scholarly journals Identification of Potential Chemical Compounds Able to Trigger Enucleation of Immortalized Human Erythroid Cell Lines

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 951-951
Author(s):  
Svetlana Soboleva ◽  
Ryo Kurita ◽  
Fredrik Ek ◽  
Rita Alves ◽  
Roger Olsson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Chemical Compounds ◽  
Potential Method ◽  
Ips Cells ◽  
Erythroid Cell ◽  
Specific Cell ◽  
Growth Capacity ◽  
M344 Treatment

Ex vivo production of functional red blood cells (RBCs) is a potential method to provide abundant RBCs which therefore is expected as a solution to improve the current shortage of blood supply in donor-dependent transfusion therapies. Immortalized human erythroid cell lines are expected to be an alternative source for ex vivo production of RBCs, as these cells are already committed to the erythroid lineage and still keep a limitless growth capacity. We have previously established immortalized erythroid cell lines derived from human UCB-derived CD34+ cells (HUDEP) and human iPS cells (HiDEP) by ectopically overexpressing human papilloma virus E6/E7 gene (Kurita et al., PLoS ONE, 2013). HUDEP/HiDEP sustain the infinite growth capacity, express erythroid specific cell surface markers (e.g. Glycophorin-A) and produce functional hemoglobin. However, in similar to erythroid cells directly differentiated from ES/iPS cells, these cells do not efficiently enucleate and easily cause cell death upon the induction of differentiation. In this study, we employed an imaging-based high throughput screening system (Cellomics ArrayScan) combined with two distinct DNA dyes (SYTO16 and SYTOXRed) to capture morphological changes of the immortalized erythroid cell lines. Among >1,200 chemical compounds, we identified multiple histone deacetylase (HDAC) inhibitors (HDACi) that largely increased the enucleation of HiDEP. In particular, Fluoro-SAHA and M344 achieved 9.0 fold increase in the enucleation rate. The exert of enucleation was confirmed by morphological examinations using cytospin and motional observations by time-lapse imaging. These assay finally observed that aprrox. 20 % of HiDEP cells enucleated upon Fluoro-SAHA treatment. However, we noticed that a large proportion of enucleated cells were fragile, and a similar number of enucleated cells with damaged membrane were also found. Addition of pan-caspase inhibitor, QVD-OPH, improved the viability of enucleated cells but also blocked enucleation, suggesting that HDACi-induced enucleation is also a caspase-dependent process. Gene expression profiling revealed that the Fluoro-SAHA and M344 treatment commonly induced cytoskeletal genes including kinesins, e.g. KIF3A, indicating cytoskeletal rearrangement occurred upon Fluoro-SAHA/M344 treatment. Of note, one of main erythroid-membrane components, SPTA1, was significantly down-regulated, whereas non-erythrocytic spectrin, SPTAN1, was abnormally induced. This α-spectrin switching was considered to result in the high fragility of enucleated cells. We therefore generated HiDEP cells with enhanced expression of SPTA1 using CRISPR-activation system. Activation of SPTA1 expression significantly improved viability of the enucleated cells and as the consequence 30 % of cells successfully enucleated while damaged cells were decreased to less than 10 %. Our data propose a potential method to induce enucleation of immortalized erythroid cell lines which would lead to a future large-scale ex vivo RBC production. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of potential chemical compounds enhancing generation of enucleated cells from immortalized human erythroid cell lines

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Svetlana Soboleva ◽  
Ryo Kurita ◽  
Fredrik Ek ◽  
Hugo Åkerstrand ◽  
Rita Silvério-Alves ◽  
...  
Keyword(s):  
Cell Lines ◽  
Histone Deacetylase Inhibitors ◽  
Ex Vivo ◽  
Chemical Compounds ◽  
Erythroid Cell ◽  
Specific Cell ◽  
Cell Membrane Protein ◽  
Immortalized Cell ◽  
Promising Source ◽  
Hdaci Treatment

