Cell Line TF-1 Research Articles

Expression of Erythroid Transcription Factors Are Decreased in a RPS19 Knockdown Cell Line Model of Diamond Blackfan Anaemia

AbstractAbstract 3473 Introduction/Background:Diamond Blackfan Anaemia (DBA) is a rare bone marrow failure disorder characterised predominantly by severe erythroid (red blood cell) failure. While the causative mutation remains unknown in approximately 50% of DBA patients, a mutation resulting in haploinsufficient expression of one of a number of ribosomal proteins (RPS or RPL) has been identified in the remaining 50% of cases, leading to DBA being classified as a ‘ribosomopathy'(Dianzani & Loreni Haematologica. 2008). Activation of the p53 pathway in response to ribosomal disruptions and nucleolar stress has also been implicated in both animal and cellular models of DBA (Danilova et al. Blood 2008; McGowan et al. Nat. Genet. 2008; Fumagalli et al. Nat. Cell. Bio. 2009). Haploinsufficiency of ribosomal proteins appears to be the major cause of DBA although a recent study (Sankaran VG, et al. J. Clin. Invest. 2012) reported the discovery of mutations in the transcription factor GATA-1, in absence of any known ribosomal protein mutation. While GATA-1 mutations have only been detected in 2 DBA pedigrees to date, this exception to the ‘ribosomopathy' rule may help identify the link between ribosomal protein disruption and the tissue-specificity of the erythroid defect. Results:We have developed a Doxycycline-inducible shRNA RPS19 knockdown model of DBA in the human erythroleukaemic cell line TF1.8. Two independent shRNA triggers targeting RPS19 (the most common gene affected in DBA patients), and a vector-alone control were introduced into cells. RPS19 knockdown was validated by Q-RTPCR which showed a 5-fold decrease in RPS19 mRNA levels upon the addition of 0.25ug/ml Dox (Figure 1). Expression at the protein level was validated by western blotting. Other ribosomal proteins, for example RPL11, are unaffected.The recent discovery of GATA-1 mutations in 2 unrelated DBA pedigrees led us to investigate whether the expression of GATA-1, or other key erythroid transcription factors, may be affected in our RPS19 DBA cell line model. Q-RTPCR results show decreased expression of two key transcription factors (GATA-1 and Myb) in cells with RPS19 knockdown compared to the controls (Figure 1). Furthermore, the expression of the GATA-1 target gene, FOG-1 is decreased compared to controls. MYBBP1A, a Myb target gene involved in ribosomal stress pathways (Yamauchi et al. Genes. Cells 2008) is increased, while another Myb target, GFI-1b is decreased in this model. Discussion/Conclusions:We have generated an inducible RPS19-knockdown model of DBA in TF1.8 cells, and have showed that several key transcription factors important in erythropoiesis (including GATA-1) display down-regulation associated with RPS19-knockdown. This suggests that changes to GATA-1 levels may be important not only in the rare cases of DBA where GATA-1 is mutated, but also in classical DBA associated with ribosomal protein haploinsufficiency. Based on the observation that GATA-1 associates with and inhibits p53 during normal erythropoiesis (Trainor et al. Blood 2009) we propose that the decreased GATA-1 expression may contribute to activation of the p53 pathway during impaired erythropoiesis in Diamond Blackfan Anaemia.Further studies are being undertaken to test this model using assays for transcription factor activity, which will then be further characterised in a DBA model based on CD34+ cord blood cells infected with our validated RPS19 knockdown constructs. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

CD109 Deficiency Induces Preferential Proliferation of the Piga Mutant Leukemia Cell Line TF-1 in the Presence of TGF-β

Abstract 4399 Background:An increase in glycosylphosphatidylinositol-anchored protein-deficient (GPI-AP−) blood cells is a significant predictor of a good response to immunosuppressive therapy in patients with aplastic anemia (AA) and low-risk myelodysplastic syndrome (MDS). Immune mechanisms are therefore thought to be involved in the increase of GPI-AP− cells in such bone marrow (BM) failure, though the exact mechanisms are unknown. The lack of GPI-APs due to PIGA mutations may render hematopoietic stem cells (HSCs) resistant to myelosuppressive cytokines such as TGF-b. This may lead to preferential activation of PIGA mutant HSCs in immune-mediated BM failure, although little is known about the GPI-APs involved. Objectives/Methods:We assessed the roles of GPI-APs in TGF-b-mediated inhibition of cell proliferation, cell cycle, and signal transduction in the myeloid leukemia cell line TF-1, TF-1 with a PIGA mutation (PNH-TF-1), and TF-1 cells transfected with siRNA against CD109, a GPI-AP that serves as a TGF-b co-receptor in human keratinocytes. We used flow cytometry to study expression of CD109 in BM CD34 cells of healthy individuals and other hematopoietic cell lines. Results:TGF-b inhibited PNH-TF-1 proliferation to a lesser degree (percentage of inhibition, 19±13%) than wild-type TF-1 cells (67±3%) in an MTT-based proliferation assay. Transfection of PIGA into PNH-TF-1 cells restored GPI-AP expression as well as sensitivity to TGF-b (53±10% versus 19±13% in PNH-TF-1 cells). CD109 coimmunoprecipitated with TGF-b in TF-1 cells, and its expression was confirmed on BM CD34+ cells of healthy individuals, particularly on CD34+CD38+CD123−CD45RA− megakaryocyte-erythroid progenitor cells, and also on TF-1 and KG1a cells. CD109 knockdown rendered TF-1 cells less sensitive to TGF-b-induced inhibition of proliferation (26±10% versus 67±3%), TGF-b induced cell cycle arrest (a relative increase in the G0/G1 phase of the cell cycle, 6.9% versus 20.7%, Figure) and TGF-b-induced SMAD2 phosphorylation (a relative increase in pSMAD2 MFI, 1±0.3% versus 16%±3%) compared to non-transfected TF-1 cells. Conclusions:The lower sensitivity of PIGA mutant TF-1 cells to TGF-b compared to wild-type TF-1 cells is partially due to CD109 deficiency. This deficiency may also account for the preferential activation of PIGA mutant HSCs in immune-mediated BM failure, in which TGF-b suppresses activation of wild-type HSCs. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Production and characterization of human granulocyte–macrophage colony-stimulating factor (hGM-CSF) expressed in the oleaginous yeast Yarrowia lipolytica

