Rapamycin (slows MEP cell cycle) induces a Mk bias from 19% to 52%. The combination of Ro5-3335 plus Rapa results in increased Er from 27% to 45% suggest that RUNX1 influences MEP fate specification downstream of the cell cycle machinery. In summary, the phosphorylation status of RUNX1 in primary human hematopoietic progenitors regulates MEP fate commitment. Detailed mechanisms linking the cell cycle machinery to RUNX1 protein levels and function toward Mk vs E specification are being explored. Disclosures No relevant conflicts of interest to declare.

Starvation promotes Dictyostelium development by relieving PufA inhibition of PKA translation through the YakA kinase pathway

Development ◽  
1999 ◽  
Vol 126 (14) ◽  
pp. 3263-3274 ◽  
Author(s):  
G.M. Souza ◽  
A.M. da Silva ◽  
A. Kuspa
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mrna Levels ◽  
Wild Type ◽  
Mobility Shift ◽  
Developmental Defects ◽  
C Protein ◽  
Protein Levels ◽  
Cell Extracts ◽  
Puf Protein

When nutrients are depleted, Dictyostelium cells undergo cell cycle arrest and initiate a developmental program that ensures survival. The YakA protein kinase governs this transition by regulating the cell cycle, repressing growth-phase genes and inducing developmental genes. YakA mutants have a shortened cell cycle and do not initiate development. A suppressor of yakA that reverses most of the developmental defects of yakA- cells, but none of their growth defects was identified. The inactivated gene, pufA, encodes a member of the Puf protein family of translational regulators. Upon starvation, pufA- cells develop precociously and overexpress developmentally important proteins, including the catalytic subunit of cAMP-dependent protein kinase, PKA-C. Gel mobility-shift assays using a 200-base segment of PKA-C's mRNA as a probe reveals a complex with wild-type cell extracts, but not with pufA- cell extracts, suggesting the presence of a potential PufA recognition element in the PKA-C mRNA. PKA-C protein levels are low at the times of development when this complex is detectable, whereas when the complex is undetectable PKA-C levels are high. There is also an inverse relationship between PufA and PKA-C protein levels at all times of development in every mutant tested. Furthermore, expression of the putative PufA recognition elements in wild-type cells causes precocious aggregation and PKA-C overexpression, phenocopying a pufA mutation. Finally, YakA function is required for the decline of PufA protein and mRNA levels in the first 4 hours of development. We propose that PufA is a translational regulator that directly controls PKA-C synthesis and that YakA regulates the initiation of development by inhibiting the expression of PufA. Our work also suggests that Puf protein translational regulation evolved prior to the radiation of metazoan species.

scholarly journals The Molecular Signature of Megakaryocyte-Erythroid Progenitors Reveals a Role for the Cell Cycle in Fate Specification

Cell Reports ◽  
2018 ◽  
Vol 25 (11) ◽  
pp. 3229 ◽  
Author(s):  
Yi-Chien Lu ◽  
Chad Sanada ◽  
Juliana Xavier-Ferrucio ◽  
Lin Wang ◽  
Ping-Xia Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Signature ◽  
Erythroid Progenitors ◽  
Fate Specification

Preclinical Evaluation of the BET-Bromodomain (BET-BRD) Inhibitor OTX015 in Leukemia Cell Lines Harboring the JAK2 V617F Mutation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 873-873
Author(s):  
Maria Eugenia Riveiro ◽  
Lucile Astorgues-Xerri ◽  
Charlotte Canet-jourdan ◽  
Mohamed Bekradda ◽  
Esteban Cvitkovic ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Mrna Levels ◽  
Protein Levels ◽  
V617f Mutation