AbstractImmortalized erythroid cell lines are expected to be a promising source of ex vivo manufactured red blood cells (RBCs), however the induction of enucleation in these cell lines is inefficient at present. We utilized an imaging-based high-throughput system to identify chemical compounds that trigger enucleation of human erythroid cell lines. Among >3,300 compounds, we identified multiple histone deacetylase inhibitors (HDACi) inducing enucleated cells from the cell line, although an increase in membrane fragility of enucleated cells was observed. Gene expression profiling revealed that HDACi treatment increased the expression of cytoskeletal genes, while an erythroid-specific cell membrane protein, SPTA1, was significantly down-regulated. Restoration of SPTA1 expression using CRISPR-activation partially rescued the fragility of cells and thereby improved the enucleation efficiency. Our observations provide a potential solution for the generation of mature cells from erythroid cell lines, contributing to the future realization of the use of immortalized cell lines for transfusion therapies.

Leukemic Blasts With The PNH Phenotype: Correlation With Cytogenetics In ALL

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2628-2628
Author(s):  
David J Araten ◽  
Mignon L. Loh ◽  
Meenakshi Devidas ◽  
Andrew J. Carroll ◽  
Nyla A. Heerema ◽  
...  
Keyword(s):  
Cell Lines ◽  
Rare Variants ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Initial Step ◽  
Rapid Screening ◽  
Published Data ◽  
Median Frequency ◽  
Single Mutation ◽  
Cell Bank

Abstract It has been proposed that hypermutability is essential for the generation of malignancies that require more than a few oncogenic mutations and may also promote the emergence of drug-resistant clones by contributing to intratumoral diversity. However, most genes are not suitable as a reporter for quantitating the frequency of spontaneous somatic inactivating mutations. A notable exception is the PIG-A gene, which encodes an enzyme catalyzing an initial step in the biosynthesis of glycosylphosphatidylinositol (GPI), which links certain membrane proteins to the cell surface. Of note, the PIG-A-mutant phenotype can be readily demonstrated by flow cytometry, using antibodies specific for GPI-linked proteins (e.g., CD55 and CD59), as well as the FLAER reagent (which binds to GPI directly), enabling the rapid screening of large populations for rare variants that are GPI-negative. It is known from the condition paroxysmal nocturnal hemoglobinuria that a very broad spectrum of PIG-A mutations can produce this “PNH phenotype”, and because the gene is X-linked, this requires only a single mutation. Apart from patients with PNH, there is evidence that PIG-A mutations are growth-neutral in most all other circumstances. We have previously demonstrated rare spontaneously arising cells with the PNH phenotype and genotype in blood cells of normal donors and in B-lymphoblastoid cell lines (BLCLs); similar populations can be demonstrated in mice, with marked increases after mutagen exposure. We have also shown that malignant cell lines often demonstrate an elevated frequency (f) of spontaneously arising cells with the PNH phenotype. Recently, we have reported similar findings in ex vivo leukemic blasts derived from patients with ALL: in samples from 10 of 19 patients, the median frequency of blasts with the PNH phenotype was low, with a median f value of 13 per million, which was similar to that of BLCLs from normal donors. The remaining 9 samples demonstrated an elevated f value (median 566 per million). Based on our preliminary subset analysis, along with published data demonstrating a higher mutation burden in leukemias with the ETV6-RUNX1 (TEL-AML1) translocation compared with hyperdiploid samples, we hypothesized that we would find a higher mutant frequency in the former group. Therefore, we have now analyzed in a blinded manner a new panel of blast cells derived from ficolled pre-treatment marrow samples from patients with B-lineage ALL obtained from the Children’s Oncology Group ALL Cell Bank. To determine f, samples were stained sequentially with an Alexa-488 conjugate of the FLAER reagent and then a mixture of mouse anti-CD55 and anti-CD59 antibodies, followed by FITC-conjugated rabbit anti-mouse immunoglobulin secondary antibody (registering on FL1), and then PE-conjugated anti-CD45 antibody (registering on FL2). The blast population was identified based on forward and side scatter, expression of CD45, and exclusion of propidium iodide. GPI-negative cells were defined as expressing <4% of the mean level of FL1 fluorescence as the GPI-positive population, and >10% of its mean FL2 fluorescence. Overall, among 26 ALL samples, the f values spanned 5 orders of magnitude and had a median value of 49 per million. For the 8 ETV6-RUNX1 samples, the median f value was 2243 per million, compared with 18 per million for the 11 hyperdiploid samples (p <0.003). Among 7 samples with neither abnormality, the median f value was 60 per million. The one sample with a t(1;19) translocation that we analyzed demonstrated an f value of 330 per million. In comparison, the median f value among 11 BLCLs from normal donors was ∼ 5 per million, and f was ∼1850 per million for HBL2A, a mantle cell lymphoma line that exhibits hypermutability. Based on the data for BLCLs, we used a value of 50 per million as a cutoff: 7 of 8 ETV6-RUNX1 samples demonstrated hypermutabilty, compared with 2 of 11 hyperdiploid samples, and 4 of 7 samples with neither abnormality. These data demonstrate that hypermutability is frequently seen in ALL and is associated with, but not restricted to samples with the ETV6-RUNX1 fusion. These data also confirm that hypermutability—at least as detected by this assay, is not always a requirement for leukemogenesis. We predict that it will be possible to find associations between other cytogenetic subsets and hypermutability, which may correlate with the number of mutations required to produce the malignancy. Disclosures: No relevant conflicts of interest to declare.