Since its isolation, the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) has been proposed as a new class of therapeutic biological products in the treatment of various diseases. However, the toxicity of this cytokine towards its expression host constitutes a major obstacle to bioprocess development for large-scale production. In this work, the optimized gene encoding hGM-CSF was expressed in the yeast Yarrowia lipolytica in one and two copies under the control of the fatty acid-inducible POX2 promoter. Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): preXALip2. After 48 h of induction, Western blot analysis revealed the presence of a nonglycosylated form of 14.5 kDa and a trail of hGM-CSF hyperglycosylated varying from 23 kDa to more than 60 kDa. The two-copy transformants produced hGM-CSF level which was sevenfold higher compared to the single-copy ones. Deglycosylation with PNGase F showed two forms: a mature form of 14.5 kDa and an unprocessed form of 18 kDa. The addition of two alanines to the signal sequence resulted in correct hGM-CSF processing. The production level was estimated at 250 mg/l after preliminary optimization studies of the cultivation and induction phases. The purified hGM-CSF was identified by N-terminal sequencing and LC-MS/MS analysis; its biological activity was confirmed by stimulating the proliferation of TF1 cell line. This study demonstrated that Y. lipolytica is a promising host for the efficient production of active toxic proteins like hGM-CSF.

On the Nature of the Tumor-Initiating Cell

Certain aspects of tumors that may influence areas of basic biology and medicine are reviewed. The hypothesis that malignant stem cells evolve from normal stem cells, is considered. Information is being accumulated on the possibility that certain cell populations that can be propagated as cell lines in vitro can produce cells with features of differentiated cells in addition to others that maintain the line and, in some cases may also initiate tumor formation in vivo. Up to the present time, there is evidence to show that cancer stem cells persist in many cell lines. Tyrosine kinase inhibition produces combinations of autophagy and apoptosis in the human erythroleukemia cell line TF-1 hinting at a heterotypic aggregation of cells containing cancer stem cells. Finally, the mechanisms of cancer development, invasion and metastasis are operatively defined. The purpose of this paper is to review some of the salient features of cancer stem cells in support of the proposal that research in neoplasia be increased. Rather than presenting details of various studies, we have attempted to indicate general areas in which work has been done or is in progress. It is hoped that this survey of the subject will demonstrate a variety of opportunities for additional research in human neoplasia.

RNAi Screening Identifies BCL-XL As An Erythroid Lineage-Specific 5-Azacytidine Sensitizer While the BCL-2/BCL-XL/BCL-W Inhibitor ABT-737 Results in More Universal Sensitization in Leukemia Cells,

Abstract 3513To identify targets for rational combination therapies with 5-Azacytidine (5-Aza) in myeloid malignancies, we utilized high-throughput RNA-interference (RNAi) viability screening. A siRNA library targeting 572 kinases and a custom collection of 289 putative cancer targets, including cell cycle and apoptosis regulatory genes, were screened alone and in combination with 5-Aza in TF-1 and ML-2 myeloid leukemia cell lines to identify synergistic interactions in reducing cell viability. Of the 572 kinases that were individually silenced, less than 1% significantly increased sensitivity to 5-Aza. The kinase library was also screened in combination with 5-Aza in a third myeloid cell line, THP-1, confirming that few kinases sensitize to 5-Aza when inhibited. While few kinases sensitized to 5-Aza, the anti-apoptotic Bcl-2 family of genes emerged as potent sensitizers to 5-Aza from RNAi screens. Therefore, silencing by siRNA of BCL-XL, BCL-2, BCL-W, MCL-1 and BFL-1 was evaluated in combination with 5-Aza treatment in an expanded panel of myeloid cell lines including TF-1, HEL, THP-1, ML-2 and MDS-L. BCL-XL validated as a vulnerability and potent sensitizer to 5-Aza in erythroid leukemia cell lines TF-1 and HEL, whereas MCL-1 was a strong vulnerability in the monocytic leukemia cell line THP-1 and also a moderate sensitizer to 5-Aza in ML-2, THP-1, TF-1 and HEL. Published proteomics data from our group indicate that M6 and M7 leukemias exhibit higher levels of BCL-XL, while additional unpublished data suggest elevated levels of MCL-1 in M4 and M5 leukemias, supporting our functional observations. Additionally, data from the public database Oncomine suggest that BCL-XL expression is elevated in M6 and M7 leukemias while MCL-1 shows a trend towards elevation in M4 and M5 leukemias. Based on RNAi screening results, siRNA validation experiments and proteomic/mRNA expression data, we evaluated the BCL-2/BCL-XL/BCL-W inhibitor ABT-737 in combination with 5-Aza. ABT-737 resulted in dose-dependent sensitization to 5-Aza in all AML-derived cell lines examined (including M7, M6, M5, M4 and M2 FAB subtypes) and in the MDS cell line MDS-L; however, no sensitization was observed in the CML cell line K562. In extensive ex vivo experiments with 17 primary specimens, potent synergy between 5-Aza and ABT-737 was observed in AML, MDS and MPN samples, but not in most CML samples examined. Calculations with CalcuSyn software demonstrate synergy, with combination index values as low as 0.2, between 5-Aza and ABT-737 both ex vivo and in vitro. The combination of 5-Aza with ABT-737 resulted in substantial induction of apoptosis, measured by the induction of cleaved caspase 3 in TF-1 and HL-60 cells, as compared to either compound alone. Interestingly, although siRNA silencing of MCL-1 in combination with 5-Aza was potent across several cell lines, and silencing of BCL-XL preferentially in an erythroid differentiation background, ABT-737 with 5-Aza sensitized across a variety of cell lines and all myeloid primary specimens ex vivo. We suggest that inhibition of anti-apoptotic Bcl-2 family members is a most promising rational combination strategy with 5-Aza for the treatment of leukemias. Our results also highlight the potential utility of more specific anti-apoptotic Bcl-2 family inhibitors in the lineage-specific treatment of myeloid malignancies. Disclosures:Off Label Use: AraC in AML. Experimental Agent MK1775. Mesa:Incyte: Research Funding; Lilly: Research Funding; SBio: Research Funding; Astra Zeneca: Research Funding; NS Pharma: Research Funding; Celgene: Research Funding.