Abstract Background: Exposure of cancer cells to BET-BRD protein inhibitors has been associated with a significant downregulation of C-MYC expression, leading to suppression of the transcriptional program linked to proliferation and survival. C-MYC mRNA expression, mediated by STAT5 activation, is induced by the JAK2 (V617F) mutation (JAK2mu) in transfected BA/F3 cells (Funakoshi-Tago, et al. 2013). We selected JAK2mu leukemia-derived cell lines for preclinical evaluation of OTX015 (Oncoethix, Switzerland), a selective orally-bioavailable inhibitor of BET-BRD proteins with promising early results in an ongoing phase I study in hematologic malignancies (Herait et al, AACR 2014, NCT01713582). Material and Methods: Antiproliferative effects of OTX015 and JQ1 were evaluated in three established JAK2mu human myeloid leukemia cell lines (SET2, MUTZ8, HEL 92.1.7). GI50 (OTX015 concentration inducing 50% growth inhibition) and Emax (% cell proliferation at 6 µM OTX015) values were determined by MTT assay after 72h exposure. Protein levels were analyzed by Western blot, and RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For cell cycle analysis, cells were stained with propidium iodide and analyzed with a FACScan flow cytometer. Induction of apoptosis was evaluated by Annexin-V. Simultaneous schedules of OTX015 combined with ruxolitinib, a JAK2 inhibitor, were evaluated. Combination index (CI) was determined using the Chou & Talalay method; CI<1 reflects synergy, CI=1 additivity and CI>1 antagonism. Results: After 72h exposure, SET2 was the most sensitive cell line (GI50=0.12 µM and Emax=15%), and HEL92.1.7 cells had a GI50=1.9 µM with an Emax=23%. MUTZ8 was the most resistant cell line with an Emax=61%. Similar GI50 and Emax values are observed with JQ1. A significant increase in the fraction of apoptotic cells was observed in SET2 cells after 72h 500 nM OTX015 exposure. Non-significant increases in Annexin-positive cells were seen in HEL92.1.7 and MUTZ8 cells. Cell cycle analysis revealed a significant increase in the percentage of SET2 cells in subG0/G1 after 24, 48, and 72h 500 nM OTX015, correlating with the increase in apoptosis. Conversely, an increase in the percent cells in the G1 phase was observed in HEL 92.1.7 cells. After 4h 500 nM OTX015, BRD2 mRNA levels were significantly increased in all three cell lines, whereas BRD3 levels were not modified. BRD4 mRNA levels increased significantly after 48h in SET2 cells. OTX015 treatment induced a transitory reduction of C-MYC mRNA levels after 4h with an increase at 24h in all cell lines. At the protein level, C-MYC decreased substantially in SET2 cells after 4h, with complete disappearance after 48h without recovery, while in the less sensitive MUTZ8 cell line, the decrease in C-MYC protein levels was transitory. Conversely, this proto-oncogene was not modified in HEL92.1.7 cells. In addition, p-STAT5 protein was downregulated by OTX015 in SET2 cells, but was increased in MUTZ8 cells after longer exposure time. Furthermore, BCL2 mRNA and protein levels decreased in SET2 cells, correlating with the apoptosis induction seen with OTX015 treatment. In HEL92.1.7 cells, P21 mRNA levels and cyclin D1 protein levels increased after 4h and 48h OTX015 treatment, respectively. Moreover, concomitant combination of OTX015 with ruxolitinib showed a highly antagonist effect (CI>7) in SET2 cells, the most sensitive cell line to both agents. On the other hand, very strong synergy was observed in HEL92.1.7 (CI=0.19) and MUTZ8 (CI=0.41), despite their low sensitivity to single agent OTX015. Conclusions. Our findings demonstrate that OTX015 exhibits potent activity against cultured leukemic cells expressing the JAK2 V617F mutation, inducing apoptosis or cell cycle arrest at submicromolar concentrations. This activity correlates with modulation of C-MYC, p-STAT5, BCL2, P21 and cyclin D1 mRNA and protein levels following OTX015 treatment. Our study highlights the novel and synergistic activity of the combination of a BRD antagonist and a JAK inhibitor in human leukemic cells harboring the JAK2 V617 F mutation, supporting the rationale for in vivo testing of OTX015 in combination with JAK inhibitors in leukemic JAK2mu models. Disclosures Riveiro: Oncoethix SA: Research Funding. Astorgues-Xerri:Oncoethix SA: Research Funding. Canet-jourdan:Oncoethix SA: Research Funding. Bekradda:Oncoethix SA: Research Funding. Cvitkovic:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Shareholder and CSO Other. Herait:Oncoethix SA: CMO and Shareholder Other. Raymond:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Automated cell cycle and cell size measurements for single-cell gene expression studies

2017 ◽  
Author(s):  
Anissa Guillemin ◽  
Angelique Richard ◽  
Sandrine Gonin-Giraud ◽  
Olivier Gandrillon
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Single Cell ◽  
Cell Size ◽  
Cell Tracking ◽  
Erythroid Progenitors ◽  
Expression Variability ◽  
Unbiased Gene ◽  
Transcriptomic Level ◽  
Cell To Cell Variability