Mutations in the Poly(A)-Specific Ribonuclease (PARN) Impair Telomerase RNA 3' End Maturation in Dyskeratosis Congenita Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 669-669 ◽  
Author(s):  
Diane H. Moon ◽  
Matthew Segal ◽  
Baris Boyraz ◽  
Eva Guinan ◽  
Inga Hofmann ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Ips Cells ◽  
Dyskeratosis Congenita ◽  
Telomere Maintenance ◽  
Telomerase Rna ◽  
Primary Role ◽  
Mrna Metabolism ◽  
Deficient Cell ◽  
Non Coding Rna

Abstract Mutations in 11 genes have been described in patients with dyskeratosis congenita (DC) and related telomere diseases, and account for ~60% of cases. Amongst these, loss-of-function mutations in the poly(A)-specific ribonuclease (PARN) have most recently been found in patients with DC and idiopathic pulmonary fibrosis. PARN has no known role in telomere biology. Why PARN deficiency should mimic telomere diseases remains unclear. Based on its 7-methylguanylate (m7 G) cap recognition and poly(A) deadenylase functions, PARN's primary role is considered to be in regulating mRNA metabolism. Recent studies implicate PARN in non-coding RNA biogenesis, including maturation of small nucleolar RNAs (snoRNAs) via the deadenylation of oligo-adenylated (oligo(A)) intermediates. In two DC patients with biallelic defects in the PARN gene, we found decreased levels of the telomerase RNA component (TERC). TERC is the essential non-coding RNA template and scaffold of the telomerase holoenzyme. TERC possesses an m7 G cap but unlike mRNAs does not have a long poly(A) tail; rather, TERC contains a box H/ACA motif as is found in some snoRNAs. We hypothesized that TERC is regulated by PARN in a manner similar to snoRNAs, given their shared 3′ box H/ACA architecture. Here, usingsomatic cells and induced pluripotent stem (iPS) cells from DC patients with PARN mutations, we show that PARN is required for the 3′ end maturation and accumulation of TERC RNA. In PARN-mutant iPS cells and PARN-deficient cell lines, we find impaired telomerase activity and telomere maintenance. Deep sequencing of TERC 3' ends reveals that PARN is required for the removal of genomically-encoded extensions and post-transcriptionally acquired oligo(A) tails that target nuclear RNAs for destruction. In keeping with this, TERC transcripts from PARN-mutant patient cells decay at an accelerated rate. The diminished TERC steady-state levels, increased oligo(A) forms of TERC, and defects in telomere maintenance in patient cells are rescued by ectopic expression of PARN. Global transcript analysis by RNA-Seq in patient cells and PARN-deficient cell lines compared to controls reveals no protein coding mRNAs that consistently manifest a fold-change exceeding the change in TERC levels. Our data indicate that PARN functions in TERC biogenesis via deadenylation of oligo-adenylated nascent transcripts, which promotes 3' end maturation and stability. Given these findings and the phenotype of patients with PARN mutations, we speculate that a major, non-redundant function of PARN in human cells is regulating the maturation of TERC and other non-coding RNAs, more so than mRNA metabolism. Our results reveal a novel role for PARN in the biogenesis of TERC, and provide a mechanism linking PARN mutations to defective telomere maintenance in DC and related diseases. Disclosures No relevant conflicts of interest to declare.