BCR-ABL Regulates Death Receptor Expression for TNF-Related Apoptosis-Inducing Ligand (TRAIL) in Philadelphia Chromosome-Positive Leukemia

Abstract 2740Allogeneic stem cell transplantation (allo-SCT) is potentially curative therapy for CML and Ph+ ALL patients. Graft-versus-leukemia (GVL) effect plays a central role in eradication of residual leukemia after allo-SCT, and GVL effect is mediated by cytotoxic T-lymphocytes (CTLs) and NK cells though cytotoxic ligands. TNF-related apoptosis-inducing ligand (TRAIL) is one of cytotoxic ligands expressed on CTLs and NK cells, and it has been shown that TRAIL mediates GVL effect in mice model. We and others previously demonstrated that Ph+ leukemic cells frequently express death-inducing receptors (DR4 and DR5) for TRAIL resulting in high sensitivity to pro-apoptotic activity of recombinant human soluble TRAIL (rhsTRAIL), suggesting that TRAIL-DR4/DR5 interaction mediates GVL effect against Ph+ leukemia cells at least in part. Despite the fact that TRAIL mediates tumor-specific immunity and plays a role in GVL effect, mechanisms of the DR4 and DR5 expression in leukemia cells have yet to be elucidated fully. Since we found that Ph+ leukemia cells express DR4 and/or DR5 more frequently than Ph- leukemia cells, we assumed that BCR-ABL plays a role in DR4 and DR5 expression in Ph+ leukemia cells. To verify this assumption, we analyzed the effect of imatinib on DR4 and DR5 expression in Ph+ leukemia cells. Of note, imatinib effectively downregulated gene and cell surface expression of DR4 and DR5 in all of the 12 Ph+ leukemia cell lines tested, while it did not in Ph- leukemia cell lines. Moreover, imatinib downregulated DR4 and DR5 expression on primary samples from Ph+ ALL patients. Not only imatinib but also second-generation TKIs, dasatinib and nilotinib, downregulated DR4 and DR5 expression of Ph+ leukemia cell lines. Imatinib also downregulated promoter activities of the DR4 and DR5 gene in luciferase assay. In contrast, imatinib failed to downregulate gene and cell surface expression of DR4 and DR5 in Ph+ ALL cell line having T315I mutation of BCR-ABL. These observations strongly suggested that BCR-ABL plays a role in DR4 and DR5 expression of Ph+ leukemia cells. To further verify this possibility, we transfected shRNA against bcr-abl using lentivirus vector into Ph+ leukemia cell line, and found downregulation of gene and cell surface expression of DR4 and DR5. Moreover, transfection of bcr-abl into Ph- leukemia cell line TF-1 induced gene and cell surface expression of DR4 and DR5 that was abrogated by imatinib treatment. Since BCR-ABL promotes growth and survival of Ph+ leukemia cells though activation of the MAPK, PI3K, and JAK/STAT pathways, we tested the effect of inhibitor of each pathway on the DR4 and DR5 expression of Ph+ leukemia cell lines. U0126, an inhibitor of the MAPK pathway, and LY294002, an inhibitor of the PI3K pathway, but not SD1029, an inhibitor of the JAK/STAT pathway, partially downregulated gene and cell surface expression of DR4 and DR5. These observations demonstrated a unique finding that an oncogenic fusion product derived form chromosomal translocation is implicated in DR4 and DR5 expression on leukemia cells, and provide new insight into the mechanisms of tumor-specific cytotoxic activities of TRAIL. Considering that imatinib downregulates cell surface expression of DR4 and DR5 in Ph+ leukemia cells, it is assumed that the pro-apoptotic activity of TRAIL against Ph+ leukemia cells may be functionally impaired by pretreatment with imatinib. Thus, we analyzed the effect of imatinib on the sensitivity of Ph+ leukemia cell lines to rhsTRAIL, and found that imatinib pretreatment impaired the pro-apoptotic activity of rhsTRAIL in 6 of the 7 TRAIL-sensitive Ph+ leukemia cell lines tested. Of note, imatinib pretreatment of Ph+ ALL cell line, in which DR4 and DR5 expression was not downregulated due to the T315I mutation, did not impair TRAIL sensitivity whereas imatinib pretreatment of its parental cell line with intact BCR-ABL substantially impaired TRAIL sensitivity. These observations suggested that imatinib partially attenuate the in vivo activity of CTLs and NK cells against Ph+ leukemia cells that is mediated by the TRAIL-DR4/DR5 interaction. Since imatinib is now widely used for prophylaxis of relapse and controlling hematological relapse of patients with CML and Ph+ ALL after allo-SCT, our findings are important from the clinical point of view, and suggest that careful observation is required in the clinical use of imatinib during the post-transplant period. Disclosures:No relevant conflicts of interest to declare.