AbstractRecent rise of single-cell studies revealed the importance of understanding the role of cell-to-cell variability, especially at the transcriptomic level. One of the numerous sources of cell-to-cell variation in gene expression is the heterogeneity in cell proliferation state. How cell cycle and cell size influences gene expression variability at single-cell level is not yet clearly understood. To deconvolute such influences, most of the single-cell studies used dedicated methods that could include some bias. Here, we provide a universal and automatic toxic-free label method, compatible with single-cell high-throughput RT-qPCR. This led to an unbiased gene expression analysis and could be also used for improving single-cell tracking and imaging when combined with cell isolation. As an application for this technique, we showed that cell-to-cell variability in chicken erythroid progenitors was negligibly influenced by cell size nor cell cycle.

scholarly journals Blood Cell Fate Decisions: Insights from Single-cell RNA-seq

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-20-SCI-20
Author(s):  
Merav Socolovsky
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Cell Fate ◽  
Terminal Differentiation ◽  
S Phase ◽  
Erythroid Cell ◽  
Hematopoietic Progenitors ◽  
Erythroid Progenitor ◽  
Cell Fate Decisions ◽  
Computational Predictions

The manner by which multipotent hematopoietic progenitors commit to the erythroid lineage, and the subsequent processes that govern early erythroid progenitor development, are not well understood. Part of the challenge for investigating these was the lack of a rigorous strategy for isolating directly from tissue the early erythroid progenitors, which are functionally defined as the cell 'units' that give rise to erythroid colonies (CFU-e) or bursts (BFU-e) in culture. Indeed, the early erythroid trajectory, that starts with multi-potential progenitors and gives rise to BFU-e, CFU-e and to erythroblasts undergoing terminal differentiation, was not fully elucidated. We addressed these gaps using single cell transcriptomics, combined with functional assays that validated computational predictions 1. These showed that early hematopoietic progenitors form a continuous, hierarchical branching structure, in which the erythroid and basophil/mast cell fates are unexpectedly coupled. We delineated a novel flow-cytometric strategy that prospectively isolates CFU-e and BFU-e progenitors with high purity, and in combination with computational predictions, identified novel growth factor receptors that regulate early erythropoiesis. We further discovered that early erythroid development entails profound remodeling of both G1 and S phases of the cycle, resulting in cell cycle specializations that orchestrate the developmental process, including a gradual shortening of G1 during the CFU-e phase, followed by a sharp increase in the speed of S phase during the S-phase dependent activation of the erythroid terminal differentiation program 1-3(Figure 2). 1. Tusi BK, Wolock SL, Weinreb C, et al. Population snapshots predict early haematopoietic and erythroid hierarchies. Nature. 2018;555(7694):54-60. 2. Hwang Y, Futran M, Hidalgo D, et al. Global increase in replication fork speed during a p57KIP2-regulated erythroid cell fate switch. Science Advances. 2017;3:e1700298. 3. Pop R, Shearstone JR, Shen Q, et al. A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression. PLoS Biol. 2010;8(9). Disclosures No relevant conflicts of interest to declare.

scholarly journals ATF6 knockdown decreases apoptosis, arrests the S phase of the cell cycle, and increases steroid hormone production in mouse granulosa cells

2017 ◽  
Vol 312 (3) ◽  
pp. C341-C353 ◽  
Author(s):  
Yongjie Xiong ◽  
Huatao Chen ◽  
Pengfei Lin ◽  
Aihua Wang ◽  
Lei Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Er Stress ◽  
Granulosa Cells ◽  
Physiological Function ◽  
Steroid Hormone ◽  
Follicular Development ◽  
S Phase ◽  
Mrna Levels ◽  
Hormone Production ◽  
Protein Levels