scholarly journals Plasticity of Cells andEx VivoProduction of Red Blood Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Takashi Hiroyama ◽  
Kenichi Miharada ◽  
Ryo Kurita ◽  
Yukio Nakamura
Keyword(s):  
Red Blood Cells ◽  
Cell Lines ◽  
Blood Cells ◽  
Progenitor Cell ◽  
Ex Vivo ◽  
Es Cells ◽  
Erythroid Cell ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Erythroid Progenitor Cell

The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If transfusable RBCs could be produced abundantly from certain resources, it would be very useful. Our group has developed a method to produce enucleated RBCs efficiently from hematopoietic stem/progenitor cells present in umbilical cord blood. More recently, it was reported that enucleated RBCs could be abundantly produced from human embryonic stem (ES) cells. The common obstacle for application of these methods is that they require very high cost to produce sufficient number of RBCs that are applicable in the clinic. If erythroid cell lines (immortalized cell lines) able to produce transfusable RBCsex vivowere established, they would be valuable resources. Our group developed a robust method to obtain immortalized erythroid cell lines able to produce mature RBCs. To the best of our knowledge, this was the first paper to show the feasibility of establishing immortalized erythroid progenitor cell lines able to produce enucleated RBCsex vivo. This result strongly suggests that immortalized human erythroid progenitor cell lines able to produce mature RBCsex vivocan also be established.

scholarly journals Glucocorticoid Resistance in IKZF1-Deleted BCP-ALL: It Is PTEN Again

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4088-4088
Author(s):  
Laurens Van Der Meer ◽  
Rene Marke ◽  
Miriam Butler ◽  
Dorette van Ingen Schenau ◽  
Blanca Scheijen ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Membrane Integrity ◽  
Single Copy ◽  
Leukemic Cells ◽  
Pten Gene ◽  
Akt Inhibitor ◽  
Hes1 Expression