The inositol 5-phosphatase SHIP1 is a nucleo-cytoplasmic shuttling protein and enzymatically active in cell nuclei

The inositol 5-phosphatase SHIP1 is a negative regulator of signaling processes in hematopoietic cells. SHIP1 mediates its regulatory function after relocalization from the cytoplasm to the plasma membrane where it converts its substrate PI(3,4,5)P3 to PI(3,4)P2 thereby terminating PI3-kinase mediated signaling. In addition, SHIP1 converts Ins(1,3,4,5)P4 to Ins(1,3,4)P3 thereby regulating inositol phosphate metabolism. Here we report, that SHIP1 can be detected in nuclear puncta of Jurkat cells by confocal microscopy after expression of SHIP1 from a tetracycline inducible vector. SHIP1-containing nuclear puncta partially co-localize with FLASH, a multifunctional nuclear protein that has been linked to apoptotic signaling and transcriptional control. Nuclear localization was confirmed for endogenously expressed SHIP1 in the myeloid leukemia cell line TF1. In addition, enzymatically active SHIP1 was found in nuclear fractions of Jurkat cells with a similar specific activity as cytoplasmic SHIP1. Further analysis revealed that SHIP1 is a nucleocytoplasmic shuttling protein which is actively imported into and exported out of the nucleus. Nuclear import is mediated by two canonical nuclear localization signals (NLS) i.e. K327KSK and K547KLR. Mutational inactivation of each NLS motif inhibited nuclear import and reduced the proliferation of cells indicating a functional role of nuclear SHIP1 for cell growth. Our data indicate that SHIP1 is partly localized in the nucleus and suggest that SHIP1 plays a role for nuclear phosphoinositide and/or nuclear inositol phosphate signaling.

Transcriptional regulation of the chemokine co-receptor CCR5 by the cAMP/PKA/CREB pathway

The cyclic adenosine monophosphate (cAMP)-dependent signaling pathway directs the expression of several genes involved in diverse neuroendocrine, immune, metabolic, and developmental pathways. The primary effectors of this pathway are members of the cAMP response element binding (CREB) family of transcription factors, in particular the CREB-1 and cAMP response element modulator (CREM). Both these genes encode alternative splice variants that serve as activators or repressors in a context- and position-specific manner. Although the β-chemokine receptor CC chemokine receptor 5 (CCR5) has been identified on progenitor cells in the bone marrow, the regulatory mechanisms orchestrating its expression are not fully understood. Previous reports have identified putative cAMP response elements in the CCR5 promoter and have described a suppressive role of cAMP in CCR5 expression. In this study, the CD34+CD4+CCR5+ human bone marrow progenitor cell line TF-1 was used to investigate the detailed kinetics of CCR5 transcription in response to the elevation of intracellular cAMP levels and the underlying molecular events. We hypothesize that CCR5 transcription follows an asymmetrical sinusoidal pattern in TF-1 cells that parallels a protein kinase A-dependent alternating change in the ratio of activator pCREB-1-α,Δ to repressor pCREM-α,β isoforms. However, elevated CCR5 mRNA levels do not correlate with enhancement in infectivity with respect to the R5 human immunodeficiency virus type 1 (HIV-1) strain. Our results lend critical insight into the precise mechanism governing the cAMP-CCR5 axis in progenitor cells and pose interesting questions regarding its functional role in HIV-1 infection.

Cloning, soluble expression and purification of high yield recombinant hGMCSF in Escherichia coli.

Expression of human granulocyte macrophage colony stimulating factor (hGMCSF), a cytokine of therapeutic importance, as a thioredoxin (TRX) fusion has been investigated in Escherichia coli BL21 (DE3) codon plus cells. The expression of this protein was low when cloned under the T7 promoter without any fusion tags. High yield of GMCSF was achieved (∼88 mg/L of fermentation broth) in the shake flask when the gene was fused to the E. coli TRX gene. The protein was purified using a single step Ni2+-NTA affinity chromatography and the column bound fusion tag was removed by on-column cleavage with enterokinase. The recombinant hGMCSF was expressed as a soluble and biologically active protein in E. coli, and upon purification, the final yield was ∼44 mg/L in shake flask with a specific activity of 2.3 × 108 U/mg. The results of Western blot and RP-HPLC analyses, along with biological activity using the TF-1 cell line, established the identity of the purified hGMCSF. In this paper, we report the highest yield of hGMCSF expressed in E. coli. The bioreactor study shows that the yield of hGMCSF could be easily scalable with a yield of ∼400 mg/L, opening up new opportunities for large scale production hGMCSF in E. coli.

Inhibition of TET2 Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine Disturbs Myelopoiesis and Granulo-Monocytic Differentiation.