Activating transcription factor 6 (ATF6), a sensor protein located in the endoplasmic reticulum (ER) membrane, is an important factor in the ER stress signaling pathway. ER stress is known to be involved in folliculogenesis, follicular growth, and ovulation; however, the physiological function of ATF6 in mouse granulosa cells remains largely unknown. The aim of this study was to assess the role of ATF6 in mouse granulosa cells with respect to apoptosis, the cell cycle, and steroid hormone production, as well as several key genes related to follicular development, via RNA interference, immunohistochemical staining, real-time quantitative PCR, Western blotting, flow cytometry, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) assay, and ELISA. Immunohistochemical staining revealed that ATF6 was extensively distributed in the granulosa cells of various ovarian follicles and oocytes in adult female mice. FSH or LH treatment significantly increased ATF6 protein levels in mouse granulosa cells. In the meantime, a recombinant plasmid was used to deplete ATF6 successfully using short hairpin RNA-mediated interference technology, which was verified at both the mRNA and protein levels. Flow cytometry and TUNEL assay analysis indicated that ATF6 depletion decreased apoptosis and arrested the S phase of the cell cycle in mouse granulosa cells. Consistent with these results, p53, caspase-3, B cell lymphoma 2 (Bcl-2)-associated X protein, CCAAT-enhancer-binding protein homologous protein, cyclin A1, cyclin B1, and cyclin D2 mRNA expression decreased, whereas Bcl-2 and glucose-regulated protein 78 kDa mRNA expression increased. Interestingly, ATF6 knockdown obviously increased progesterone and estradiol production in mouse granulosa cells. Cytochrome P450 1b1 ( Cyp1b1) mRNA levels were downregulated, whereas Cyp11a1, steroidogenic acute regulatory, and Cyp19a1 mRNA levels were upregulated, in keeping with the changes in steroid hormones. Furthermore, ATF6 disruption remarkably increased insulin-like growth factor binding protein 4 ( Igfbp4) expression and decreased hyaluronan synthase 2 ( Has2), prostaglandin-endoperoxide synthase 2 ( Ptgs2), and prostaglandin F receptor ( Ptgfr) expression in mouse granulosa cells, which are proteins crucial for follicular development. But, after treating with tunicamycin, the levels of Has2, Ptgs2, and Ptgfr increased relatively, whereas Igfbp4 expression decreased. Collectively, these results imply that ATF6, as a key player in ER stress signaling, may regulate apoptosis, the cell cycle, steroid hormone synthesis, and other modulators related to folliculogenesis in mouse granulosa cells, which may indirectly be involved in the development, ovulation, and atresia of ovarian follicles by affecting the physiological function of granulosa cells. The present study extends our understanding and provides new insights into the physiological significance of ATF6, a key signal transducer of ER stress, in ovarian granulosa cells.

Apoptosis in Response to Inhibition of Dipeptidyl Peptidase 2 (DPP2) Defines Low Risk Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3094-3094
Author(s):  
Alexey V. Danilov ◽  
Olga V. Danilova ◽  
Andreas K. Klein ◽  
Brigitte T. Huber
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
B Cells ◽  
Treatment Regimen ◽  
Protease Activity ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Dipeptidyl Peptidase ◽  
Mrna Levels ◽  
Protein Levels

Abstract Maintenance of the G0 state is a key to survival of CLL B-cells. Heterogeneity of prognosis suggests that CLL is not a uniform disease. Molecules expressed in CLL with unfavorable prognosis, such as ZAP-70, Lyn, CD38 and others, provide stimuli which, coupled with B-cell receptor signaling, may alter cell cycle progression and delay apoptosis. We studied mechanisms of apoptosis in CLL B-cells via inhibition of Dipeptidyl Peptidase 2 (DPP2). DPP2 is a serine protease cloned in our lab, which is involved in the maintenance of the G0 (quiescent) state. Inhibition of DPP2 triggers apoptosis in healthy B-cells. 50 patients with B-CLL were recruited from the Hematology clinics at Tufts-NEMC (Boston, MA). Median time from diagnosis of CLL to enrollment in the study was 7 years with median follow up of 10.5 years. 24 patients (48%) have received treatment in the course of their disease. CLL B-cells were isolated from peripheral blood with standard Ficoll-Hypaque technique, treated with ValboroPro (Point Therapeutics), a non-specific inhibitor of DPPs, and/or AX8819 (ActivX), a DPP2-specific inhibitor, incubated for 16 hours and stained with anti-CD19 antibodies, propidium iodide and Annexin V. Expression of DPP2 and ZAP-70 mRNA was assessed by RT-qPCR, p27 protein - by western blot analysis. By blocking DPP2 protease activity, we distinguished two subsets of CLL - sensitive (S-CLL) and resistant (R-CLL) to DP-P2 inhibition-induced apoptosis. In 30 cases (60%), inhibition of DPP2 resulted in caspase-dependent apoptosis of CLL B-cells (S-CLL). 70–90% of B-cells stained Annexin V-positive after incubation with AX8819. Pre-incubation with Rituximab (Biogen Idec) did not enhance cell death. In the remaining 20 cases (40%) inhibition of DPP2 did not cause cell death (R-CLL). R-CLL demonstrated higher expression of ZAP-70 mRNA (p<0.001) and lower levels of p27 protein (p<0.01) than S-CLL. ZAP-70 mRNA levels strongly correlated with resistance to apoptosis (χ2=26.7, p<0.0001). Inhibition of DPP2 resulted in a decrease in p27 protein levels in S-CLL, but not in R-CLL. Both groups expressed higher DPP2 mRNA levels than B-cells derived from healthy donors. In the R-CLL subgroup, 18 patients (84%) required treatment for their disease, 14 received more than one treatment regimen, 2 underwent allogeneic transplant and 2 died of complications of CLL. Among the S-CLL cohort, 10 patients (33%) initiated treatment, 4 received more than one treatment regimen. R-CLL cohort required treatment earlier than S-CLL (3.5 vs. 9.9 years from diagnosis). Thus, DPP2 inhibition discriminates two subsets of CLL based on their ability to undergo apoptosis upon disruption of the quiescent program. We postulate that DPP2 is critical for the maintenance of G0 in S-CLL. Its inhibition leads to inappropriate cell cycle entry, as evidenced by a decrease in p27 protein levels and cell death. The R-CLL subgroup expresses high ZAP-70 mRNA levels which portends a worse clinical course. In this subgroup, ZAP-70 kinase activity may provide an additional tonic signal that pushes CLL B-cells into late G0/early G1. At this stage, DPP2 protease activity is no longer required for survival. DPP2 inhibition may serve as an easy-to-perform prognostic test, as well as a novel therapeutic approach.