Abstract Single copy deletions of IKZF1, which occur in 10-15% of all B cell precursor acute lymphoblastic leukemia (BCP-ALL) cases, are associated with a poor outcome. We previously showed that loss of IKZF1 dictates resistance to glucocorticoids (GC) in BCP-ALL cell lines, a knockout mouse model and ex-vivo analysis of primary leukemic cells. When we analyzed the initial response to prednisolone therapy, we found that pediatric patients who suffer from an IKZF1 deleted leukemia are strongly enriched in the poor responder group (14 vs 7%, p<0.05). Although we were able to show that glucocorticoid receptor (GR)-mediated transcription regulation is attenuated in IKZF1 deleted cells, in remained unclear how loss of IKZF1 affects GR signaling. In T-ALL, GC resistance is frequently induced by genomic mutations that result in hyperactivation of AKT. AKT mediated phosphorylation of the GR results in suppression of GR function and resistance to GC treatment. Although the genomic loci that are frequently mutated in T-ALL resulting in hyperactivation of AKT, such as PTEN, are unaffected in BCP-ALL, we tested whether aberrant activation of AKT may also cause resistance to GC in BCP-ALL. Indeed, both CRISPR/Cas9 induced knockout and shRNA mediated knockdown of IKZF1 resulted in activation of AKT in human BCP-ALL cell lines. This hyperactivation was also observed in splenic B cells isolated from Ikzf1+/- mice. Active AKT in turn promotes phosphorylation of the GR on Ser143. This phosphorylation inhibits GR function, at least in part, by preventing shuttling of the ligand bound protein to the nucleus. In order to understand how a single copy loss of IKZF1 activates AKT, we tested expression of upstream regulators of AKT activity and found a near complete loss of PTEN expression, the phosphatase that inhibits PI3K and AKT signaling. Indeed, shRNA mediated knockdown of PTEN in wildtype cells resulted in activation of AKT and induced resistance to GCs. Furthermore, in IKZF1+/- cells we observed an upregulation of HES1, a transcriptional factor that is known to repress expression of the PTEN gene. Of note, HES1 was previously identified as a target of IKZF1 mediated transcriptional repression in erythroid and T cells. To test whether this AKT mediated mechanism of GC resistance could be reverted through pharmacological inhibition, we tested the effect of AKT inhibitors on prednisolone induced apoptosis of human BCP-ALL cell lines (figure A). In contrast to control cells, where inhibition of AKT had little to no effect on the sensitivity to GC, in IKZF1 deleted cells AKT inhibition reverted the resistance phenotype to a level comparable to control cells. Importantly, also in patient derived xenografts we observed that AKT inhibition was able to sensitize cells to GC treatment (Figure B). In conclusion, we identified hyperactivation of AKT, as a result of aberrant HES1 expression, as the mechanism causing GC resistance in IKZF1 deleted BCP-ALL. The availability of AKT inhibitors that are under clinical evaluation, may allow the development of combination therapies that restore the response to GCs in IKZF1-deleted BCP-ALL. Figures: A) IKZF1, PTEN, AKT, pAKT(Ser473), pGR(Ser134) and GR protein expression levels of NALM6 IKZF1+/- cell lysates were analyzed by western blot. Non-targeting controls were used as control and actin was used as a loading control. B) NALM6 parental, non-targeting controls or IKZF1+/- cells were treated for 48 hours with increasing concentrations of prednisolone with or without 0.5µM AKT inhibitor MK2206 and analyzed using an MTT based viability assay. All values were normalized to untreated NALM6 cells. Error bars represent ± standard error of the mean (SEM). (C) hTERT immortalized MSCs were seeded in a 96 wells format and allowed to settle for 24 hours prior to the addition of BCP-ALL patient-derived xenografts (PDX). PDX cells were allowed to settle for 24 hours before prednisone was added in increasing concentrations with or without AKT inhibitor MK2206. After 3 days, sensitivity to treatment was measured by flowcytometry using an amine staining to test for membrane integrity as a measure of cell viability. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Expression Cloning of Genes Enabling Erythropoietin -Independent Erythropoiesis in Vitro.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3608-3608
Author(s):  
Tomoko Yokoo ◽  
Ryo Kurita ◽  
Atsushi Takahashi ◽  
Michiyo Okada ◽  
Hirotaka Kawano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Apoptotic Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Expression Cloning ◽  
Hematopoietic Stem

Abstract Abstract 3608 Poster Board III-544 Erythropoietin (EPO) is essential during both ontogeny and the course of erythropoiesis. In primitive (yolk sac) erythropoiesis, however, the role of EPO is not fully understood. Elucidation of such role in primitive erythropoiesis would be very helpful for the development of ex vivo red blood cell expansion system from embryonic stem (ES) cells. Recently, we reported the establishment of the ex vivo induction of hematopoietic stem cells from common marmoset ES cells using lentiviral-Tal1/Scl gene transfer in the absence of any stromal cells (Stem Cells 24: 2014-2022, 2006). This method should also be applicable to both of human ES cells and induced pluripotential stem (iPS) cells, but the efficiency will be very critical for its clinical application. In the present study, we proceed further to find out unknown factors which accelerate ex vivo proliferation and differentiation of erythroid cells, constructed the human fetal liver cDNA expression lentiviral library (Mol Cell Biochem 319: 181-187, 2008) and screened for cDNAs which confer EPO independency to an EPO-dependent cell line, UT-7/Epo ( Blood 82: 456-464, 1993). Among twenty-two candidate genes cloned after screening of 6×10∧5 cDNA, we particularly focused on two full-length genes, ribosomal protein L11 (RPL11) and retinol dehydrogenase 11 (RDH11). Two candidate gene-transduced-UT-7/Epo cells, respectively named LV-RPL11 and LV-RDH11, showed complete EPO-independent survival and proliferation, increased expression of fetal γ-globin, and decreased expression of adult β-globin compared with parental UT-7/Epo cells in the presence of EPO. Cell cycle and apoptosis analyses showed decreased apoptotic cell death and increased S/G2/M cells in LV-RPL11 and LV-RDH11 cells compared with UT-7/Epo cells in the absence of Epo. Moreover, STAT5 phosphorylation and upregulation of its target genes, c-Myc, cyclin D and Pim, were observed in LV-RPL11, LV-RDH11 cells in the absence of EPO. In conclusion, the findings suggest that RPL11 and RDH11 play a role in EPO-independent erythropoiesis and might be applicable to ex vivo expansion of red blood cells from ES/iPS cells. Disclosures: No relevant conflicts of interest to declare.