AbstractAbstract 1584TET2, a gene frequently mutated in myeloid malignancies, encodes an oxygenase that may convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in hematopoietic cells. Using high performance liquid chromatography coupled to tandem mass sprectrometry (HPLC-MS-MS), we found lower levels of 5hmC in erythroblasts from patients with myeloproliferative neoplasms and TET2 mutations than in erythroblasts from non mutated patients. To study the function of TET2 during myelopoiesis, we used lentiviruses expressing short hairpin RNA (shRNA) designed to inhibit TET2 expression in CD34+ cells from normal bone marrow or umbilical cord blood. TET2 mRNA was knocked down by 56 to 74% in several human leukemia cell lines expressing shRNA-TET2, compared to cell lines expressing scramble shRNA. TET2 expression was then assessed in these cell lines by western blot, which showed a 49% reduction of TET2 protein in TF1 cell line, and 80 to 90% reduction in Kasumi, Uke1, and Mo7E cell lines. In addition, knock down of TET2 led to a 73% decrease in 5hmC in Mo7E cell line DNA whereas 5mC and cytosine contents remained unchanged as measured by HPLC-MS-MS. These results indicate that TET2 has a role in the hydroxylation of 5mC in human cells of hematopoietic origin. We then studied the consequences of TET2 knock-down in umbilical cord blood CD34+ cells in vitro. We observed a skewing of CD34+CD38- progenitor differentiation toward the myeloid lineage (52 +/− 4% of the TET2 knock down cells versus 35 +/− 3% of the control cells, n=3, p=0.001) at the expense of lympho-myeloid development and B cell and natural killer (NK) lymphoid lineages in a B/myeloid/NK culture condition. Methylcellulose in the presence of EPO, IL3, SCF, G-CSF showed greater numbers of CFU-G/GM (66 +/− 4 versus 58 +/− 3 colonies per 1000 input CD34+ cells) and lower numbers of BFU-E (60 +/−19 versus 78 +/− 19) in TET2 knock-down samples than in control samples (n= 4, p<0.05) indicating that there was also a skewing toward the granulo-monocytic differentiation at the expense of the erythroid lineage. In addition, in presence of G-CSF, IL3, and FLT3-ligand granulocytic differentiation was delayed in TET2 knock-down cells with a relative excess of monocytic cells at day 10 of culture as assessed by the analysis of cell morphology (47 +/− 4% monocytic cells versus 37 +/− 3%, n=5, p=0.006) and immunophenotype (52 +/− 3% of CD14+ cells versus 39+/−8%, n=5, p=0.002). We then analyzed the expression of a set of transcription factors at the mRNA level that confirmed that some transcription factors specific to granulocytic differentiation were under-expressed in TET2 knocked down cells between day 5 and day 10 of culture. Together, our results show that TET2 participates to the conversion of 5mC to 5hmC in hematopoietic cells, and suggest that TET2 inactivation may have a role in the pathogenesis of myeloid malignancies through the disturbance of myeloid differentiation. Disclosures:No relevant conflicts of interest to declare.

Leukemia Inhibitory Factor (LIF) Is Secreted by Marrow-Derived Human Mesenchymal Stem Cells (huMSC) and Inhibits Umbilical Cord Blood (UCB) Hematopoietic Stem Cell (HSC) Proliferation at Early Time Points During In Vitro Expansion.

AbstractAbstract 3843 Background.The defining characteristic of stem cells is their ability for asymmetric division to provide progenitors for specific tissue generation, while maintaining renewal of the stem cell population. LIF protein has been shown to be critical for maintaining embryonic stem cells in an undifferentiated state. UCB HSC in vitro expansion using cytokines has been pursued to augment hematopoietic recovery following UCB transplantation. Our previous studies have shown that a feeder layer of huMSC inhibits UCB HSC proliferation and differentiation during short-term cytokine-driven expansion. We sought to determine whether LIF is secreted by huMSC and if so, at what concentrations, and to elicit its role, if any, in inhibiting cytokine-driven UCB HSC proliferation. Methods.Third passage huMSC were cultured at density 2×10e6/ml in DMEM supplemented with 10% FBS. Supernatant was collected at 24, 36, 48, and 72h and analyzed for LIF secretion by ELISA. UCB was obtained, and MNC were separated on a Histopaque-1077 density gradient. UCB CD133+ cells were isolated using AutoMACS magnetic cell sorter (Miltenyi Biotec) and surface stained for LIF receptor (LIF-R) using anti hLIF-Rα antibody (R&D Systems). LIF-R expression by isolated UCB CD133+ HSC was confirmed by Western blot (n=3). Isolated UCB CD133+ HSCs were plated in 24 well plates at density 3.3×10e3/ml and cultured in StemPro™ media supplemented with 10% FBS, L-glutamine, penicillin, streptomycin and amphotericin B. UCB CD133+ HSCs were culture-expanded for 96h with or without recombinant human LIF (10ng/ml) in a combination of cytokines including: IL-3, IL-6, Flt-3L, SCF, G-CSF, and EPO. At 0, 48, and 96h cell counts were obtained. Given variability of LIF & LIF-R expression by primary human CD133 HSC, we compared the expression of key factors in the LIF signaling pathway between the TF-1 cell line (CD34+/CD38+) and the tumorigenic sub-clone, TF-1a cell line which displays a more primitive HSC phenotype (CD34+/CD38−), using quantitative PCR (qPCR). TF-1 and TF-1a cells (ATCC) were propagated as defined by ATCC. TF-1 cultures were supplemented with human GM-CSF (required for growth of these factor-dependent cells). Cell cultures were maintained at density range 0.3–5×10e5/ml for TF-1 cells and 0.3–3×10e6/ml for TF-1a per ATCC guidelines. Culture aliquots containing equivalent numbers of suspended cells from TF-1 and TF-1a cultures were collected by centrifugation and total RNA prepared (Trizol). RNA was quantified by spectrophotometry and equivalent amounts were used to prepare cDNA by reverse transcription. qPCR was performed in 96-well plates using commercially available Taq-Man primers, with GAPDH used as endogenous control. The assays were run on an ABI 7500 Fast system and SDS software. Comparisons to the control transcript were performed using the 2-(ddCt) method, after verifying baseline, threshold, and completion of reactions as indicated by the plateau phase of the amplification curve. Results.huMSC secreted LIF at all 4 time points, with peak secretion at 48h (mean 52.1(±3.3) pg/ml) (n=3). Surface expression of LIF-R on gated CD133+ cells was 2.61%. At 48h in vitro expansion, higher UCB CD133+ cell counts in cultures without LIF were noted [6.3×10e4 (±0.9)/ml], versus cultures with LIF [4.4×10e4 (±0.8)/ml] suggesting LIF inhibits UCB HSC proliferation. We observed little difference in LIF transcript secreted by TF-1 and TF-1a cells, but a significant down-regulation of LIF-R in TF-1a (RQ=-6.37) compared to TF-1 cells. Similarly, there was a reduction of SOCS3 transcript in TF-1a cells (RQ=-5.23) compared to TF-1 cells. However, expression of Myc, a primary downstream target of LIF-JAK/STAT signaling, did not differ between TF-1 and TF-1a. Conclusions.LIF secretion by huMSC peaks at 48h at a concentration 3 logs lower than that previously used to inhibit embryonic stem cell differentiation (10ng/ml). LIF exerted inhibitory effects on UCB HSC proliferation at early time points (48h) in cytokine-driven in vitro expansion studies. We observed a down-regulation of the LIF-R and the SOCS3 transcript as might be expected. These data are the first to demonstrate and characterize LIF secretion by huMSC. Further studies are ongoing to further clarify cellular pathways involved in the regulation of LIF signaling on UCB CD133+ HSC differentiation during short-term in vitro cytokine expansion. Disclosures:No relevant conflicts of interest to declare.