Role of Survivin in Drug Resistant B-Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 10-10
Author(s):  
Eugene Park ◽  
Enzi Jiang ◽  
Gregor von Levetzow ◽  
Cihangir Duy ◽  
Lars Klemm ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Resistance ◽  
B Cell ◽  
Real Time Pcr ◽  
Mrna Levels ◽  
Drug Resistant ◽  
Novel Therapies ◽  
Protein Levels ◽  
Ic50 Value

Abstract Abstract 10 Survivin/BIRC5, an inhibitor of apoptosis (IAP) protein,is critical for the survival and proliferation of cancerous cells and has become the target of an increasing number of preclinical novel therapies against primarily solid tumors. Survivin is expressed in AML and ALL cells and has been implicted in leukemia relapse. Here we test the hypothesis that Survivin is critical to chemotherapeutic drug resistance in ALL. To test this hypothesis, we initially compared survivin mRNA levels in both patient-derived B-ALL cells and B-ALL cell lines versus normal B-cells in various stages of development using real-time PCR. ALL cells encompassing various cytogenetic subgroups showed significantly greater mRNA levels of Survivin versus normal B cell precursors ranging from 2 to greater than 20-fold versus controls. To determine whether Survivin contributes to drug resistance, we lentivirally overexpressed Survivin in primary B-ALL and B-ALL cell lines. Overexpression of Survivin attenuated the effect of Vincristine on ALL cell proliferation when compared to ALL cells transduced with empty vector controls. Vincristine IC50 value determination for the control was 0.1 nM, whereas Survivin overexpression resulted an IC50 value of 10 nM (p<0.01). Similarly, significantly higher concentrations of L-Asparaginase (>0.01 U/l vs ab >0.1; p<0.05) and Dexamethasone (0.1 nmol/l vs >10 nmol/l; p<0.013) were needed to achieve drug cytotoxicity. We conclude that Survivin overexpression decreases sensitivity of ALL cells to standard chemotherapy. Conversely, lentivirally-mediated survivin shRNA knockdown of the same cell type significantly sensitized a greater percentage of the leukemia to 10nM vincristine versus non-silencing controls. Survivin shRNA targeting resulted in a 30.2% greater affected population than controls, assayed using MTT (p<0.003). Overexpression or knockdown of Survivin was confirmed by Western Blot and real-time PCR. Of note, expression levels of Survivin is heterogeneous within the same ALL sample: Transduction of patient-derived ALL cells with a Survivin-GFP reporter construct revealed that a small subpopulation of Survivin-GFP high ALL exhibiting approximately 7-fold higher levels of endogenous Survivin than bulk leukemia cells. Consistent with cell cycle-dependent regulation of Survivin, Survivin-GFP high ALL were enriched in G2/M phase of the cell cycle. In addition, endogenous variations of Survivin protein levels were in the same range as changes in Survivin protein levels in overexpression and knockdown experiments, which indicates that these experiments tested physiologically occurring levels of Survivin. Next, we determined whether Survivin contributes to self-renewal of drug resistant ALL cells. Survivin overexpression of primary samples yielded three times more colonies, than controls in a primary plating CFU assay (p<0.019). Taken together, our data suggests that targeting Survivin in B-ALL may sensitize to chemotherapy and highlights the role of Survivin in drug resistant B-ALL as a target for novel therapies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ddx41 inhibition of DNA damage signaling permits erythroid progenitor expansion in zebrafish