Inhibition Of Wee1 Enhances The Anti-Leukemic Effects Of Antimetabolites In Vitro and In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1281-1281
Author(s):  
James B. Ford ◽  
Susan Fosmire ◽  
Annemie van Linden ◽  
Dmitry Baturin ◽  
Christopher C. Porter
Keyword(s):  
Combination Therapy ◽  
Mouse Model ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Single Agent ◽  
Parp Cleavage

Abstract While some patients with acute leukemia are cured, for many subsets of patients current therapeutic strategies are not adequate. Novel therapeutic approaches are needed for patients with higher risk leukemias, including T-ALL and AML. We and others identified Wee1 as a potential target in AML cells using RNAi screening. We have validated chemosensitization to cytarabine by genetic and pharmacologic inhibition of Wee1 in AML cell lines and primary patient samples ex vivo. A Wee1 inhibitor, MK1775, is in clinical development. We sought to further our findings with a wider range of conventional anti-leukemia agents, to determine whether the functionality of p53 influences chemosensitization, and to determine the tolerability and efficacy of MK1775 in combination with cytarabine in a mouse model of leukemia. We have found that MK1775 synergistically inhibits proliferation of the T-ALL Jurkat cell line with several antimetabolite chemotherapeutics including cytarabine, 6-thioguanine, and methotrexate. In contrast, MK1775 does not sensitize Jurkats to doxorubicin or etoposide, suggesting specific sensitization to antimetabolites. The addition of MK1775 enhances the antimetabolite induced apoptosis, as measured by Annexin V/7-AAD staining, and PARP cleavage measured by Western blotting. As expected, the addition of MK1775 enhances DNA damage induced by cytarabine as measured by γH2AX staining and flow cytometry, although preliminary data suggest that this is not the only mechanism of enhanced cell death, as a substantial proportion of cleaved PARP+ cells does not stain for γH2AX. In addition, we have found that AML cell lines with both wild-type and mutated TP53 are sensitive to chemosensitization by Wee1 inhibition. Furthermore, in isogenic models of p53 dysfunction, we have found that the functionality of p53 does not influence chemosensitization. Lastly, in an aggressive mouse model of AML, we observed enhanced disease control and survival in mice treated with MK1775 and ARA-C as compared to ARA-C alone. Hematotoxicity associated with treatment was related to the duration of combination therapy, but was tolerated well with intermittent dosing. Taken together, these data indicate that Wee1 inhibition may enhance the efficacy of several clinically relevant anti-leukemia agents, particularly the antimetabolites, but not topoisomerase inhibitors. Further, they suggest caution about the use of p53 mutation as a biomarker predictive of response to Wee1 inhibition. Moreover, we show that the addition of MK1775 to cytarabine is tolerable and more effective than cytarabine alone in vivo. Ongoing studies are aimed at better understanding the mechanism of combinatorial effect and to determine whether combination therapy is more efficacious than single agent therapy in xenograft models of leukemia. These data provide justification for early phase clinical trials of MK1775 in combination with antimetabolites in patients with high risk acute leukemia. Disclosures: No relevant conflicts of interest to declare.

Characterization of Healthy and Tumor Oral Cell Lines of Human Origin. The preliminary stage in the assessment of relevant chemical compounds with impact on dentistry

2018 ◽  
Vol 69 (10) ◽  
pp. 2889-2894
Author(s):  
Ion Virgil Corlan ◽  
Adelina Cheveresan ◽  
Delia Berceanu Vaduva ◽  
Cristian Nica ◽  
Alin Faur ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Cell Lines ◽  
Squamous Cell ◽  
Chemical Compounds ◽  
Tongue Squamous Cell Carcinoma ◽  
Gingival Fibroblasts ◽  
Human Origin ◽  
Preliminary Stage