Phosphatase RNA-Interference Lethality Screening with Suberoylanilide Hyrdoxamic Acid In Myeloid Cell Lines

AbstractAbstract 1851 Background:In previous work we performed RNAi sensitizer screening of epigenetic modulation with 5-Azacytidine and the human kinome. Few kinase hits were identified. Therefore we asked if phosphatases might be involved in modulating epigenetic therapies. Thus in order to identify potentiators of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) we performed high-throughput RNA-interference (RNAi) lethality screening using the myeloid cell lines HEL, THP-1 and TF-1 in combination with SAHA. Methods:A small-interfering RNA (siRNA) library targeting 206 phosphatases with 4 sequences per target gene was used for this study. siRNA were transfected in a single-siRNA-per-well format in 384-well plates using commercial lipid-based transfection reagents. Non-silencing siRNA were included as negative controls to access non-specific siRNA toxicity and universal lethal siRNA were included as positive controls to indirectly assess functional transfection efficiency. After a 96 hour incubation period, cell viability/proliferation was measured with Cell Titer Glo. For analysis of screen data, log2 values of (SAHA + siRNA treatment/siRNA treatment only) were calculated. Potentiation was defined as two standard deviations from the median log2 ratio of all siRNA (excluding positive controls) on a plate-by-plate basis. Hits were defined as ≥ 2/4 siRNA sequences meeting potentiation criteria. Results:Transfection efficiency was 95, 90 and 70% for HEL, THP-1 and TF-1, respectively. All three cell lines were similarly sensitive to SAHA with IC50 values of 0.3, 0.4 and 0.6 μM for HEL, THP-1 and TF-1, respectively. Resulting SAHA concentrations for screens were between the IC5 to IC30. For THP-1, 5 phosphatases qualified according to the hit selection criteria applied indicating strong potentiation to SAHA. Of these 5 hits, 3 did not exhibit strong reductions in viability with siRNA alone, suggesting a SAHA-dependent relationship. For HEL and TF-1, although no phosphatases met the strict selection cutoff, there were several phosphatases exhibiting potentiation, suggesting biological relevance. The primary screening data and preliminary analysis will be validated with rigorous secondary experimentation and top hits for all cell lines will be investigated and advanced. Conclusions:While there are numerous kinase inhibitors in the clinic and in preclinical development, phosphatases are much less explored as therapeutic targets. Validated potentiating targets from this RNAi screen can facilitate the development of rational SAHA combination therapies. Disclosures:No relevant conflicts of interest to declare.

Erythropoietin-Inducible MicroRNA-362 Contributes to Erythropoiesis Via the Suppression of Histone Deacetylase 3.

AbstractAbstract 2603Erythropoietin (EPO) is crucial for various aspects of erythropoiesis, such as proliferation, differentiation and maturation through the regulation of downstream genes including anti-apoptotic regulator, Bcl-XL. However, there is still no answer about the detail list of the downstream genes of EPO. Therefore, we tried to identify EPO-inducible microRNAs (miRNAs) which are a novel class of regulatory molecules. MiRNAs fine-tune gene expression that is likely to be important for a wide range of cellular functions, including the hematopoietic system. We screened specific miRNAs expressed in human leukemia cell lines, UT-7 and its sublines UT-7/GM, UT-7/EPO and UT-7/TPO which are all differentiated from UT-7 cells by the cytokine stimulation. In an EPO-dependent subline, UT-7/EPO, miR-188 and miR-362 were highly expressed compared to the other sublines. An increased expression of both miR-188 and miR-362 in response to stimulation by EPO was observed in human erythroleukemia cell line TF-1 cells and a EPOR expressing FDCP2 cells. Furthermore, we investigated the expression of miR-188 and miR-362 in enhanced erythropoiesis following PHZ-induced hemolytic anemia in mice. The expression of both miRNAs was increased 3- to 6-fold in spleen cells of PHZ-treated mice. In addition, the expression of miR-188 was gradually up-regulated, whereas the expression of miR-362 was down-regulated during erythroid differentiation in the primary culture. These results showed that the expression of miR-188 and miR-362 was induced by EPO stimulation and indicated the involvement of both miRNAs in post-transcriptional regulation during erythropoiesis. To narrow down the putative mRNA targets of miR-362, we used in silico algorithms (TargetScan, Sanger MiRBase, and MiRTarget2), restricting to the erythropoietic process-related genes. According to these criteria, histone deacetylase 3 (HDAC3) emerged as a candidate. Previous study reported that HDAC3 represses the transcription factor GATA-2 that plays essential roles in hematopoietic stem and progenitor cell compartments. Transfection of miR-362 mimic molecule decreased the expression of HDAC3 compared to the negative control (NC) in UT-7 and UT-7/EPO cells. To prove that miR-362 directly recognizes the identical predicted target site in the 3' UTR of HDAC3, that target sequence was cloned into the 3′ UTR of a Renilla luciferase reporter vector to generate HDAC3-UTR-report. MiR-362 mimic molecule significantly downregulated Renilla luciferase activity, demonstrating that miR-362 represses gene expression by directly recognizing the predicted target sequence in the 3′ UTR of HDAC3. Consequently, our study shed the light on a new aspect of EPO mediated erythropiesis, suggesting that EPO-inducible miR-362 regulates the epigenetic status of erythrocyte through the suppression of HDAC3 and the activation of GATA-2. Disclosures:Miyazaki:Kyowa Hakko Kirin Co., Ltd: Employment.