Haematologica ◽  
2021 ◽  
Author(s):  
Joshua T. Weinreb ◽  
Varun Gupta ◽  
Elianna Sharvit ◽  
Rachel Weil ◽  
Teresa V. Bowman
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Critical Role ◽  
Hematologic Malignancies ◽  
In Vivo Model ◽  
Erythroid Progenitors ◽  
Critical Function ◽  
Erythroid Progenitor ◽  
Genomic Stress

DEAD-box Helicase 41 (DDX41) is a recently identified factor mutated in hematologic malignancies whose function in hematopoiesis is unknown. Using an in vivo model of Ddx41 deficiency, we unveiled a critical role for this helicase in regulating erythropoiesis. We demonstrated that loss of ddx41 leads to anemia caused by diminished proliferation and defective differentiation of erythroid progenitors. Misexpression and alternative splicing of cell cycle genes is rampant in ddx41 mutant erythroid progenitors. We delineated that the DNA damage response is activated in mutant cells resulting in an Ataxia-telangiectasia mutated (ATM) and Ataxiatelangiectasia and Rad3-related (ATR)-triggered cell cycle arrest. Inhibition of these kinases partially suppressed ddx41 mutant anemia. These findings establish a critical function for Ddx41 in promoting healthy erythropoiesis via protection from genomic stress and delineate a mechanistic framework to explore a role for ATM and ATR signaling in DDX41-mutant hematopoietic pathologies.

Induction of Apoptosis in K562 Human Chronic Myeloid Leukemia Cells by Tetra-Arsenic Tetra-Sulfide.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4375-4375
Author(s):  
Qidong Ye ◽  
Long-Jun Gu ◽  
Yanxia Zhao ◽  
Jincai Zhao ◽  
Wengao Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Morphological Changes ◽  
K562 Cells ◽  
Morphologic Change ◽  
Mrna Levels ◽  
Protein Levels ◽  
Reverse Transcription Pcr ◽  
M Phase

Abstract Realgar has been used as a traditional medicine in China for more than 1500 years. Some studies found that tetra-arsenic tetra-sulfide (As4S4), the main ingredient of realgar, used alone was highly effective and safe for all stages of acute promyelocytic leukemia. To explore the effects of As4S4 in treatment of human chronic myeloid leukemia K562 cells, we used microculture MTS assay to measure the growth inhibition of K562 cells. The morphologic change was determined by Wright’s staining and Hoechst33342 assay. Cell apoptosis was evaluated by DNA agarose gel electrophoresis. The apoptotic rate and cell cycle were measured by flow cytometry. The changes of transcript and protein levels were determined by real-time reverse transcription-PCR and Western blot analysis, respectively. As4S4 had signigicant cytotoxicity on K-562 cells. At the concentration of 2.0μmol/L, the cell viability decreased significantly after 24 hours cultured with the reagent. When the concentration was lower than 0.5μmol/L, As4S4 had little effect on K562 cells. The effect of As4S4 on K562 was time and concentration dependent. After cultured with As4S4 at the concentration of 2.0μmol/L for 24 to 48 hours, K562 cells appeared typical morphological changes of apoptosis. At a concentration greater than or equal to 2.0μmol/L, As4S4 could induce apoptosis significantly. After 12 hours of incubation with 2.0μmol/L As4S4, the apoptosis rate increased from 2.05% to 12.03%. At the same time, the percentage of cells in G1 phase decreased from 69.65% to 50.53%, whereas the percentage of G2/M phase increased from 9.56% to 25.91%. The mRNA levels of BCL-2, BCL-XL, BAD and BAX, and the protein levels of Akt and pAkt down-regulated after the inhibition of As4S4. The transcript and protein levels of BCR-ABL had no change after incubation with As4S4. These results indicated that As4S4 can inhibit the growth of K562 cells efficiently through inducing apoptosis and cell cycle arrest. It seems that As4S4 interferes with Akt pathway and down-regulate BCL-2, BCL-XL, BAD and BAX, which may be involved in the response of K562 to this agent. As4S4 could be beneficial for treatment of CML in combination with conventional drugs.

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