The present study was aimed to evaluate the confluence percentage of three oral cell lines, namely primary gingival keratinocytes (PGK), primary gingival fibroblasts (HGF) and tongue squamous cell carcinoma (SCC-4). All cells have been monitored at different passages for 21 days. Evaluation of confluence percentage reveals the fact that primary gingival keratinocytes and tongue squamous cell carcinoma at small passages requires a period of about two weeks to reach a confluence of approximately 80% while for the gingival fibroblasts a period of about three times smaller is satisfactory.

scholarly journals Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Leeann Klassen ◽  
Greta Reintjes ◽  
Jeffrey P. Tingley ◽  
Darryl R. Jones ◽  
Jan-Hendrik Hehemann ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Ex Vivo ◽  
Rapid Assessment ◽  
Vital Role ◽  
Strain Level ◽  
Metabolic Phenotype ◽  
Specific Cell ◽  
Bacterial Cells ◽  
Carbohydrate Utilization

AbstractGut microbiomes, such as the microbial community that colonizes the rumen, have vast catabolic potential and play a vital role in host health and nutrition. By expanding our understanding of metabolic pathways in these ecosystems, we will garner foundational information for manipulating microbiome structure and function to influence host physiology. Currently, our knowledge of metabolic pathways relies heavily on inferences derived from metagenomics or culturing bacteria in vitro. However, novel approaches targeting specific cell physiologies can illuminate the functional potential encoded within microbial (meta)genomes to provide accurate assessments of metabolic abilities. Using fluorescently labeled polysaccharides, we visualized carbohydrate metabolism performed by single bacterial cells in a complex rumen sample, enabling a rapid assessment of their metabolic phenotype. Specifically, we identified bovine-adapted strains of Bacteroides thetaiotaomicron that metabolized yeast mannan in the rumen microbiome ex vivo and discerned the mechanistic differences between two distinct carbohydrate foraging behaviors, referred to as “medium grower” and “high grower.” Using comparative whole-genome sequencing, RNA-seq, and carbohydrate-active enzyme fingerprinting, we could elucidate the strain-level variability in carbohydrate utilization systems of the two foraging behaviors to help predict individual strategies of nutrient acquisition. Here, we present a multi-faceted study using complimentary next-generation physiology and “omics” approaches to characterize microbial adaptation to a prebiotic in the rumen ecosystem.

scholarly journals Pre-Clinical Evaluation of the Proteasome Inhibitor Ixazomib against Bortezomib-Resistant Leukemia Cells and Primary Acute Leukemia Cells

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 665
Author(s):  
Margot S.F. Roeten ◽  
Johan van Meerloo ◽  
Zinia J. Kwidama ◽  
Giovanna ter Huizen ◽  
Wouter H. Segerink ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Unmet Need ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Cross Resistance ◽  
Proteasome Subunit

At present, 20–30% of children with acute leukemia still relapse from current chemotherapy protocols, underscoring the unmet need for new treatment options, such as proteasome inhibition. Ixazomib (IXA) is an orally available proteasome inhibitor, with an improved safety profile compared to Bortezomib (BTZ). The mechanism of action (proteasome subunit inhibition, apoptosis induction) and growth inhibitory potential of IXA vs. BTZ were tested in vitro in human (BTZ-resistant) leukemia cell lines. Ex vivo activity of IXA vs. BTZ was analyzed in 15 acute lymphoblastic leukemia (ALL) and 9 acute myeloid leukemia (AML) primary pediatric patient samples. BTZ demonstrated more potent inhibitory effects on constitutive β5 and immunoproteasome β5i proteasome subunit activity; however, IXA more potently inhibited β1i subunit than BTZ (70% vs. 29% at 2.5 nM). In ALL/AML cell lines, IXA conveyed 50% growth inhibition at low nanomolar concentrations, but was ~10-fold less potent than BTZ. BTZ-resistant cells (150–160 fold) displayed similar (100-fold) cross-resistance to IXA. Finally, IXA and BTZ exhibited anti-leukemic effects for primary ex vivo ALL and AML cells; mean LC50 (nM) for IXA: 24 ± 11 and 30 ± 8, respectively, and mean LC50 for BTZ: 4.5 ± 1 and 11 ± 4, respectively. IXA has overlapping mechanisms of action with BTZ and showed anti-leukemic activity in primary leukemic cells, encouraging further pre-clinical in vivo evaluation.

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