CDDO-Im is a stimulator of megakaryocytic differentiation

Although the triterpene CDDO and its potent derivatives, CDDO-Im and CDDO-Me, are now in phase I/II studies in the treatment of some pathological conditions, their effects on normal hematopoiesis are not known. In the present study we provide evidence that CDDO-Im exerts in vitro a potent inhibitory effect on erythroid cell proliferation and survival and a stimulatory action on megakaryocytic differentiation. The effect of CDDO-Im on erythroid and megakaryocytic differentiation was evaluated both on normal hemopoietic progenitor cells (HPCs) induced to selective erythroid (E) or megakaryocytic (Mk) differentiation and on erythroleukemic cell lines HEL and TF1. The inhibitory effect of CDDO-Im on erythroid cell survival and proliferation is mainly related to a reduced GATA-1 expression. This conclusion is supported by the observation that GATA-1 overexpressing TF1 cells are partially protected from the inhibitory effect of CDDO-Im on cell proliferation and survival. The stimulatory effect of CDDO-Im on normal megakaryopoiesis is seemingly related to upmodulation of GATA2 expression and induction of mitogen-activated protein kinases ERK1/2.

PIM1 kinase is destabilized by ribosomal stress causing inhibition of cell cycle progression

PIM1 is a constitutively active serine/threonine kinase regulated by cytokines, growth factors and hormones. It has been implicated in the control of cell cycle progression and apoptosis and its overexpression has been associated with various kinds of lymphoid and hematopoietic malignancies. The activity of PIM1 is dependent on the phosphorylation of several targets involved in transcription, cell cycle and apoptosis. We have recently observed that PIM1 interacts with ribosomal protein (RP)S19 and cosediments with ribosomes. Defects in ribosome synthesis (ribosomal stress) have been shown to activate a p53-dependent growth arrest response. To investigate if PIM1 could have a role in the response to ribosomal stress, we induced ribosome synthesis alterations in TF-1 and K562 erythroid cell lines. We found that RP deficiency, induced by RNA interference or treatment with inhibitor of nucleolar functions, causes a drastic destabilization of PIM1. The lower level of PIM1 induces an increase in the cell cycle inhibitor p27(Kip1) and blocks cell proliferation even in the absence of p53. Notably, restoring PIM1 level by transfection causes a recovery of cell growth. Our data indicate that PIM1 may act as a sensor for ribosomal stress independently of or in concert with the known p53-dependent mechanisms.

Investigating cryoinjury using simulations and experiments. 1: TF-1 cells during two-step freezing (rapid cooling interrupted with a hold time)

There is significant interest in designing a cryopreservation protocol for hematopoietic stem cells (HSC) which does not rely on dimethyl sulfoxide (Me 2SO) as a cryoprotectant. Computer simulations that describe cellular osmotic responses during cooling and warming can be used to optimize the viability of cryopreserved HSC; however, a better understanding of cellular osmotic parameters is required for these simulations. As a model for HSC, the erythroleukemic human cell line TF-1 was used in this study. Simulations, based on the osmotic properties of TF-1 cells and on the solution properties of the intra- and extracellular compartments, were used to interpret cryoinjury associated with a two-step cryopreservation protocol. Calculated intracellular supercooling was used as an indicator of cryoinjury related to intracellular ice formation. Simulations were applied to the two-step cooling protocol (rapid cooling interrupted with a hold time) for TF-1 cells in the absence of Me 2SO or other cryoprotectants and optimized by minimizing the indicator of cryoinjury. A comparison of simulations and experimental measurements of membrane integrity supports the concept that, for two-step cooling, increasing intracellular supercooling is the primary contributor to potential freezing injury due to the increase in the likelihood of intracellular ice formation. By calculating intracellular supercooling for each step separately and comparing these calculations with cell recovery data, it was demonstrated that it is not optimal simply to limit overall supercooling during two-step freezing procedures. More aptly, appropriate limitations of supercooling differ from the first step to the second step. This study also demonstrates why high cell recovery after cryopreservation could be achieved in the absence of traditional cryoprotectants.

Abstract LB-128: Synthetic lethal RNAi screening identifies inhibition of Bcl-2 family members as sensitizers to 5-Azacytidine in myeloid cells

Abstract In order to identify novel targets and clinically exploitable vulnerabilities, we performed high-throughput RNA-interference (RNAi) synthetic lethality screening using myeloid cell lines TF-1 and ML-2 in combination with the hypomethylating agent 5-Azacytidine. A custom small interfering RNA (siRNA) library targeting nearly 1000 cancer-associated genes, including the human kinome, was utilized. Bcl-XL emerged as the strongest 5-Azacytidine sensitizing target in the erythroid cell line TF-1 but not in ML-2 cells. Additional siRNA sequences targeting Bcl-XL were transfected into a panel of myeloid cell lines and tested in drug dose response experiments using a ten-point serial dilution of 5-Azacytidine. The erythroid differentiated myeloid cell lines TF-1 and HEL both exhibited a 3 to 4-fold reduction in the IC50 of 5-Azacytidine upon knockdown of Bcl-XL, whereas IC50 values for non-erythroid, myeloid cell lines ML-2, THP-1 and MDS-L were unchanged. Furthermore, we tested lentiviral delivery of short-hairpin RNA (shRNA) targeting Bcl-XL in additional cell lines. Lentiviral knockdown of Bcl-XL confirmed 2 to 3-fold 5-Azacytidine sensitization in TF-1 and HEL cells while U937, HL-60, ML-2, THP-1 and MDS-L were not sensitized by Bcl-XL knockdown. Interestingly, while Bcl-XL knockdown with shRNA potentiated the effects of 5-Azacytidine in TF-1 and HEL, Bcl-XL knockdown alone resulted in considerable reductions in cell viability in TF-1 and HEL cells. These results demonstrate erythroid lineage-specific responses to Bcl-XL knockdown and suggest that Bcl-XL may represent an erythroid lineage-specific therapeutic vulnerability. To begin to explore this possibility we examined the putative BH3 mimetics Gossypol, Obatoclax and ABT-737 with 5-Azacytidine drug dose response experiments in myeloid cell lines. Neither Gossypol nor Obatoclax potentiated the effects of 5-Azacytdine. In striking contrast, ABT-737 resulted in a 2 to 4-fold reduction in the IC50 of 5-Azacytidine in all cell lines tested to date (TF-1, HEL, ML-2 and THP-1). ABT-737 sensitization to 5-Azacytidine was dose dependent. Furthermore, Combination Index (CI) values calculated with Calcusyn software demonstrate synergy between ABT-737 and 5-Azacytidine at clinically relevant concentrations. These results suggest that inhibition of anti-apoptotic Bcl-2 family members with a BH3 mimetic may represent an effective strategy for 5-Azacytidine combination therapy that can be directly translated into design of combination clinical trials in patients with myeloid leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-128.

Abstract LB-127: Synthetic lethal RNAi screen of the human kinome with cytarabine (AraC) in leukemia cells

Abstract Cytarabine is the backbone of therapy for acute myeloid leukemia (AML) patients, yet due to a high incidence of relapse and treatment-related complications novel, less toxic treatment regimens incorporating AraC need to be designed. To increase the therapeutic effect of cytarabine we developed and performed an RNAi synthetic lethal screening assay using siRNA, targeting 572 human kinases in combination with AraC in human myeloid cell lines TF-1 and THP-1. Several genes that enhance the effectiveness of cytarabine were identified. Among the top candidate genes, a trend became apparent in which several key regulators of the cell cycle as well as DNA repair genes where among the strongest sensitizers (CHEK1, PKMYT, CDC2, AURKB and ATR). The effectiveness of targeting these genes in combination with AraC was verified using a highly robust siRNA drug-dose response assay (siDDR) that validated CHEK1 as strong sensitizer, PKMYT as moderate sensitizer, and did not confirm CDC2 or AURKB as sensitizers. Since CHEK1 is emerging as a potential treatment target in combination chemotherapy in solid tumors, we obtained two commercially available CHEK1 inhibitors, PD 407824 and SB 218078 (both Tocris). PD 407824 strongly sensitized to AraC in the range of 2 to 10 fold decrease in several lines (MV 4;11, THP-1, HL60, HEL, ML-2) whereas in a few no sensitization was observed (MDS-L, U937). Calcusyn software was utilized to ensure the effect was more than simply additive in effect. Less effect was observed for SB 218078, which was surprisingly found to be antagonistic in some lines (TF-1, U937, ML-2, HL-60). Both compounds have slightly different target spectrum's that include CHEK1 plus other kinases. Interestingly, the sequence of addition of the compounds dramatically affected the outcome, with the greatest effects seen when CHEK1 inhibitors were added before AraC. In cases where AraC was added first or simultaneously, the effect was less pronounced or was not seen at all. Herein we present the “first-in-class” human kinome synthetic lethal screen in leukemia suspension cells in combination with AraC. In this unbiased functional genomics screen a strong convergence of hits emerged around G2/M cell cycle and DNA repair control. This information can immediately inform clinical drug development, channel resources and serve to direct development of new therapies in acute myeloid leukemias combining AraC with the first clinical available CHEK1 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-127.

Putative innate immunity of antiatherogenic paraoxanase-2 via STAT5 signal transduction in HIV-1 infection of hematopoietic TF-1 cells and in SCID-hu mice.

Paraoxanase-2 (PON2) activity was increased upon HIV-1 infection of the CD34+CD4+ hematopoietic cell line TF-1. Thymocytes derived from the human fetal conjoint thymus/liver hematopoietic organ of SCID-hu mice also exhibited an increase in PON2 activity. Additionally, a remarkable increase of PON2 mRNA expression was also observed in both TF-1 and thymocytes following HIV-1 infection. The phosphorylation of STAT5 was decreased in TF-1 cells upon HIV-1 infection. Interestingly, phosphorylation of STAT5 does not occur in GM-CSF "starved" TF-1 cells; however, PON2 protein, activity and mRNA expression are increased under these conditions, similar to HIV-1 infection. We conclude that PON2 is induced in HIV-1 infection through a mechanism that may involve STAT5 inactivation.

Effect of cell lysates on retroviral transduction efficiency of cells in suspension culture

Recombinant retroviruses are effective vectors able to integrate transgenes into the target cell's genome to achieve longer-term expression. This study investigates the effect of cell lysis products, a common cell culture by-product, on the transduction of suspension cells by gammaretroviral vectors. Cell lysates derived from human and murine suspension cell lines significantly increased the transduction of human TF-1 and K-562 cell lines by gibbon ape leukemia virus-pseudotyped retroviral vectors without altering tropism. The transduction efficiency of TF-1 cells increased as a function of lysate concentration and decreased with increasing target cell concentrations. This was adequately predicted using a saturation equation based on the lysed-to-target cell concentration ratio, R, where: Fold increase = 1+Fold_(Max) (R/(K_(L)+R)). Lysate completely masked the effects of fibronectin when the two were added in combination. With protamine sulfate, the transduction efficiency was increased by lysate to 58% from 20% for protamine sulfate alone. Overall, the presence of cell lysate significantly influenced the outcome of the transduction process, either alone or in the presence of protamine sulfate or fibronectin.